Date: March 7-8, 2018
Venue: 6th Floor, UGSAS Building, Gifu University, JapanThe United Graduate School of Agricultural Science, Gifu University

Organizing Committee
Gifu University
Prof. Masateru SENGE(Dean of UGSAS-GU)

Prof. Kohei NAKANO(Vice Dean of UGSAS-GU)

Prof. Ken HIRAMATSU(Faculty of Applied Biological Sciences)

Assoc. Prof. Masafumi SHIMIZU(Faculty of Applied Biological Sciences)

Assoc. Prof. Haruhisa SUGA(Life Science Research Center)

Prof. Koji KAGEYAMA(River Basin Research Center)

Mr. Akiharu NAKAMINE(Section Chief, Administrative Section of
UGSAS-GU)

Ms. Ayano IMAI

(Administrative Section of UGSAS-GU)

Ms. Satoko YOSHIDA(Administrative Section of UGSAS-GU)

Ms. Tomoko KASAI(Administrative Section of UGSAS-GU)* This symposium is supported by IC-GU12.

General Information
Venue
The United Graduated School of Agricultural Science (UGSAS), Gifu University
1-1 Yanagido, Gifu 501-1193, Japan
Tel: +81-58-293-2984 (or, 2985)
E-mail: renno@gifu-u.ac.jp
Home page: http://www.ugsas.gifu-u.ac.jp/eng/

AccessDaily schedule
March 7th(Wed)

9:30–10:00

Registration

10:00–10:05

Opening remarks
Dr. Masateru Senge(Dean of UGSAS, Gifu University)

10:05–10:10

Welcome speech
Dr. Fumiaki Suzuki(Executive Director and Vice President of Gifu University)

10:10–10:30

Special guest speech
Dr. Shirley C. Agrupis(President of Mariano Marcos State University)

March 8th(Thu)

10:30–10:40

Photo session

10:40–11:40

Poster presentation & free discussion

11:40–12:10

Plenary lecture 1

12:10–13:30

Lunch break & poster viewing

13:30–14:00

Plenary lecture 2

14:00–15:15

Oral session 1

15:15–15:30

Coffee break& poster presentation

15:30–17:10

Oral session 2

17:40–19:00

Dinner meeting

9:30–10:00

Registration

10:00–10:30

Plenary lecture 3

10:30–11:45

Oral session 3

11:45–12:45

Lunch break & poster viewing

12:45–13:15

Plenary lecture 4

13:15–14:55

Oral session 4

14:55–15:10

Coffee break & poster presentation

15:10–16:50

Oral session 5

16:50–17:00

Closing remarks
Dr. Kohei Nakano(Gifu University)

Plenary lectures (Main seminar room, 6 floor of UGSAS-GU Building)thPL-1 (March 7th, 11:40–12:10)
Chair: Dr. Haruhisa Suga (Gifu University)

Dr. Sotaro Chiba (Nagoya University)“Farmer Field Schools leading to sustainable management of insect pests in
Cambodian rice fields”

PL-2 (March 7th, 13:30–14:00)
Chair: Dr. Koji Kageyama (Gifu University)

Dr. Masafumi Shimizu (Gifu University)“Endophytic Streptomyces: attractive biocontrol agents”

PL-3 (March 8th, 10:00–10:30)
Chair: Dr. Koji Kageyama (Gifu University)

Dr. Haruhisa Suga (Gifu University)“Molecular characterization of Fusarium fujikuroi in Japan”

PL-4 (March 8th, 12:45–13:15)
Chair: Dr. Masafumi Shimizu (Gifu University)

Dr. Shigenobu Yoshida (National Agriculture and Food Research

Organization)“Perspective on the development of biopesticides applicable to both
agricultural insect pests and disease”

Oral sessions –Day 1– (Main seminar room, 6 floor of UGSAS-GU Building)th

OS I : Current status and management of crop diseases in Indonesia

March 7th, 14:00–15:15
Chair: Dr. Yuyun Fitriana (Lampung Univ.)
OS I-1(14:00–14:25)

Dr. Achmadi Priyatmojo (Gadjah Mada University)“Current status and management of Rhizoctonia solani, the causal

pathogen of sheath blight disease on rice and maize in Indonesia”

OS I-2

Ms. Hanifah Ihsaniyati (Sebelas Maret University)(14:25–14:50)“Indonesian farmers problems in implementing integrated pest
management (IPM)”

OS I-3

Ms. Dwiwiyati Nurul Septariani (Sebelas Maret University)(14:50–15:15)“Taxonomical studies of blood disease bacterium of banana”

OS II : Plant probiotic bacteria

March 7th, 15:30–17:10
Chair: Dr. Md. Motaher Hossain (BSMRA Univ.)
OS II-1

Dr. Tri Joko (Gadjah Mada University)(15:30–15:55)“Bacterial endophytes isolated from orchids and their influence on
plant health”

OS II-2

Dr. Radix Suharjo (Lampung University)

OS II-3

Dr. Hadiwiyono (Sebelas Maret University)

OS II-4

Dr. Md. Rashidul Islam (Bangladesh Agricultural University)(15:55–16:20)(16:20–16:45)(16:45–17:10)“Potential of endophytic bacteria as plant growth promoter andantagonist against pineapple-fungal plant pathogen in Indonesia”“Endophytic Bacillus as biological control agent of banana wilt”“Molecular based identification and formulation of cyanogenic
Pseudomonas spp. controlling Phytophthora infestans”

Oral sessions –Day 2– (Main seminar room, 6 floor of UGSAS-GU Building)th

OS III : Natural product-based pesticides and physical control measures

March 8th, 10:30–11:45
Chair: Dr. Tri Joko (Gadjah Mada Univ.)
OS III-1

Dr. Siti Subandiyah (Gadjah Mada University)

OS III-2

Dr. Pongphen Jitreerat (King Mongkut’s University of Technology(10:30–10:55)“Utilization of chitosan and glukomanan for fruit coating of chili
againts antrachnose disease”(10:55–11:20)

Thonburi)“Antifungal effects of ethanolic shellac - Modified coconut oil (ESMCO) combined with physical treatments against postharvest
diseases of mango and mangosteen”

OS III-3

Dr. Kanlaya Sripong (King Mongkut’s University of Technology(11:20–11:45)

Thonburi)“Enhancing plant defense in mango fruit by hot water and UV-C
treatments”

OS IV : Plant probiotic fungi

March 8th, 13:15–14:55
Chair: Dr. Achmadi Priyatmojo (Gadjah Mada Univ.)
OS IV-1

Dr. Moslama Aktar Maya (British American Tobacco Bangladesh(13:15–13:40)

Limited)“Management of fusarium wilt in cyclamen plants using multiple
soil microbes (AMF and Piriformospora indica)”

OS IV-2

Dr. Maria Viva Rini (Lampung University)(13:40–14:05)“Mycorrhizal oil palms seedlings response to different sources of
Ganoderma boninense as the causal agent of basal stem rot
disease”

OS IV-3

Dr. Purnomo (Lampung University)(14:05–14:30)“Potency of watery extract compost plus Beauveria sp. after storage
for controlling planthopper and rice bug”

OS IV-4

Dr. Yuyun Fitriana (Lampung University)“Low pH-tolerant mutant of Trichoderma spp. induced by EMS,

(14:30–14:55)

gamma rays and UV irradiation”

Oral sessions –Day 2– (Main seminar room, 6 floor of UGSAS-GU Building)th

OS V : Resistant cultivars

March 8th, 15:10–16:50
Chair: Dr. Pongphen Jitareerat (KMUTT)OS V-1

Dr. Abu Shamim Mohammad Nahiyan (Advanced Seed Research(15:10–15:35)& Biotech Centre)“Management of rice and wheat blast pathogen in Bangladesh”

OS V-2

Dr. Triwidodo Arwiyanto (Gadjah Mada University)

OS V-3

Dr. Md. Motaher Hossain (Bangabandhu Sheikh Mujibur Rahman

OS V-4

Dr. Nandariyah (Sebelas Maret University)“In vitro selection of sugarcane (Saccharum officinarum L) for(15:35–16:00)(16:00–16:25)(16:25–16:50)“Control of eggplant and tomato bacterial wilt by grafting inIndonesia”

Agricultural University)“Detection and characterization of Asia soybean rust in
Bangladesh”

Fusarium-pokah bung (Pb) resistance”

Poster session (Seminar room, 6 floor of UGSAS-GU Building)th

P-1

Fumonisin production recovery in a Fusarium fujikuroi strain bycomplementation of FUM21, FUM6 and FUM7 genes

Sharmin Sultana, Hironori Kobayashi, Ryuou Yamaguchi, Masafumi Shimizu, Koji
Kageyama, Haruhisa Suga

P-2

Genetic mapping of chromosome No.1 region associated withpathogenicity in Fusarium head blight pathogen

Rina Okumura, Maho Ikawa, Yuki Hirata, Masafumi Shimizu, Koji Kageyama, Haruhisa
Suga

P-3

Isolation of plant probiotic Bacillus spp. from tea rhizosphere

P-4

Microbial basis of Fusarium wilt suppression by Allium-cultivated soils

P-5

Isolation of novel deoxynivalenol-degrading microorganisms from Poaceae
planted soils

Nusrat Ahsan, Tomoki Nishioka, Haruhisa Suga, Hiroyuki Koyama, Masafumi Shimizu
Tomoki Nishioka, Malek Marian, Haruhisa Suga, Masafumi Shimizu

Hiroyuki Morimura, Sotaro Chiba, Daigo Takemoto, Kazuhito Kawakita, Ikuo Sato

P-6

Plant growth-promoting traits of rhizospheric Flavobacterium and Chryseobacterium
Fumiya Mizutani, Tomoki Nishioka, Haruhisa Suga, Koji Kageyama, Masafumi Shimizu

P-7

Establishment of global Phytophthora database for quarantine control

P-8Morphological and molecular identification of causal agent of cocoa pod
rot disease in Indonesia

Ayaka Hieno, Mingzhu Li, Kayoko Otsubo, Haruhisa Suga, Koji Kageyama

Masanto, Ayaka Hieno, Arif Wibowo, Siti Subandiyah, Masafumi Shimizu, Haruhisa Suga,
Koji Kageyama

P-9

Biocontrol of tomato bacterial wilt using Ralstonia and Mitsuaria species

P-10

Comprehensive evaluation of the resistance of root-stock-used Cucumis
melo stock to Meloidogyne incongnita

Malek Marian, Tomoki Nishioka, Hiroyuki Koyama, Haruhisa Suga, Masafumi Shimizu

Wanxue BAO

P-11

Population genetics analysis of Phytopythium helicoides in Japan

P-12

Study of a transcriptional regulator of plant pathogenic genes in a soft rot
disease causing bacterium, Dickeya dadantii

Auliana Afandi, Emi Murayama, Ayaka Hieno, Haruhisa Suga, Koji Kageyama

Dina Istiqomah, Naoto Ogawa

Poster session (Seminar room, 6 floor of UGSAS-GU Building)
th

P-13

Identification of freshness marker of stored soybean sprouts

P-14

Studies on acetaldehyde tolerance system in the budding yeast using myoinositol

Syukri, D., Thammawong, M., Kuroki, S., Tsuta, M., Yoshida, M., Nakano, K.

Annisyia Zarina Putri, Mizuho Inagaki, Masaya Shimada, Takashi Hayakawa, Tomoyuki
Nakagawa

P-15

Identification of bioaerosols from environmental samples in the AIST,
Tsukuba, Japan

Panyapon Pumkaeo, Wenhao Lu, Youki Endou, Tomohiro Mizuno, Junko Takahashi,
Hitoshi Iwahashi

P-16

The effect of persimmon (Diospyros kaki) on the prevention of sarcopenia

P-17

Transcriptional biomarkers for managing pulse crop production in acid soil
regionNayla Majeda Alfarafisa, Tomio Yabe

Raj kishan Agrahari, Hiroyuki Koyama

P-18

The accumulation of carotenoid in mango during fruit maturation

P-19

Augmented nuclease resistance and gene silencing with 3’-end modified
small interfering RNAs and dendrimer based drug delivery

W. Yungyuen, T.T. Vo, G. Ma, L.C. Zhang, P. Jitareerat, A. Uthairatanakij, M. Kato

Akash Chandela, Yoshihito Ueno

P-20

Protein-based functional analysis of renin and (pro)renin receptor genes inhypertensive and diabetic Bangladeshi population: Pursuing theenvironment-induced molecular traits
Jobaida Akther, A. H. M. Nurun Nabi, Tsutomu Nakagawa, Fumiaki Suzuki, Akio Ebihara

P-21

Proposals for countermeasures to reduce risk of hydraulic fracturing
adjacent to culvert – A case study

Duy Quan Tran, Shinichi Nishimura, Masateru Senge, Tatsuro Nishiyama, Fumitoshi
Imaizumi

P-22

Droughts hotspot distribution by long term assessment the Standardized
Precipitation Index (SPI) in Indonesia
Yudhi Pramudya, Takeo Onishi

P-23

The role of floral volatiles for attracting pollinators and reproductive
isolation in Mimulus species
Muhammad Arifin, Tomoko Okamoto

Plenary Lecture Abstracts

-1-

-2-

PL-1
Farmer Field Schools leading to sustainable management of insect pests
in Cambodian rice fields
1,2
2,3
Sotaro CHIBA , Chhay NGIN , Seng SUON4, Toshiharu TANAKA1,2, Editha C CEDICOL2,
Akira YAMAUCHI1,5, Kazuhito KAWAKITA1(1Graduate School of Bioagricultural Sciences, Nagoya University, 2Asian Satellite Campuses Institute, Nagoya
University, 3General Directorate of Agriculture, Ministory of Agriculture, Forestry and Fisheries of Cambodia, 4Center
for Development-Oriented Research in Agriculture and Livelihood Systems, 5International Cooperation Center for
Agricultural Education, Nagoya University)

SUMMARY
Agro-chemicals has brought benefit in rice productivity and management, especially for easy control of insect pests byintensive treatments of insecticides. However, undesired side-effects of agro-chemicals are appeared problematic:
environmental degradation, elimination of natural biocontrol agents, poisoning pesticide applicators, and declined foodsafety. This study demonstrates effectiveness of Farmer Field School (FFS) as a farmer education system for
implementing Integrated Pest Management (IPM) in Cambodian rice fields, to accomplish improved profitability by
reducing insecticide cost and to sustain field natural enemies for insect pest control. Field observation leveled an
intriguing fact that most of Cambodian rice fields are rich enough in natural enemy population to autonomously control
insect pest. The IPM-FFS also contributed to decrease farmers’ exposure risk to toxic chemicals. This paper is based on
the thesis of Dr. Chhay Ngin who had studied under the Transnational Doctoral Program for Leading Professionals in
Asia, Nagoya University Asian Satellite Campuses Institute.

Introduction

ways to achieve this, we bereave, is by implementing

Rice is the staple food of Asia and is central to thesuitable Integrated Pest Management (IPM) practices tofood security of about half of the world’s population.

reduce reliance on pesticide use and moving towards

Rice production is an important source of livelihood forfood production methods based on the effective use of

approximately 140 million rice-farming households. As

ecosystem services that are regenerative and that

Cambodia’s staple food crop, rice has provided income

minimize negative impacts. Such an implementation of

to majority of the rural population and has contributed to

IPM in Cambodian farmers has been attempted by thethe national economy. It is predominantly grown on moregovernment-leading farmers’ education program called

than three million hectares comprising 75% of the‘Farmer Field School’ (FFS).

agricultural land in Cambodia.

In this study, we assessed impacts of IPM-FFS in terms

Major challenges include the need to produce moreof natural enemy populations in the rice fields, rice

rice to meet the rising demand from world population

farmers’ profitability (cost effectiveness), and reduction

growth that is expected to reach 9.6 billion in 2050;

in farmers’ risk of exposure to toxic pesticides. To thisenvironmental degradation; decline in rice biodiversity;end, three independent but co-related subjects wereclimate change and increasing competition for land,established and conducted as follows: 1) investigation on

labour and water. Increase in crop yield has often beeneffect of insecticide-treatments in rice filed on arthropod

attained through the exhaustive application of pesticides

population and on economic balance, 2) elucidating

and fertilizers. However, the indiscriminate use of theseeffectiveness of technology intervention by FFS-training

chemicals damages the environment and human healthon rice production, cost performance, and farmers’

while jeopardising agricultural production.

profitability, and 3) unveiling long-term impact of FFS

For increasing crop production profitability while

by interviewing FFS-participants whether they retain

reducing the risks from pesticides to humans and thetheir capability developed during FFS, those briefly

environment, new ways need to be explored. One of theschematically represented in Fig. 1.

-3-

IPM practice directed in Cambodian FFSs
The FFS is used as an extension tool to facilitate
farmers’ skill on IPM and modern agriculture. In FFS,
farmers learn about knowledge-intensive management
techniques/approaches with on-site participatory learning
style. In Cambodia, the first implementation of FFS wasin 1996. While it was said that the FFS program in
Cambodia has been successfully improved farmers, there
was no scientific evidence provided before. Thus, thestudy assessed effectiveness of IPM–FFS training on the
production efficiencies (yields and profitability) of rice
farming in three provinces by comparing the technicallyFig. 1 Research scheme of three independent but
related studies.

recommended practices (TRs) with the traditional farmer

Rich natural enemy population controllinginsect pests in Cambodian rice fields

operated during dry, early-wet, and wet seasons inIn three provincial research stations, IR66 rice variety

In general, TR-plot received higher rice yield per ha

was planted during dry season (January-April) in 2015

than FP-plots in all provinces, seasons and years, and itwith either of treatments: non-treatment (control),showed reverse trends for total expenses. This resulted in

IPM-treatment, and insecticide- (Cypermethrin; 2 L/ha)

greater net profit in TR-plot, i.e., maximum difference

treatments with 3- or 4-times spray. Then major pest

between TR- and FP-plots was 399 USD/ha in dry

insects (caseworms, stem borers, leaffolders, gall midges,

season (785 vs 386, USD/ha). The cost factors thatbrown planthoppers, and rice bugs) and natural enemies

FP-plots expensed more were seeds (15-45 USD/ha(spiders, dragonflies/damselflies, lady beetles, grounddifference) and pesticide (22-42 USD/ha dif.), andbeetles/earwigs,

grasshoppers/crickets,

TR-plots conversely fairly did for fertilizers (5-13

mirid bugs, and water bugs) were each counted weekly

USD/ha dif.). To determine cost factors contributing tofrom 15 to 67 days after transplanting (DAT) (Fig. 1).

rice yields in TR-plots, multiple regression analysis was

long-horned

practices (FPs). Data from 270 randomly selected FFSs,
2012-2014 were used for statistical analyses (Fig. 1).

In brief, total numbers of insect pests were always

performed, and it revealed that the costs for field

below that of natural enemies, and rice productivities

management (land leveling, watering, etc.) and fertilizer

were at similar levels between non-/IPM-treated plots

application (fertilizer and labour hiring costs) wereand insecticide-treated plots regardless of spray times insignificantly contributed, but not pesticide application

two provinces. An incident of caseworm was occurredcosts. Thus, together with right management of fields

and damaged rice plants in another province, and thisand fertilizers, the IPM-based insect pest control

decreasedincontributed to gain higher profit. These results suggested

insecticide-treated plots. However, IPM-based coping tothat FFSs conducted in Cambodia 2012-2014 were

caseworm successfully controlled the damage and gained

effective enough to convincingly encourage participants

equivalent harvest with insecticide-treated plots. Overall,

for applying IPM technology in their own farms.

production

compared

with

those

representative rice fields in Cambodia are covered byunder autonomous control of them. Even though some

IPM practices taken root in Cambodian
FFS-participants

pest outbreak occurs, an appropriate IMP-treatment can

The development of a sustainable agricultural program

manage this and sustain rice production. Nevertheless,is a challenging task. Although abovementioned FFS

saving the cost for insecticides directly reflected higher

program in Cambodia was effective to demonstrate aprofitability

inmerit of IPM and modern agricultural practice to farmers,

insecticide-treated plots. This encouraging fact highlights

whether this leads to capacity building of farmers has not

the benefit of conservation of natural enemies inbeen evaluated since the first introduced of IPM-FFS in

Cambodian rice fields, as classically stated this important

Cambodia in 1996. As poisoning of farmers by toxic

natural resource and function ‘ecosystem services.’

pesticides have long been serious concern, the IPM-FFS

rich population of natural enemies and pest insects areinnon-/IPM-treated

plots

than

-4-

is expected to be one of solutions which guide farmers totechniques based on the field realities. In this study, we

reduce pesticide use and protect themselves as well. In

evidenced that the IPM-FFS is one of the best

this study, we examined short-term (2-year, 2010) andeducational paradigms for improving rice production in

long-term (7-year, 2015) impacts of an FFS in Prey Veng

Cambodia in a sustainable way which includes technical,

province, by comparing baseline data obtained during

economic, social and environmental aspects. ThisFFS in 2008 (Fig. 1). To this end, three farmers groups

combined approach can be empowered by appropriate(30 farmers each) were interviewed and comparably

strategy and conducive policy on ecology-based

analyzed: FFS-trained farmers, non-trained farmers inagriculture production. We strongly hope that thethe same village and in a distant village where FFS has

Cambodian government pays attention to the researchnever been implemented.

outcome and leads to a more sustainable andBy looking at trends of farmers’ behavior on

cost-effective rice production, a risk reduction to public

protection of themselves from toxic pesticides, allhealth in terms of reduced toxic residues in food, an

farmers groups showed significant increases in the rate

improvement in field biodiversity and marketability ofofboots;

the products contributing to enhanced rice productivity,

head-protective items are almost 100% used from thean improvement in the farmers’ livelihoods and,beginning). Likewise, rate of farmers adopting theeventually, acceleration of national economic growth.

using

protective

ware(grovesandpractices of burying/burning pesticide waste, the rate of

Acknowledgment

pesticide applicators experienced the pesticide poisoning,This study is supported in part by Southeast AsianRegional Center for Graduate Study and Research inAgriculture (SEARCA, Philippines), the Transnational
doctoral program for leading professionals in Asian
countries (Nagoya University ASCI), and a grant-in-aid
for Scientific Research (A) from the Japanese Ministry of
Education, Culture, Sports, Science, and Technology(KAKENHI). We express sincere thanks to Dr. Buyung
Hadi (IRRI, Philippines), Mr. Jan Willem Ketelaar (FAO,
Thailand), Dr. Ikuo Sato, Dr. Daigo Takemoto (Nagoya
University), and Provincial IPM trainers (Cambodian
National IPM Program) for their kind support andfruitful discussions.

and calculated potential risk of pesticide applicators werealso commonly decrease year by year. Of note is, thesefour parameters were always better in FFS-trained
farmers than non-trained farmers in the same or outervillage, with one exception of FFS-trained farmers thatexperienced poisoning at baseline survey. This exception
might come from fear to pesticide toxicity after taking
education in the FFS.
A remarkable difference was observed in the amount of
total pesticide use. That is, while FFS-trained farmers
had a reduced pesticide usage over the short-term andRelated articles

continued to reduce their usage over the long-term, two

Ngin C, Suon S, Tanaka T, Yamauchi A, Cedicol CE,
Kawakita K, Chiba S. (2017) Rice productivity
improvement in Cambodia through application oftechnical recommendation in Farmer Field School.
International Journal of Agricultural Sustainability
Vol. 15 (1), 54-69.

control farmers groups had a reduced pesticide usage
over the short-term but showed an increased usage at thelong-term survey. In the meantime, FFS-trained farmers
sustained greater net profit which gradually increased
2008-2015, seemingly by decreased pesticide usage.

Ngin C, Suon S, Tanaka T, Yamauchi A, Kawakita K,
Chiba S. (2017) Effects of mechanical defoliation anddetillering at different growth stages on rice yield in
dry season in Cambodia. International Journal of
Agriculture and Environmental Research 3 (4),3452-3470.

Therefore, working towards a non-toxic environment is a
viable option for achieving sustainable production,
maintaining a healthy environment, protecting humanhealth and attaining income security for rice producers in

Ngin C, Suon S, Tanaka T, Yamauchi A, Kawakita K,
Chiba S. (2017) Impact of insecticide applications onarthropod predators and plant feeders in Cambodian
rice fields. Phytobiomes journal (in press, publishedon-lineaheadofprint:
https://doi.org/10.1094/PBIOMES-01-17-0002-R)

Cambodia.

Concluding remarks
IPM provides a knowledge-intensive integrated cropand pest management approach, whereas FFS training

Ngin C, Suon S, Chou C, Tanaka T, Kawakita K, Chiba S.
Long term impact of Farmer Field School on
pesticide reduction and productivity improvement in
Cambodian rice cropping. (submitted)

provides opportunities for farmers to learn and develop
their technical capacity and informed decision-making
skills to become local experts to build economically
viable and environmentally friendly crop management

-5-

-6-

PL-2
Endophytic Streptomyces: Attractive biocontrol agents
Masafumi SHIMIZU(Faculty of Applied Biological Sciences, Gifu University)

Abstract
Almost all terrestrial plants are known to harbor large and diverse microbial communities within their bodies.
Microbes that have an ability to endophytically colonize plant tissues play important roles in the growth and health oftheir host plants. Recent metagenomics analysis revealed that actinobacteria, particularly the genus Streptomyces, are oneof the most abundant groups in the endophytic bacterial communities present in a wide variety of plant species.
Streptomyces species are ubiquitous in the natural environment, particularly in soils, and are well-known producers of
bioactive secondary metabolites, including antimicrobials, that are effective against plant pathogens. Therefore, since along time, antagonistic Streptomyces species have often been isolated from soils and used for the biocontrol of soil-borne
diseases. Over the last two decades, the global attention on the biocontrol of plant diseases using mutualistic endophytic
Streptomyces has been increasing. Although the importance of a vast majority of the endophytic Streptomyces species inplant growth, development and ecological fitness is still unclear, some of them have been found to have a great potential
to defend host plants from pathogens. We isolated and characterized several endophytic Streptomyces species that caneffectively suppress various plant diseases.
In this lecture, the speaker will describe an example of the biocontrol of plant diseases using our isolate ofendophytic Streptomyces and its possible mode of action.

-7-

-8-

PL-3
Molecular characterization of Fusarium fujikuroi in Japan
Haruhisa SUGA
( Life Science Research Center, Gifu University )SUMMARY
Rice bakanae is a serious plant disease in rice growing country. Fusarium fujikuroi is known as a fungal pathogen of
the rice bakanae disease and also as an important mycotoxin producer such as fumonisins. This fungal species has been
isolated not only from rice but also from other crops. We obtained 95 strains of F. fujikuroi most of which were fromvarious source in Japan and performed phylogenetic analyses based on AFLP finger printing pattern to demonstrate
genetic relationship of individual strains. F. fujikuroi strains used in this study were divided into two geneticallydifferent groups and difference of fumonisin and gibberellin producibility was observed between them. These result
indicates F. fujikuroi comprises two different hazardous groups in the sense of plant pathogens and food safety.
species Fusarium verticillioides is the predominant

Introduction
Rice is one of the most important grains andfungal contaminant in the corn in the world. Basically,

approximately 480 million tons of rice is produced in thefumonisin production can be detected in all F.

world every year. Rice bakanae disease occurs in almost

verticillioides strains while in case of F. fujikuroi,

all rice growing area. Diseased rice are slender, a palefumonisin production can be detected only in someyellowish color leaves bending and taller than undiseased

strains (Suga et al. 2014). In this study, strains that do notplants.

produce detectable level of fumonisins under in vitro

Fusarium fujikuroi, a fungal pathogen of the riceculturing are tentatively referred to as fumonisinbakanae disease, is one of the species in Fusarium

non-producers. Clustering of sixteen genes involving

fujikuroi species complex (Ff complex). Ff complex

fumonisin biosynthesis has been revealed in F.

includes more than 50 phylogenetical species, in which

verticillioides (FUM cluster) and a similar cluster was

thirteen independent mating populations (MPs) havedetected in the whole genome sequence of F. fujikuroi

been discovered and MP-C corresponds to F. fujikuroi. F.

(Wiemann et al. 2013). However, the FUM cluster was

fujikuroi produce a large variety of secondary

detected not only in fumonisin producers but also inmetabolites such as bikaberin, fusaric acid and fusarbin.

non-producers. Therefore, fumonisin production ofAmong them, gibberellins and fumonisins have been

non-producers of F. fujikuroi is still ambiguous andintensively studied. Gibberellin currently well known as

genetic elucidation of fumonisin non-production isa plant hormone was originally discovered as a virulence

required for accurate assessment of fumonisin

factor of F. fujikuroi to rice. Clustering of seven genes

producibility in F. fujikuroi.

involving gibberellin biosynthesis has been revealed in F.
fujikuroi (GIB cluster) (Wiemann et al. 2013). A similar

Material and Method

cluster has been detected from other species in Ff

Phylogenetic analysis
AFLP Microbial Fingerprinting kit was used forcomplex though many genes are putatively inactive andactual gibberellin production was observed only inobtaining binary matrix for phylogenetic analysis. Binary

several strains belong to Fusarium proliferatum, Fusarim

data of totally 66 AFLP markers from 96 taxa including

konzum and Fusarim sacchari (Studt and Tudzynski

an outgroup, F. proliferatum MAFF236459 and2014)

SUF1207 were compiled into a single data matrix.
Neighbor-joining analysis was performed with PAUP.

Fumonisin, a polyketide mycotoxin, is associated withhuman esophageal cancer in South Africa and causes

The strength of the internal branches from the resulting

leucoencephalomalacia in horses and lung edema intree was tested by bootstrap analysis with 1,000

swine. F. fujikuroi that is frequently isolated from rice isreplications.

known to produce fumonisin though its closely related

Gibberellin analysis-9-The strains were grown in 10 % ICI medium for 7

by PCR and sequencing for CPR , P450-4 in GIB cluster,days on a reciprocal shaker at 25 °C. GA3 and GA4/7 in

FUM1 and FUM18 including FUM18 / FUM19

culture filtrate were analyzed with a TLC silicagelintergenic region in FUM cluster. Data of five SNP

60F254 plate. GA1, GA3, GA4 and GA7 concentrations

markers (TEF_T618G in Suga et al. 2014, CPR_C1152A,

was quantified by LC-MS/MS analysis using an

P4504_C842T, FUM1_G423A, FUM18_G51T) wereACQUITY UPLCTM system coupled to a Xevo QTof

obtained by allele-specific primer extension reactions.

mass spectrometer. Ionization was conducted with ESI

Reaction products were finally detected with a Luminex

probe in negative polarity.

100 flow cytometer for determination of SNPs. MAT

Fumonisin analysis
The strains were grown in sterile cracked maize seeds

type data was obtained by PCR.

Result and Discussions

at 25 °C for 10 days. Fumonisin in cracked maize
cultures were extracted with methanol:water (3:1, v/v). A

Phylogenetic tree
In order to investigate intraspecific differentiation of F.

ELISA using RIDASCREEN®FAST Fumonisin kit

fujikuroi, phylogenetic analysis of 95 strains based on(R-Biopharm, Darmstadt, Germany) was performed and

AFLP markers was conducted. F. fujikuroi strains

fumonisin concentrations were determined on the basis

divided into two phylogenetic groups. Fumonisinof standard curves made with authentic fumonisin (0.2 toproduction was detected only from one group by ELISA

6.0 ppm), as provided with the kit. Fumonisin

and we designated it as F-group (Fig. 1).

purification was performed
MAFF2 3594 9
MAFF2 3595 1
Gfc0 6250 08

by a strong anion exchange

6083
62 APF0GL24

column before LC-MS/MS
analysis. FB1, FB2 and FB3
concentrations wasquantified by LC-MS/MS
analysis. Ionization was
conducted with ESI probe
in positive polarity.
Crossing
Crossing progenies
between a F-group strain
Gfc0825009 (MAT 1-1
type) and a G-group strain
Gfc0801001 (MAT 1-2
type) was obtained. They
were transplanted tosterilized rice straws in a
beaker. Perithecthicia
developed in two weeks
later were thoroughly
rinsed by sterile water.
Ascospores discharged ona glass slide were collectedby several hundred μl ofsterile water and spread to100

F．fujikuroi

a MMTS medium.
Linkage analysis
SNPs between
Gfc0825009 andGfc1 0340 01
GL25
GL27
GL28
58 Gfc8 7071
23
Gfc8 7071 82
56 Gfc0MYG9210
9250 1074 Gfc0 9010 02
Gfc0 9010 05
Gfc9
4247
02
73
Gfc9 4247 03
Gfc0 8250 01
Gfc0 8250 02
89 Gfc0 8250 03
Gfc0 8250 04
Gfc0 8250 05
Gfc0 8250 06
Gfc0 6250 01
Gfc0 9010 09
Gfc0 9250 05
Gfc8 7071 17
73 Gfc0 9250 11
Gfc0 9250 12
Gfc1 0040 01
M6883
Gfc0 8010 01
71
51
Gfc0 8010 03
Gfc0 8010 02
SMN8 62
Gfc8 7072 49
Gfc8 7076 42
Gfc1 0040 02
MIE924
Gfc9 4247 07
Gfc0 6250 02
696250 04
100 Gfc0
Gfc0 6250 05
Gfc0 6250 06
Gfc0 6250 07
Gfc0 6250 10
Gfc1 0040 03
411 1666
411 08
Gfc1 0060 01
Gfc1 0190 04
Mo136
Gfc1 0060 02
Gfc1 0190 03
Gfc1 0190 01
Gfc0 9210 34
Gfc0 9210 41
Gfc1 0250 91
417 9
Mo309
Gfc0 9210 01
90
Gfc1 0430 32
Gfc1 0430 46
Gfc1 0430 47
Gfc1 0430 37
90
Gfc1 0430 45
Mo78
Gfc0 9210 02
Gfc0 9210 09
Gfc0 8250 07
Gfc0 0091 17
Gfc0 9210 14
Mo175
Gfc1 0340 02
IBR891
Mo141
Gfc0 0091 10
Gfc0 8250 11
Gfc0 0091 05
Gfc1 0060 07
99 Gfc1 0250 290250 37
76 Gfc1
Gfc1 0410 03
88
Gfc1 0410 11
Gfc1 0410 10
Gfc0 8210 04
Gfc0 8250 09
Gfc1 0430 35
Gfc0 0090 63
Gfc0 9210 39
Gfc1
0160
90 53 Gfc1 016022
24
Gfc1 0160 25
73 Gfc1 0160 26
Gfc0 9210 40
Mo80
418 4
MAFF2 3546 3

76

MAFF2 3645 9 F．proliferatum（outgroup）
SUF120 7

G-group

F-group0.01 changes

Fig. 1Phylogenetic tree of F. fujikuroi based on 66 AFLP markers (NJ method)

Gfc0801001 were obtained- 10 -

Gibberellin producibility
All 21 strains from bakanae diseased rice were

Gfc0801001. Complete linkage was observed between afumonisin non-producers (Suga et al 2014) and, therefore,

gibberellin low producibility (Table 1). The lack ofgibberellin production of 95 stains was investigated by infumonisin producibility showed complete linkage to thevitro culturing. Gibberellin was not detected in F-group

SNP markers (FUM1_G423A and FUM18_G51T) in the

by TLC, while it was detected from all the strains

FUM cluster (Table 1).

belonging to another group.We designated it as G-group

Conclusion(Fig. 1).

F. fujikuroi strains divided into F-group with fumonisin

LC-MS/MS analyses of a representative strain of

producibility / gibberellin low producibility and andG-group and F-groupG-group with the lack of fumonisin producibility /SNP marker (P4504_C842T) in the GIB cluster andPreliminary results of fumonisin and gibberellin

gibberellin high producibility. Linkage analysis indicatedproducibility obtained by ELISA and TLC were further

that gibberellin low producibility attributes to the GIB

confirmed by LC-MS/MS analyses using strain

cluster in Gfc0825009 and the lack of fumonisin

Gfc0801001 and strain Gfc0825009 as a representative

producibilityof G-group and F-group, respectively.

Gfc0801001.

As for fumonisins, FB1 (182.2 ppm), FB2 (121.5 ppm)
Gfc0825009 but they are undetectable level in the
G-group strain Gfc0801001. As for gibberellins, GA3(37.8 ppm), GA7 (2.0 ppm), GA4 (1.2 ppm) and GA1 (1.2
ppm) were detected from the G-group strain Gfc0801001.
the F-group strain Gfc0825009 by TLC, low level ofGA3 (2.2 ppm), GA7 (0.4 ppm) were detected from thisstrain.
Based on these results, it was considered that F-grouphas fumonisin producibility / gibberellin low
producibility and G-group has lack of fumonisin
producibility / gibberellin high producibility.
Linkage analysis
In order to reveal the cause of gibberellin low
producibility in the F-group strain Gfc0825009 and thelack of fumonisin producibility in the G-group strain
Gfc0801001, linkage analysis was conducted using
crossing progenies between Gfc0825009 and

to

the

FUM

cluster

Suga H, Kitajima M, Nagumo R, Tsukiboshi T, Uegaki R,
Nakajima T, Kushiro M, Nakagawa H, Shimizu M,
Kageyama K, Hyakumachi M, 2014. A single
nucleotide polymorphism in the translation
elongation factor 1α gene correlates with the ability
to produce fumonisin in Japanese Fusarium fujikuroi.
Fungal Biology, 118: 402-412.
Wiemann P, Sieber CMK, von Bargen KW, Studt L,
Niehaus E-M, Espino JJ, Huß K, Michielse CB,
Albermann S, Wagner D, Bergner SV, Connolly LR,
Fischer A, Reuter G, Kleigrewe K, Bald T, Wingfield
BD, Ophir R, Freeman S, Hippler M, Smith KM,
Brown DW, Proctor RH, Münsterkötter M, Freitag M,
Humpf H-U, Güldener U, Tudzynski B, 2013.
Deciphering the cryptic genome: genome-wide
analyses of the rice pathogen Fusarium fujikuroi
reveal complex regulation of secondary metabolism
and movel metabolites. PLoS Pathog 9:e1003475.
Studt L and Tudzynski B, 2014 Gibberellins and the red
pigments bikaverin and fusarubin. In: Biosynthesis
and Molecular Genetics of Fungal Secondary
Metabolites. Series “Fungal Biology”; Martín, J.-F.
García-Estrada, C., Zeilinger, S.(Eds.); Springer New
York 2014.

Table 1 Result of SNP analyses of a part of 42 progenies between Gfc0825009 and Gfc0801001
ProgenyinReferenceand FB3 (3.7 ppm) were detected from the F-group strain

Though GA3 and GA7 /GA4 spots were not detected from

attributes

SNP data

MAT type

GA3
prodetection by
TLC

Fumonisindetection by
ELISA+

TEF_T618G

CPR_C1152A

P4504_C842T

FUM1_G423A

FUM18_G51T

Gfc0825009 (parent)

T

C

C

G

G

1-1

-

Gfc0801001 (parent)

G

A

T

A

T

1-2

+

-

Gfc①CP91002

G

A

C

G

G

1-2

-

+

Gfc①CP91005

T

C

C

A

T

1-1

-

-

Gfc①CP91007

T

C

T

A

T

1-1

+

-

Gfc①CP91008

G

A

C

A

T

1-2

-

-

Gfc①CP91009

G

A

T

G

G

1-1

+

+

Gfc①CP91011

T

C

T

G

G

1-1

+

+

Gfc①CP91017

T

C

T

G

G

1-1

+

+

Gfc①CP91019

T

A

T

A

T

1-1

+

-

Gfc①CP91020

G

C

C

G

G

1-2

-

+

Gfc①CP91022

T

C

C

A

T

1-1

-

-

- 11 -

- 12 -

PL-4
Perspectives on the development of biopesticides applicable toboth agricultural insect pests and diseases
Shigenobu YOSHIDA(Central region of agricultural research center, NARO)

SUMMARY
Biopestiscides effective against both agricultural insect pests and diseases have been implicated as promising materials
to promote IPM practices and environmental friendly agriculture with cost- and labor-saving in Japan. We recently
found that several microbes hitherto known to control agricultural insects also showed inhibitory effects against
soilborne plant diseases such as bacterial wilt of tomato. Here we will mention outline on the research developmenton biocontrol using of such ‘dual control’ agents.
Biocontrol is a promising control measure to solve

microbes against significant diseases of Solanaceae, such

issues on agricultural chemical residues and emergenceas bacterial wilt, Fusarium wilt, and crown and root rot.

of drug resistant insects and microbes. For biocontrol

Through the research project, our research group foundagainst agricultural insects, bioinsecticides have been

Bacillus thuringiensis, which is known as an effective

developed and various products have been on the market

bio-insecticide, has an ability to activate plant defensesoincludesystem on tomato plants, leading to suppression of

microorganisms or their derivatives effective against

bacterial wilt. Hyakumachi et al. 6) reported that when

agricultural insects, reports that such microbes also showroot of tomato seedlings were treated with B.

beneficial functions against plant diseases caused bythuringiensis culture and challenge-inoculated withvarious plant pathogenic microbes have been increased.

Ralstonia solanacearum, development of wilt symptoms

For example, a famous bioinsecticidial bacterium,

were suppressed to less than one third of the control.

Bacillus thuringiensis, has been reported to showThis

inhibitory effects against plant pathogenic fungi, such as

reproduced by pretreating their roots with a cell-freefar.

Althoughthese

insecticides

1)

diseasesuppression

intomatoplantswas

Fusarium oxysporum and Phytophthora megasperma f.

filtrate (CF) that had been fractionated from B.

sp. medicaginis 2), based on secretion of antifungal

thuringiensis culture by centrifugation and filtration.

substances. In addition to such insecticidial bacteria,

Theysimilar inhibitory effects have also been reported onchallenge-inoculated

several insecticidial fungi; Beauveria bassiana has been

pretreatment with CF, the growth of R. solanacearum inreported to suppress damping off of tomato seedlingstem tissues clearly decreased, and expression of

caused by Pythium myriotylum and Rhizoctonia solani

defense-related genes such as PR-1, acidic chitinase, and

3,4), and several isolates belonging to genus Lecanicillium

β-1,3-glucanase was induced in stem and leaf tissues.

inhibited occurrence of various diseases such as powdery

Furthermore, the stem tissues of tomato plants with their

5)

also

found

that,
with

R.

in

tomatoplantssolanacearum

after

mildew of cucumber, tomato and strawberry . Thus,

roots were pretreated with CF exhibited resistanceapplication of insecticidial microbes against plant

against direct inoculation with R. solanacearum. Taken

diseases is feasible, and development of the control

together, they suggested treatment of tomato roots withagents suppressing both agricultural insects and plant

the CF of B. thuringiensis systemically suppresses

diseasesbacterial wilt through systemic activation of the plant

contribute

cost

and

labor-saving

pest

managements, which is recently more necessary to

defense system.
In addition to the above report, Takahashi et al. 7)

promote in Japanese agriculture system.
From 2011 to 2014, we carried out a research projectfurther elucidated the details of the systemic activation offunded by Ministry of Agriculture, Forestry and Fisheries

the plant defense system by B. thuringiensis.

to develop novel microbial agents which can lead tothey carried out comparative analysis of the expression

biopesticides, and methods for application of theof a marker gene for induced resistance to pathogens, in- 13 -

Whenvarious tissues of tomato seedlings treated with CF on

From these results, the disease inhibitory activity by thetheir roots, B. thuringiensis-induced defense system wasstrain T1 was suggested to be based on the systemic

activated in the leaf, stem, and main root tissues, but not

activation of the plant defense system.

in the lateral root tissue. In addition, the growth of theSuch disease inhibitory activity was confirmed incausal pathogen was significantly suppressed in theseveral field experiments. When the tomato seedlings

CF-treated main roots but not in the CF-treated lateral

drenched with 500-folds dilution of the commercial

roots. This distinct activation of the defense reaction andGottsu A were transplanted in bacterial wilt-infested test

suppression of R. solanacearum were reflected by thefields,differences in the transcriptional profiles of the main andsuppressed on the transplanted plants by the treatment.

lateral tissues in response to the CF. In CF-treated main

Also, the suppression tended to be more stable byroots, but not CF-treated lateral roots, the expression of

additional drenching of the dilution into the base of theseveral salicylic acid (SA)-responsive defense-related

plants every two weeks.

genes was specifically induced, whereas jasmonic acidbioinsecticide is also applicable as a promising novel(JA)-related gene expression was either down-regulated

biopesticide for bacterial wilt, and further fieldor not induced in response to the CF.

From thesedemonstration on the disease suppression by the

results, they concluded the co-activation of SA

insecticidial fungal product are ongoing to lead todependent

registration as the biopesticide.

signaling

pathway

with

ET-dependent

thediseaseoccurrencewassignificantly

Thus, the P. tenuipes

signaling pathway and suppression of JA-dependent
signaling pathway might play key roles in B.

Concept that plural control of both agricultural insects

thuringiensis-induced resistance to R. solanacearum inand plant diseases is generally called as ‘dual control’ 5).

tomato.

As described above, this concept could be suitable to

Because the insecticidial bacterium, B. thuringiensis

solve issues arising in the recent Japanese agricultural

has verified to show another inhibitory potential against

systems, cost and labor-saving pest managements, since

the plant disease, we further screened to find novel

using products containing such wider spectral biocontrol

biocontrol agents effective against both agricultural

agents reduce the kinds of products farmers should

insects and plant diseases from bioinsecticide products

prepare for pest managements.

on the market in Japan.

When dilutions of eachmentioned B. thuringiensis and P. tenuipes are promising

candidate product were irrigated into the potted tomato

to be used as biopesticides against a soilborne disease,seedlings

Although here we

wasbacterial wilt of tomato, biopesticides against soilborne

challenge-inoculated, several bioinsectisides showeddiseases have less been developed in Japan, rather thaninhibitory

disease.

in other countries, such as Europe and USA. Hence,

Paecilomyces

more commercialization of such dual control agents

tenuipes (Gottsu A) showed the superior inhibitory

would also contribute to more provision of biologicalactivity.

control measures against soilborne diseases.

andactivity

Particularly,

Ralstonia
againstcommercial

solanacearum
theproduct

bacterialof

The product contains P. tenuipes strain T1

isolated from soil in Japan, and is known to be effective
against whiteflies and aphids of vegetables. Gottsu A

Referencehas been registered as a bioinsecticide since 2008.

1) Raddadi N, Belaouis A, Tamagnini I, Hansen BM,
Hendriksen NB, Boudabous A, Cherif A, Daffonchio D(2009) Characterization of polyvalent and safe Bacillus
thuringiensis strains with potential use for biocontrol. J
Basic Microbiol.
2) Zhou Y, Choi YL, Sun M, Yu Z (2008) Novel roles ofBacillus thuringiensis to control plant diseases. Appl
Microbiol Biotechnol, 80:563–572.
3) Ownley, BH, Bishop, DG and Pereira, RM. (2000)
Biocontrol of Rhizoctonia damping-off of tomato with
Beauveria bassiana, Phytopathology 90, S58.
4) Clark, MM, Gwin, KD and Ownley BH. (2006)
BiologicalcontrolofPythium
myriotylum,
Phytopathology 96, S25.
5) Koike, M. (2014) Dual control using Entomopathogen.
Proceedings of PSJ Biocontrol Workshop XIII, 32-39.

To verify the mode of actions in the diseasesuppression by the strain T1, experiments using potted
tomato seedlings were carried out. Consequently, thefollowing results were obtained: 1) tomato seedlings
irrigated with CF of strain T1, as well as the fungal
propagules, showed inhibitory effects against bacterial
wilt, while the CF did not inhibit the growth of the causal
pathogen in vitro. 2) expression of SA dependent marker
genes (PR-1 and PR-2), responsible for induced
resistance to pathogens of tomato, was increased on themain roots of tomato seedlings dipped with the CF.

- 14 -

6) Hyakumachi, M, Nishimura, M, Arakawa, T, Asano, T,
Yoshida, S, Tsushima, S and Takahashi, H. (2013)
Bacillus thuringiensis suppresses bacterial wilt diseasecaused by Ralstonia solanacearum with systemic
induction of defense-related gene expression in tomato,
Microbes Environ. 28: 128-134.
7) Takahashi, H, Nakaho, K, Ishihara, T, Ando, S, Wada,
T, Kanayama, Y, Asano, S, Yoshida, S, Tsushima, S andHyakumachi, M. (2014) Transcriptional profile of tomato
roots exhibiting Bacillus thuringiensis-induced resistanceto Ralstonia solanacearum. Plant Cell Rep. 33: 99-110.

- 15 -

- 16 -

Oral Session Abstracts- 17 -

- 18 -

OS I-1
Current status and management of Rhizoctonia solani, the causal pathogen of sheath blightdisease on rice and maize in Indonesia
Achmadi PRIYATMOJO(Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia)

SUMMARY
Rhizoctonia solani, a fungal soil-borne pathogen, has vary wide host range. In Indonesia, R. solani has been reported
causing significantly yield losses on rice and maize. The causal agent was mostly identified as R. solani anastomosis
group 1-IA (AG 1-IA) on the basis of colony cultural morphology on PDA. However other Rhizoctonia spp. were also
isolated from rice and maize but anastomosis grouping could not be established because of lack of the AG tester isolatesof R. solani for anastomosis test. Moreover, determination by colony cultural morphology is very difficult because of its
high variation even in isolate belonging to the same AG or subgroup of AG. At present in Indonesia, many of isolates of
R. solani obtained from other crops have not been differentiated into AG due to lack of the tester isolates. Precise
identification of R. solani into AG is very useful for control recommendation. Currently, sheath blight on rice and maizecaused by R. solani has been managed by controlling water for irrigation, applying proper fertilizers, utilizing biologicalcontrol agents, and and spraying chemical fungicides. For both enviromental friendly and healthy product reasons, theuse of chemical fungicides should not be as a primary control strategy.

Introduction

and pathogens of many plant species (7,24,40). The

Rice is an important staple food in Indonesia. Rice

fungus survives in unfavorable conditions by forming

production is dominated by the small-holder farmers

dormant mycelia or sclerotia (43). Sclerotia can survive

which each farmer holding an average of land area lessfor 2 years in soil and are spread during field preparation

than 0.5 hectares. Small-holder farmers account forand irrigation. These sclerotia can become source ofapproximately 90 percent of Indonesia’s rice productiondisease inoculum for the next planting (21).

(20). In some regions of Indonesia, maize is chosen as

Rhizoctonia sp. is rare to produce sexual spore or

staple food for some reasons such as climate and pricebasidiospore in field. Many attempts have beenfactors. Moreover, maize is also used for animal feeddeveloped to induce sexual state of Rhizoctonia species(46).

in the laboratory (1,12,49). However, some of AGs or
Many decades Indonesia Government has been

subgroups of Rhizoctonia sp. remain could not becreated many programs to reach rice and maize

induced the formation of basidiospores by applying theseself-sufficiency in order to maintain political, social andartificial techniques.

economic stability. However, many constraints have been

On the basis of number of nuclei per hyphal cell,appeared for increasing rice and maize production in

Rhizoctonia sp. is divided into uninucleate, binucleate,most of the rice and maize growing area of Indonesia, forand multinucleate which contains one, two, and more

examples low educational of farmers, unfavorable

than two nuclei per hyphal cell, respectively. At present,

climate condition, attacking pests, and occurring diseases.

uninucleate Rhizoctonia isolates have been reported only

One of the most important pathogens on rice and maizefrom Norwegian and Finnish conifer seedlings (8,39).

is Rhizoctonia solani causing sheath blight disease (34).

Moreover,bothbinucleateandmultinucleate

Therefore disease control measures are necessary to

Rhizoctonia are grouped under species level based on

prevent sheath blight disease development.

hyphal fusion or hyphal anastomosis reaction (3,5,23,25).
Currently, 21 anastomosis groups (AGs) of binucleate

Grouping of Rhizoctonia Species

Rhizoctonia and 13 AGs of multinucleate Rhizoctonia

Rhizoctonia species is a universal filamentous

solani have been reported (6,10,18,37). On the otherfungal soil exist as saprophytes, mycorrhizal symbionts

hand, multinucleate Rhizoctonia circinata is divided into

- 19 -

R. circinata var. circinata, R. circinata var. oryzae, and R.

stadium) in a rice crop and include oval or ellipsoidal

circinata var. zeae based on differences of colony

greenish gray lesions, usually 1-3 cm long, on the leaf

morphology of the vegetative state (15,26).

sheath, under favorable conditions, these initial lesions
andmultiply and expand to the upper part of the sheaths

multinucleate Rhizoctonia solani are further divided into

(Figure 1) (22,34). In the absence of protection measures,subgroups or types based on cultural morphology,

sheath blight disease on rice causes 10-30% yield loss

pathogenicity,and may reach up 50% under favorable environmental

Some

AGs

of

hyphalbinucleate

fusionRhizoctonia

frequency,DNAcomplementarity, fatty acid analysis, and zymogram

condition for disease development (17,51).

patterns (9,11,16,27-32).

Occurrence of Rhizoctonia Species inIndonesia
Rhizoctonia sp. obtained from various plants has
been reported from many places in Indonesia. These
isolates of Rhizoctonia sp. belong to binucleate andmultinucleate. None of isolates belong to uninucleate
Rhizoctonia. The pathogen attacks rice, maize, wheat,sorghum, peanut, soybean, pepper, tomato, potato,
tobacco, cotton, cabbage, lettuce, strawberry, cacao,
coffee, pine and other economically crops (33-35,44).
In addition to pathogenic Rhizoctonia sp., many
beneficial Rhizoctonia sp. have been obtained from many
places in Indonesia. These beneficial Rhizoctonia sp. are
predominantly by binucleate Rhizoctonia (7,40,45).
Due to the lack of AG tester isolates of Rhizoctonia

Figure 1. Sheath blight symptom on rice.

sp. in Indonesia, most of Rhizoctonia sp. isolates fromvarious places and crops have not been grouped yet into

Rhizoctonia solani on Maize

AG system (41), except for isolates of mycorrhizal

Banded leaf and sheath blight (BLSB) on maize

Rhizoctonia obtained from healthy vanilla roots which

are commonly found in the center of maize production inreported belong to binucleate Rhizoctonia AG-F based

Indonesia. Rhizoctonia solani, a soil-borne disease, hason anastomosis reaction and PCR-RFLP. The study was

been reported as causal pathogen of BLSB. It is a major

held in Gifu University, Gifu, Japan in 2009 (7). The

disease on maize and can cause serious damage reach up

remaining isolates of Rhizoctonia sp. are still unknown

to 100% yield loss (34,38,47). The initially infection is

AG. However, other approaches applying culturalfrom the basal sheath and then spread to the developing

morphology, pathogenesis, and molecular techniques

ear under favorable environmental conditions. The

have been taken in order to placing isolates ofdeveloping ear is completely damaged and dries up

Rhizoctonia sp. into proper AG. Recent study reveal thatprematurely (Figure 2).

R. solani collected from rice showing sheath blight
symptom and from potato showing black scurf symptom
could be placed into AG 1-1A and AG 3, respectively(2,4).

Rhizoctonia solani on Rice
Rhizoctonia solani, the causal agent of sheath blightdisease on rice, is often occur in rice fields and easily
isolated from diseased tissue plant or from sclerotia that
produced around disease symptom. Symptoms areusually observed from tillering to milk stage (generative

Figure 1. Sheath blight symptom on maize.

- 20 -

Sheath Blight Disease Management

strategies should be applied and creating resistant variety

Sheath blight disease on rice and maize in Indonesia

should be strongly supported in controlling the disease.

is very difficult to be controlled because of continuing
rice or maize planting a whole year, favorable
environmental condition, almost lack of resistant variety
of rice or maize to the disease, and existing of sclerotia
which can survive over 2 years. Resistant variety of riceand maize to sheath blight has not been reported inIndonesia (19).

Acknowledgement
The author is grateful to UGSAS, Gifu University,
Gifu Japan for providing financial support to attend
International Conference on Innovative Crop Protection
for Sustainable Agriculture.

Reference
1) Adams GC and Butler EE (1983) Influences ofnutrition on the formation of basidia andbasidiospores in Thanatephorus cucumeris.
Phytopathology, 73:147-151.
2) Aminatullailiyati, Widiastuti A and Priyatmojo A(2016) Characterization of Rhizoctonia solani
isolated from potato tuber and antagonistic test
using Trichoderma sp. and Penicillium sp. The 4th
National Seminar on Agricultural Research Results.
Faculty of Agriculture, Universitas Gadjah Mada,
Yogyakarta, Indonesia. September 24, 2016
(Abstract).
3) Anderson NA (1982) The genetics and pathology
of Rhizoctonia solani. Ann Rev Phytopathol 20,
329-347.
4) Bintang AS, Wibowo A, Priyatmojo A and
Subandiyah S (2017) Morphological and molecularcharacterization of Rhizoctonia solani isolates fromtwo different rice varieties. Jurnal Perlindungan
Tanaman Indonesia, 21 (impress).
5) Carling DE (1996) Grouping in Rhizoctonia solani
by hyphal anastomosis reaction. In: Rhizoctonia
species: taxonomy, molecular biology, ecology,
pathology and disease control (Sneh B., Jabaji-Hare
S., Neate S., Dijst, G., eds.). Kluwer Academic
Publishers. Dordrecht. pp. 35-47.
6) Carling DE, Baird RE, Gitaitis RD, Brainard KA
and Kuninaga S (2002) Characterization of AG-13,
a newly reported anastomosis group of Rhizoctonia
solani. Phytopathology, 92:893-899.
7) Haryuni H, Hadisutrisno B, Priyatmojo A and
Widada J (2010) Relationship among mycorhizzal
Rhizoctonia from vanilla on the basis of
PCR-RFLP analysis. Jurnal Agroteknologi, 12:34-38.
8) Hietala AM, Sen R and Lilja A (1994) Anamorphic
and teleomorphic characteristics of a uninucleate
Rhizoctonia sp. isolated from the roots of nursery
grown
coniferseedlings.
Mycol
Res,
98:1044-1050.
9) Hyakumachi M, Mushika T, Ogiso Y, Toda T,
Kageyama
K
and
Tsuge
T
(1998)
Characterization of a new cultural type (LP) of
Rhizoctonia solani AG2-2
isolated fromwarm-season turfgrasses, and its genetic
differentiation from other cultural types. Plant
Pathol, 47:1-9.
10) Hyakumachi M, Priyatmojo A, Kubota M andFukui H (2005) New anastomosis groups, AG-T
and AG-U, of binucleate Rhizoctonia spp.
causing root and stem rot of cut-flower andCultural Control
As a tropical country, rice can be planted 2 or 3
times a year depend on water availability for irrigation.
This condition is favorable for sheath blight diseasedevelopment because many sclerotia will be produced asa source of inoculum for the next successive rice planting(21). Therefore, in the rice field with the history ofoccurring sheath blight disease, it is not recommended tocultivate rice or other crop belonging to Poaceae family
continuously (34). Study on the host range of R. solani
obtained from rice, wheat, and maize showed that R.
solani from each crop could infect each other crops (36).
Chemical Control
After establishment of integrated pest management(IPM) program in 1995, chemical control by applying
fungicides should be the last choice after other control
techniques

cannotprotector

prevent

diseasedevelopment (50). Some of active ingredients offungicide used to control R. solani on rice and maize arepropiconazole,

difeconazole,

benomyl,mancozeb,validamycin, and methyl thiophanate.
Biological Control
Biological control technique using antagonistic
microorganisms

includingfungi,

bacteria

andactinomyces has been increased in recent year. Comparetochemical

control, biological control is moreenvironmental friendly and provide quality health forhuman. Bacillus sp., Trichoderma sp. and Gliocladium
sp. are among the most biological control agents used tocontrol sheath blight disease on rice and maize(14,19,41,42).

Conclusion
Sheath blight disease on rice and maize is an
important disease causing serious damage and can reachup to 50-100% yield loss. Many compatible control- 21 -

miniature roses Phytopathology, 95: 784-792.
11) Johnk, Janell Stevens, and Roger K. Jones. 2001.
Differentiation of three homogeneous groups of
Rhizoctonia solani anastomosis group 4 by analysis
of fatty acids. Phytopathology, 91:821-830.
12) Kangatharalingam N and Carson ML (1988)
Technique to induce sporulation in Thanatheporus
cucumeris. Plant Dis, 72:146-148.
13) Kuninaga S, Carling DE, Takeuchi T and
Yokosawa R (2000) Comparison of rDNA-ITS
sequences between potato and tobacco strains inRhizoctonia solani AG-3. J of Gen Plant Pathol,
66:2-11.
14) Kusuma A, Widiastuti A and Priyatmojo A (2017)
Induction of resistance using Trichoderma spp. and
Penicillium sp. against banded leaf and sheathblight (BLSB) caused by Rhizoctonia solani in
Maize. Proceeding of the 1st International
Conference on Tropical Agriculture. Springer,
Cham, p:3-11.
15) Leiner RH and Carling DE (1994) Characterizationof Waitea circinata (Rhizoctonia) isolated fromagricultural soils in Alaska. Plant Dis, 78:385-388.
16) MacNish GC, Carling DE, Brainard KA (1993)
Characterization of Rhizoctonia solani AG 8 frombare patches by pectic isozyme (zymogram) andanastomosis
techniques.
Phytopathology,
83:922-927.
17) Meng QZ, Liu ZH, Wang HY, Zhang SS, Wei SH(2001) Research progress in rice sheath blight. (inChinese with English abstract) J. Shenyang AgricUniv, 32:376–381.
18) Misawa T and Toda T (2013) First report of black
scurf on carrot caused by binucleate Rhizoctonia
AG-U. J of Gen Plant Pathol, 79:86-88.
19) Muis A (2007) Management of banded leaf andsheath bligh disease caused by Rhizoctonia solani
Kuhn on corn. Jurnal Litbang Pertanian,
26:100-103.
20) Nuryanti S (2016) Rice self-sufficiency inIndonesia: an Analysis on budget allocation and theachievement.
ProceedingofInternational
Conference on Agribusiness: Development for
Human Welfare. UMY, Yogyakarta.
21) Nuryanto B, Priyatmojo A, Hadisutrisno B andSunarminto BH (2010) Relationship between initial
pathogen inoculums with disease development ofsheath blight on ciherang rice variety. Jurnal
Perlindungan Tanaman Indonesia, 19:55-61.
22) Nuryanto B, Priyatmojo A, Hadisutrisno B andSunarminto BH (2011) Rice sheath blight(Rhizoctonia solani Kühn) development on theintensive rice productions of Central Java andYogyakarta Provinces. Jurnal Budidaya Pertanian,
7: 1-7.
23) Ogoshi A (1975) Grouping of Rhizoctonia solani
Kühn and their perfect states. Rev Plant Prot Res
Japan 8, 98-103.
24) Ogoshi A (1996) Introduction of the genus
Rhizoctonia: Taxonomy, Molecular Biology,
Ecology, Pathology and Disease Control. Kluwer
Academic Publishers, Dordrecht, NL.

- 22 -

25) Ogoshi A, Oniki M, Araki and, Ui T (1983)
Studies on the anastomosis groups of binucleate
Rhizoctonia and their perfect states. J Fac Agric
Hokk Univ 61, 244-260.
26) Oniki M, Ogoshi A, Araki T, Sakai R and Tanaka S(1985) The perfect state of Rhizoctonia oryzae andR. zeae and the anastomosis group of Waitea
circinata. Trans. Mycol. Soc. Japan, 26:189-198.
27) Priyatmojo A, Escopalao VE, Tangonan NG,
Pascual CB, Suga H, Kageyama K, Hyakumachi
M (2001) Characterization of a new subgroup of
Rhizoctonia solani anastomosis group 1 (AG-1-ID),
causal agent of a necrotic leaf spot on coffee.
Phytopathology, 91:1054-1061.
28) Priyatmojo A, Yamauchi R, Carling DE, Kageyama
K and Hyakumachi M (2002) Differentiation of
three varieties of Rhizoctonia circinata; var.
circinata, var. oryzae and var. zeae on the basis ofcellular fatty acid compositions. Journal of
Phytopathology, 150: 1-5.
29) Priyatmojo A, Yamauchi R, Kageyama K and
Hyakumachi M (2001) Grouping of binucleate
Rhizoctonia anastomosis group D (AG-D) isolates
into subgroups I and II based on whole-cell fatty
acid compositions. Journal of Phytopathology, 149:
421-426.
30) Priyatmojo A, Yamauchi R, Kageyama K.,
Hyakumachi M (2002) Whole-cell fatty acidcomposition to characterize and differentiate
isolates of Rhizoctonia species associated with
turfgrass diseases in Japan. Journal of General Plant
Pathology, 68: 1-7.
31) Priyatmojo A, Yamauchi R, Naito S, Kageyama K
and Hyakumachi M (2002) Comparison ofwhole-cell fatty acid compositions of isolates of
Rhizoctonia solani AG 2 from tobacco (Nt-isolates)
and tulip (AG2-t), AG 2-1 and AG-BI. Journal of
Phytopathology, 150: 283-288.
32) Priyatmojo A, Yotani Y, Hattori K, Kageyama K,
Hyakumachi M (2001) Characterization of
Rhizoctonia spp. causing root and stem rot ofminiature rose. Plant Disease, 85:1200-1205.
33) Semangun H (1989) Diseases on Horticultural
Crops in Indonesia. Gadjah Mada University Press,
Yogyakarta.
34) Semangun H (1990) Diseases on Food Crops inIndonesia. Gadjah Mada University Press,
Yogyakarta.
35) Semangun H (2000) Diseases on Estate Crops inIndonesia. Gadjah Mada University Press,
Yogyakarta.
36) Setyawan B, Priyatmojo A and Sumardiyono C(2009) Characterization and pathogenicity of
Rhizoctonia solani on wheat, rice and maize.
Widyariset 12:53-61.
37) Sharon M, Kuninaga S, Hyakumachi M, Naito S,
and Sneh, B (2008) Classification of Rhizoctonia
spp. Using rDNA-ITS sequence analysis supports
the genetic basis of the classical anastomosis
grouping. Mycoscience, 49: 93-114.
38) Singh A and Shahi JP (2012) Banded leaf andsheath blight: an emerging disease of maize.
Maydica, 57:215–219.

39) Sneh B, Burpee L and Ogoshi A (1991)
Identification of Rhizoctonia Species. APS Press,
St. Paul.
40) Soelistijono S, Christanti S., Priyatmojo A andEndang A (2011) Characterization of pathogenic
and mycorrhizal Rhizoctonia on Spathoglottis
plicata. Biota 16: 371-380.
41) Soenartiningsih S, Akil M and Andayani NN(2015) Soil borne fungus (Rhizoctonia solani) the
pathogen of sheath blight disease of maize andsorghum and its control measures. Iptek Tanaman
Pangan, 10:85-91.
42) Soenartiningsih S (2015) Utilization of arbuscular
mycorrhizal fungi in controlling sheath blightdisease on maize. Iptek Tanaman Pangan, 8:48-53.
43) Sumner DR (1996) Sclerotia formation by
Rhizoctonia species and their survival: Taxonomy,
Molecular Biology, Ecology, Pathology andDisease Control. Kluwer Academic Publishers,
Dordrecht, NL.
44) Suryantini R, Priyatmojo A, Widyastuti SM and
Kasiamdari RS (2011) Characteristic of
Rhizoctonia spp. from Pine (Pinus Merkusii Jungh.
Et De Vriese) Forest Soil. Jurnal Budidaya
Pertanian, 7: 8-13.
45) Suryantini R, Priyatmojo A, Widyastuti S.M,
Kasiamdari RS (2012) Hypovirulent binucleate
Rhizoctonia as biocontrol agent of Rhizoctonia
solani in tusam seedling (Pinus merkusii). Jurnal
Budidaya Pertanian, 8: 27-30
46) Swastika DKS, Kasim F, Sudana W, Hendayana R,
Suhariyanto K, Gerpacio RV and Pingali PL (2004)
Maize Production in Indonesia: Production Systems,
Contrains, and Research Priorities. Cymmyt,
Mexico.
47) Tang HT, Rong TZ and Yang JP (2004) Researchadvance on sheath blight (Zea mays L.) in maize. J
Maize Sci, 12:93–96.
48) Toda T, Mghalu JM, Priyatmojo A andHyakumachi M (2004) Comparison of sequencesof the internal transcribed spacer region in
Rhizoctonia solani AG 1-ID and other subgroup of
AG 1. Journal of General Plant Pathology, 70:
270-272.
49) Tu CC and Kimbrough JW (1975) A modified
soil-over-culture method for inducing basidia inThanatephorus
cucumeris.
Phytopathology,
65:730-731.
50) Untung K (1993) Introduction to Integrated Pests
Management. UGM Press, Yogyakarta.
51) Xie XW, Xu MR, Zang JP, Sun Y, Zhu LH, et al.
(2008) Genetic background and environmental
effects on QTLs for sheath blight resistancerevealed by reciprocal introgression lines in rice.
Acta Agron Sinica 34:1885-1893.

- 23 -

- 24 -

OS I-2
Indonesian farmers problems in implementing integrated pest management (IPM)
Hanifah IHSANIYATI(Faculty of Agriculture, Sebelas Maret University）

SUMMARY
Food occupies an important position in Indonesian agricultural sector. As food producers, farmers are often faced withproblems caused by pests. As the enemy of farmers, pests can damage food crops and eventually decrease production.
The best solution for pest problems is Integrated Pest Management (IPM). Although some research indicates that
information related to IPM has already been familiar to farmers, the practice of IPM has not been implemented by all
farmers in Indonesia. There are several problems faced by Indonesian farmers in implementing IPM. This article aims at
providing an overview of the problems faced by Indonesian farmers in the implementation of IPM. This paper ispresented descriptively based on several articles and previous research on IPM.
Keywords. Farmers, problems, pests, management, food

Introduction

outbreak of brown planthopper in 1975-1977 (RolingIntegrated Pest Management (IPM) has been aand Elske van de Fliert, 1994). It was then declared as adominant crop protection paradigm promoted globallynational policy in 1986, and its implementation has had asince the 1960s (Parsa et al., 2014). The fundamental

profound effect on national agricultural production

principles of pest management include 1) understanding(SEAMEO, 1999 in Utama, 2003). The concept of IPM

the ecology of the agroecosystem and the populationin Indonesia is also described in the Law Number 12 ofdynamics of the species in the ecosystem, 2) recognizing

1992 (Indonesian : Undang-Undang Nomor 12 Tahunand using economic-injury thresholds, and 3) integrating

1992) on Agricultural Cultivation System and the Decree

all appropriate control tactics into a rational system ofof the Minister of Agriculture/the Chairman of BIMAS

management (Robbins et al., 1974 in Minnick , 1976);

Controlling(Hammond et al., 2006); (Chandler, 2011); (Minnick,

SK/Mentan/Bimas.XII/1990 on the guidelines of the1976). An analysis of 62 IPM research and development

implementation of integrated pest control (Sulistiyono etprojects in 26 countries, covering over 5 million farmal. , 2008).

AgencyNumber14/

households, showed that IPM leads to substantial

In the beginning, farmers have made efforts to

reductions in pesticide applications (Chandler, 2011).

control Plant Pest Organisms (Indonesian: Organisme

However, its adoption by developing country farmers is

Pengganggu Tanaman or OPT) both in physical andsurprisingly low (Parsa et al., 2014).

mechanical ways. However, as the development of

An understanding of the various methods employed

science and technology, the use of pesticides is

in managing pests is essential in obtaining a perspective

considered more effective in controlling pests (theof integrated pest management. An inventory of the more

Directorate General of Agricultural Facility (Indonesian :prominent control tactics includes chemical control,

Ditjen Bina Sarana Pertanian), 2005 in Sulistiyono et al.,attractants and growth regulators, preventive measures,2008). There is a common view that the success of

autodial control, host-plant resistance, cultural andagricultural development cannot be separated from thephysical control, and biological control. Cultural control

use of pesticides. It is better to use more pesticides

are usually associated with such standard agricultural

because it can increase the production of agricultural

practices as site selection, irrigation, fertilization, tilling,

products. This is a general perception that still prevails incrop rotation, mixed or trap crops, sanitation, and drying

today’s world, including in Indonesia (Sulistiyono, 2004).

(Minnick, 1976); (Effendi, 2009).

It is supported by the significant increase of pesticides

The effort to introduce IPM started in the early 1979,

registered at the Directorate General of Agricultural

after Indonesia had experienced its first nationwide

Production Facility of the Ministry of Agriculture.

- 25 -

Looking at the data of registered trademark, there werepesticides after China and India (Soerjani, 1996 in

770 formulations in 2000, it then increased to 1,298

Wahyuni, 2010).
Generally, the high use pesticides in handling pests

formulations in 2005 (the Directorate General ofand diseases cannot be separated from the old paradigm

Agricultural Facility, 2005 in Sulistiyono et al, 2008 ).
The constraints in the implementation of IPM inseeing that the success of agricultural production is a

Indonesia are mainly due to the actions of farmers

result of the role of pesticides. The support fromthemselves, such as the use of uncontrolled pesticides.

government policy has encouraged farmers to use

Focusing on the use of pesticides, some of thepesticides through a subsidy regulation of 80% of the

problematic actions that farmers undertake in theprice of pesticides in 1987. In addition, this condition is

implementation of IPM in Indonesia will be described inreinforced by the creation of a chance circle between

this paper, including the attitudes of farmers as a motive

knowledge gaps of farmers in pest control and thethat affects the action. Attitude is an important key toincessant promotion of pesticides efficacy, also the weakunderstanding long-term behaviour and it affects

supervision and law enforcement and the policy to

behaviour (Newcomb et al., 1985); (Gibson et al., 1994).

achieve the target of agricultural production programs

Therefore, the attitude formation as a stage in the(Sulistiyono, 2004).

adoption of technology needs to be given serious

The most intensive and intensive use of pesticides

attention to the coaches because it determines the speed

in agricultural activities is in the types of holticulture andof farmers in adopting technology (Herman et al., 2006).

palawija (such as corn, sorghum, green beans, soybeans)

There have been a number of reviews on the issues ofcultivation activities (Abadi et al, 1993 in Rario et al,

IPM implementation and pesticide use by farmers;2005).

however, it is still necessary to re-sharpen our knowledge

The benefits of pesticides are proven to be so large

and awareness of IPM and the underlying factors thatthat they become the determinant factor in increasing

affect farmers’ lack of actions in supporting thecrop productivity and improving product quality, as

implementation of IPM. It is hoped that this paper can bereflected in every package of programs or agriculturala reference in determining the program of assistance andactivities that always include pesticides as part ofempowerment

production inputs (Wahyuni, 2010).

ofIndonesian

farmersinthePesticides

implementation of IPM.

areconsidered

as

the

saviourofMaterial and Method

agricultural production (Sulistiyono, 2004). However,This paper is arranged based on the compilation ofpesticides not only provide benefits to agriculture, butseveral research papers and books written by the experts

also have negative impacts. The negative effects ofin the field of IPM and attitudes. It is presented

pesticide use have been widely reported in various

descriptively in discussing the problems of farmers in thestudies. These impacts may include ecosystem instability,

implementation of IPM, which focuses on the attitudes

residuals on crops and their processed materials,

of farmers in Indonesia as a fundamental factor thatenvironmental pollution and poisoning, and even deathaffects farmers’ lack of actions in supporting thein humans (Wahyuni, 2010).

implementation of IPM

Result and Discussions

D.R.Minnick (1976) stated that chemical compounds
are indiscriminate in their pesticidal properties; they kill

Although conceptually the use of pesticides ismany non-target species that help to control pest

positioned as the last alternative in controlling Plant Pest

populations. The environmental effects of pesticide

Organisms (OPT) in the IPM concept, in reality,

residues have been well documented (Graham 1970;

pesticides are often used as the main and most common

Reese 1972; Robbins et al. 1974; Turk et al. 1975). Somechoice of farmers. Pesticides have been widely used bypesticides, when introduced in large quantities and on a

farmers in overcoming pests. This condition has become

continual basis into the biosphere, are responsible fora tradition and existed up to the present day among

disrupting life processes in non-target food chains.

farmers in running their system of farming (Sulistiyono,

Residues incorporated into human tissues, for example,

2004); (Untung, 1996 in Effendi, 2009); (Effendi, 2009).

may prove harmful (EPA 1975; Malibach et al. 1971).

In Asia, Indonesia is a country that uses a lot of

Several insecticides used extensively in rice in numerous- 26 -

countries are extremely toxic to birds and are expected toprotecting crops from pest attacks; their products can because frequent and largely unavoidable mortality

sold quickly at competitive prices so that maximum(Parsons et al., 2010).

profits can be achieved (Sulistiyono, 2004).

Having a closer look at its impacts on human lives,

Farmers may not realize that pesticides are actually

the WHO data also photograph at least 20,000 people per

toxic. They even make them as a primary choice andyear died from pesticide poisoning. About 5,000 toconsider it as medicine. Sometimes, farmers are still

10,000 people per year are affected by its side effects,

spraying vegetables that have been harvested and readysuch as cancer, disability, infertility, and liver disease.

to be transported, fearing that their harvests will be

Pesticides are one of the examples of Hazardous andattacked by pests. The ease of getting pesticides also

Toxic Materials (Indonesian: Bahan Berbahaya dan

encourages farmers to use excessive pesticides (Wahyuni,

Beracun or B3) that are often used by farmers (Riyadi,2010).

1995 in Wahyuni, 2010). FAO surveys in Tegal and

The lack of farmers' support in the implementation ofBrebes conducted in 1991-1992 found 69 pain symptoms

IPM is influenced by their perception and attitude.

in which 43 of them were known as typical symptoms of

Sulistyono et al. (2008) stated that the direct application

pesticide poisoning, such as shortness of breath,of pesticides in the field is still constrained by several

dizziness, nausea, and vomiting. The symptoms werefactors, such as knowledge of farmers about pesticides,

apparent after farmers performed 21% of the frequency

farmers’ attitudes toward the regulation of pesticide use,of spraying. Unfortunately, such a phenomenon isand the action of its use. A research conducted by Dwi

ignored by the farmers (Wahyuni, 2010).

Sadono (2001) in Kabupaten Karawang West Java

Experts state that in its practice, farmers are spraying

revealed that there is a significant relationship between

chemical pesticides on schedule, inaccurately targeted,the perception of farmers and the level of its adoption.

with inaccurate doses (they likely tend to mix some

Sadono (1999) in Herman et al (2006) also said that thepesticides and use more chemical pesticides than theyinternal factors of farmers that correlate significantly

should), without using self-protection equipment andwith the application of integrated pest control (IPM) arelack of environmental awareness ( Sulistiyono et al.,the education and perception of farmers towards IPM.

2008); (Ningrum, 2012); (Ruhs et al., 1999 andElske van de Fliert's (1993) study on IPM in Central

Kartaatmadja et al., 1997 in Wahyuni, 2010). The

Java

appropriate dosage of pesticide use by onion farmers

management, including: 1) “Hama jangan sampai

who have attended Integrated Pest Management School

merajalela” (Do not let pests break out), is an often(Indonesian:Hamaheard expression from rice farmers in the eight villages

Terpadu or SLPHT) in Nganjuk District were; 4.17%

in Grobogan, which clearly indicates the prevalentappropriate and 95.83% not appropriate, whereas theattitude towards pests in the rice crop: one insect pest indata garnered in the non-SLPHT participants showed thatthe crop is the forerunner for the others if the outbreak is

1.04% appropriate and 98.96% not appropriate. This way

uncontrollable. 2) “Sedia payung sebelum hujan “ (Haveof using pesticides has generated very serious ecologicalan umbrella ready before it starts raining), though often

impacts (Sulistiyono, 2004).

heard, does not exactly describe the actual perception of

SekolahLapangPengendaian

mentions

farmers’

perception

about

pest

Another issue of concern according to Pimentel andmany farmers towards (chemical) pest control. A more

Khan (1997) is a consumer demand that makes farmers

appropriate expression would be: “Use an umbrella evenconsider pesticides as the main alternative. Until today,

when it does not rain”, explaining the habits of many

consumers assess the quality of horticultural products

farmers in using pesticides as their preventive actions, as

especially from the enchanting appearance of thea result of the exposure of rice intensification program

products, usually known as “Cosmetic Appearance”.

for over twenty years. Christopher Vaughan (1988);

Because “Cosmetic Appearance” is still serving as the

Irham and Joko Maryono (2011); Dwi Sadono, (2001)

main assessment of consumers, it is necessary to create aalso stated that farmers were often spraying their fields

climate of competition among horticultural crop farmers

habitually – regardless of whether the fields wereto keep the appearance of the products that attract

infested – and that overuse of pesticides actuallyattention. The farmers' efforts are to use pesticides inincreased the risk and severity of pest infestation in the- 27 -

rice fields.

that they likely tend to conduct the spraying on schedule.

A research conducted by Herman et al. (2006) in

3) awareness, there is farmers’ low awareness in theWest Sulawesi showed that the application of pest

implementation of IPM, 4) behaviour intention, farmers

control technology (cocoa) is influenced by four factors,

intend to conduct IPM because of the support ofincluding farmers’ attitudes, level of income, assistancecognitive aspects, but its implementation is strongly

of guide/coach, and space of garden. The attitudes ofinfluenced by their surroundings so that it will restrict

farmers have a positive effect on farmers' actions inthem to act based on the rules. Azwar (2012) explained

adopting cocoa pod borer pest control technology. Anggi

that a person who is considered important in making

Khoirudin Siregar (2014) study stated that there is still a

decisions will greatly influence the formation of attitudes,

lack of knowledge and attitude towards integrated pest

including: parents, peers, close friends, co-workers,

management (IPM) in District of Rancabungur, Bogor

family, people with a higher social status, neighbours.

Regency. This is influenced by the perception of farmers

Individuals tend to have the similar attitudes with those

who still believe that the use of pesticides is the one andwho are considered important. According to Rogers

only effective way in controlling pests. Although most of(2003), farmers’ innovative decision-making is also

farmers (82.5%) agree that pesticides endanger the

influenced by the behaviours or decisions taken by social

environment and users (farmers), as many as 70% of theinstitutions

respondents agree that pesticides are effective infarming communities, such as neighbours, relatives,controlling pests and diseases.

farmer groups, and religious groups, 5) internal conflict,(groups/organizations)existing

withinBaron and Byrne (2003) also explained that,the most influential internal factors are the fulfilment ofaccording to a social psychologist, attitudes are veryneeds and the constraints of farming, severe pest attacks

important factors. Not only because it is difficult toraise fears and cause strong feelings of anxiety thatchange, but it is also seen as the central issue in the field

encourages farmers to apply pesticides in a reckless way.

of social psychology, for the following reasons: 1)

Instead of being instantly changed, the development

attitudes deeply influence social thinking, although theyof attitudes likely tends to need a relatively long time andare not always reflected in apparent behaviour, 2)

it is commonly accompanied by repetitive efforts that

attitudes often affect behaviour, this is especially true

result in positive attitudes toward its newly introduced

when the attitudes are strong and steady.

technology, which will then be followed by the stability

Luluk Sulistiyono et al. (2008); (Utama, 2003)

in adopting the technology (Tjitropranoto, 2005).

mentioned that farmers’ attitudes in using pesticides are

Therefore, although it is not easy, farmers’ empowerment

formed from information that is believed to be true and itpractitioners need to develop strategies to shape farmers'isthroughpositive attitudes toward IPM activities and change theircommunication between farmers. Wahyuni (2010) said

mindset of using excessive pesticides so that they willthat the activity of controlling pest intruders is a work

have a right mindset on how to deal with plant pests andthat takes a lot of time, effort, and cost. Besides being

diseases.

easy to use, the efficacy of pesticides is reliable.

Conclusion

Furthermore, in addition to its high success rate,

Integrated Pest Management (IPM) aims at limiting thepesticides are available and easy to get at relatively low

use of synthetic insecticides by introducing the concept

and affordable prices. These conditions allow the farmersof economic threshold as a basis for determining pest

to expose their plants with pesticides easily, and evencontrol. This approach encourages the replacement ofexcessively.

chemical pesticides with alternative control technologies,

inheritedeitherinprivatewayorLuluk Sulistiyono et al. (2008) stated that there arewhich use more biological materials and methods,

several factors causing a weak relationship between

including natural enemies, biological pesticides, andattitude and action, they are: 1) anxiety, it happens whenpheromones.

farmers feel very anxious if there is crop failure so thatthoroughly implemented by farmers in Indonesia. There

they cannot return the investment value. 2) forecasting, it

are several obstacles or problems in the implementation

is a lack of farmers’ ability in predicting what pests andof IPM in Indonesia, and they are mainly due to the lackdiseases that will attack during the growing season, so

of support from the farmers themselves. These actions- 28 -

Unfortunately,

this

still

cannotbeare caused by their attitudes toward IPM, especially theiruse of pesticides in overcoming pests and plant diseases.

9)

Acknowledgement
Farmers’ empowerment practitioners need to realizethat the actions of farmers in the implementation of IPM
are influenced by their attitudes and mindset. Therefore,

10)

although it is not easy, the farmers' coach or
empowerment practitioners need to carry out a specificstrategy to form farmers’ positive attitudes toward IPM

11)

so that in the future they are able to use pesticides more
wisely.

Reference
1)
2)

3)

4)

5)

6)

7)

8)

Baron, R and D. Byrne. 2003. Social Psychology(in Indonesian). Edisi Sepuluh. Jakarta . Erlangga
Chandler, D et al. 2011. The Development,
regulation and use of biopesticides for integrated
pest management.doi : 10.1098/rstb.2010.0390.
http://rstb.royalsocietypublishing.org/content/366/1
573/1987.full.html#related-uris.
Accessed
December 2017
Effendi, B.S. 2009. Integrated Pest Control Strategy
of Paddy Plant in Perspective of Good Agricultural
Practices.Pengembangan Inovasi Pertanian 2 (1)2009.
http://pustaka.litbang.pertanian.go.id/publikasi/ip02
1095.pdf. Accessed December 2017
Fliert, E. van de. 1993. Integrated Pest Management
farmer Field Schools Generate Sustainable
Practices A case study in Central Java evaluating
IPM Training.Thesis. Agricultural University
Wageningen
Netherlands.
http://library.wur.nl/WebQuery/wurpubs/fulltext/13
3364. Accessed December 2017
Gibson, J.L., J.M. Ivancevicg, J.H. Donnely. 1994.
Organization
andManagement(inIndonesian).Jakarta. Erlangga
Hammond, C.M et al.2006. Adoption of Integrated
Pest Management Tactics by Wisconsin Farmers.
Weed Technology, Vol 20, No 3 (Jul-Sep 2006).
Published by Weed Science Society of America andAllen Press. http:/www.jstor.org/stable/4495750.
Accessed December 20107
Herman et al. 2006. Analysis of Factors Influencing
Adoption of Technology in Cocoa Pod Borer Pest
Control : A case study in west Sulawesi (inIndonesian). Pelita Perkebunan 22(3) 2006
http://iccri.net/download/Pelita%20Perkebunan/vol_22_no_3_desember_2006/Analisis%20Faktor-Fak
tor%20yang%20Mempengaruhi%20Adopsi%20Te
knologi%20Pengendalian%20Hama%20Penggerek%20Buah%20Kakao%20Studi%20Kasus%20di%2
0Sulawesi%20Barat.pdf.Accessed
December2017
Irham and J.Mariyono. 2001. The Change ofChemical Pesticides Use Decision Making in Ricce
by Integrated Farms (in indonesian). Manusia dan
Lingkungan Vol VIII No 2 Agustus 2001.
https://journal.ugm.ac.id/JML/article/view/18576.

12)

13)

14)

15)
16)

17)

18)

19)

20)

- 29 -

Accessed December 2017.
Minnick, D.R.1976. Integrated Pest Management.
The American Biology Teacher, Vol 38, No 4 (April,
1976).Published by University of California Presson behalf of the National Association of Biology
Teachers.http://www.jstor.org/stable/4445558.
Accessed December 2017
Newcomb T.M., R.H. Turner.,P.E.Converse.1985.
Social Psychology (in Indonesian).CV Diponegoro.
Bandung.Indonesia
Ningrum, N.T. 2012. An Evaluation Survey ofIntegrated Pest Management (IPM) Socialization
Program of Rice Farmers in Tambun Utara
Subdistrict of Bekasi (in Indonesian). Skripsi.
Bogor.Bogor Agriculture University
Parsa, S et al.2014.Obstacles to Integrated Pest
Management Adoption in Developing Countries.
PNAS
March
11,
2014.
www.pnas.org/cgi/doi/10.1073/pnas.1312693111.
Accessed December 2017.
Parsons, K.C., P.Mineau, and R.B.Renfrew. 2010.
Effects of Pesticide use in Rice Fields on Birds.
Waterbirds, 33 (sp 1).The Waterbird Society.
http://dx.doi.org/10.1675/063.033.s.115.http://www.
bioone.org/doi/full/10.1675/063.033.s.115.
Accessed December 2017
Rario, B., Kasto, and S.Ritohardoyo. 2005.
Farmer’s Perception and Behavior Toward the
Risk of Pesticide to the Environment in
Kalampangan Village Sabangan Sub District
Palangka Raya City Indonesia (in Indonesian).
Manusia dan Lingkungan Vol 12 No 1 March 2005.
https://journal.ugm.ac.id/JML/article/view/18633 .
Accessed December 2017.
Rogers, E.M. 2003. Diffusion of Innovations.Fifth
Edition..New York. Free Press
Roling, N and E.van de Fliert. 1994. Transforming
extension for sustainable agriculture: The case of
integrated pest management in rice inIndonesia..Environmental Values. Agriculture and
Human
Values-Spring
Summer.
https://link.springer.com/article/10.1007/BF015304
51.Accessed December 2017
Sadono, D. 2001. Adoption Innovation ofIntegrated Pest Management by Farmers : Case
Study in Karawang Regency West Java (inIndonesian). Mimbar Sosek.Jurnal Sosial Ekonomi
Pertanian Volume 14 No 1 April 2001.
Siregar, A.K. 2014. Knowledge, Attitudes, andAction of Farmers against Pest Organisms Palawija
and Horticulture in sub District Rancabungur
Bogor.Skripsi.Bogor..Bogor Agriculture University
Sulistiyono, L. 2004. Dilemma of Pesticide Use in
Horticultural Crops System in Indonesia (inIndonesian).http://www.rudyct.com/PPS702-ipb/08
234/luluk_sulistiyono.pdf. Accessed December
2017.
Sulistiyono, L et al. 2008. Knowledge of Attitudes
and Action of Red Onion Farmers in Pesticide Use(Case Study in Nganjuk District East Java
Province)(in Indonesian). J.Agroland 15 (1), Maret

21)

22)

23)

24)

2008.
ISSN
:
0854-641X.
http://jurnal.untad.ac.id/jurnal/index.php/AGROLA
ND/article/view/37. Accessed December 2017.
Tjitropranoto, P. 2005. Self-Understanding, Self
Potential / Readiness, and Innovation Introduction(in Indonesian).Jurnal Penyuluhan Vol 1 No 1
September2005.
http://ilkom.journal.ipb.ac.id/index.php/jupe/article/
view/2103. Accessed December 2017.
Utama, S.P. 2003. Study on Technical Efficiency ofRice Farming on Farmers Participants of Integrated
Pest Control Field School (SLPHT) in West
Sumatera (in Indonesian). Jurnal Agrisep Vol 2 No
1
September2003.
http://repository.unib.ac.id/7881/1/B4%20Satria.pdf.
Accessed December 2017.
Vaughan, C. 1988. Disarming Farming’s Chemical
Warriors. Science News, Vol 134, No 8 (Aug.20
1988). Published by Society for Science & the
Public.http://www.jstor.org/stable/3973133.
Accessed December 2017.
Wahyuni, S.2010. Behavior of Red Onion Farmers
in the Use and Handling of Pesticides and Their
Impact on the Environment (Case Study in
Kemukten Village, Kersana Sub-district, Brebes
District)(inIndonesian).
Thesis.
Semarang.Universitas Diponegoro.

- 30 -

OS I-3
Taxonomical studies of blood disease bacterium of banana
Dwiwiyati Nurul Septariani, Hadiwiyono(Faculty of Agriculture, Sebelas Maret University)

SUMMARY
Ralstonia solanacearum is considered a species complex-a heterogeneoius group of related but genetically distinct
strains. The term “species complex” was used first to describe the high genetic variation between isolates, and theconcept of R. solanacearum species complex was expanded by including two closely related species fromIndonesia, Ralstonia syzygii (a pathogen of clove trees) and the agent of blood disease of banana, known as the BDB.
Within the R. solanacearum group, BDB and R. syzygii are most similar to the R. solanacearum strains fromIndonesia, and thus belong to phylotype IV. The four phylotypes encompass three different species, thereby
justifying the use of the term “species complex”, defined as a cluster of closely related isolates whose individual
members may represent more than one species. However, the genetic basis for this variation is unknown, andoriginal culture deposited as the type strain no longer exists si the name is taxonically invalid. For the past four
decades, two different systems, race and biovar, have been widely used to differentiate R. solanacearum strains. The
race structure of R. solanacearum is poorly defined and not taxonomically useful. Comparative genomic analysis isneeded to defined phylogenetic of R. solanacearum species complex.

Introduction

tip-over disease (Erwinia carotovora ssp. carotovora and

Banana is a leading food crop in terms of production

E. chrysanthemi), bacterial rhizome and pseudostem wet

value. Its total tarde value stood at some USD 8 billion inrot (Dickeya paradisiaca formerly E. chrysanthemi pv.

2016 with some 15 percent of global production exported

paradisiaca). Other bacterial diseases of less widespreadand making bananas the largest traded fruit crop in value

importance include: Javanese vascular wilt, bacterial wilt

terms [1]. In Indonesia, that is not only rich in banana

of abaca and bacterial fingertip rot (probably caused bydiversity, but also is a center of banana’s distribution.

Ralstonia spp., unconfirmed) [4].

Banana production in Indonesia is one of the largest in the

Banana blood disease is thought to have originated on

world. However, 90% of the products are used on national

Salayar Island near Sulawesi, where it was first reported

scale only [2]. It caused productivity particularly in terms

after the introduction of dessert bananas in the earlyof the quality of the fruit. An indication that result in a

1900s [5,6]. The disease was confined to Salayar for

decerase of quality is pest and disease problems at the

many years due to the strict quarantine regulations

time of its growth [3].

implemented by the Dutch. However, it had become

Independent of region and production system, pests andwidespread on local cooking banana cultivars in southerndiseases have been considered the main constraints

Sulawesi (formerly Celebes) by 1920 [7,8], and thenresponsible for yield losses and low productivity ofprobably spread throughout the island until its discovery

bananas. The fungal diseases black leaf streak diseasein Java in the late 1980s [6].

(commonly known as black Sigatoka), (Mycosphaerella

Unfortunately, the pathogen has since continued its spreadfijiensis) and Fusarium wilt (Fusarium oxysporum f. sp.

to most of the larger Indonesian islands, where averagecubense) have always been considered as the mostyield losses often exceed 35% [9]. These outbreaks were

important banana diseases globally and have therefore

associated with the transmigration of people from Java toreceived more attention. However, bacterial diseases

less populated islands in Indonesia [10]. The banana

cause

blood disease is currently spreading in peninsularsignificant

impactsonyield

globallyandmanagement practices are not always well known [4].

Malaysia where it coexists with the Moko and Fusarium

Bacterial diseases of banana and enset can be classified

wilt diseases [11]. The disease has been first detected in

into three distinct groups: i) Ralstonia-associated diseases

the province of Perak and more recently in the province(Moko/Bugtok disease caused by Ralstonia solanacearumof Selangor [11,12,13]. Banana blood disease has also

and banana blood disease caused by R. syzygii subsp.

been observed on the island of New Guinea [14]. Severecelebesensis); ii) Xanthomonas wilt of banana and enset,

destruction due to banana blood disease was noted incaused by Xanthomonas campestris pv. musacearum andSouth Sulawesi, where 70–80% of plantations were lostiii) Erwinia-associated diseases (bacterial head rot or[15], and in West Java, where 27–36% plantation loss was- 31 -

than 20,000 tons of banana, with an estimated value ofpoly-hydroxybutyrate intracellularly. Most strains
denitrify and produce a diffusible brown-red pigment on

US$1 million, were lost to banana blood disease [17].

rich medium. It does not grow below 40C or above 400C,

Losses will most likely escalate with disease spread. If theand there is little or no growth in 2% NaCl [25, 26, 27,disease 1 day arrives on the South-East Asian mainland

28].

there would be no barriers to its eventual/gradual

Ralstonia solanacearum is a heterogeneous species, as

movement to the Indian subcontinent [10,15].

demonstrated by its large host range, pathogenic

One disease have caused production tend to stagnant or

specialization and physiological and cultural properties,

decline was blood bacterial. It was seem that blood
bacterial wilt disease caused by blood disease bacterium

as well as its phylogeny [29]. Despite being classified as asingle species, it has been reported that different strains of(BDB) have involved in the case of low production of

R. solanacearum may have DNA–DNA relatedness

bananas [9]. The national loss of banana production due
to blood bacterial wilt disease was estimated around 36%

values below 70% [30] and therefore could possibly bemembers of different species. The term ‘species complex,’in 1991 [19]. The damage of banana mats was extremely

which refers to ‘a cluster of closely related bacteria whose

serious in certain districts in where ABB genomic groupindividual members may represent more than one

were planted such as Bondowoso and Lombok, thespecies,’ was then proposed for R. solanacearum [31].

disease incidence could reach over 80% [9]. Now thepathogen has been distributed in 90% of Provinces in

It is assumed that R. solanacearum originated, adaptedand evolved in widely different places, resulting in greatIndonesia with various disease incidences from 10

geographic and pathogenic diversity and translating inthousand to millions of banana clusters [20].

variable disease expression and disease potentials for

BDB is existent in all of the sample tissue point andeach host/parasite genotype interaction [32,33]. However,sistemically distributed in all parts of infected plants.

recent studies suggest that R. solanacearum most likely

Specific symptoms showed hanging drop of leaves on

originated in Oceania/Indonesia, and migrated to Africa,

vegetative stage, dried inflorescence of flower and wilt onSouthgenerative stage, and dried inflorescence of flower

fragmentation of the ancestral continent Gondwana

extending to upper parts or fruits on bunch. Internal[34,35].

symptoms showed browning in vessel cells occurred in

The R. solanacearum species complex is subdivided into

the most part of plants, pulp, stalk, fruit shelter, pedundle,

four distinct phylotypes, largely correlating with the

middle peduncle and pseudostem, basal peduncle, midrib,

geographic origin and evolutionary past of the strains.

petiole, corm, and root; and yellowing leaf lamina [21].

Strains are assigned to the Asian (phylotype I), American

Ralstonia solanacearum was first described and classified(II), African (III), and Indonesian (IV) phylotypes

as Bacterium solanacearum by Erwin F. Smith at the end[36,37,38]. Phylotype IV hosts the two closely relatedof the 19th century [22]. The causal agent of bacterial wilt

bacteria R. syzygii (the causal agent of Sumatra disease of

was then successively named P. solanacearum, and more

clove) and the ‘blood disease bacterium (BDB)

recently,[28,34,38,39,40].

recorded [16]. In Lampung Province (Sumatra), moreby

Burkholderia

applicationofsolanacearumDNA-based
[23]

and

methods,
finallyR.

America

and

Asia,

possibly

beforetheUsing a polyphasic taxonomic approach, the R.
solanacearum species complex was merged into threesolanacearum [24].

Material and Method

species: R. solanacearum corresponding to phylotype II

Strains and genomic DNA extraction, sequencing andstrains (including Moko strains); R. pseudosolanacearum

assembly, genomic annotation, and comparative genomic

corresponding to phylotypes I and III; and R. syzygii

analyses.

corresponding to phylotype IV. R. syzygii sp. nov is

Result and Discussions

further divided into three subspecies: the broad host range

The genus Ralstonia belongs to the family Burkholderia

strains are R. syzygii subsp. indonesiensis subsp. nov.; the(class Betaproteobacteria) that includes nine genera andstrains causing Sumatra disease of cloves as R. syzygii

many humanand plant-pathogenic species and several

subsp. syzygii subsp. nov.; and the BDB strains causing

symbionts. Ralstonia is an aerobic, Gram-negative rod

the banana blood disease as R. syzygii subsp. celebesensis

with a polar flagella tuft. It is oxidase positive, arginine

subsp. Nov [41]. Comparative analysis of 29 whole

dihydrolase

genomes by MUMi and the use of protein profiling on anegative,andaccumulates- 32 -

larger set of bacterial strains by matrix-assisted laser

genome size may be a step in the evolution of thesedesorption/ionization-time of flight mass spectrometry

specialized bacteria via genome decay under selective(MALDI-TOF-MS), support the division of the R.

pressures within the host. The critical questions about thesolanacearum species complex into three species

unique biology of these two microorganisms remain toconsistent with genomic and proteomic data as well as
biological differences [42].

be experimentally addressed. The two genome sequences

Ralstonia

studies.

syzygii subsp. celebesensis (BDB) was

will be a valuable tool for these subsequent functional

historically described and named P. celebensis in 1921,

Referencebut the name became invalid when the original type strain

1)

was lost [5,7,43] suggested that the blood disease

United Nations) (2017) Food Outlook Biannual report on

pathogen coevolved with banana. Buddenhagen (2009)

globalhowever, indicated that this was unlikely due todifferences in when and where the disease first appeared.

http://www.fao.org/3/a-i7343e.pdf.

Blood disease was first observed where wild bananas

Pisang Indonesia. Bunga Rampai Forum Peneliti Muda

were not found (Rijks 1916), supporting the suggestion

Indonesia 2017. Bandung (ID): Penerbit ITB.

that the bacterium originated on other plant species than3) Riastiwi I (2017) Inventarisation of banana plantbanana [33]. Colonies of the ‘banana blood disease’

diseases in germplasm collection garden, Cibinong

strains are smaller than those of R. solanacearum causing

Science Center-BG. Jurnal Mikologi Indonesia 1(1):Moko and are slow-growing, nonfluidal on Kelman’s

38-44

TZC

medium4) Blomme G, Dita M, Jacobsen KS, Vicente LP, Molina(commonly used for R. solanacearum) and have smoothA, Ocimati W, Poussier S, Prior P (2017) Bacterial

margins with a dark-red center [44]. Genetic analyses, bydisesase oh bananas and enset: Current state ofwhole genome RFLP groupings, comparison of partial

knowmledge

16s ribosomal DNA sequences and analysis of tRNAsustainable management. Front Plant Sci 8:1290.

consensus primer amplification products, indicate a close
relationship, but distinctly different from other strains of5) Eden-Green SJ (1994b) Banana Blood Disease.

the R. solanacearum species complex [5,39,45]. Genetic

And Agriculture Organization of The United Nation.

analyses revealed that there is little diversity among

6) Thwaites R, Eden-Green SJ, and Black R (2000)

strains of BDB [38,46], suggesting few introductory and“Diseases caused by bacteria,” in Diseases of Banana,founder events as well as a recent evolutionary past on

Abacá and Enset, ed. D. R. Jones (Wallingford: CAB

banana [10].

International), 213–239.

Conclusion

7) Gäumann E (1921) Onderzoekingen over deThe(Triphenyl

genomes

Tetrazolium

of

the

Blood

Chloride)Diseass

Bacterium

FAO (Food and Agriculture Organization of thefoodmarkets.

Availableat:

http://

2) Dwivany F, Nurrahmah A (2017) Pentingnya Dataandintegrated

approaches

towardINIBAP Musa Disease Fact Sheet No. 3. Roma: Food

bloedziekte der bananen op Celebes I. Mededelingen van

and R. syzigii are closely related to R.solanacearum

het. Instituut voor Plantenziekten 50, 1–47.

strains from Indonesia. Comparative genomic analysis8) Stover RH and Espinoza A (1992) Blood disease ofdemonstrated that these specialized organisms, whichbananas in Sulawesi. Fruits 47, 611–613.

belong to R. solanacearum phylotype IV, are part of the9) Supriadi (2005) Present status of blood disease insame genomic species. R. solanacearum species complex

Indonesia. In Allen C, Prior P, Hayward AC (eds)should be divided into three genomic species. The

Bacterial wilt disease and the Ralstonia solanacearum

pathogenic

species complex. APS Press, Minnesota.

behaviorofR.

syzygii

very unusualin the R. solanacearum species complex, may have10) Ploetz RC, Kema GHJ and Ma L-J (2015) Impact of

resulted from ecological adaptation and genomic

diseases on export and smallholder production of banana.

convergence during vertical evolution. The pathogenicAnn.

and life-style traits may have been horizontally acquired

10.1146/annurev-phyto-080614-120305

from uncharacterized microbes. BDB and R.syzygii11) Teng SK, Aziz NAA, Mustafa M, Laboh R, Ismail IS,

represent

R.

Sulaiman SR, et al. (2016) The occurrence of blood

solanacearum species complex although the genomes are

disease of banana in selangor, Malaysia. Int J Agric Biol

relatively large for bacterial genomes. This reduction in

18, 92–97.doi: 10.17957/IJAB/15.0067.

theshortest

genomeswithinthe- 33 -

Rev.

Phytopathol.

53,

13.1–13.20.

doi:12) Heng SB (2012) Blood Disease, Banana: Malaysia.

nov. and transfer of seven species of the genus

ProMedPseudomonas homology group II to the new genus, with

20120106.10021287.

Available

at:http://www.promedmail.org/

the type species Burkholderia cepacia (Palleroni and13) Kogeethavani R, Sulastri NJ, Mazanah M, Rozeita L,
and Mohamad Roff MN (2013) First Report of Blood

Holmes 1981) comb. nov. Microbiol. Immunol. 36,

Disease Bacterium on Banana in Malaysia. Malaysia:24) Yabuuchi E, Kosako Y, Hano I, Hotta H, andMalaysian Agricultural Research and Development

Nishiuchi Y (1995) Transfer of two Burkholderia and an

Institute.
14) Davis RI, Moore NY, and Fegan M (2001) “Blood

Alcaligenes species to Ralstonia gen. nov.: proposal of
Ralstonia pickettii (Ralston, Palleroni, and Douderoff

disease and panama disease: two newly introduced and1973) comb. nov., Ralstonia solanacearum (Smith 1896)

grave threats to banana production on the island of New

comb. nov. and Ralstonia eutropha (Davis 1969) comb

Guinea,” in Proceedings of the Papua New Guinea Foodnov.

and Nutrition 2000 Conference (Canberra: ACIAR),

10.1111/j.13480421.1995.tb03275.x.

26–30.

25) Baharuddin B, Rudolph K, and Niepold F (1994)

15) Roesmiyanto LH and Hutagalung L (1989) Blood
disease (P. celebesis) on banana in Jeneponto – Sulawesi

Production of monospecific antiserum against the blood
disease bacterium affecting banana and plantain.

Selatan [abstract in English]. Hortikultura 27, 39–41.

Phytopathology 84, 570–575.doi: 10.1094/Phyto-84-570.

16) Subijanto M (1991) “Status of banana disease in26) Taghavi M, Hayward C, Sly LI, Fegan M (1996)

Indonesia,” in Proceedings of Technical Meeting on
Diseases Affecting Banana and Plantain in Asia and theAnalysis of the phylogenetic relationships of strains ofPacific: Banana Diseases in Asia and the Pacific, Brisbane,

the blood disease bacterium of banana based on 16S

Australia, eds RV Valmayor, BE Umali, and CP BejosanorRNA gene sequences. Int J Syst Bacteriol 46, 10–15.doi:(Los Baños: INIBAP), 44–49.

10.1099/0020771346-1-10.

17) Nurhadi M and Harlion R (1994) Serangan bakteri27) Coenye T and Vandamme P (2003) Simple sequence

dan cendawan pada tanaman pisang di propinsi dati I

repeats and compositional bias in the bipartite Ralstonia

lampung [in indonesian]. Info Hortik. 2, 37–40.

solanacearum GM11000 genome. BMC Genomics18) Jones DR (2013) “Emerging banana diseases: new

4:10.doi: 10.1186/1471-2164-4-10.

threats from old problems,” in Proceedings of the XX28) Villa JE, Tsuchiya K, Horita M, Opina N,

Internationl Meeting ACORBAT, Fortaleza, 79–90.

Hyakumachi M (2005) Phylogenetic relationships of19) Muharom A and Subijanto (1991) Status of banana

Ralstonia solanacearum species complex strains from

disease in Indonesia. In: banana disease in Asia andAsia and other continents based on 16S rDNA,

Pacific. Proceeding of technical meeting on diseaseendoglucanase, and hrpB gene sequences. J. Gen. Plant

affecting banana and plantain in Asia and the Pacific,

Pathol. 71, 39–46. doi: 10.1007/s10327-0040156-1.

Brisbane, 15th-18th Augustus 1991.

29) Hayward AC (1991) Biology and epidemiology of20) Subandiyah S, Hadiwiyono, Nur E, Wibowo A, Feganbacterial wilt caused by Pseudomonas solanacearum.

M, Taylor P (2006) Survival of blood disease bacterium

Annu. Rev. Phytopathol. 29, 65–87. doi: 10.1146/

of banana in soil. In: Proceeding of the 11th international
conference on plant pathogenic bacteria, Edinburgh,

annurev.py.29.090191.000433.
30) Palleroni NJ and Doudoroff M (1971) Phenotypic

10-14 July 2006.

characterization and deoxyribonucleic acid homologies of21) Hadiwiyono (2011) Blood bacterial wilt disease of

Pseudomonas solanacearum. J. Bacteriol. 107, 690–696.

banana: the distribution of pathogen in infected plant,31) Gillings MR and Fahy P (1994) “Genomic

symptoms, and potentially of diseased tissues as source offingerprinting:towardsaunified

view

of

the

infective inoculums. Bioscience 3(3): 112-117.

Pseudomonas

solanacearum

species

complex,”in22) Smith EF (1896) A bacterial disease of the tomato,

Bacterial wilt: the Disease and Its Causative Agent,

eggplant, and Irish potato (Bacillus solanacearum n. sp.).

Pseudomonas solanacearum, eds A. C.

Bull. Div. Veg. Physiol. Pathol. U.S Dep. Agric. 12, 1–28.

32) Buddenhagen IW (1986) “Bacterial wilt revisited,” in23) Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta H,

Bacterial wilt Disease in Asia and the South Pacific.

Hashimoto Y, et al. (1992) Proposal of Burkholderia gen.

Proceedings of an international workshop held at1251–1275.doi: 10.1111/j.1348-0421.1992.tb02129.x.

Microbiol.

Immunol.

39,

897–904.doi:

Burkholderia solanacearum, Pseudomonas syzygii, and- 34 -

PCARRD, Los Baños, Philippines, 8 to 10 October 1985.

as Ralstonia syzygii subsp. Syzygii subsp. nov., R.

ACIAR proceedings no. 13, ed. G. J. Persley (Canberra:solanacearum phylotype IV strains as Ralstonia syzygii

Australian Centre for International Agricultural Research),subsp. indonesiensis subsp. nov., banana blood disease

126 –143.

bacterium strains as Ralstonia syzygii subsp. celebesensis

33) Buddenhagen IW (2009) Blood bacterial wilt ofsubsp. nov. and R. solanacearum phylotype I and III

banana: history, field biology and solution. Acta Hort. 828,

strains as Ralstonia pseudosolanacearum sp. nov.

57–68.doi: 10.17660/ActaHortic.2009.828.4.

International Journal of Systematic and Evolutionary34) Remenant B, de Cambiaire JC, Cellier G, Jacobs JM,

Microbiology

Mangenot S, Barbe V, et al. (2011) Ralstonia syzygii, the10.1099/ijs.0.066712-0.

blood disease bacterium and some Asian R. solanacearum42) Prior P, Ailloud F, Dalsing BL, Remenant B, Sanchez

strains form a single genomic species despite divergent

B and Allen C (2016) Genomic and proteomic evidence

lifestyles.

supporting the division of the plant pathogen Ralstonia

PLoS

ONE

6:e24356.doi:

64,

3087–3103.

doi:

10.1371/journal.pone.0024356.

solanacearum into three species. BMC Genomics 17:90.

35) Wicker E, Lefeuvre P, de Cambiaire JC, Lemaire C,

doi:10.1186/s12864-016-2413-z.

Poussier S, and Prior P (2012) Contrasting recombination

43) Jones DR (2000) Diseases of Banana, Abaca andpatterns and demographic histories of the plant pathogen

Enset. Oxfordshire: CABI.

Ralstonia solanacearum inferred from MLSA. Int. Soc.

44)

Microb. Ecol. J. 6, 961–974.doi: 10.1038/ismej.2011.160.

Compendium. Ralstonia solanacearum Race 2. Available36) Fegan M (2005) “Bacterial wilt diseases of banana:

at:http://www.cabi.org/isc/datasheet/44999/aqb.

evolution and ecology,” in Bacterial wilt Disease and the45) Eden-Green SJ (1994a) “Diversity of Pseudomonas

Ralstonia solanacearum Species Complex, eds C.

solanacearum and related bacteria in southeast asia: new37) Prior P and Fegan M (2005) Recent developments indirections for Moko disease,” in Bacterial wilt: The

the

Diseasephylogeny

solanacearum.

and
Acta

classification

of

Hort.

127–136.

695,

Ralstonia
doi:

CAB

and

Internationalits(2014)

Causative

Invasive

Agent,

Species

Pseudomonas

solanacearum, eds A. C.

10.17660/ActaHortic.2005. 695.14.

46) Thwaites R, Mansfield J, Eden-Green S, and Seal S38) Fegan M and Prior P (2006) Diverse members of the(1999) RAPD and rep PCR-based fingerprinting ofRalstonia solanacearum species complex cause bacterial

vascular bacterial pathogens of Musa spp. Plant Pathol.

wilts of banana. Aust. Plant Pathol. 35, 93–101. doi:

48, 121–128.doi: 10.1046/j.1365-3059.1999.00321.x

10.1186/s12864-016-2413-z.
39) Seal SE, Jackson LA, Young JPW, and Daniels MJ(1993) Differentiation of Pseudomonas solanacearum,
Pseudomonas syzygii, Pseudomonas pickettii and theblood disease bacterium by partial 16S rRNA sequencing:
construction of oligonucleotide primers for sensitive
detection by polymerase chain reaction. Microbiololgy
139, 1587–1594. doi: 10.1099/00221287-139-7-1587
40) Vaneechoutte M, Kämpfer P, De Baere T, Falsen E,
and Verschraegen G (2004) Wautersia gen. nov., a novel
genus accommodating the phylogenetic lineage including
Ralstonia eutropha and related species, and proposal of
Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990)
comb. nov. Int. J. Syst. Evol. Microbiol. 54, 317–327. doi:
10.1099/ijs.0. 02754-0
41) Safni I, Cleenwerck,x I, De Vos P, Fegan M, Sly L,
and Kappler U (2014) Polyphasic taxonomic revision of
the Ralstonia solanacearum species complex: proposal toemend the descriptions of Ralstonia solanacearum and
Ralstonia syzygii and reclassify current R. syzygii strains- 35 -

- 36 -

OS II-1
Bacterial endophytes isolated from orchids and their influence on plant health
Tri JOKO1,3, Dewi Nur ANGGRAENI2, Mery Irianti3, Budi Setiadi DARYONO2, Jaka WIDADA1,3,
Siti SUBANDIYAH1,3(1Faculty of Agriculture, Universitas Gadjah Mada, Indonesia; 2Faculty of Biology, Universitas Gadjah Mada,
Indonesia; 3Research Center for Biotechnology, Universitas Gadjah Mada, Indonesia)

SUMMARY
The intimate relationship between endophytic bacteria and their host may influence the physiologicalmechanisms of plants. In this study, a hundred and six bacterial endophytes were isolated from tissues of twelve orchid
species, and screened according to in vitro antagonistic activity, indole-3-acetic acid (IAA) production, and phosphatesolubilization. Among 106 isolates able to inhibit soft-rot pathogen, 79 were found to produce IAA and 92 were able tosolubilize inorganic phosphate. Thirty selected isolates were then subjected to ERIC and rep-PCR to investigate their
genetic diversity. Three promising isolates (AkOc1, DnAr4, and TbPh7) were then applied for in vitro growth andchallenged against soft rot bacterial pathogen. It was found that introduction of endophytic isolates to plantlets of
Phalaenopsis amabilis increased significant growth of plantlet as well as disease suppression. Characterization by
partial 16S rRNA gene sequences analysis revealed that the isolates were closely related to Raoultella ornithinolytica,
Klebsiella michiganensis, and Bacillus amyloliquefaciens subsp. plantarum respectively. The ability of the isolates toinhibit soft rot bacterial pathogen as well as to promote plant growth makes them potential for enhancing plant health.
growers, but it was not very successful. Rather, chemical

Introduction
Some bacteria were known to live and associateapplication might impact on environmental problems.

with plants, either live in rhizosphere or inside plants

Therefore, alternatives to control soft rot disease should

tissues. Bacteria on roots and in the rhizosphere benefit

be the alternative way out to encounter this problem. The

from root exudates, but some are capable of entering thepresent investigations have been focused on the ability ofplant as endophytes that could establish a mutualistic

endophytic bacteria from orchid to growing stimulation of1

association .

Endophytic

bacteria

aredefinedasassociated bacteria found and colonized within the plant

Phalaenopsis amabilis plantlets and as an alternative tochemical in plant disease management.

tissue without causing harmful effect for host plants.
These bacteria can be isolated from surface-sterilized

Materials and Methods

plant tissue. The growth enhancement by the bacterial

Endophytic bacterial strains and growthendophytes can be a consequence of the synthesis ofconditions. Investigated bacterial strains were originallyphytohormones, biocontrol of phytopathogens in the root

isolated from root, pseudobulb, leaf, stalk, and flower

zone (through production of antifungal or antibacterial

tissues of twelve orchid species collected from nature

agents, siderophore production, nutrient competition andand nurseries. Isolation and characterization of theinduction of systematic acquired host resistance, or

strains based on their phenotypic characteristics, as well2

immunity) or by enhancing availability of minerals .

as their screening for antibiosis were described earlier.

Soft rot disease is one of the most common andimportant limiting factors for growing orchid in many

The strains were routinely maintained at 4C after
growth at room temperature on Tryptic soy broth (TSB)

nurseries worldwide. Several reports have indicated theor Nutrient agar (NA). Cultivation in liquid media wasbacterial soft rot was the most disastrous disease and madeperformed in 50 ml shake flasks; 20 ml of experimental

significant economic losses. Soft rot disease in Indonesia

medium was inoculated with a 1% of inoculums, grownexists in many orchid growing regions and the diseasein the same medium for 48 h. Bacterial growth was

3,4intensity reach up to 46% . Until recently, the application

estimated by measuring optical density at 600 nm

of chemical spray still the most common disease control by(OD600).

- 37 -

Colorimetric assay for indole acetic acid (IAA)

DNA sequencing and phylogenetic analyses. To

estimation. To confirm the presence of IAA in theconfirm the identities of the isolates, PCR amplification

supernatant, one hundred and six bacterial isolates wereand sequencing of the 16S rRNA gene were performed.

grown in 5 ml liquid medium (YPB) in a sterile flask

The 16S rRNA genes were PCR-amplified from the

supplemented with L-Tryptophan. One ml of suspension

genomic DNA using the bacterial universal primer set ofbacterial from each flask was centrifuge to separate the27f (5'-AGA GTT TGA TCC TGG CTC AG-3') andsupernatant and pellet. One ml of the supernatant was

1492r (5'-GGC TAC CTT GTT ACG ACT T-3'), whichremoved and mixed with 4 ml of Salkowski’s reagent (50were also used for sequencing9. The reaction mixture

ml of 35% HClO4 + 1 ml of 0.5 M FeCl3 x 6H2O) andcontained 1µl of the purified genomic DNA, 1µl of eachallowed to stand for 20 min at room temperature. The

primer and 25µl GoTaq®Green master mix (Promega,

positive reaction for IAA production was observed as theUSA) in a total volume of 50 µl. Amplification was done5

development of a pink-red color . IAA amounts wereindetermined at 550 nm in a Microplate Manager Bio-Rad

denaturation at 94°C for 2 min was followed by 34 cycles

Lab, Inc.

of denaturation at 94°C for 15 sec, annealing at 55°C forathermocycler(MyCycler,

Bio-Rad).

Initialactivity.

30 sec and elongation at 68°C for 30 sec. After 34 cycles,

Quantitative screening of phosphate-solubilizing bacteria

there was a final 5-min extension at 72°C and then cooled

was carried out by culturing the bacterial isolates onand held at 4°C. The amplified PCR products were

solid National Botanical Research Institute`s Phosphateanalyzed by 1% (w/v) agarose gel electrophoresis10.

Growth (NBRIP) medium containing (per liter): 10 g ofSequencing of the amplified DNA fragments was

glucose, 5 g of Ca3(PO4)2, 5 g of MgCl2.6H2O, 0.25 g ofperformed

MgSO4.7H2O, 0.2 g of KCl, and 0.1 g of (NH4)2SO3, and(http://base-asia.com/dna-sequencing-services).

Phosphatesolubilization

(PSB)

using

the

services

of

1st

BASE

then incubated for 3 days at room temperature. The

Screening of selected bacteria for in planta

presence of clearing zone around associated bacterial

biocontrol activity and growth enhancement. Three6

spot was measured .

selected bacterial endophytes were tested for the ability to

Genomic DNA extraction. Genomic DNAs fromprotect plants from infection with soft rot bacterial

the bacterial strains were prepared as describedpathogen, as well as to evaluate the effect on plant growth7

previously . The bacterial isolates were grown overnightby using the orchid plantlet P. amabilis. Plantlets grownin 5 ml of TSB in a rotary shaker at 28°C. Total DNA wasin tissue culture were dipped in bacterial endophyte

isolated using the Wizard Genomic DNA Purification Kit

cultures (108 cells per ml) and subsequently transplanted(Promega, USA) in accordance with the instructions ofonto MS agar11. After incubation for 1 week in a growth

the manufacturer.

chamber at 22°C with a light period of 14 h per day,

Rep-PCR analysis. Thirty orchid associatedplantlets were dipped in a culture of Pectobacterium

bacteria were selected to characterize by rep-PCR

carotovorum (108 cells per ml) and again transplanted.

technique8. The DNA sequences of the primers used for

After 3 weeks of incubation in the growth chamber, plants

DNA fingerprinting were as follows:

were scored for disease symptoms. Nonbacterized

5´-CTACGGCAAGGCGACGCTGACG-3 (BOX A1R),

controls were dipped in either a 0.9% sterile NaCl

5´-CCGCCGTTGCCGCCGTTGCCGCCG-3´(MBO

solution or the pathogen only. The effect of endophytic

REP1),5´-AAGTAAGTGACTGGGGTGAGCG-3´(ERICbacterization on the growth of plantlets was evaluated 22). PCR reactions were carried out in a final volume of 25months later.

μL consisting of GoTaq green master mix (Promega). The
reaction conditions were as follows: After the initial

Results and Discussion

activation step (15 min, 95 °C), 35 cycles at 94 °C for 1

Production of secondary metabolites. All

minute, at 53 °C for 1 minute, and at 72 °C for 2.5 minbacterial isolates tested in this study were previously

were run followed by a final elongation step at 72 °C forshown

10 min. Ten microliters of the PCR reactions werecarotovorum with variation range of inhibition. When

analyzed on a 1% agarose gel. The DNA bands were

testing for the production of metabolites that may

stained with ethidium bromide and visualized by UV

facilitate plant growth, 79 isolates of orchids associatedlight.

bacteria (74.5% of total isolates) were found to produce- 38 -

tohave

antagonistic

activity

againstP.

IAA and 92 (86.79%) were able to solubilize inorganic

isolate AkOc1 determined to be close relatives tophosphate. Bacterial isolates were varied greatly in theirRaoultella ornithinolytica JCM7251 (GenBank accession

efficiency of IAA production. Strain AkOc1 was thenumberhighest in producing IAA, whereas strain TbPh7 which

sequence-similarity. The partial 16S rDNA sequence of

showed the most effective in antibiosis assay, was also

isolate DnAr4 showed 98.6% identity with the sequences

the most efficient PSB strain.

of Klebsiella michiganensis W14 (GenBank accession

Rep-PCR characterization of bacterial isolates

AJ251467)

with

98.7%

16S

rDNA

number JQ070300). In the phylogenetic tree, strain

using BOXA1R, MBO REP1, and Eric-2 primers. The

TbPh7

was

most

amplification profiles of thirty selected isolates examined

amyloliquefaciens subsp. plantarum SN13 (GenBankby rep-PCR analysis using 3 different primers wereaccession number KC293995) with 98.05% 16S rDNA

described on Fig. 1. Three selected bacterial isolates (no.

sequence-similarity.

22, 29, 30) which showed different unique rep-PCR
patterns

andpromisingproduction

of

(A)
2

3

4

5

6

7

8

related

10

11 12

13

14

15

M2

M1 1

2

3

4

5

6

7

8

Bacillus

Raoultella ornithinolytica JCM7251

secondary

DnAr4
98 Klebsiella michiganensis W14
TbPh7

(B)
9

to

AkOc1

75

metabolites were identified and used for further assay.
M1 1

closely

9

10

11 12

13

14

15

M2

100

Bacillus amyloliquefaciens subsp. plantarum SN13

0.02
M1 16

17

18 19 20

21 22

23 24 25 26 27

28

29

30

M2

M1 16

17

18 19 20

21 22

23 24 25 26 27

28

29

30

M2

Figure 2. Phylogenetic tree based on partial 16S rDNA
sequences, showing the relationship between the isolated
strains and other species. The tree was constructed using
the CLUSTAL-X and neighbour-joining method. Scalebar corresponds to 0.01 subscriptions per nucleotide

(C)
M1 1

2

3 4

5

6

7 8

9 10 11 12 13 14 15 M2

position. Numbers at nodes indicate levels of bootstrap
support (%) determined from 100 re-sampled data.
Monitoring the effect of the isolated bacterial

M1 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M2

endophytes on disease suppression and plant growth.
The beneficial effect of bacterial endophytes was
described earlier with potato11, clove12, and other crops13.
In the present study, all the tested bacterial endophytes
Figure 1. Characteristic rep-PCR fingerprint patterns of

could suppress soft rot disease on plantlets of P. amabilis

endophytic bacteria isolated from different species of(Fig. 3). The highest reduction of both disease incidence

orchids in Yogyakarta and west Java, Indonesia using

and intensity was found on P. ambilis co-cultured with B.

BOX A1R primers (A), MBO-REP1-primers (B), andamyloliquefaciens subsp. plantarum TbPh7 (Table 1).

ERIC2-primers (C).

The presence of bacterial endophytes has advantages of
escaping microbial competition as well as influencing the

16S

rDNA

sequence

similarity

andhost’s response to pathogens attack.

phylogenetic analysis. Molecular identification of theisolated strains was carried out based on 16S rDNA
sequence analysis. The 1.5 kb sequences obtained fromthe strains were aligned with all the presently available(A)

16S rDNA sequences in the GenBank database. As a

(B)

(C)

(D)

result, a phylogenetic tree was constructed using the

Figure 3. In vitro responses of P. amabilis plantlets after

neighborjoining method, and is shown in Fig. 2.

infection only with P. carotovorum (A), or previously

Phylogenetic analysis using the 16S rDNA sequences

co-cultured with R. ornithinolytica AkOc1 (B), K.

indicated that the three bacterial isolates belonged tomichiganensis DnAr4 (C), B. amyloliquefaciens subsp.

different genera. From the branching pattern of the tree,

plantarum TbPh7 (D).

- 39 -

Table 1. Disease progress after 3 weeks inoculation of P.

Referencescarotovorum
Treatment

Disease incidence (%)

Disease intensity (%)

Control

100

55

AkOc1

20

15

DnAr4

20

5

TbPh7

0

0

Indeed, the growth enhancement of P. amabilis
plantlets was also influenced by the application ofbacterial endophytes. As shown in the Table 2, plantlets
co-cultured with R. ornithinolytica AkOc1 grew fasterand had significantly more roots and length. These
effects were not observed when P. amabilis plantlets
were inoculated with sterile ddH2O or K. michiganensis
DnAr4, meaning that not all endophytic introduction will
have beneficial effect on growth enhancement.
Table 2. The growth of P. amabilis 2 months after
inoculation of bacterial endophytes
Treatment

Height (cm)

Root length (cm)

Root number

Control

1.3b

1.33b

2.67ab

AkOc1

1.62a

3.43a

3.00a

DnAr4

1.26ab

2.73ab2.00ab

TbPh7

1.4ab

1.73ab

2.67abConclusion
In conclusion, from the orchid tissues, we isolated
bacterial endophytes that were evaluated as microbial
inoculants for disease management and also forstimulation of plant growth. Our data demonstrate that invitro bacterization of orchid plantlets can be used as a
new strategy for preventing the spread of soft rot disease;
however, a further evaluation of the disease control
effectiveness of these strains and the design of abiocontrol formulation and application must be conductedunder field conditions.

Acknowledgements
This study was supported by Ministry of Research,
Technology and Higher Education of the Republic ofIndonesia through national competitive research grants.
The authors also thank the United Graduate School of
Agricultural Science, Gifu University for providing
financial assistance during “International Symposium on
Innovative Crop Protection for Sustainable Agriculture”

1) Rosenblueth A and Martínez-Romero E (2006)
Bacterial endophytes and their interactions with hosts.
Molecular Plant Microbe Interaction, 19: 827–837.
2) Sturz AV, Christie BR and Nowak J (2000) Bacterial
endophytes: Potential role in developing sustainablesystems of crop production. Critical Review in Plant
Science, 19: 1–30.
3) Joko T, Kiswanti D, Subandiyah S and Hanudin(2011) Occurence of bacterial soft rot of Phalaenopsis
orchids in Yogyakarta and West Java, Indonesia, pp.
255–265. In: Y. Koentjoro (ed.), Proceeding ofInternational Seminar on “Natural Resources, Climate
Change, and Food Security in Developing Countries”,
June 27−28. Surabaya, Indonesia.
4) Joko T, Subandi A, Kusumandari N, Wibowo A andPriyatmojo A (2014) Activities of plant cellwall-degrading enzymes by bacterial soft rot of orchid.
Archives of Phytopathology and Plant Protection, 47:
12391250.
5) Tsavkelova EA, Cherdyntseva TA, Botina SG &Netrusov AI (2007) Bacteria associated with orchid roots
and microbial production of auxin. MicrobiologicalResearch, 162: 69–76.
6) Mehta S and Nautiyal CS (2001) An efficient methodfor qualitative screening of phosphate-solubilizing
bacteria. Current Microbiology, 43: 5156.
7) Joko T, Hirata H and Tsuyumu S (2007) Sugar
transporter (MfsX) of major facilitator superfamily isrequired for flagella-mediated pathogenesis in Dickeya
dadantii 3937. Journal of General Plant Pathology, 73:
266273.
8) Genersch E and Otten C (2003) The Use of repetitiveelement PCR Fingerprinting (rep-PCR) for genetic
subtyping of German field isolates of Paenibacillus
larvae subsp. larvae. Apidologie, 34: 195–206.
9) Joko T, Koentjoro MP, Somowiyarjo S, Rohman MS,
Liana A and Ogawa N (2012) Response of rhizobacterial
communities in watermelon to infection with cucumber
green mottle mosaic virus as revealed bycultivation-dependent RISA. Archives of Phytopathology
and Plant Protection, 45: 18101818.
10) Joko T, Kusumandari N and Hartono S (2011)
Optimization of PCR method for the detection of
Pectobacterium carotovorum, a causal agent of soft-rot
disease on orchid. Jurnal Perlindungan Tanaman
Indonesia, 17: 5459.
11) Reiter B, Pfeifer U, Schwab H and Sessitch A (2002)
Response of endophytic bacterial communities in potato
plants to infection with Erwinia carotovora subsp.
atroseptica. Applied and Environmental Microbiology,
68: 22612268.
12) Dwimartina F, Arwiyanto T and Joko T (2017)
Potential of endophytic and rhizobacteria as an effective
biocontrol for Ralstonia syzygii subsp. syzygii. Asian
Journal of Plant Pathology 11: 191–198.
13) Halmann J, Quadt-Hallmann A and Kloepper JW(1997) Bacterial endophytes in agricultural crops.
Canadian Journal of Microbiology, 43: 895914.

in Gifu, Japan.

- 40 -

OS II-2
Potential of endophytic bacteria as plant growth promoter and antagonist against
pineapple-fungal plant pathogen in Indonesia
1
Radix SUHARJO , Titik Nur AENY1, Udin HASANUDIN1, TE SUKMARATRI2, Ruslan
KRISNO2, Thoriq KHOIRONI1, Diyan Adinda SAFITRI1(1Faculty of Agriculture, Lampung University, Indonesia; 2Great Giant Food Company, Lampung, Indonesia)

SUMMARY
This study was aimed to investigate potential of 15 endophytic bacteria (3C, AK, CH, GKSKK, GBSH, AM, B1,
GKSKC, GBSK3, GKSKW, GKSKP, A31, GKSKKn, NS and AP) that were isolated from healthy leaves of pinneaple
as plant growth promoter and antagonist of pineapple-fungal plant pathogens. The isolates were investigated on their
hipovirulence, ability as plant growth promoter and ability to inhibit three pineapple-fungal pathogens, namely
Phytophtora sp., Curvularia sp. and Thielaviopsis sp. The result showed that 10 out of 15 isolates were hipovirulent.
Among 10 hipovirulence isolates, 7 isolates had potential as plant growth promoter (3C, AK, GKSKK, AM, B1,
GKSKC and GBSK3). In the case of their antagonistic capability, the isolates which were produced the highest
percentage of inhibition against Phytoptora sp. and Curvularia sp. were GKSCK at 72,48% and 66,08% of inhibition,
respectively. Meanwhile, the highest percentage inhibition against Thielaviopsis sp. was obtained by CH at 64,82% of
inhibition. In this study, we found that some of the endophytic bacteria can be plant growth promoter or antagonist or
both as plant growth promoter and antagonist.

Introduction

2013; Lanna-Filho et al., 2013; Yi et al, 2013;

Pineapple is one of the most important fruit comodities

Egamberdieva et al., 2017; Leiwakabessy et al., 2018)

in Indonesia. Recently, although not very significant,and antagonist of many kinds of plant pathogens (Duffy

pineapple production in Indonesia continues to decline.

and Defago, 1999; Gaiero et al., 2013).

This is due to the decreased of soil fertility and infection

Fifteen endophytic bacteria were successfully isolatedof some plant pathogens. Three important plantfrom healthy leaves of pineapple. However, study on

pathogens that have been reported causing severe

their potential as agricultural bio agents has not been

economic losses are Phytophtora sp., Curvularia sp. andperformed. This study was conducted in order toThielaviopsis sp.. Application extra chemicals fertilizer

investigate virulence, ability as plant growth promoter

and fungicides to solve the problems can cause more

and antagonist of the fifteen isolates of above mentioned

severe harms to the environment and future cultivation

endophytic bacteria against three pineapple fungal

and efforts to improve pineapple production. Thus, itpathogens, namely Phytophtora sp., Curvularia sp., andneed to find alternative methods that is safe to be used toThielaviopsis sp..

solve the complications, and one of which are reducing

Material and Method

the chemicals and using bio agents.

Endophytic bacteria. As much as 15 isolates ofEndophytic bacteria is one of the promising bio agentsendophytic bacteria used in this study. All the strainsthat can be used to improve production of cultivatedwere isolated from healthy leaves of pineapple.

plants, including pineapple. Endophytic bacteria is

Hypovirulence test. Hypovirulence test was performed

bacteria that live internally inside the plant tissue, can beusing method developed by Worosuryani (2005). Sprouts

isolated from the plant after surface desinfection andof cucumber were used as indicator. Inoculation each ofdoes not cause negative effects on plant growth (Wilson,

endophytic bacteria was repeated 3 times. Observation

1995; Gaiero et al., 2013). It has been reported thatwas performed until 14 days after inoculation. Disease

endophytic bacteria has capability as plant growth

severity Index (DSI) was calculated using formula :promoter (Gaiero et al., 2013; Santoyo et al., 2016), plant(∑N/Z); N : total score of disease severity on eachresistance inducer against plant diseases (Costa et al.,individu, Z : total individu used. Score of the disease- 41 -

severity that was used can be explained as follows: 0 :their hypovirulence, capability as plant growth promoter

healthy, there was no infection on hypocotyl; 1 : one or

and antagonist against 3 pineapple plant pathogens,two brown spot observed with <0.25 cm of diameter; 2 :namelybrown spot observed with < 0.5 cm of diameter withThielaviopsis sp.. The result showed that 10 out of 15<10% of wetness area of hypocotyl; 3 : brown spot

isolates showed hypovirulent (Table 1). Among those 10

observed with > 1 cm of diameter with 10%<x<100% of

hipovirulent isolates, 7 isolates showed potential as plant

wetness area of hypocotyl; 4 : black spot observed, wilt

growth promoter. Application of the bacterial isolates

and sprouts death. The endophytic bacteria with DSI <2

resulting better growth on indicator plant compared to

was put in the group of hypovirulent bacteria.

the untreated plants. Application of endophytic bacteria

Investigation on its capability as plant growthconsistently improve plant height, greenish leaves, wetpromoter. Cucumber plant was used as indicator plant.

and dry weight of shoot and root and root length (Fig 1).

Investigation was conducted using methods developed

Gaiero et al (2013) and Santoyo et al. (2016) stating thatby Worosuryani (2005). As much as 10 ml of bacterial

endophytic bacteria also could promote growth of theirsuspension (~108

CFU/ml) was pured into planting

host

Phytophtora

plant.

The

sp.,

bacteriasp.

Curvularia

releaseandphytohormones

medium of plant indicator. Inoculation each of the(Bloemberg & Lugtenberg, 2001) that can improve plant

bacteria was repeated 3 times. Observation of plant

growth such as 1-aminocyclopropane-1-carboxylate

height was performed every two days. Greenish leaves(ACC) deaminase (Gaiero et al., 2013; Santoyo et al.,level was conducted once at 16 days after inoculation

2016), jasmonate, indole acetic acid, and abscisic acidusing chlorophyll content meter CCM 200 plus (opsi(Patten and Glick, 2002; Forchetti et al., 2007 ). Besidescience) at the 3 of leaves position i.e. top, midle andtheir ability as growth promoter, endophytic bacteria wasbottom. Weight of wet and dry of shoot and root wasalso reported as plant resistance inducer (Romeiro et al.,conducted at 21 days after inoculation. In the case of dry

2005; Lanna-Filho et al., 2013). Application ofweight of shoot and root, the fresh shoot and root wereendophytic bacteria has been reported can improve plantoput into envelope and it was incubated at 80 C for 3 days.

resistance against plant diseases such as bacterial leaf

After incubation, it was weight using digital balance EG

spot of pepper (Yi et al, 2013), bacterial leaf spot of

4200-2NM (Kern).

Table 1 Disease severity index resulted byinoculation of the bacterial isolates on cucumber
sprouts and its role as plant growth promoter
Disease Severity
Role as plant
Isolates
Index
growth promoter

Antagonistic capability agains pineapple fungal-plant
pathogens. Three pineapple fungal plant pathogens usedin this study i.e. Phytophtora sp., Curvularia sp., andThielaviopsis sp.. Antagonistic test was performed byscraping the bacteria using inoculating loop with adistance of 2 cm from the edge of petridish (diam 9 cm)
contains Potato Sucrose Agar (PSA) medium (Potato
extract 1000 ml, Sucrose 20 g, Agar 20 g) in both side.
One culture of 7 old days of each of the fungal pathogens(diam 0.5 cm) was placed in the midle of petridish. As
control, one culture of each of plant pathogens was put inthe midle of petridish contains PSA medium without any
endophytic bacteria. All the petridish were incubated atroom temperature. Observation was conducted at 1, 3, 5
and 7 days after inoculation on the wide of fungal colonythat was measured in milimeter. Percentage of inhibition
was calculated using formula : [L1-L2/L1]x100%. L1:
wide of fungal colony without endophytic bacteria, L2 :wide of fungal colony with endophytic bacteria.

AP

2.75

Not tested

GKSKKn2.58

Not tested

NS

2.50

Not tested

A31

2.50

Not tested

GKSKP

2.42

Not tested

3C

2.00

Yes

AK

1.92

Yes

CH

1.83

No

GKSKK

1.67

Yes

GBSH

1.67

No

AM

1.33

Yes

B1

1.17

Yes

GKSKC

1.00

Yes

GBSK3

0.75

Yes

GKSKW

0.33

No

Kontrol

0.00-

Result and Discussions

tomato (Lanna-Filho et al., 2013), bacterial leaf blight ofIn this study, 15 endophytic bacteria was investigated onrice (Leiwakabessy et al., 2018), damping off on- 42 -

was endophytic bacteria that play a role as plant growthpromoter or antagonist or both plant growth promoter
and antagonist. The best inhibition to Phytoptora sp. andCurvularia sp. were produced by GKSCK, meanwhile,BA

the highest inhibition against Thielaviopsis sp. was
obtained by CH.

Acknowledgement
We thank to Great Giant Food Company for providingA

B

Fig.1 Indicator plant after application of bacterial
isolates. A. control, B. treated plants
cucumber (Costa et al., 2013) and root rot of chickpea(Egamberdieva et al., 2017).

A

A

A

Some of endophytic

bacteria have also been reported produce anti microbial
compounds, such as siderophore and antibiotics, that caninhibit growth of plant pathogens (Duffy and Defago,
1999; Gaiero et al., 2013) such as Enterobacter,
Pseudomonas sp., Bacillus sp.. (Muzzamal et al., 2012),
Fusarium oxysporum f.sp. lycopersici (Shahzad et al.,2017), Phytophtora capsici, Alternaria panax and
Botrytis cinerea (Paul et al., 2013).
In this study, we found that some of endophytic bacteria

B

Fig. 2 Antagonist test of endophytic bacteria against 3
pineapple fungal pathogens 7 days after inoculation.
From left to right : Phytophtora sp., Curvularia sp.,
Thielaviopsis sp.. A. Endophytic bacteria that had
antagonistic capability, B. Endophytic bacteria that
did not has antagonistic capabilitycapability.

used had capability to inhibit Phytophtora sp.,

Table 2 Percentage of inhibition of endophytic
bacteria against thee pineapple fungal pathogens
Percentage of inhibition (%)
Isolates
Phytophtora Curvularia Thielaviopsis

Curvularia sp. and Thielaviopsis sp.. (Fig. 2). Inhibition
was in the range of 5.13 to 72.48% (Phytophtora sp.),
2.33 to 66.08% (Curvularia sp.) and 1.33 to 64.82%(Thielaviopsis sp.). The best capability to inhibit
Phytophtora sp., and Curvularia sp. was produced by
GKSCK.

Meanwhile,thehighestinhibition

ofThielaviopsis sp. was produced by CH (Table 2). It was
shown that one endophytic bacteria can inhibit more than
one pathogens.
Ability of endophytic bacteria to inhibit more than one
kinds of pathogens have also been reported. Endophytic
bacteria isolated from potato (Berg et al., 2005) and chilli
pepper (Paul et al., 2013) have been proven to beantagonist of more than one kinds of pathogens.
Study performed by Berg et al. (2005) revealed that
endophytic bacterium isolated from potato could inhibit
Verticillium dahliae or Rhizoctonia solani. Paul et al.
(2013) stated that edophytic bacteria isolated from chilli
pepper can inhibit Fusarium oxysporum or Alternaria
capsici or Botrytis cinerea.
In conclusion, not all endophytic bacteria used in thisstudy were plant growth promoter and antagonist. There

AP*

14.76

30.18

5.58

GKSKKn*

0.00

27.77

1.53

NS*

0.00

16.73

5.44

A31*

61.85

13.35

46.15

GKSKP*

62.74

6.37

61.85

3C*

0.00

15.40

63.71

AK

64.30

29.94

4.94

CH

67.15

2.40

64.82

GKSKK

0.00

30.53

25.87

GBSH

20.00

14.48

29.68

AM

0.00

5.67

1.50

B1

0.00

9.40

8.72

GKSKC

72.48

66.08

4.88

GBSK3

5.13

2.33

0.00

GKSKW

64.11

24.88

1.33

Kontrol
0.00
0.00
0.00
* Isolates which were virulent (DSI>2) on the resultof hypoverulence test

panax or Colletotrichum acutatum or Phytophtora

Conclusion

BB

financial support and material required for this research.
We also thank to Lampung University for many support
provide during research.

- 43 -

Reference

bacteria antagonistic to biofilm formers. Pakistan Journal1) Berg G, Krechel A, Ditz M, Sikora RA, Ulrich A andof Zoology, 44:249-257.

Hallmannectophytic

11) Patten CL and Glick BR (2002) Role of Pseudomonas

potato-associated bacterial com- munities differ inputida indole acetic acid in development of the host plantstructureJ
and(2005)

Endophytic

antagonisticandfunctionagainstplant

root system. Applied and Environmental Microbiology,

pathogenic fungi. FEMS Microbiology Ecology, 51:

68: 3795–3801.

215–229.

12) Paul NC, Ji SH, Deng JX and Yu SH (2013)

2) Bloemberg GV and Lugtenberg BJJ (2001) Molecular

Assemblages of endophytic bacteria in chili pepper

basis of plant growth promotion and biocontrol by(Capsicum annuum L.) and their antifungal activity

rhizobacteria. Current Opinion in Plant Biology 4:

against phytopathogens in vitro. Plant Omics Journal,

343–350.

6:441-448.

3) Costa FG, Zucchi TD and de Melo IS (2013) Biological13) Romeiro RS, Filho L, Junior JRV, Silva HSA,

Control of Phytopathogenic Fungi by Endophytic

Baracat-Pereira

Actinomycetes Isolated from Maize (Zea mays L.).

Rhizobacterium as Elicitors of Systemic Resistance inBrazilian Archives of Biology and Technology, 56:

Tomato to Bacterial and Fungal Pathogens. Journal of

948-955.

Phytopathology, 153:120-123.

4) Duffy BK and Défago G (1999) Environmental factors

14) Santoyo G, Moreno-Hagelsieb G, Orozco-Mosqueda

modulating antibiotic and siderophore biosynthesis by

MdC and Glick BR (2016) Plant growth-promoting

Pseudomonas fluorescens biocontrol strains. Applied andbacterial

Environmental Microbiology, 65: 2429–2438.

183:92-99.

5) Egamberdieva D, Wirth SJ, Shurigin VV, Hashem A15) Shahzad R, Khan AL, Bilal S, Asaf S and Lee IJ

and Abd_Allah (2017) Endophytic bacteria improve plant(2017) Plant growth-promoting endophytic bacteria

growth, symbiotic performance of chickpea (Cicer

versus pathogenic infections: an example of Bacillus

arietinum L) and induce suppression of root rot caused by
Fusariumsolani

under

salt

stress.

Frontier

in

MC

and

endophyte.

Carvalho

MicrobiologicalMG(2005)

Research,amyloliquefaciens RWL-1 and Fusarium oxysporum f. sp.
lycopersici

intomato.

PeerJ

5:e3107;

DOI

Microbiology, 8:1887 doi: 10.3389/fmicb.2017.01887.

10.7717/peerj.3107.

6) Forchetti G, Masciarelli O, Alemano S, Alvarez D and16) Yi HS, Yang JW and Ryu CM (2013) ISR meets SAR

Abdala G (2007) Endophytic bacteria in sunflower

outside: additive action of the endophyte Bacillus pumilus(Helianthus annuus L.): Isolation, characterization, andINR7 and the chemical inducer, benzothiadiazole, on

production of jasmonates and abscisic acid in culture

induced resistance against bacterial spot in field-grownmedium. Applied Microbiology and Biotechnology, 76:

pepper.

1145–1152.

10.3389/fpls.2013.00122.

7) Gaiero JR, Mccall CA, Thompson KA, Day NJ, Best

17) Wilson D (1995) Endophyte: The evolution of a term,

AS and Dunfield KE (2013) Inside the root microbiome:and clarification of its use and definition, Oikos 73:

bacterial root endophytes and plant growth promotion.

274–276.

American Journal of Botany, 100: 1738–1750.

18) Worosuryani C (2005) Uji kemampuan berbagai

8) Lanna-Filho R, Souza RM, Magalhães MM, Villela L,

jamur tanah yang diisolasi dari lahan pasir sebagai PGPF

Zanotto E, PRibeiro-Júnior PM and Resende MLV (2013)

dan agens pengendali hayati penyakit layu fusarium pada

Induced defense responses in tomato against bacterial

semangka. Master Thesis. Universitas Gadjah Mada.

spot by proteins synthesized by endophytic bacteria.

Yogyakarta.

Tropical Plant Pathology, 38:295-302.
9) Leiwakabessy C, Sinaga MS, Mutaqien KH,
Trikoesoemaningtyas and Giyanto (2018) The endophytic
bacteria, salicylic acid, and their combination as inducersof rice resistance against Xanthomonas oryzae pv. oryzae.
AGRIVITA Journal of Agricultural Science, 40: 25–35.
10) Muzzamal H, Sarwar R, Sajid I and Hasnain S (2012)
Isolation, identification and screening of endophytic- 44 -

Frontier

in

Plant

Science,

4:122

doi:

OS II-3
Endophytic Bacillus as biological control agent of banana wilt
HADIWIYONO(Faculty of Agriculture, Universitas Sebelas Maret)
SUMMARY
Banana wilt caused by blood disease bacterium (BDB) and Fusarium oxysporum f.sp. cubense (FOC) is a couple
important threaten in cultivation of banana in Indonesia. The use of free pathogen seedling from tissue culture isrecommended as a component of integrated control of the disease. However, the seedling resulted by tissue culture ismore susceptible to pathogen infection due to by loss of bacterial endophyte, the inducer of resistance of banana. One ofendophyte bacterial group is Bacillus spp. This paper would like to report the potentiality of endophytic Bacillus asbiocontrol agent of the banana wilts.

Based on the results of some tests in laboratory and screen house, showed thatsome isolates of Bacillus showed potential antagonistic to the pathogens and could induce resistance of banana plantlet
to both banana wilts. The isolates of endophytic Bacillus could release volatile and nonvolatile compound of pathogen
growth inhibitor, growth regulator hormone especially IAA, and enzyme of chitinase and pectinase that are considerable
to be involved in the antagonism of the Bacillus to the pathogens or inducing resistance of banana planlets to thediseases.

Introduction

bacteria are seen predominantly in asymptomatic

Wilt disease is very important in banana cultivation inbananas. It is suspected that the bacteria are as theIndonesia. Banana wilt disease is caused by bacterial

resistance inducer of the asymptomatic banana.

blood disease (BDB) and or Fusarium oxysporum f.sp.

popular that one of the most effective endophytic

cubence. Incidence of wilt disease in the field can reachbacteria as plant disease control agents is Bacillus

above 80%. Both pathogens often attack together so

spp.10,11,12. This paper is a report of some researches

that the damage to the plant becomes more severe1. So

results

far, both pathogens are still difficult to control.

endophytic Bacillus of banana as biological control agent

Controlof both diseases must be done in an integrated way.

about

the

effectiveness

and

It is

mechanismof banana wilt.

One of the most effective and practical integrated control
components is the planting of superior variety, to date theMaterials and Method

resistant banana varieties against both pathogens are not

Endophytic Bacillus isolates. A mount of isolate of

yet available2,3,4. Therefore, the use of healthy seedlings

endophytic Bacillus was isolated from healthy banana

becomes an important integrated control component.

sampled from endemic wilt fields. The Bacillus wereHealthy seedlings in the field are difficult to detect

isolated from 3 peaces ±(0.2 x 0.5 x 10 mm) the bit ofvisually because 20-30% of healthy-looking plants areinternal tissues of peduncle, pseudostem, and rhizome

5infected with BDB . For producing pathogen freedipped in 5 mL sterile water.

seedlings have been successfully developed with tissue

incubated for 12 hours and shaken with Vortex Shaker. A

culture technology6,7. Smith et al.8 reported that the tissue

volume of 100 µl suspension was plated on Nutrient

culture seedlings were susceptible to wilt pathogens, due

Agar (NA) with spread technique on medium in thedish

to aseptic conditions during the production in vitro

with composition 8 g Nnutrient Broth and 15 g agar

culture process to be free of endophytic bacteria involved

Bacto in 1 L waters. The dishes were incubated for 24

in resistance mecahnims of banana. This report is inhours. Single colony was sub-cultured using the sameline with analysis results of endophytic bacterial total

medium.

based on the pattern of DNA fragments of themedium for stock13.

asymptomatic banana through ribosomal integenic

Antagonism tests in vitro. Antagonistic Bacillus to thespecer region analysis (RISA) that is different from

pathogens in vitro was tested on Nutrient Agar (NA)8,9symptomatic ones . Some DNA fragments of certain

The samples were

The pure isolates were streaked on a slant NA

incubated 72 hours to BDB and Potato Dextrose Agar

- 45 -

(PDA) incubated 5 days to FOC. The zone of inhibition

also reported that endophytic Bacillus could be isolated

was observed as antagonistic variable.

from vasculer tissues of veries plants17,18,19. Azevedo et

Antagonism tests in vivo. The resistance test of banana

al.20 reported that many endophytic bacteria such Bacillus

seedlings to BDB was performed by placing plantlets

could be cultured purely from their plant hosts.
The results of antagonism tests of Bacillus to BDB

having been acclimatized for 3 months on sterilized soil
infested by BDB and Bacillus at 108cfu g-1 in polybags.

and FOC on agar medium showed that all of isolates wereBanana resistance test to FOC is performed by planting

antagosnitic to the pathogens indicated by performing

seedlings on infested sterile soils by pathogen spore

clear zone of growth inhibition to both. The clear zone is5-1

suspension with density at 10 spores g of soil. The

formed by toxic compound released by the antagonist.

disease severity was observed weekly for 7 weeks for

The capability of Bacillus to produce toxic compound

evaluating the resistance performance of the seedlings.

was also showed by growth inhibition caused by filtrate

Antagonistic mechanism of Bacillus to pathogens.

of culture medium.

One of the possible antagonistic mechanisms of Bacillus

medium, Bacillus released toxic compound into medium.

It indicates that in the liquid

to pathogen is antibiosis through production some toxic

The results of antagonism test in vivo showed thatvolatile or nonvolatile compounds released around theall of isolates of endophytic Bacillus were antagonistic

medium. The toxic nonvolatile compound was based on

and could reduce the wilt intensity caused by both thetoxicity test of filtrate medium. Sterilized filtrate medium

pathogens. Bacillus could reduce wilt intensity caused

harvested from bacterial culture incubated for 4 days wasby BDB with range of 45.08-71.45 and caused by FOC

tested in the capability to inhibit the growth ofthewith range of 13.95-72.1014,15. Sunaina et al.21 did a

The sterile filtrates were mixed in NA

research with B. subtilis treatment on tuber that could

medium for BDB and PDA for FOC. Toxic activity wascontrol bacterial wilt of potato and increase the yields ofevaluated by observation of zone inhibition of the

tuber reaching 160%. Whereas Georg and Kloepper22

pathogens performed by the filtrate after incubating for areported thatweek. The inhibition observation was similar withstrain GB03 had capability to promote the growth of plantantagonism test procedure. The volatile toxic compoundand minimizing disease severity

pathogens.

B. pumilus strain INR7 and B. subtilis

test was prepared by culturing endophytic Bacillus on theChen et al.23 also reported at least 10 isolates of

NA medium and at the same time filter paper ( 5 mm)
dyed in BDB inoculum suspention 108cfu mL-1 and FOC

cotton endophytic bacteria that could suppress the diseasecolony ( 5 mm) were cultured on the other platecontaining the NA or PDA culture medium. The bothf.sp. vasinfectum through bacterialization of cotton seeds.

plate were covered each other with BDB or FOC culture

induced resistance of plant to disease infection but also to

upside down and silted using isolative plastic. The

insect pests.

severity of cotton wilt caused by Fusarium oxysporum
Haiyan et al.19 reported that Bacillus could not only

growth colony diameter of BDB and FOC were observed

Based on the test of capability of endophytic

every 2 days for 8 days.

Bacillus to produce growth regulator hormone showed

The capability of Bacillus to produce enzim wasthat all isolates could produce indole acetic acid (IAA). It

based on the capability of the bacteria to grow on thehas been reported that some Bacillus could produce IAA .

medium containing certain enzim. A filter paper used forPatel and Patel24 revealed that endophytic Bacillus could

inoculum was dipped in a suspension of Bacillus (108 cfu

produce IAA. IAA was involved in promoting growthmL-1) prepared using King's B liquid medium incubatedof plant. Asari et al.25 found endophytic Bacillus with

with shakering for 24 h. Each isolate was placed in thecharater as plant growth promoting bacteria.

center of a Petri dish containing chitin and pectin agar as

spp. was known well as

14

treatment and without chitin and pectin as a control .

Bacillus

Plant Growth Promoting

Rhizobacteria (PGPR) due to by the capability of
producing IAA taking a role in biological control
mechanism through inducing systemic resistance11,26.

Results and Discussions
At least 30 isolates of endophytic Bacillus had beencollected and 10-15 isolates were tested farther toevaluate the potential antagonism15,16. Some researchers

The capability of Bacillus to produce IAA is
believed that the compound involves in promoting the
growth and inducing resistant of plant. The role of IAA in
inducing resistance of plant is important especially to- 46 -

weak pathogen such Fusarium that the infection willreported that

establish more in weak condition of plant. Waller andtogether with IAA is important in multiplication and27

production of chitinase and pectinase

confirmed that Fusarium constituts weakcolonization of the antagonistic bacteria in or on the hosts.

pathogen. It means that FOC will be more infective on

Pectinase has been reported as one of the determinants of

weak banana that can be induced by varies biotic or

induced systemic resistant40,41,42.

Brayford

abiotic stresses. Some researchers explain that Bacillus
can produce indole-3-acetic acid (IAA), which isConclusion

fungsional to help nitrogen fixation from the atmosphere,

The isolates of endophytic Bacillus could release volatileiron

production,and nonvolatile compound of pathogen growth inhibitor,

solubilization of potassium (K), zinc (Zn), and phosphate

growth regulator hormone especially IAA, and enzyme(P) from the soil, and increasing the soil porosity28,29,30,31.

of chitinase and pectinase that are considerable to be

Along with the micro- and macro-nutrient supply,

involved in the antagonism of the Bacillus to theendophytic Bacillus will protect the plants host from

pathogens or inducing resistance of banana planlets tofixationthroughsiderophore

28

phytopathogenic and take roles in antagonism . It had

the diseases.

been also reported that B. subtilis isolate A47 could
activate biological control to Gram-negative bacterium

Acknowledgements

due to by a metabolite compound the group of iturin

This manuscript was supported effectively by ADP Putra

hydrolysis and thermo-stable31.

and A.Widyantoro, the student of Agrotechnology

Bacillus spp. had been also reported as an important

Department, Faculty of Agriculture, Universitas Sebelas

biological control agents through producing toxic volatile

Maret.

being resistant to11,24

and soluble compound of plant. Several species ofBacillus have been described to produce different
antimicrobial lipopeptides. The compounds including
antibiotic

subtancesandextracellulardegradative

enzymes32,35,34,35,36. Perez et al.34 confirmed that Bacillus
showed the best potential for the production of antifungal
and antibacterial substances.
Many Bacillus produce lipopeptides that can bedivided into three major families that are surfactins,
iturins and fengycins or plispastatins. Surfactins anditurins are composed by cyclic heptapeptides, whichcontain a β-hydroxy fatty acid and β-amino fatty acid,respectively35,36,37. Besides the antimicrobial activity,
Surfactin

is

also

an

outstanding

surface-active

21,23

property. Iturins are a group of lipopeptides that are

remarkable antifungal activity to many microorganism
including bacterial and fungal plant pathogens23,26.
All isolates of endophytic Bacillus also could
produce extracellular degradative including chitinase andpectinase. Zhao et al.26 reported that there was positive
correlation between pectinase activity and inhibition rate
on Fusarium oxysporum on the medicinal plant Lonicera
japonica. It has been revealed that biological control ofReferences
1) Hadiwiyono et al. 20072) Mulyadi and Hernusa T. (2002) Intensitas penyakit
darah pada tanaman pisang yang disebabkan bakteri
Pseudomonas
solanacearumdiKabupaten
Bondowoso. Dalam: Prosiding Kongres XVI dan
Seminar Nasional Perhimpunan Fitopatologi
Indonesia. Jur. HPT F. Pertanian IPB, PFI, Bogor,
p:304-305.
3) Sudirman and Supeno (2002) skrining beberapa
varietas pisang terhadap infeksi penyakit darah
pisang. In: Purwantoro A, Sitepu D, Mustika I,
Mulya K, Sudjono MS, Mahmud M, Hidayat SH,
Supriadi, and Widodo (Eds). Prosiding Kongres XVIAnd Seminar Nasional Perhimpunan Fitopatologi
Indonesia. Jurusan Hama & Penyakit Tumbuhan
Fakultas Pertanian IPB & PFI, Bogor, P.311-312.
4) Wibowo A, Subandiyah S and Sumardiyono C (2007)
Pathogenic races of Fusarium oxysporum f.sp.
cubense Strains from Java. In: Proceedings of theThird Asian Conference on Plant Pathology on
August, 22-24, 2007 in Yogyakarta, p:83-84
5) Hadiwiyono (2010) Penyakit Darah pada Pisang:
Infeksi dan Keanekaragaman Genetika Patogen.
Disertasi Program Doktor Sekolah Pascasarjana
UGM.
6) Sunaryono HH (2002). Budidaya Pisang dengan Bibit
Kultur Jaringan. Penebar Swadaya, Jakarta.
7) Nisa C and Rodiah (2005) Kultur jaringan

beberapa kultivar buah pisang (Musaparadisiaca L.) dengan pemberian campuran
NAA dan kinetin.
Bioscientiae 2 (2): 23-36

some soil-borne fungal diseases has been correlated with39

chitinase production .
The play role of chitinase and pectinase in antagonism to plant pathogenic bacteria are still poorly
understood. The indirect role is possible. It has been8) Smith L, O’Keefe D, Smith M, and Hamill SS (2003)
The benefits of appliying Rhizoctonia to tissue
cultured bananas. DPI Newsletters 8(7): 1-4.

- 47 -

9) Hadiwiyono, Subandiyah S, Sumardiyono C, Widada
J, Fegan M, and Taylor P (2009) Analysis ofCommunity Structure of Endophytic Bacteria on
Infected Bananas by Blood Disease Bacterium with
AsymptomaticandSymptomatic
through
Independent Cultureable Approach. The manuscript
presented in the International Seminar and the 20th
Congress of Indonesian Phytopathological Society on
August 4-7, 2009 in Makassar, Indonesia.
10) Hung, P.Q. & K. Annapurna. 2004. Isolation andcharacterization of endophytic bacteria in soybean(Glycine sp.) Omanrice 12: 92-104.
11) Compant S, Duffy B, Nowak J, Clement C, andBarka EA (2005) Mini review: Use of plant
growth-promoting rhizobacteria for biocontrol ofplant diseases: principles, mechanism of action andfuture prospect. Appl Environ Microbiol 71: 4951 –4959.
12) Lee KD, Bai Y, Smith D, Han HS, and Supanjani(2005)
Inolationofplant-growth-promoting
endophytic rhizobacteria from bean nodules. Res. J.
Agric Biol Sci 1(3): 231-236.
13) Hadiwiyono and S. Widono, 2012. Endophytic
Bacillus: the potentiality of antagonism to wilt
pathogen and promoting growth to micro-plantlet of
banana in vitro. Biomirror, 3(06):1-4.
14) Salvador S, Fontana RC and Silveira MM, 2005.
Influence of pectin and glucose on growthandpolygalacruronase production
byAspergillus niger in solid-state cultivation. J. Ind.
Microbiol. Biotechnol. 32: 371-377.
15) Hadiwiyono (2012) Endophytic Bacillus: thepotentiality of antagonism to wilt pathogen andpromoting growth to micro-plantlet of banana in vitro.
BIOMIRROR: 3(06):1-4
16) Hadiwiyono and Widono S (2017) Effectivenessand mechanism of endophytic Bacillus in suppressing
wilt intensity of banana seedling inoculated by blood
disease bacterium. Plant Pathology Journal 16: 89-95.
17) Chun W and Vidaver AK (2001) Gram-Positive
Bacteria (Bacillus spp.). In: N.W. Schaad, J.B. Jones,
and Chun W (Eds.) Laboratory Guide for
Identification of Plant Pathogenic Bacteria. APS
Press, St. Paul. Minnesota. p. 250-260.
18) White JF, Torres MS, Sullivan RF, Jabbour RE, Chen
Q, Tadych M, Irizarry I, Bergen MS, Havkin-Frenkel
D, Belanger FC. 2014. Microscopy research andtechnique: Occurrence of Bacillus amyloliquefaciens
as a systemic endophyte of vanilla orchids. Microsc
Res Tech. doi:10.1002/jemt.22410.
19) Haiyan Li, Marcos A. Soares, Mónica S. Torres,
Marshall Bergen & James White Jr. (2015)
Endophytic bacterium, Bacillus amyloliquefaciens,
enhances ornamental host resistance to diseases andinsect pests. Journal of Plant Interactions 10(1):224-229, DOI: 10.1080/17429145.2015.1056261
20) Avecedo JL, Maccheroroni JrW, Pereira JO, and deAraujo WL (2000) Endophytic microorganism: areview on insect control and recent advances on
tropical plants.
Electronic J. Biotechnol.
3(1):40-65.
21) Sunaina V, Kishore V, and Shekhawat GS (2003)
Biocontrol of bacterial Wilt of potato of Ralstonia

solanacearum
and
other
bacteria.
<http://www.inra.Fr/internet/
Departements/PATHOV/2nd-IBWS/B5html>.
Accessed on 10 May 2010.
22) Georg RS and Kloepper JW (1998) Mixture of plant
growth-promoting rhizobacteria enhance biologicalcontrolofmultiple
cucumberpathogens.
Phytopathology 88: 1158-1164.
23) Chen H, Wang L, Su CX, Gong GH, Wang P, Yu ZL(2008). Isolation and characterization of lipopeptide
antibiotics produced by Bacillus subtilis. Lett Appl
Microbiol 47:180-186.
24) Patel MV and Patel RK. 2014 Indole-3acetic acid(IAA) production by endophytic bacteria isolated
from saline dessert, the little runn of Kutch. J
Microbiol 3(2):17-28.
25) Asari S, Matzen S, Petersen MA, Bejai S, andMeijer J. (2016) Multiple effects of Bacillus
amyloliquefaciens volatile compounds: plant growthpromotion and growth inhibition of phytopathogens.
FEMS Microbiology Ecology 92:1-11.
26) Zhao L et al. (2015) Screening and characterizationof endophytic Bacillus and Paenibacillus strains frommedicinal plant Lonicera japonica for use as
potential plant growth promoters. Braz. J. Microbiol.
46:977–989, doi:10.1590/S1517-838246420140024
27) Waller IM and Brayford D. (2008) Fusarium diseases
in the tropics. Tropical Pest Management 36(3):181-194 https://doi.org/10.1080/09670879009371470
28) Zahid M, Abbasi MK, Hameed S and Rahim N.
(2015) Isolation and identification of indigenous
plant growth promoting rhizobacteria from
Himalayan region of Kashmir and their effect on
improving growth and nutrient contents of maize(Zea mays L.). Front. Microbiol. 6:207,
doi:10.3389/fmicb.2015.00207.
29) Defez R, Andreozzi A, and Bianco C (2017) The
Overproduction of Indole-3-Acetic Acid (IAA) in
endophytes upregulates nitrogen fixation in bothbacterial cultures and inoculated rice plants. Microb
Ecol
74(2):441-452.
doi:
10.1007/s00248-017-0948-4.
30) Hantke K (2001). Iron and metal regulation inbacteria. Curr Opin Microbiol 4:172–177,
doi:10.1016/S1369-5274(00)00184-3 (2001).
31) Ciampi L, Fuentes R, Schobitz R, and Bernal G.
(2003) Ten years of biocontrol of bacterial wilt of
potato in Chile. International Bacterial Wilt
Symposium.http://www.inra.fr/ Internet/Departments/
phatov/2nd-IBWS/B1.html>.
32) Stein T (2005) Bacillus subtilis antibiotics: structures,
syntheses and specific functions. Mol. Microbiol. 56
845–857. 10.1111/j.1365-2958.2005.04587.x
33) Caldeira AT, Arteiro JMS., Coelho AV, Roseiro JC(2011). Combined use of LC–ESI-MS and antifungal
tests for rapid identification of bioactive lipopeptides
produced by Bacillus amyloliquefaciens CCMI 1051.
Process.
Biochem.
46
1738–1746.
10.1016/j.procbio.2011.05.016
34) Perez KJ, Viana J dos S, Lopes FC, et al. 2017
Bacillus spp. isolated from puba as a source ofbiosurfactants and antimicrobial lipopeptides.
FrontiersinMicrobiology.
8:61.

- 48 -

doi:10.3389/fmicb.2017.00061.
35) Bonmatin JM, Laprévote O, and Peypoux F (2003).
Diversity among microbial cyclic lipopeptides:
iturins and surfactins. Activity-structure relationships
to design new bioactive agents. Comb. Chem. HighThroughput
Screen6
541–556.
10.2174/138620703106298716.
36) Hsieh FC, Lin TC, Meng M, Kao SS (2008).
Comparing methods for identifying Bacillus strains
capable of producing the antifungal lipopeptide iturinA.
Curr.
Microbiol.
56
1–5.
10.1007/s00284-007-9003-x
37) Ongena M, and Jacques P. (2008). Bacillus
lipopeptides: versatile weapons for plant diseasebiocontrol. Trends Microbiol. 16 115–125.
10.1016/j.tim.2007.12.009
38) Shaligram NS, Singhal RS (2010). Surfactin – areview on biosynthesis, fermentation, purification
and applications. Food Technol Biotechnol 48
119–134.
39) Herrera-Estrella AL and Chet I. (1999) Chitinases
in biological control. EXS. 87:171-84.
40) Nagpure AL, Choudhary B, and Gupta RK. (2014)
Chitinases: in agriculture and human health care. Crit
Rev
Biotechnol.
34(3):215-32.
doi:
10.3109/07388551.2013.790874.
41) Ma B-C, Tang L-Y, Ma, Li L-L, Zhang L-B and Zhu
S-J (2009) The role of chitinase gene expression in
the defense of harvested banana
againstanthracnose disease. J. Amer. Soc. Hort. Sci.,
134(3):379–386. 2009.
42) Peng X, Xie X and Bai Z (2006) Purification andinduced resistance of pectinases from Penicillium
oxalicum BZH-2002. Chin J Appl Environ Biol
12(6):750-753.

- 49 -

- 50 -

OS II-4
Molecular based identification and formulation of cyanogenic Pseudomonas spp. controlling
Phytophthora infestans
Md. Rashidul ISLAM, Md. Nizam UDDIN and Mohammad Monirul Hasan TIPU
(Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh-2202)

SUMMARY
Late blight caused by Phytopthora infestans is one of the devastating diseases of potato and tomato worldwide. Every year
the disease incurs a loss estimated around $16 billion in the developing countries and in Bangladesh the loss estimated around$ 880 million. Recently, bacterial bioagents such as Pseudomonas spp. and Bacillus spp. have been reported to control P.
infestans. In this study, a total of 200 bacterial isolates obtained from phylloplane of potato plants were evaluated for the
production of hydrogen cyanide and only six bacterial isolates were identified as cyanogenic. The primers PsEG30F (5ʹATYGAAATCGCCAARCG-3ʹ) and PsEG790R (5ʹ-CGGTTGATKTCCTTGA-3ʹ) specific to the conserved sequences of rpoD
gene (the sigma 70 factor subunit of DNA polymerase) producing a 760bp amplicon confirmed the bacterial isolates are
Pseudomonas spp. Sequence analyses revealed that these Pseumonas spp. are closely related to Pseudomonas spp.(SJK83154),
Pseudomonas putida (CCO02553), Pseudomonas fulva (CBI68359), Pseudomonas
parafulva (CBG22656) and
Pseudomonas monteilii (CBG22644). In vitro growth inhibition assay showed that all cyanogenic Pseudomonas spp. inhibitedthe growth (17.43 to 78.71%) of P. infestans. The bacterial isolates were formulated in Talcum powder and the bacterial cells
were found viable for up to six months or more. The growing ability of these cyanogenic Pseudomonas spp. at cool to warm
temperature (8-30°C) suggesting its potentiality in controlling late blight of potato.
effective non-chemical alternatives to protect potato andtomato fields against their most threatening foliar disease.
The market for non-conventional fungicide products,
specifically biological control agent formulations, isgrowing at a rapid pace. Biological control is a routine partof disease management. Several commercial formulationsof biocontrol agents are available and many others are
currently being developed (Fravel 2005) and several
commercial formulations of biocontrol agents have beentested for efficacy against late blight. Of many trials
involvingdifferent
microorganisms,
including
Trichoderma harzianum, Bacillus subtilis, Streptomyces
sp.,Coniothy-rium minitans and a pool of undetermined
effective microorganisms (EM 5), the most effective was
the B. subtilis based-product (Stephen et al. 2005).
Cyanogenesis in bacteria seems to be restricted to a
relatively small number of species and most hydrogencyanide-producing bacteria have been identified within
the
genus
Pseudomonas
belongingtotheGammaproteobacteria (Ryall et al. 2009). Potato
associated cyanogenic Pseudomonas spp. displaying
volatile-mediated high potential against P. infestans (DeVrieze et al. 2015; Hunzike et al. 2015). The present study
has been undertaken to identify and formulate the effective
bioagents against late blight disease of potato and tomato.

Introduction
Potato (Solanum tuberosum L.) and tomato (Solanum
lycopersicum) are the important vegetable crops in
Bangladesh where it is primarily grown by resource-poor
farmers. Late blight caused by Phytophthora infestans(Mont.) De Bary limits the production of potato andtomato especially in cool weather worldwide. P. infestans(Mont.) De Bary, is an oomycete which is well known for
its explosive development when environmental conditions
are suitable and host plants susceptible to infection(Mizubuti and Fry 2006). Estimated worldwide economic
losses due to the control cost and disease vary from 3 to 5
billion dollars annually (Judelson and Blanco 2005;
Haldar et al. 2006). In Bangladesh, the yield losses havebeen estimated approximately by 25-57% due to lateblight disease of potato (Ali and Dey 1994). Moreover,late blight is considered one of the five highest ranking
priorities for potato research in Asia, Africa and Latin
America (Fuglie, 2007).
Late blight management has been heavily based onfungicide application and fungicide applications haveincreased over the last decade due to the introduction of
new and more aggressive genotypes of the pathogen (Katoet al. 1997). At the same time, two counter- balancing
factors have also grown: societal pressure for reducing
pesticide use on crops and acreage of organically-grown
food crops, potato and tomato included (Ghorbani et al.
2004). Therefore, innovative and effective control
measures are needed if fungicide use is to be reduced or,as in the case of organic production of potato and tomato
which is based on some modern and copper-based
fungicides for control of late blight. However, there has
been increasing pressure to find substitutes for theseproducts because of environmental contamination causedby these chemical residues. Thus, there is enormousinterest in finding

Materials and methods
Isolation of Pseudomonas spp.: Representative potato
plants were collected with their root systems and adheringsoil from the farmer’s fields. The leaves and stems(referred to here as “shoots”) as well as root tissues wereseparated and treated as follows: shoots was ground in adisinfected ceramic mortar using 5 ml of sterile water;roots and adhering soil will be shaken in sterile water tocollect the rhizosphere soil, while the roots- 51 -

spp.. The PCR products were purified using SV Gel and
PCR clean up system (Promega, Madison, USA). The
sequencing was done directly from PCR products using
primers (Ps30F) according to the standard protocol for
ABI3500 genetic analyzer (Applied Biosystems, Foster
City, CA, USA) with BigDye Terminator v1.1 and 3.1
Cycle Sequencing Kits. The quality of nucleic acidsequences was evaluated using Chromas (Version 2.6) toavoid the use of low quality bases.

themselves were discarded. The samples were
homogenized by shaking and pipetted into a test tube witha cut tip. These suspensions were 10-fold serially diluted
in sterile water and plated on King’s B medium. All plates
were incubated at 20°C for at least 6 days. In order to coveras much of the cultivable diversity as possible, single
colonies with different morphologies (rhizosphere versus
phyllosphere) were picked for isolation. Strains were thenpreserved in glycerol stock at-80˚C.

Analysis of nucleotide sequence: The nucleotide
sequences were analyzed using online bioinformatics tools.
The amino acid sequences translated from the nucleotide
sequences were compared with other amino acidsequences of rpoD gene of Pseudomonas spp. available in
the NCBI database using Basic Local Alignment Search
Tool (BLAST) algorithm to identify closely relatedsequences. Multiple sequence alignments were performed
using aligner tool from www.justbio.com. The
phylogenetic tree was constructed based on rpoD genesequences of different closely isolate of Pseudomonas spp.
deposited in the NCBI database using CLUSTALW
software.

Analysis of HCN: The emission of volatile hydrogencyanide (HCN) was assessed qualitatively by growing
bacterial isolates in LB medium containing 0.5 g glycine(a precursor for HCN production) per litre medium.
Gaseous hydrogen cyanide released by bacterial isolates
was detected via a color change from yellow to orange ofa filter paper (impregnated with picric acid solution). The
filter papers (90 mm Whatman®) were autoclaved,
saturated with 1% picric acid solution, let dry in thelaminar flow bench overnight and wetted with 10%
sodium carbonate solution prior to final use. The filter
papers were fixed to the lid of a petridish containing
culture medium on which the tested bacteria growon.

Formulation of cyanogenic Pseudomonas spp
controlling late blight of potato: The identified potential
cyanogenic Pseudomonas spp. was formulated on two
carrier material -talcum powder and sodium alginate. One
hundred grams of each carrier material was placed in a
metal tray under aseptic conditions and the pH adjusted to
7.0 by adding CaCO3 at the rate of 15 g/kg. Carboxy
methyl cellulose (CMC) was then be added at the rate of
10g/kg and mixed well. Finally, the mixtures were packed
into a polythene bag and autoclaved for 1h at 121oC(15lb/inch2). After autoclaving, 400 ml of bacterial
suspension (1×108 cfu/ml) was added to the sterilized
carrier material (1kg) followed by thorough mixing understerile conditions. The formulation thus prepared wasallowed to dry aseptically and was then ground to powder.
They were then packed in sterile polythene bags and stored
at 4°C. The viability of the formulated bacterial cells wasevaluated following dilution plate technique for a periodof six months.

Antagonistic assay of Pseudomonas spp. against P.
infestans: The antagonism test was carried out on Luria
Bertani (LB) agar medium. Bacterial isolates werestreaked on LB agar medium twenty four hours ago in atriangle. Then P. infestans isolate was inoculated using a
5 mm cork borer at the centre of the triangle. The plates
were incubated at 20ºC for two weeks. This test was donein triplicates. Radial growth inhibition of P. infestans wasassessed 14 days later by measuring the radial growth ofthe pathogen in the dual and control plates.
Identification and detection of hydrogen cyanide
producing Pseudomonas spp.: The Pseudomonas spp.
wereidentified
using
primers
PsEG30F(ATYGAAATCGCCAARCG)
and 736PsEG790R(CGGTTGATKTCCTTGA) designed based on rpoD
gene sequence specific to Pseudomonas spp. as described
previously by Mulet et al. (2009). HCN producing strains
were confirmed by PCR using degenerated primers
HCNC-F (5' GARTTYYTNTGYGAYCAYCA 3') and
HCNC-R (5' ATYTCNCCRTTRTCYTTYTG 3')
designed for the hcnC gene, which is part of the hcnABC
gene cluster that encodes for the HCN synthase (Dürr,
2014). The PCR was carried out with an initialdenaturation at 95°C for 3 minutes, followed by 40 cycles
of denaturation at 95°C for 30 seconds, annealing at 58°C
for 45 seconds, and elongation at 72°C for 1 minute. The
PCR was completed by a final elongation step at 72°C for
10 minutes.

Results and Discussions
Identification of Pseudomonas spp.: Bacterial colonies
were primarily identified as Pseudomonas spp. based oncolony morphology and gram reactions. All the colonies
were yellowish in colour and showed gram negative
reactions as tested by KOH solubility test. A total of 200
bacterial colonies were primarily identified as
Pseudomonas spp..
Analysis of HCN: The emission of volatile hydrogencyanide (HCN) by the bacterial isolates assessed
qualitatively as described above. Gaseous hydrogencyanide released by bacterial isolates was detected via a

Sequencing of rpoD gene: Partial nucleotide sequencing
of DNA polymerase sigma factor rpoD was performed
from the amplified products of different Pseudomonas- 52 -

identified as cyanogenic out of 200 isolates (Data notshown).

color change from yellow to orange of a filter paper(impregnated with picric acid solution). Six isolates were

Fig. 1 A. In vitro growth inhibition of P. infestans by different isolates of cyanogenic Pseudomonas spp., B. Radial growthof P. infestans against different cyanogenic Pseudomonas spp. and C. Per cent growth inhibition of P. infestans by different
cyanogenic Pseudomonas spp.
In vitro growth inhibition of P. infestans by
Pseudomonas spp.: In vitro growth assay showed that the
all Pseudomonas spp. inhibited the growth of P. infestans(Fig. 1A & 1B). The results revealed Pscya 4 isolate
inhibited highest (78.71%) growth of P. infestansfollowed by isolate Pscya 1 (78.36%) and Pscya 5(78.61%) (Fig.1C). The isolates Pscya 2 and Pscya 3
inhibited 77.46 and 75.03% growth of P. infestans,respectively.The minimum growth inhibition of P.
infestans was exhibited by Pscya 6, Pscya 7 and Pscya 8.
The variation in growth inhibition of P. infestans bydifferent isolates of Pseudomonas spp. might be due totheir potential in production of some volatile organic
compounds. Growth inhibition of P. infestans by
Pseudomonas spp. through synthesis of volatile organic

compounds was also observed (De Vrieze et al. 2015;
Hunziker et al. 22015).
Molecular based detection: A PCR -based approach wasdeveloped for species identification of Pseudomonas
strains and for the direct detection of Pseudomonas
populations natural environment (Mulet et al. 2009). A
highly selective set of primers (PsEG30F and PsEG790R),
giving an amplicon of 760 nucleotides in length, wasdesigned based on the internal conserved sequences of
rpoD gene sequences (the sigma 70 factor subunit of the
DNA polymerase) of Pseudomonas type strains. In thisstudy, the cyanide producing Pseudomonas spp. wasidentified by PCR using primers PsEG30F and PsEG790R.
An amplicon size 760 bp confirmed the isolates werePseudomonas spp. (Fig. 2A).

PCR based assay for identification of cyanogenic
Pseudomonas spp.: In addition to the functional
qualitative screening methods for cyanogenic bacterial
isolates, a PCR based molecular assay was performed. For
this purpose, degenerated primers were designed for the
hcnC gene, which is part of the hcnABC gene cluster that
encodes for the HCN synthase. All isolates were also
tested with the HCNC PCR. Primer design was carried out
by Gajender Aleti (AIT) by ClustalW aligning (Thompson
et al., 1994) of Pseudomonas aeruginosaprotein
sequences of the HCNC subunit (P. aeruginosa strain
PAO1 with NCBI accession number NP_250885.1 asstarting sequence for initial protein

blast). Using wobble codes, degenerate primers were
designed for the most conserved regions. With the HCNC
PCR set-up described above, amplification was recorded
for all isolates, which were detected as producers ofhydrogen cyanide with both functional screenings
methods. The PCR products could always be detected witha sequence length of approximately 360bp when analyzed
with agarose gel electrophoresis. Moreover, noamplification occurred for any of the acyanogenic isolates(Fig. 2B).

- 53 -

Fig. 2 A. PCR based detection of cyanogenic Pseudomonas spp. using primers specific to rpoD gene. 1: Pscya 1, 2: Pscya
2, 3: Pscya 4, 4: Pscya 6, 5: Pscya 7 and 6: Pscya 8. B. PCR based detection of HCNC genes in Pseudomonas spp. 1: Pscya
1, 2: Pscya 2, 3: Pscya 4, 4: Pscya 6, 5: Pscya 7, 6: Pscya 8, 7: Pscya 9 (Negative control), 8: Pscya 10 (Negative control)and 9: Water control
99% sequence identity with the corresponding nucleotide
sequences of sigma factor 70 (rpoD gene) sequences of
Pseudomonas fulva (GeneBank accession # FN599525
and CP023048) (Table 1).

Identification of cyanogenic Pseudomonas spp. by
sequencing: Nucleotide sequences of Pseuodomonas spp.
were compared with nucleotide sequences of other
Pseudomonas spp. available in the NCBI database using
Basic Local Alignment search Tool (BLAST) algorithm.
Blast homology of nucleotide sequences showed 97-

Table 1. Closest relatives of cyanogenic Pseudomonas spp. with other Pseudomonas spp. based on rpoD genesequence
Isolates Accession no.
Closest relatives (accession no.)Identity
Homology (%)
Pscya 1
Pseudomonas fulva (FN599525)
682/685
99
Pscya 2
Pseudomonas fulva (FN599525)
680/687
99
Pscya 4
Pseudomonas fulva (CP023048)
690/715
97
Pscya 6
Pseudomonas fulva (FN599525)
677/687
99
Pscya 7
Pseudomonas fulva (FN599525)
676/685
99
Pseudomonas fulva (FN599525)
677/686
99
Pscya 8bootstrap value of 96% and consist of two subgroups
supported by bootstrap values of 79 and 83%. Group II
includes 7 strains (Six cyanogenic Pseudomonas spp. and
one Pseudomonas fulva (CBI68359) of South Korea andwas supported by bootstrap value 92%. This groupconsists of two subgroups and was supported by bootstrap
value 100 and 96%. Group III represents two
Pseudomonas spp. from Japan (BAB17556) and China(AKC04323) These results clearly indicated that the
Bangladeshi cyanogenic Pseudomonas spp. seems
different from other Pseudomonas spp. available in the
NCBI database (Fig. 3A & B). However, Bangladeshi
cyanogenic Pseudomonas spp. were distributed in two
subgroups. This might be due to their variation in
suppressing the growth of P. infestans as well as theirvariation in genetic constituent of RpoD protein.

Phylogenetic relationship among the isolates of
Pseudomonas spp.: Blast-P homology search showedamino acid sequence translated from the partial nucleotide
sequence of rpoD gene of six cyanogenic Pseudomonas
spp. showed similarity with the RpoD protein of
Pseudomonas strains reported in the NCBI database suchas 99% homology with Pseudomonas fulva (CBI68359),
97% with P. fulva (BAB17556) and P. fulva (AKC04323),
93% with P. putida (CCO02553) and 92% with P. putida(BAA25357), Pseudomonas sp. (SJK83154) and P.
monteilii (CBG22644). Phylogenetic relationship of six
cyanogenic Pseudomonas spp. and their close relatives
analyzed using CLUSTALW program based on aminoacid sequences of rpoD gene revealed that all these
Pseudomonas spp. were distributed in three different
groups. Group I was supported by- 54 -

A

B

Fig. 3 A. Amino acid sequence alignment of conserved region of sigma factor 70 (RpoD) protein of six Pseudomonas spp.
with other close relatives Pseudomonas spp. available in the NCBI database B. Phylogenetic trees constructed from partiallytranslated amino acid sequences of sigma factor 70 (rpoD gene), using the maximum likelihood method. The evolutionary
distances were computed using MEGA (version 5.220). The analysis involved amino acid sequences including six cyanogenic
Pseudomonas spp. collected from phylloplane of potato and representatives of Pseudomonas spp. available in the NCBI
database. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted inbacterial cells up to six month or more at room
temperature and at 4-8˚C. Although it was observed thatthe formulated bacterial cells lost their viability with theincrease of storage time at room temperature (Fig. 4). Talc
based formulation has been reported for the managementof several crop diseases in India (Samiyappan et al., 1998).
Talc based formulation of P. fluorescens was found to beeffective as seed treatment on the control of fusarium wiltof tomato plants (Sarma et al., 2011) and foliar applicationon the control of rust and leaf spots of groundnut (Meena
et al., 2002).

Formulation of cyangenic Pseudomonas spp.: Talc is anatural mineral and chemically it is referred as magnesium
silicate (Mg3Si4O10(OH)2). It has very low moistureequilibrium, relative hydrophobicity, chemical inertness,
reduced moisture absorption and prevents the formation of
hydrate bridges that enable longer storage periods. Owing
to the inert nature of talc and easy availability as raw
material from soapstone industries, it is used as a carrier
for formulation development. In this study, one of theselected isolates was formulated in talc powder and wasstored at 4ºC until six months. The results revealed thesurvival ability of the formulated

Bacterial CFU g
formulation

1E+13

4-8˚C

RT

8E+12
6E+12
4E+12
2E+12
0
30 days post
formulation

60 days post
formulation

90 days post
formulation

Fig. 4 Colony forming unit (CFU) of cyanogenic Pseudomonas spp. in the talcum formulation. The CFU was counted bydilution plate technique at different days post formulation.

- 55 -

Conclusion

Pseudomonas spp. available in the NCBI database. In vitro
test confirmed the Pseudomonas spp. inhibited the growthof late blight pathogen, P. infestans. The Pseudomonas
spp. was then formulated in talcum powder and thebacterial cells become viable up to six month or more atroom temperature and at 4-8ºC.

Potential cyanogenic Pseudomonas spp. was identified
from the phylloplane of potato plants. Pseudomonas spp.
was confirmed by PCR and sequencing of sigma factor 70
DNA polymerase (rpoD gene). Sequence analyses of
rpoD gene revealed the identified Pseudomonas spp. arevery close to someAcknowledgement
This research work was funded by the Bangladesh Academy of Sciences-United States Department of Agriculture (BASUSDA) (Grant No.: BAS-USDA PALS BAU-CR-38) to Dr. Md. Rashidul Islam.

References
Judelson HS and Blanco FA (2005) The spores of
Phytophthora : weapons of the plant destroyer.
Nature Reviews Microbiology 3: 47-58.
Kato M, Mizubuti ESG, Goodwin SB, Fry WE (1997)
Sensitivity to protectant fungicides andpathogenic fitness of clonal lineages of
Phytophthora infestans in the United States.
Phytopathology 87:973-978
Mizubuti ESG., Fry WE (2006) Potato late blight. In:
Cooke BM, Jones DG, Kaye B (Eds) The
Epidemiology of Plant Diseases (2ndEdn),
Springer-Verlag, Dordrecht, pp 445-471
Mulet M, Bennasar A, Lalucat J, Garcı´a-Valde´s E.
(2009) An rpoD-based PCR procedure for theidentification of Pseudomonas species and fortheir detection in environmental samples.
Molecular and Cellular Probes. 23: 140–147
Ryall B, Mitchell H, Mossialo D, and Williams HD(2009): Cyanogenesis by the entomopathogenic
bacterium Pseudomonas entomophila. Lett Appl
Microbiol, 49:131-135.
Stephan D, Schmitt A, Carvalho SM, Seddon B, Koch E(2005) Evaluation of biocontrol preparations andplant extracts for the control of Phytophthora
infestans on potato leaves. European Journal of
Plant Pathology, 112:235- 246.
Samiyappan R, Jayashree K., Raghuchander T andNarasimhan, V (1998) Fluorescent
pseudomonads for the management of sheathblight in rice. Indian Farmers Digest 31: 28–29.
Sarma MVRK., Kumar V, Saharan K, Srivastava R,
Sharma AK, Prakash A, Sahai V and Bisaria VS(2011) Application of inorganic carrier-based
formulations of fluorescent pseudomonads andPiriformospora indica On tomato plants andevaluation of their efficacy. Journal of Applied
Microbiology, 111: 456–466.
Meena B, Radhajeyalakshmi R, Marimuthu T and
Velazhahan R (2002) Biological Control of
Groundnut Late Leaf Spot and Rust by Seed andFoliar Applications of a Powder Formulation of
Pseudomonas fluorescens. Biocontrol Scienceand Technology, 12(2):195-204.

Ali, M.S and T.K. Dey (1994) Pathological research. on
tuber crops in Bangladesh. In. Proc. Workshop
on Transfer Technology of CDP crops under
Research.-Extension. Linkage Programme, heldon Oct. 22-27, 1994. 159-165.
De Vrieze M, Pandey P, Bucheli TD, Varadarajan AR,
Ahrens CH, Weisskopf L and Bailly A (2015)
Volatile Organic Compounds from Native
Potato-associated Pseudomonas as Potential
Anti-oomycete Agents. Front. Microbiol. 6:1295.
Dürr SR (2014)Development of high-throughput methods
for the detection of hydrogen cyanide- producing
bacteria for the application in biocontrol.
Master’s Thesis. Department of Forest- and Soil
Sciences, Institute of Soil Research, Universityof Natural Resources and Life Sciences, Vienna.
Fravel DR (2005) Commercialization and implementationof biocontrol. Annual Review of Phytopathology,
43: 337-359.
Fuglie KO (2007) Priorities for potato research indeveloping countries: Results of a survey.
American Journal of Potato Research, 84 (5):353-365.
Ghorbani R, Wilcockson SJ, Giotis C, Leifert C (2004)
Potato late blight management in organic
agriculture. Outlooks on Pest Management
15:176-280.
Haldar K, Kamoun S, Hiller, NL, Bhattacharje S, van Ooij
C (2006) Common infection strategies ofpathogenic eukaryotes. Nature Reviews
Microbiology 4: 922-931.
Hunziker L, Bönisch D, Groenhagen U, Bailly A, Schulz
S, Weisskopf L (2015) Pseudomonas strains
naturally associated with potato plants produce
volatiles with high potential for inhibition of
Phytophthora infestans. Appl Environ Microbiol
81:821–830.
Hunziker L, Bönisch D, Groenhagen U, Bailly A, Schulz
S and Weisskopf L (2015) Pseudomonas strains
naturally associated with potato plants produce
volatiles with high potential for inhibition of
Phytophthora infestans. Appl Environ Microbiol
81:821–830.

- 56 -

OS III-1
Utilization of chitosan and glukomanan for fruit coating of chili
againts antrachnose disease
Chaieydha Noer Afiefah1, Ani Widiastuti1,2, Arif Wibowo1,2, and Siti Subandiyah1,2(1Dept. Of Plant Protection, UGM; 2Research Center for Biotechnology, UGM. Yogyakarta, Indonesia)

ABSTRACT
Chili (Capsicum spp.) is the most important vegetable in Indonesia since it is needed for dairy food aschili souce or as hot spice in many culinary cuisines. The cultivation period of chili is mostly in dry
season, however the product is needed whole year therefore the supply during rainy season is usually
insufficient leading to highly increase of the price in the market. Low production of chili in the wet
season is especially due to fungal and bacterial diseases and antrachnose disease is devastating for the
fruit product. Antrachnose of Chili is caused by Colletotrichum spp., especialy C. gloeosporioides andC. accutatum group. The fruits may have been infected in the field however the symptom is becoming
more severe after harvesting during the ripening period. This research was conducted to protect post
harvest chili fruits using nano chitosan, glucomanan, and the mixture of both polymers. Threedifferent chili varieties were used in the experiment, Capsicum annum var Hot Chili and var Phoenix
and C. fruitescens var. RM 28. The antrachnose pathogen of C. gloeosporioides was cultivated in the
Potato Dextroxa Brorth and the conidium suspension in PBS at the concentration of 106/ml was used
for spraying inoculation. Disinfected wounded freshly harvested chili fruits were coated before thepathogen inoculation. Fruiit coating application was done using different mixter of 0.1% chitosan,
0.1% gluocoman, and the mixture of 0.1% chitosan and 0.1% glucomanan by immergimg the fruits
for 2 min in the solution and air dried before iniculation. The biopolymers were also used forconidium germination and colony growth analysis. The reuslts suggested that chitosan andglucomanan fruit coating of chili could suppress post harvest antrachnose disease. Chitosan gave
better protection than glucoman and the mixture of both polymers. While chitosan suppres conidium
germination on the other hand glucomanan suppress the colony development in vitro.
This research was funded by Australian Center for International Agricultural Research (ACIAR) HORT 2009/056
Key words: post harvest, biopolymer coating, chili fruit, antrachnose disease- 57 -

- 58 -

OS III-2
Antifungal effects of ethanolic shellac - Modified coconut oil (ES-MCO) combined withphysical treatments against postharvest diseases of mango and mangosteen
Jitreerat PONGPHEN*, Sripong KANYALA*, Vo Thi THOUNG*, Apiradee UTHAIRATANAKIJ*,
Sontaya LIMMATVAPIRAT**(*School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Thailand, **Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University, Thailand）

SUMMARY
Postharvest losses has been known a major problems of tropical fruits which resulting to economic losses. Currently
control of postharvest diseases is based on fungicide application. However, environmental and health has been now
concerned. Therefore, safe alternative methods were studied. This experiment was to evaluate the antifungal properties of
ethanolic shellac-modified coconut oil (ES-MCO) in vitro test and to investigate the combined effects of ES-MCO andphysical treatments on controlling postharvest disease of mango and mangosteen (in vivo test). Antifungal property of
ES-MCO, ES and MCO was measured by paper disc method. 2% MCO and ES-MCO completely inhibited the growth ofColletotrichum gloeosporioides, a causal agent of anthracnose disease in mango. In vivo test, antifungal effect of
ES-MCO combined with hot water (HW) and UV-C treatments (HW+UV-C+ MCO) against anthracnose disease ofmango was tested in compared with treatments of HW+UV-C or ES-MCO. HW+UV-C+ES-MCO treatments showed themost effective in reducing anthracnose disease and delayed fruit ripening. In mangosteen, application of ES-MCO
combined with 1-MCP and low density polyethylene (LDPE) bag could delay fruit rot diseases as same as fungicide
treatment, and also showed the greatest effective in delaying the changes of calyx and pericarp color, weight loss,
hardening of the pericarp and ethylene production. These results suggest that combined effect of ES-MCO and physicaltreatments may serve as an alternative method for controlling postharvest disease of mango and mangosteen.

Introduction

the quality of fruit (Lurie, 1998; Mercier et al., 2001;

Mango (Mangifera indica L.) and mangosteen (Garcinia

Sripong et al., 2015b). Otherwise, some physicalmangostana L.) are exotic tropical fruit which Thailand

treatments such as modified atmosphere packaging

produces to serve for the domestic and export markets.

(MAP) and wax coating has indirectly effect to delay the

However, postharvest diseases is the major loss during

diseasestorage and marketing period. Anthracnose disease(Pranamornkith et al., 2003; Accaseavorn et al., 2006). In

caused by Colletotrichum gloeosporioides Penz. is aaddition,main problem of mango, whereas fruit rot disease caused

1-methylcyclopropene (1-MCP), which is an ethylene

by C. gloeosporioides, Lasiodiplodia theobromae,

inhibitor (Watkins, 2006) has been shown to prolong thePestalotiopsis sp., and Phomopsis sp. are the major

shelf life of many fresh produce and delay postharvest

problem of mangosteen (Khewkhom et al., 2012).

disease development such as mangosteen and jujube

Fungicides are known as the most effective treatment to(Piriyavinit et al., 2011; Zhang et al., 2012).

developmentinpostharvest

severalharvestedtreatment

fruitswith

control plant disease but they have high potentiallyharmful on human health and environment (Ma andWax coatings are commonly used to maintain the quality

Michailides,

areof fruit by preventing moisture loss, providing the barrier

non-chemical method such as treatments with hot waterto oxygen transfer, and also be able to prevent theand UV-C are shown high efficiency to control disease

penetration of microbes resulting in an extend shelf lifeand maintain the fruit quality. Pre-heat treatment and)Pramod et al., 2016(. Shellac (SH) wax is a natural

UV-C treatments have directly suppressed pathogens,product from an insect (Laccifer lacca) used widely toelicited the natural plant resistance, and also maintained

extend the shelf life of several fresh produce such as2005).

Physicaltreatments- 59 -

mangosteen and lime )Accaseavorn et al., 2006(. Inbyrecent years, wax coatings have been developed with the(HW+UV-C+ES-MCO), and 4) untreated as the control.

additionagainstSamples were then placed in plastic basket covered withpostharvest diseases. A mixture of shellac wax witha polyethylene bag (PE) at 13°C, 85-90%RH for 15 days.

modified coconut oil )ES-MCO( solution could reduce

Each treatment contained three replicates and eachanthracnose disease and maintain the quality of mango

replicate consisted of 4 fruits. Disease incidence, disease

fruit (Sripong et al., 2015a).

severity (Chantrasri et al. , 2007) , fruit quality (weightofantimicrobial

compounds

to

UV-C

and

then

coatedwith

ES-MCOloss, firmness, and hue angle color), and respiration rateof mango were determined every 3 days interval.

Coconut oil is edible oil extracted from kernel of maturecoconut. In coconut oil contains the fatty acids whichhave the function of antimicrobial property (DebMandal

Combined effect of ES-MCO, 1-MCP and LDPE bag

and Mandal, 2011). Whereas modified coconut oil

on fruit rot disease of mangosteen)MCO( that obtained by glycerolysis of the free fatty

Mangosteen fruits at maturity stage 3 )reddish pink( wereacid of coconut oil with glycerol. It contains monolaurin

coated with ES-MCO and/or 500 ml/l carbendazim

and lauric acid that can inhibit various bacteria )Oyi et al.,(fungicide), packed in LDPE bags containing a 1-MCP

2010; Tangwatcharin and Khopaibool, 2012( and fungi

sachet (ethyl-gone® a.i. 1.7861 μg/g). Samples were then

)Altieri et al., 2009(. Therefore, this research was tostored at 13°C, 90-95% RH for 42 days. Non-treated andpresent the effects of ES-MCO combined with various

non-packed fruits, carbendazim treated fruits, andphysical treatments and postharvest treatments against

ES-MCO coated fruits alone were served as the controls.

postharvest diseases and maintain the quality ofFruit quality (color change of pericarp and calyx, weight

harvested mango and mangosteen.

loss), physiological changes (ethylene prodcution) anddisease index were evaluated every 7 days. EachMaterial and Method

treatment had 4 replicates with 5 fruits in each replicate.

Antifungal property of MCO, SH, ES-MCO against

The experiment was arranged as completely randomized

C. gloeosporioides by paper disc method
ES-MCO was prepared from 2% MCO and 8% shellac

design )CRD( and the means of each treatment were

) SH( and dissolved in 95% ethanol (ETOH). Spore)DMRT( at p ≤ 0.05.

compared using the Duncan’s new multiple range test

suspension of C. gloeosporioides was dropped on to
black paper disc, dried on air, and then dropped with an

Result and Discussions

equal volume of 2% MCO, 8% SH, ES-MCO and

Antifungal property of MCO, SH, ES-MCO against

95% ETOH on the same position. Dropping with sterile

C. gloeosporioides

distilled water was used as the negative control. All

MCO, SH, ES-MCO and ETOH treatments completely

treated discs were incubated in a high moisture chamber,

inhibited the mycelail growth of C. gloeosporioides on

and observed mycelium growth on disc surface after 2

paper disc, whereas the control showed 100%

weeks. The survival of spores was detected by placing

mycelail growth. The spore survival was tested on PDA

the treated paper disc onto the surface of PDA plates, andplate. It was found that MCO and ES-MCO treatments

incubated at 25°C for 2 weeks. Paper disc showed

completely killed the fungal spore, while spore treatedmycelial growth on PDA was indicated as the survival ofwith ETOH and SH still grew as same as the control

the fungal spores. Each treatment contained three) Table 1( . In general, MCO is obtained by glycerolysis

replicates and each replicate consisted of 4 plates.

process of fatty acid of coconut oil )Luo et al., 2014(. In
thisCombined

effectsof

ES-MCOandphysical

experiment

MCO

monolaurin and 5. 22%

contained

with

of

23. 65%luaric acid that can against

treatments on anthracnose disease of mango.
Naturally infected mango cv. Chok-Anan at 75-80%

various food borne pathogens and postharvest fungal

were treated as the follows; 1) surface coated with

Khopaibool, 2012; Altieri et al. , 2009; Sripong et al.

ES-MCO ( ES-MCO) for 1 min, 2) dipped in hot water

2015a( . Previous studies, reported that molecule of(HW) at 55°C for 5 min followed by irradiating with 6.16

monoglycerides and fatty acids can penetrate into lipid

kJ/m2 UV-C (HW + UV-C), 3) dipped in HW followed

phase and physically disrupt cell membranes, and inhibit

pathogens (Oyi et al. , 2010; Tangwatcharin and- 60 -

enzymes involved in energy production and nutrient

The quality of treated mango shows in Fig.1 and Fig.2.

transfer of microbe, that may lead to the death of

Change of peel color )as indicated by hue angle( andmicrobial cell )Luo et al., 2014(.

firmness of mango fruit delayed when the fruits weretreated

Combined

effectsof

ES-MCO

andwith

ES-MCOorHWT+UV-C+ES-MCO)Fig.1(. Especially HWT+UV-C+ES-MCO treatment

physicaltreatments on anthracnose disease of mango.

showed the most effective than ES-MCO alone. The

Effect of ES-MCO coating combined with HW andlower hue angle changes of coated fruit may be

UV-C treatments on anthracnose disease and quality of

associated with the coating of ES-MCO may help tomango was investigated. This result found that ES-MCO,

create modified atmospheres condition within the fruit.

HW+UV-C,

treatments

Maftoonazad et al. )2007( demonstrated that thesignificantly reduced disease incidence and severity

and

HW+UV-C+ES-MCO

presence of CO2 in the storage atmosphere is an(Table 2). Several reports demonstrate that the effect ofimportant factor to suppress chlorophyll degradation. In

HW and UV-C on disease reduction could be explained

addition, UV-C may retard the activity of chlorophyll

as direct effect of HW and UV-C on the fungi present on

degrading enzymes )Costa et al., 2006( and cell wall

the fruit surface, and as indirect effect by eliciting plantdegrading enzymes )Barka et al., 2000( resulting in the

defense enzymes in mango, bell pepper and strawberry

delay of fruit senescence.

)Sripong et al., 2015b; Mercier et al., 2001; Pombo et al.,2011(. Moreover, ES-MCO coating may function as abarrier against fungal infection, furthermore MCO also(A)

have high antimicrobial property of monoglycerides(Rihakova et al., 2002; Wang et al., 1993(.
Table 1 Mycelial growth and survival of C.
gloeosporioides spore

after

treatedwithsteriledistilled water ) Control( , ETOH, SH, MCO and
ES-MCO by paper dice method
Treatments

Mycelial

Survival ofgrowth )%(spore )%(a(B)

100.00a

Control

100.00

ETOH

0.00b

100.00a

b

SH

0.00

100.00a

MCO

0.00b

0.00b

ES-MCO

0.00b

0.00b

Table 2 Incidence and severity of anthracnose disease
of

mango

fruit

when

coatedwith

ES-MCO)ES-MCO(, dipped in hot water followed by UV-C

Fig. 1 Hue angle (A) and firmness (B) of mango

irradiation )HW+UV-C(, dipped in hot water

when coated with ES-MCO, dipped in hot water

followed by UV-C irradiation and coated withfollowed by UV-C irradiation )HW+UV-C(, dipped in

ES-MCO

hot water followed by UV-C irradiation and coated)HW+UV-C+ES-MCO(

anduntreated)control( during storage at 13°C for 15 days.

with ES-MCO )HW+UV-C+ES-MCO( and untreatedTreatment)Control( during storage at 13°C for 15 days.

Dis. incidence

Dis. severity(%)(score)Control

100a

3.0a

Moreover, HW+ UV-C+ES-MCO treatment also had

ES-MCO80b

1.1ab

effect to reduce respiration rate which was correlated to

HWT+UV-C

40c

0.6b

lower weight loss in mango fruit when compared withHWT+UV-C+ES-MCO

30c

0.5b

other treatments )Fig. 2(. This may be explained that thecoating with ES-MCO forms thin film on mango fruit- 61 -

which can reduce the respiration rate as be ever reported

activity of MCO (Rihakova, et al. , 2002; Wang et al. ,in strawberry and avocado )Maftoonazad et al., 2007;

1993( and 1-MCP.

Perdonesa et al., 2012(
Table 4 The combined effect of ES-MCO, LDPE bag,
(A)

1-MCP sachet on disease index, weight loss (WL),
different color (E) change of calyx and pericarp, andethylene production (μl C2H4/kg.hr) of mangosteen
during storage at 13ºC for 45 days.
Dis.

WL

E of

E

index

(%)

Calyx

Pericarp

Production

Control

0.4a

5.1a

28.4a

20.6b

10.3bc

CBZ

0.3ab

1.9b

28.4a

22.8b

13.9a

ES-MCO

0.3ab

0.4c

28.9a

27.0a

8.3cd

CBZ+LDPE

0.2b

0.3d

17.9b

17.8c

12.3ab0.2b0.2e

23.9ab

15.0c

6.5d

Treatment

(B)

of

C2H4

+1-MCP
ES-MCO+LDPE+1-MCPIn fact, 1-MCP has been known an effective inhibitor of
ethylene action, and can delay a number of genes
associated with the ripening process (Watkins, 2006(.
However,

antifungal

activity

of

1-MCP

againstFig. 2 Weight loss (A) and respiration rate (B) of

anthracnose disease in harvested mango and apple had

mango when coated with ES-MCO, dipped in hot

been discovered by Xu et al. (2016) and Li et al. (2017).

water followed by UV-C irradiation )HW+UV-C(,

1-MCP showed the direct suppression effect on the spore

dipped in hot water followed by UV-C irradiation

germination and mycelial growth of C. gloeosporioides

and coated with ES-MCO )HW+UV-C+ES-MCO(

and P. expansum, and also induced reactive oxygen

and untreated )Control( during storage at 13°C for 15

species (ROS) generation in pathogenic spore. In

days.

addition, antifungal effect of 1-MCP was found that it
could induce plant disease resistance in jujube fruit

Combined effect of ES-MCO, 1-MCP and LDPE bag

against postharvest blue mold and also delayed fruiton fruit rot disease of mangosteen.

senescence (Zhang et al., 2012). Similar with our present

Rapid increase in pericarp hardening, pericarp color,

report, 1-MCP combined with ES-MCO+LDPE bag

browning of calyx, and fruit rot disease is theshowed the greatest effective in controlling fruit rot

characteristic of senescence of mangosteen fruit which

disease of mangosteen and also delaying fruit ripening as

leading to low quality and short shelf life. Our previous

indicating by the retarding of color changes of calyx andstudies, we found that 1-MCP+ LDPE could maintain the

pericarp, weight loss, ethylene production )Table 4(, andquality of mangosteen fruit by delaying the ripening

hardening of the mangosteen pericarp )data not shown(.

process. In this study, ES-MCO coating combined with
LDPE+ 1-MCP sachet was used to maintain fruit quality

Conclusionand control fruit rot disease development. The result

In conclusion, the data presented here show that MCO

showed that treatments of ES-MCO and ES-MCO+LDPE

and ES-MCO significantly killed C. gloeosporioides, abag+ 1-MCP could delay fruit rot diseases as same as

causal agent of anthracnose disease in mangoes. The

fungicide

combination

treatments(carbendazime

alone

and

treatmentof

ES-MCO

with

other

carbendazim+ LDPE+ 1-MCP( . Particularly mangosteen

postharvest treatments such as HWT, UV-C, 1-MCP andtreated with ES-MCO+ LDPE bag+ 1-MCP had a lowest

packaging can enhance the effectiveness to reduce

disease index compared with other treatments ) Table 4( .

postharvest loss by anthracnose in mango and fruit rot

Low disease index may result the effect of antifungal

disease in mangosteen. Moreover, the combined- 62 -

treatments has more effectiveness to delay fruit

blue mold of apple fruit by inhibiting the growth ofsenescence than the single treatment. Thus, their

Penicillium

application could be a promising and alternative

Technology, 125: 59–64.

approach to the use of chemicals for the control of9) Luo C Zeng Z Gong D Zhao C and Liang Q (2014)

postharvest diseases and the maintenance of quality in

Evaluation of monolaurin from camphor tree seeds for

mango and mangosteen.

controlling food spoilage fungi. Food Control, 46:

expansum.

Postharvest

Biologyand488–494.

Acknowledgement

10) Lurie S (1998) Postharvest heat treatment-Review.

MCO, SH and ES-MCO were obtained Faculty of

Postharvest Biology and Technology, 14)3(: 257-269.

Pharmacy, Silpakorn University, Thailand. Some part of11) Ma Z and Michailides TJ (2005) Advances inthis work was supported by the Thailand Research Fundunderstanding molecular mechanisms of fungicide

through the Royal Golden Jubilee Ph.D. Program (RGJ)

resistance and molecular detection of resistant genotypes(Grant No. PHD/00642554).

in phytopathogenic fungi. Crop Protection, 24: 853–863.
12) Maftoonazad N Ramaswamy HS Moalemiyan M and

References

Kushalappa AC (2007) Eﬀect of pectin-based edible

1) Accaseavorn P Ampornratana T Boonsiri A

emulsion coating on changes in quality of avocado

Siwarungsun N Prichanont S and Ampornratana T (2006)

exposedDevelopment of shellac coating for extending shelf-life ofCarbohydrate Polymers, 68: 341–349.

mangosteen and lime (cv. Pan). Agricultural Science13) Mercier J Baka M Reddy B Corcuff R and Arul J

Journal, 37 (Suppl.5) : 42-45. (in Thai)(2001) Short-wave ultraviolet irradiation for control of2) Altier C Bevilacqua A Caridillo D and Sinigaglia M

decay by Botrytis cinerea in bell pepper: induced(2009) Antifungal activity of fatty acids and theirresistance and germicidal effects. Journal of themonoglycerides against Fusarium spp. in a laboratory

American Society for Horticultural Science, 126:

medium. International Journal Food Science Technology,

128–133.

44)2(: 242-245.

14) Oyi AR Onaolapo JA and Obi RC (2010)

3) Barka EA Kalantari S Malhlouf J and Arul J ) 2000)

Formulation and antimicrobial studies of coconut )Cocos

Impact of UV-C irradiation on the cell wall-degrading

nucifera Linne( oil. Research Journal of Applied Science,

enzymes during ripening of tomato ) LycopersiconEngineering and Technology, 22)2( : 133-137.

esculentum L. ( fruit. Journal of Agricultural and Food

15) Perdonesa A Sanchez-Gonzaleza L Chiralta A andChemistry, 48, 667–671.

Vargasa M (2012) Effect of chitosan–lemon essential oil

4) Chantrasri P Sardsud V Sangchote S and Sardsud U

coatings on storage-keeping quality of strawberry.

(2007) Combining yeasts and chitosan treatment toPostharvest Biology and Technology, 70: 32–41.

reduce anthracnose fruit rot in mangoes. Asian Journal of16) Piriyavinit P Ketsa S and van Doorn WG (2011)

Biology Education, 3: 40–46.

1-MCP extends the storage and shelf life of mangosteen5) Costa L Vicente AR Civello PM Chaves AR and)Garcinia mangostana L.(. Postharvst Biology andMartinez GA (2006) UV-C treatment delays postharvest

Technology, 61: 15-20.

senescence in broccoli florets. Postharvest Biology and17) Pombo MA Rosli HG Martinez GA Civello P M

Technology, 39: 204–210.

)2011( UV-C treatment affects the expression and activity6) DebMandal M and Mandal S (2011) Coconut )Cocosof defense genes in strawberry fruit )Fragaria×ananassa,

nucifera L.: Arecaceae(: In health promotion and disease

Duch.(. Postharvest Biology and Technology, 59: 94–102.

prevention. Asian Pacific Journal of Tropical Medicine :18) Pramod KR, Nidhi A and Mitu S (2016) Edible

241–247.

coating of fruits and vegetables : A review. International

7) Khewkhom N Sangchote S and Sungsiri T (2012)

Journal of Scientific Research and Modern Education, 1

Postharvest control of fruit rot of mangosteen by plant)1( : 2455-5630.

extracts

19) Pranamornkith T Kanlayanarat S and Uthairatanakij

fromZingberaceae

family.

ISHS

Acta

to

Lasiodiplodia

theobromae

infection.

Horticulturae, 973 : 119-126.

A (2003) Effect of polyethylene film packaging on the8) Li J Lei H Song H Lai T Xu X and Shi X (2017)

storage life of mangosteen )Garcinia magotana L.(,

1-methycyclopropene (1-MCP) suppressed postharvest

Proceedings of the APEC Symposium on Postharvest- 63 -

Handling System, Bangkok. p. 377.
20) Rihakova Z Filip V Plockova M Smidrkal J andCervenkova R (2002) Inhibition of Aspergillus niger
DMF 0801 by monoacylglycerols prepared fromcoconut oil. Journal of Food, 20: 48–52.
21) Sripong K Jitareerat P Uthairatanakij A Srilaong V
Wongs-Aree C Tsuyumu S and Kato M (2015a) Effects of
hot water, UV-C and modified coconut oil treatments on
suppression of anthracnose diseases and maintenance ofquality in harvested mango cv. “Chok-Anan”. Journal of
Food and Nutrition Science, 3)1-2(: 1-8.
22) Sripong K Jitareerat P Uthairatanakij A Srilaong V
Wongs-Aree C Tsuyumu S Ma G Zhang L and Kato M(2015b)., Combined treatments with hot water and UV-C
elicited disease resistance against anthracnose andimproved quality of harvested mangoes. Crop Protection,
77: 1-8.
23) Tangwatcharin P and Khopaibool P (2012) Activityof virgin coconut oil, lauric acid or monolaurin in
combination with lactic acid against Staphylococcus
aureus. The Southeast Asian Journal of Tropical
Medicine and Public Health, 43 : 969-985.
24) Watkins CB (2006) The use of 1-methylcyclopropene)1-MCP( on fruits and vegetables. Biotechnology
Advance, 24: 389-409.
25) Wang LL Yang BK Parkin KL and Johnson EA(1993) Inhibitionof

Listeria

monocytogenes

bymonoacylglycerols synthesized from coconut oil andmilk fat by lipase-catalyzed glycerolysis. Journal of
Agricultural and Food Chemistry, 41: 1000–1005.
26) Xu X Lei H Ma X Lai T Song H Shi X and Li J
(2016) Antifungal activity of 1-methycyclopropene(1-MCP)againstanthracnose(Colletotrichum

gloeosporioides) in postharvest mango fruit and its
possible mechanisms of action). International Journal of
Food Microbiology, 214: 1-6.
27) Zhang Z Tian S Zhu Z Xu Y and Qin G (2012) Effectof 1-methycyclopropene )1-MCP( on ripening andresistance of jujube (Sizyphus jujube cv. Huping) fruit
against postharvest disease. LWT-Food Science andTechnology, 45 (1): 13-19.

- 64 -

OS III-3
Enhancing plant defense in mango fruit by hot water and UV-C treatments
Kanlaya SRIPONG*, Apiradee UTHAIRATAKIJ*, Masaya KATO**, Pongphen JITAREERAT*(*School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Thailand, **Facultyof Agriculture, Shizuoka University, Japan)

SUMMARY
This research investigated the effects of hot water treatment (HWT), UV-C irradiation and their combination on the
induction of resistance to anthracnose disease and on quality improvement in harvested mangoes. Naturally infectedmango fruit cv. Chok-Anan was used in this study. Fruit were divided into four treatments; UV-C irradiation at a dose
6.16 kJ m-2, HWT at 55°C for 5 min, HWT followed by UV-C irradiation. Untreated fruit served as the control. All
samples were kept at 13°C for 12 days. The combined treatment significantly suppressed the severity of anthracnose
symptoms compared with either single treatment. Additionally, the specific activities of key plant defense-related
enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (POD), chitinase (CHI) and β-1,3-glucanase (GLU),
increased in peel of the mango fruits, as well as the expressions of the MI-PAL, MI-POD, MI-CHI and MI-GLU genes.
The combined treatment or UV-C treatment alone significantly delayed fruit ripening by retarding firmness andmaintaining peel and pulp color of mango fruit. While, weight loss was not significantly different among the treatments.

Introduction

emerged as an alternative method to avoid chemical

Mango (Mangifera indica L.) is a major tropical fruit infungicides. Application of UV-C irradiation reduce

both the domestic and export markets of Thailand.

postharvest disease in several fresh produce such as

AnthracnoseColletotrichum

papaya, strawberry and tomato (Patricia et al., 2007;

gloeosporioides Penz. is a major problem of harvested

Marina et al., 20011; Maria et al., 2009). Control ofmango. Due to the fungus is able to infect young fruit

postharvest diseases by UV-C irradiation seems to be

and remain latent during fruit growth in the field. Thus, it

depended on two different mechanisms - a direct

is hard to prevent disease development after harvested.

germicidal effect on pathogens and an indirect effect byControl of anthracnose disease is based on fungicides but

inducing defense mechanisms in the plant tissue (Porat etchemical residues in fruit and development of pathogensal., 2000). Moreover, HWT and UV-C irradiation also

to resist chemicals may result. Thus, it is important toreported as the effective methods to improve the quality

searchthediseasecausednon-chemicalbydiseaseof fruits (Pan et al., 2004). In previous studies, thesuppression by inducing plant defense mechanisms intreatments

forcombination of these two treatments has been applied to

fruit before storage.

some crops such as strawberry (Pan et al., 2004) andeggplant (Karasahin et al., 2005), and it could reduce

Hot water treatment (HWT) is a physical method that has

postharvest disease and maintain the quality of fresh

been used for the control of postharvest diseases in

produce greater than the results by either one of theseseveral fruits such as peach, muskmelon, satsuma

treatments. The objectives of the present study was tomandarin (Liu et al., 2012; Yuan et al., 2013; Hong et al.,investigate the combined effects of HWT and UV-C

2007), and also in mango fruit (Alvindia et al., 2015).

irradiation for inducing enzymes related to the defense

HWT showed to be efficient for controlling postharvest

mechanism and improving postharvest quality of mango

diseases by directly inhibiting pathogen and activating

fruit.

the natural resistance of the host (Porat et al., 2000).
However, HWT alone cannot completely control

Material and Method

postharvest disease of fruit and it has less effective than

Mango fruits cv. Chok-Anan at 75-80% maturity was

chemical treatment (Wasker, 2005; Prakash and Pandey,

harvested

2000). Therefore, there is a need for a method combining

Ratchaburi province, Thailand. The fruit were selected

couple of methods together. UV-C irradiation has also

for uniformity of size, shape and freedom from any- 65 -

fromcommercial

orchards

locatedindefects, and then disinfected with a solution of 200 ppm

significantly (P ≤ 0.05) reduced both anthracnose diseasesodium hypochlorite and air dried at ambient temperature.

incidence and severity in mango fruit. The fruit treated

Mango fruits were divided into four groups. The firstwith the combined treatment did not show any visiblegroup was subjected to HWT; the HWT was conducteddecay until 6 day of storage, while control and fruitby submersing the fruits in a hot water bath at 55°C for 5

treated with UV-C or with HWT alone were 100%,

min. After treatment, the fruits were immediately cooled

58.3% and 25.0% of disease incidence, respectively

in tap water (10°C) for 15 min and then air dried at(Table 1). Similarly, disease severity at the end of storage

ambient temperature. The second group was treated with

was lowest in the combined treatment fruit, followed by

UV-C irradiation using General Electric 30 W G15T8

hot water treatment, UV-C treatment and control (Table

germicidal lamps. The mango fruits were placed under1). These result indicated that the combination treatment

the UV-C lamps on aluminum foil for light reflection at ahelped to reduce anthracnose disease in mango fruit

distance of 20 cm from the light source for 5 min tobetter than when either hot water or UV-C alone was-2provide a dose of 6.16 kJ m . The UV-C dose wasused. Similarly, Pan et al. (2004) found that ameasured with a digital radiometer (Cole-Parmer

combination of UV-C and heat treatment enhanced theInstrument Company, Vernon Hills, IL, USA). The third

benefits to control postharvest disease in strawberry andgroup was treated with a combination treatment

eggplant more than applying of each treatment

consisting of hot water and UV-C. They were first treated(Karasahin et al., 2005). In addition, this present resultwith hot water at 55°C, cooled, dried and then irradiatedshows that the suppression of anthracnose disease werewith UV-C as described above. A fourth group of fruit

correlated with the genes expression and activities of

was kept untreated as a control. All of the fruit samples

enzyme related with plant defense.

were placed in plastic baskets covered within apolyethylene bag (PE) and kept at 13°C under

Table 1 Incidence and severity of anthracnose disease

humidified conditions (85-90% RH) for 12 days. Eachin mango fruit when treated with UV-C irradiation attreatment had 4 replicates with 5 fruits in each replicate.

a dose of 6.16 kJ m-2, HWT at 55°C for 5 min, HWT

Before and after the treatments, sampling was carried out

followed by UV-C irradiation, and untreated (control)

every 3 days throughout the storage time to evaluate theduring storage at 13°C.

degree of anthracnose disease. Scores of disease severity

Treatments

were recorded according to Chantrasri (2001), where areControl
UV-C
HWT
HWT + UV-C
F-test
C.V. (%)

0.0
0.0
0.0
0.0
NS
0.0

Control

0.0

Days of storage
3
6
9Disease incidence (%)
0.0
100a
100a
b
0.0
58
83a
0.0
25c
41b
0.0
0.0c
33b
NS
**
**
0.0
39.2
19.7
Disease severity (score)
0.0
1.5a
2.6a

UV-C

0.0

0.0

0.6b

1.2b

2.7b

HWT

0.0

0.0

0.2c

0.6c

1.7c

0.3c

0.6d

0

0 = no disease symptoms, 1 = symptoms less than
0.1-10% of overall peel area, 2 = symptoms 10.1-20% of
overall peel area, 3 = symptoms 20.1-30% of overall peelarea, 4 = symptoms 30.1-40% of overall peel area, 5 =symptoms more than 40% of overall peel area. The
specific activities and the expression of defense-related
genes were examined, including phenylalanine ammonia
lyase (PAL), chitinase (CHI), β-1,3-glucanase (GLU),
and peroxidase (POD). The quality of the fruit, including

12
100a
83ab
66b
33c
**
23.5
4.1a

HWT + UV-C

0.0

0.0

0.0c

weight loss were also analyzed. Results were analyzed

F-test

NS

NS

**

**

**

by conducting analysis of variance using the general

C.V. (%)

0.0

0.0

31.1

35.23

49.14

color change of the peel and the pulp, firmness andlinear models procedure of SAS (SAS Institute, Cary,means.

With a column, means not followed by the same letterare significantly at ** p ≤ 0.01. The NS indicatesstatically no-significance. Data are expressed as meanof each replication assays.

Result and Discussions

The expression of gene and the activity of defence

Disease incidence of anthracnose disease
The combination of HW and UV-C treatments

related enzyme

N.C.) for completely randomized design experiments.
Data are presented as means ± standard errors of the

The expression of MI-PAL gene increased quickly and- 66 -

peaked at day 3 of storage, then declined sharply. Its

lignification. For the expression of the MI-CHI gene waslevel was significantly higher in the fruit treated with the

low in both treated and control fruit during storage for 24

combined treatment than the fruit treated with HWT orh, then the levels of mRNA increased rapidly and

UV-C alone and non-treated fruit (Fig. 1A). This was thereached a maximum on day 9 of storage. Highest levels

same as the activity of PAL which showed highestof mRNA were found in the combined treatment

expression level in the combination treatment (Fig. 1B).

followed by HWT, UV-C irradiation and in the

These results were closely associated with the levels of

non-treated fruit (control). These results were correlateddisease incidence and severity of anthracnose disease inwith the enzymatic activities, with the highest activities

the mango fruit. The increased activity of PAL wasbeing found in the combined treated fruit (Fig. 3A andcorrelated

Fig. 3B).

with

the

compounds

synthesized

bymRNA levels of MI-POD gene(arbitrary units)

phenylpropanoid pathway such as phytoalexins, lignin or
tannin (Porat et al., 2000). Thus, high activity of PAL is
implicated in a protective role against pathogens throughreinforcement of plant cell walls and direct inhibition of

80

(A)

Control

UV-C

HWT

UV-C+HWT

20
0

40

2.0

0
0

3

6

9

12

5

0

3

6

9

12

0

3
6
9
Days of storage at 13 C

12

1.5
1.0
0.5
0.0

(B)
PAL activity(Unit/mg protein)

UV-C
UV-C+HWT

40

2.5

20

Control
HWT

60

60

POD activity(Unit/mg protein)mRNA levels of MI-PAL gene(arbitrary units)

pathogen growth (Treutter, 2005).

80

4
3
2

Fig. 2 Expression of MI-POD gene (A) and the1

activity of POD in the peel of mango when treatedwith UV-C irradiation at a dose of 6.16 kJ m-2, HWT

0
0

3
6
9
Days of storage at 13 C

12at 55°C for 5 min, HWT followed by UV-C
irradiation, and untreated (control) during storage at

Fig. 1 Expression of MI-PAL gene (A) and the activity

13°C for 12 days.
mRNA levels of MI-CHI gene(arbitrary units)

of PAL in the peel of mango when treated with UV-C
irradiation at a dose of 6.16 kJ m-2, HWT at 55°C for
5 min, HWT followed by UV-C irradiation, anduntreated (control) during storage at 13°C for 12
days.
The MI-POD gene expression, the levels of mRNA

20

5
0

8

CHI activity(Unit/mg protein)increased rapidly and peaked at 6 days of storage in the

UV-C alone or HWT alone and 10-fold higher comparedto the control (Fig. 2A). Similarly, the activity of POD
treatment (Fig. 2B). POD might contribute to theeffectofreducedanthracnose

0

3

6

9

12

0

3
6
9
Days of storage at 13 C

12

6
4
2
0

was the highest in the mango treated with combination
beneficial

UV-C+HWT

10

10

combined treatment fruit was 2-fold higher compared to

UV-C

15

increased slightly until 3 days after treatment, and then
combined treatment. The MI-POD expression in theControl
HWT

diseaseFig. 3 Expression of MI-CHI gene (A) and the activity

incidence and severity by influencing cell wallof CHI in the peel of mango when treated with UV-C

- 67 -

irradiation at a dose of 6.16 kJ m-2, HWT at 55°C for

more rapidly than those of the other treatments. By the

5 min, HWT followed by UV-C irradiation, andend of the storage, the control fruit and HW treated fruit

untreated (control) during storage at 13°C for 12

remained at low level of firmness by 4.96 N and 6.05 N,

days.

while UV-C and the combined treatment showed higher
firmness by 44.66 N and 41.49 N, respectively. The

MI-GLU gene expression, the levels of mRNA increased

highest firmness in the UV-C and combined treated fruit,progressively in all treatments and decreased after 3 days

which could be associated with the effect of the radiationof storage, then increased again on day 6 of storage andon the activity of enzymes involved in cell wall

peaked at the end of storage. The higher expression of

degradation (Barka et al., 2000). In contrast result

MI-GLU gene and activity of GLU enzymes were shownshowed that HW treated mango fruit became soften at ain the combined treated fruit (Fig. 4A and Fig. 4B).

similar rate to that of the control. Therefore, this result

Which GLU can also inhibit the fungal pathogens

indicated that UV-C treatment was the main effect toindirectly by releasing oligosaccharides and eliciting

delay the firmness of mango fruit.

defense reactions, and then act synergistically with CHI

mRNA levels of MI-GLU gene(arbitrary units)

to inhibit fungal growth (Tian et al., 2007).

Table 2 Firmness, weight loss, color of peel and colorof pulp of mango fruit when treated with UV-C

20

Control

UV-C

HWT

UV-C+HWT

irradiation at a dose of 6.16 kJ m-2, HWT at 55°C for

15

5 min, HWT followed by UV-C irradiation, and10

untreated (control) during storage at 13°C.

5Treatments
0

0
0

3

6

9

12

GLU activity(Unit/mg protein)

1.5
1.2
0.9
0.6
0.3
0.0
0

3
6
9
Days of storage at 13 C

12Fig. 4 Expression of MI-GLU gene (A) and theactivity of GLU in the peel of mango when treatedwith UV-C irradiation at a dose of 6.16 kJ m-2, HWT
at 55°C for 5 min, HWT followed by UV-C
irradiation, and untreated (control) during storage at
13°C for 12 days.
The quality of mango during storage
The result found that fruit treated with UV-C irradiationand the combined treatment of UV-C and HW had
delayed ripening to a greater degree than fruit treatedwith HW alone or non-treated fruit (as indicated by fruit
firmness, weight loss and change in color peel and pulp

Control
UV-C
HWT
HWT + UV-C
F-test
C.V. (%)

84.9
84.9
84.9
84.9
NS
2.3

Control
UV-C
HWT
HWT + UV-C
F-test
C.V. (%)

-17.1
-17.1
-17.1
-17.1
NS
4.0

Control
UV-C
HWT
HWT + UV-C
F-test
C.V. (%)

-14.5
-14.5
-14.5
-14.5
NS
1.6

Days of storage
6
9
Firmness (N)
83.7
82.5
62.6b
80.5
86.7
71.5ab
88.6
78.2
66.7ab
83.1
78.5
76.4a
NS
NS
*
5.0
8.4
9.6
a* value of peel
-17.6 -15.7 -12.5ab-16.1 -15.0-15.3b
-16.6 -15.5-10.6a-16.2 -15.9-15.1b
NS
NS
**
3.6
3.6
11.6
a* value of pulp
-11.7 -11.9ab-7.1a-12.1 -13.3b-10.3b
-11.5-13.1b-6.8a-12.2-10.6a
-8.2a
NS
*
*
11.5
7.4
13.4
3

12
4.9b
44.6a
6.0b
41.4a
**
15.6
-3.0a
-14.3b-4.2a-13.5b
**
10.23
-3.2a
-6.7b
-3.3a
-6.8b
**
20.8

With a column, means not followed by the same letterare significantly at ** p ≤ 0.01 and * p ≤ 0.05. The NS
indicates statically no-significance. Data are expressed asmean of each replication assays.
Yellowing of the peel and pulp is one physicalof the fruit) (Table 2).

manifestation of ripening that can be quantitatively

Firmness of mango fruit decreased in all treatments

showed that mango fruit treated with the UV-C and HW

included non-treated fruit. However, after 9 days ofcombined with UV-C had a delay of color change in peelstorage, the control fruit and HW treated fruit softened

and pulp of mango which was slower than that in either

measured based on increases in a* value. These results- 68 -

the control or HW treated fruit. The effect of UV-C todelayed the color change were recorded in several crops
such as tomato, strawberry and red pepper (Obande et al.,2011; Pombo et al., 2011; Cuvi et al., 2011). It wasreported that UV-C irradiation can delay the chlorophyll
degradation processes (Obande et al., 2011). For theweight loss of the mango fruits in all of the treatments
increased

progressively

duringextendedstorage.

However, weight loss was not significantly different
among the treatments (data not shown).

Conclusion
The combination of HW and UV-C treatments
significantly reduced anthracnose disease and increased
the expression and specific activities of the defense
related enzymes (PAL, POD, CHI and GLU) of mango
fruit when compared with those single treatments andnon-treated. The combined treatment of HW and UV-C
or UV-C treatment alone delayed fruit ripening
significantly by maintaining fruit firmness and color ofpeel and pulp of mango fruit. While, weight loss was not
significantly different among the treatments.

Acknowledgement
This work was supported by the Thailand Research Fundthrough the Royal Golden Jubilee Ph.D. Program (RGJ)(Grant No. PHD/00642554).

References
1) Alvindia DG and Acda MA (2015) Revisiting theefficacy of hot water treatment in managing anthracnoseand stem-end rot diseases of mango cv. Carabao. Crop
Protection, 67:96-101.
2) Barka EA Kalantari S Malhlouf J and Arul J (2000)
Impact of UV-C irradiation on the cell wall-degrading
enzymes during ripening of tomato (Lycopersiconesculentum L.) fruit. Journal of Agriculture Food
Chemistry, 48:667-671.
3) Chantrasri P Sardsud V Sangchote S and Sardsud U(2001) Combining yeasts and chitosan treatment to
reduce anthracnose fruit rot in mangoes. Asian Journal of
Biology Education, 3:40-46.
4) Cuvi MJA Vicent AR Concellón A and Chaves AR(2011) Changes in red pepper antioxidants as affectedby UV-C treatments and storage at chilling temperatures.
Food Science and Technology, 44:1666-1671.
5) Hong S Lee H and Kim D (2007) Effects of hot water
treatment on the storage stability of satsuma mandarin as
a postharvest decay control. Postharvest Biology andTechnology, 43:271-279.
6) Karasahin I Pekmezco M and Erkan M (2005)
Combined hot water and UV-C treatments reduces
postharvest decay and maintains quality of eggplants.
Postharvest Technology, 5:11-18.

7) Liu J Sui Y Wisniewski M Droby S Tian S Norelli J
and Hershkovitz V (2012) Effect of heat treatment on
inhibition of Monilinia fructicola and induction of
disease resistance in peach fruit. Postharvest Biology andTechnology, 65:61-68.
8) Maria TC Kablan T Alain A and Joseph A (2009)
Physiological basis of UV-C induced resistance to
Botrytis cinerea in tomato fruit V. Constitutive defence
enzymes and inducible pathogenesis-related proteins.
Postharvest Biology and Technology, 51:414-424.
9) Marina AP Hernan GR Gustavo AM and Pedro MC(2011) UV-C treatment affects the expression andactivity of defense genes in strawberry fruit (Fragaria ×ananassa, Duch.). Postharvest Biology and Technology,
59:94-102.
10) Obande MA Tucker GA and Shama G (2011) Effectof preharvest UV-C treatment of tomatoes (Solanum
lycopersicon Mill.) on ripening and pathogen resistance.
Postharvest Biology and Technology, 62:188-192.
11) Pan J Vicente A Martinez G Chaves A and Civello M(2004) Combined use of UV-C irradiation and heat
treatment to improve postharvest life of strawberry fruit.
Journal of Science Food Agriculture, 84:1831-1838.
12) Patricia C Sergio FP Eliane AB Elisangela CC andCarlos AS (2007) Effects of gamma and UV-C
irradiation on the postharvest control of papaya
anthracnose. Postharvest Biology and Technology,
43:366-373.
13) Prakash O and Pandey BK (2000) Control of mango
anthracnose by hot water and fungicides treatment.
Indian Phytopathology, 53:92–94.
14) Pombo MA Rosli HG Martinez GA and Civello PM(2011) UV-C treatment affects the expression andactivity of defense genes in strawberry fruit(Fragaria×ananassa, Duch.). Postharvest Biology andTechnology, 59:94-102.
15) Porat R Lers A Dori S Cohen L Ben-Yehoshua S
Fallik E Droby S and Lurie S (2000) Induction ofresistance against Penicillium digitatum and chilling
injury in star ruby grapefruit by a short hot
water-brushing treatment. The Journal of Horticultural
Science and Biotechnology, 75:428-432.
16) Tian SP Yao HJ Deng X Xu XB Qin GZ and Chan
ZL (2007) Characterization and expression of
β-1,3-glucanase genes in jujube fruit induced by the
microbial biocontrol agent Cryptococcus laurentii.
Phytopathology, 97:260-268.
17) Treutter D (2005) Significance of flavonoids in plant
resistance and enhancement of their biosynthesis. Plant
Biology, 7:581-591.
18) Wasker DP (2005) Hot water treatment for disease
control and extension of shelf life of ‘Kesar’ mango(Mangifera indica L.) fruits. Acta Horticulturae,
682:1319–1324.
19) Yuan L Bi Y Ge Y Wang Y Liu Y and Li G (2013)
Postharvest hot water dipping reduces decay by inducing
disease resistance and maintaining firmness inmuskmelon (Cucumis melo L.) fruit. Scientia
Horticulturae, 161:101-110.

- 69 -

- 70 -

OS IV-1
Management of fusarium wilt in cyclamen plants using multiple soil microbes (AMF and
Piriformospora indica)
Moslama Aktar MAYA1,2*, Iris CAMEHL1, Philipp FRANKEN1(1Leibniz Institute of Vegetable and Ornamental Crops (IGZ), Kuehnhaeuser Straße 101, 99090 Erfurt, Germany,
2

Department of Horticulture, EXIM Bank Agricultural University (EBAUB), Chapainawabganj 6300, Bangladesh*Current affiliation: Agronomy manager, Operations Team, British American Tobacco Bangladesh Limited (BATB),
Dhaka 1205, Bangladesh, Email: Ecoagri.Maya@gmail.com)

SUMMARY
Biotic (soil-borne pathogens) and abiotic (high temperatures) stress factors have negative effects on plant growth anddevelopment and it limits crop production around the globe. To meet up with the vast demand of food for the increasing
population of the world we use hybrids and agrochemicals for more production. To alleviate the environmental hazards
due to excessive uses of modern varieties and the agrochemicals, sustainable agricultural production is a current
concern. As a part of this approach, a variety of beneficial soil microbes are used as bio-regulating agents in crop
production. Bio-regulating agents have been singly being used against biotic and abiotic stresses, but recent studies
indicate that mixtures of bio-regulating agents may enhance the efficacy and dependency of the bio-regulating strategies.
Based on this perspective, microbe-microbe interactions such as between AM fungi and Piriformospora indica and theirinteraction effects were investigated in Cyclamen crops. Results of this study suggest that single use of AM fungi havepromising results in controlling fusarium wilt over combined use of AM fungi and Piriformospora indica together.

Introductionthat mixtures of biocontrol agents may enhance theAgriculture is one of the most climate dependent humanefficacy and dependency of the biocontrol strategy [4].

activities and it is one of the sectors where climate

Based on this perspective, microbe - microbe interactions

change impacts are expected to be significant [1]. Stresssuch as between AM fungi and Piriformospora indica

in plants is defined as abnormal changes in physiologicaland their interaction effects were investigated inprocesses based on environmental and biological factors

Cyclamen.

or a combination of both. As an abiotic stress factor, high

The association of roots with AM fungi is effective intemperatures have a negative effect on plant growth andreducing both shoot and root diseases [5,6] and, also

development, and it limits crop production around theshows bioprotection ability against abiotic stressglobe. Therefore, research on the mechanism of heatconditions [7,8,9]. Several studies have been reported

stress in plants is important to develop heat tolerant

concerning

plants. On the other hand biotic stress agents speciallyrhizospheric microbes, and only a very few studies havesoil-borne pathogens account for enormous losses ofbeen reported on mycorrhizal interaction with plant

agricultural production throughout the world. Current

growth promoting endophytic P. indica. The utilization

control measures are based on the use of chemicalofpesticide, and those are ineffective or are banned because

plant-microbeof health risks, product safety, and damage to theecofriendly strategies both for conventional and organic

ecosystem. To alleviate the environmental hazards due toagriculture around the globe. Therefore, it is important tointeractions

microbes

andthebetweenAM

exploitationinteractions

offer

of

fungi

and

beneficial

promisingandexcessive uses of agrochemicals, sustainable agricultural

understand the interaction effects between AM and P.

production is a current concern [2]. As a part of thisindica fungi for better utilization of these two biocontrol

approach,agents in practical agricultural production.

integrated

pest

management

has

been

conceived and a variety of beneficial soil microbes are

Cyclamen is a herbaceous perennial flowering pot plantalso used as biocontrol agents in crop production.

growing throughout the temperate regions of the world.

Biocontrol agents have been singly being used against

Among all the biotic stress agents Fusarium oxysporum,

biotic and abiotic stresses [3], but recent studies indicate

causes Fusarium wilt in cyclamen, is one of the highly- 71 -

destructive soil-borne pathogen which can economicallylimit the quality cyclamen production [10].

Pathogenic infection
Initially the isolates of F. oxysporum MAFF 712100 (Fo)The objective of this study was, therefore, focuses on thewere collected from the Ministry of Agriculture, Forestry

following objectives:and Fisheries, Japan. Isolates were grown in potato

1. To observe the overall effect of AM and endophytic

dextrose agar (PDA) medium and were incubated atfungi on biomass and flower development in cyclamen.

28°C. The Fo isolates were subcultured into PDA

2. To observe the biocontrol efficacy of AM andmedium and the conidia were harvested in sterile

endophytic fungi and their interactive effects on

distilled water. The spore concentration was adjusted toresistance in cyclamen against Fusarium wilt.

107 cfu/ml and 50 ml suspension was poured in each pot

3. To elucidate the molecular mechanisms of resistancesoil for Fusarium wilt challenged treatment after 14

mediated by biocontrol agents under pathogen stressweeks of AMF

condition.

condition. Thereafter, the symptoms were checked forMaterial and Method

Fusarium wilt and the plants were raised another 2 weeks

Experimental designin phytochamber.

The experimental treatments were conducted using five
microbial inoculation (Mock AM and mock P. indica (Pi)

Disease incidence and severity
Disease symptoms were checked 2 weeks after pathogen

as control (C); Am with mock Piri as Am; Mock AM

inoculation, both shoots and roots were observed forwith P. indica as Pi; AM with P. indica as Am+Pi; )Fusarium wilt symptoms. The disease severity inalong with pathogen Fusarium oxysporum inoculation.

individual plants was rated visually on scale of 0 to 5 e.g.

Two fertilizer treatment was used (Standard phosphorus

0: no visible symptoms (healthy plants)

as +P; Deficient of Phosphorus as –P). A completely

1: <20% disease symptom (small discolored leaves

randomized design were used and the whole experimentlesion covering less than 20% of total leaves of a plant)

was replicated 4 times. Each replication has 160 plants

2: 20 to 40% disease symptoms (minor small discolored(10 plants/treatment). The experimental treatments werelesions covering 20 to 40% of leaves)conducted under both the greenhouse and phytochamber

3: 40 to 60% disease symptoms (moderate brown lesions

condition. Initially plants were raised in greenhousein 40 to 60% of leaves and 15% defoliation)

condition for 12 weeks. After that plants were transferred

4: 60 to 80% disease symptoms (mild wilt discoloration

to phyto-chamber with 20/18 degree C alternatively

covered 60 to 80% of leaves and more than 50% leaf

14/10 hrs (day and night) keeping the moisture level at

defoliation in case of Fusarium wilt)

60% and light intensity 75%. Plants were raised another

5: 80 to 100% disease symptoms (stems and leaves

4 weeks under phytochamber condition.

severely affected).

Plant materials, growth conditions and microbial

The individual ratings were converted to mean percent

inoculation
Three-month-old cyclamen (C. persicum Mill cv. Pastel)

infection using {(sum of individual plant rating

seedlings (seeds supplied by Hakusan Co., Ltd., Japan)

index for F. oxysporum was calculated using thewere planted into plastic pots (8 cm in diameter)

following formula:2

inoculation under phytochamber

values*100)/number of plants diseased} and the disease

containing autoclaved (121 °C, 1.2 kg/cm , 15 min)Disease Index (%) = {∑ (No. of plants * Severity ofcommercial

incidence) / Total no. of plants * no of maximum

potting

media.

The

seedlings

wereinoculated with (10% of total substrate) AM fungal
inoculum for mycorrhizal plants, 50 ml/plant P. indica
suspension was used as Pi inoculation, and an equalamount of both (Am+Pi) autoclaved inoculum were used
for control plants. A commercial mycorrhizal inoculum
was used, containing Rhizophagus irregularis (spore
density unknown). Plants were repotted in 12 cm indiameter after 12 weeks of inoculation. Standard fieldpractices, and regular irrigation was conducted during
the entire growth period.

severity} *100

Result and Discussions
DNA extraction and determination of infection
The specific sequences of R. irregularis and F.
oxysporum were amplified through PCR amplification
using total genomic DNA from root system and shootpart as templates. Then, 5 μL of DNA fragments was
examined using 1% agarose electrophoresis after
amplification (Fig. 1, 2). Specific sequences from F.

- 72 -

oxysporum were amplified using total genomic DNA

higher in M treated plant compared to control both

templates extracted from both the root and shoot samples.

before and after pathogen inoculation condition. Whereas,

Bright, clear bands were detected which indicates that

the Pi and MPi inoculated plants have no major

the cyclamen root system was successfully infected withdifferences than the controls in no of total leaves andF. oxysporum.

flower bud production (Fig. 3, 4). The average dryweight (DW) of M treated plants, both before and after
pathogen inoculation, was observed higher than the othertreatments (fig. 5).
Average numbers of leaves/flower buds

1 2 3 4 5 6 7 8

Fig. 3. Average number of leaves and flower buds during
12 weeks after microbial inoculation. Note. C: Control;
M: AMF; Pi: P. indica; MPi: M+Pi.
Average numbers of leaves/flower buds

Fig. 1. Molecular quantification of AMF colonization.
Note. 1: Control; 2: AMF (M); 3: P. indica (Pi); 4: M+Pi;
1-4: Deficient P condition (-P); 5-8: Standard P condition(+P).

1 2 3 4 5 6 7

Fig. 4. Average number of leaves and flower buds during
16 weeks after microbial and 2 weeks after pathogen
inoculation. Note. C: Control; M: AMF; Pi: P. indica;

Plant Dry Weight (g)MPi: M+Pi.

Fig. 2. Molecular quantification of F. oxysporum.
Note: 1: 100bp DNA ladder; 2-4: C+Fusarium; 5-7:
M+Fusarium
Plant growth responses
Average number of leaf and flower bud production wasobserved throughout the experimental period. The

Fig. 5. Average plant dry weights (g) after 16 weeks ofaverage number of leaf and flower bud production foundmicrobial inoculation. Note. C: Control; M: AMF; Pi: P.

- 73 -

indica; MPi: M+Pi.
Disease incidence and severity

Molecular observations
RNA accumulation analyses by quantitative RT-PCR

The disease incidence levels for Fusarium wilt werebased on primer pairs for genes involved in plant defense

observed higher in control plants (Fig. 6). M treatedreactions and to test the ability of AMF and of theplants showed more healthy plants (0 severity level) thannon-mycorrhizal root endophyte P. indica to increase

the controls. Regarding fusarium wilt incidence, thetolerance and/or resistance against fusarium wilt was

maximum severity level 5 (80 to100% disease symptom)

observed under this project (data not presented here).

was noticed in Pi and MPi treated plants but not in AMFcolonized plants (Fig. 6). Development of fusarium wilt
symptoms in cyclamen plants was affected by thepresence of AMF. Controls, Pi and MPi with pathogen
association showed maximum disease severity level
resulting to higher disease indices than M-colonizedplants. Which indicates the microbe-microbe interactions
to control fusarium wilt diseases in cyclamen is lesseffective than the single effects of Mycorrhiza. Pi might
have the negative interaction effects with Fusarium andwhich ultimately have negative impact on to control

Disease Index

fusarium wilt symptoms in cyclamen.

Fig. 6. The disease incidence levels for Fusarium wilt in
cyclamen plants in deficient P condition. Note. C:

Summary of disease index

Control; M: AMF; Pi: P. indica; MPi: M+Pi.

Fig. 7. Distribution of Fusarium wilt disease severity
levels in cyclamen plants under different treatments.
Note. C: Control; M: AMF; Pi: P. indica; MPi: M+Pi.

Acknowledgement
The authors sincerely acknowledge the contribution of
Dr. Siegfried ZERCHE, Leibniz Institute of Vegetable
and Ornamental Crops (IGZ), Kuehnhaeuser Straße 101,
99090 Erfurt, Germany for his generous support inplanning and management of phosphoric fertilizers
throughout the experimental period.

References
1) Hertel TW, Bruke MB and Lobell DB (2010) The
poverty implications of climate-induced crop yieldchanges by 2030. Global Environmental Change.
20:577–585.
2) Ekstrom G and Ekbom B (2011) Pest Control in
Agro-ecosystems: An Ecological Approach. Critical
Review in Plant Science. 30:74–94.
3) Roberts DP, Lohrke SM, Meyer SLF, Buyer JS,
Bowers JH, Baker CJ, Li W, de Souza JT, Lewis JA and
Chung S (2005) Biocontrol agents applied individuallyand in combination for suppression of soil borne diseasesof cucumber. Crop Protection. 24:141-155.
4) Saldajeno MGB and Hyakumachi M (2011) The plant
growth promoting fungi Fusarium equiseti and thearbuscular mycorrhizal fungus Glomus mosseae
stimulate plant growth and reduce severity ofanthracnose and damping off diseases in cucumber(Cucumis sativus) seedlings. Annals of Applied Biology.
159: 28-40.
5) Maya MA and Matsubara Y (2013) Tolerance toFusarium wilt and anthracnose diseases and changes ofantioxidative activity in mycorrhizal cyclamen. Crop
Protection. 47:41-48.
6) Matsubara Y, Li Y, Okada T and Maya MA (2012)
Factors on induced systemic disease resistance inmycorrhizal strawberry plants.
Acta Horticulturae(ISHS), 926:497-502.
7) Maya MA and Matsubara Y (2013) Influence ofarbuscular mycorrhiza on the growth and antioxidative
activity in cyclamen under heat stress. Mycorrhiza.
23:381-390.
8) Grover M, Ali SZ, Sandhya V, Rasul A, andVenkateswarlu B (2011) Role of microorganisms in
adaptation of agricultural crops to abiotic stresses. World
JournalofMicrobiology
andBiotechnology.
27:1231-1240.
9) Zhu XC, Song FB and Xu HW (2010) Arbuscular
mycorrhizae improves low temperature stress in Maize
via alterations in host water status and photosynthesis.
Plant and Soils. 331:129-137.
10) Elmer WH and McGovern RJ (2004) Efficacy ofintegrating biological with fungicides for the suppressionof Fusarium wilt of cyclamen. Crop Protection.
23:909-914.

- 74 -

OS IV-2
Mycorrhizal oil palms seedlings response to different sources of Ganoderma boninense as
the causal agent of basal stem rot disease
Maria Viva RINI，Syaifudin Nur HASAN, Kuswanta Futas HIDAYAT, Titik Nur AENY
(Faculty of Agriculture, The University of Lampung）

SUMMARY
Basal stem rot (BSR) caused by Ganoderma boninense is the important disease of oil palm in Indonesia and Malaysia.
BSR is characterized by a decay of roots and bole, production of aerial symptoms such as multiple spears andproduction of fruit bodies on the base of the trunk. These studies were aimed to evaluate the ability of arbuscular
mycorrhiza fungi in controlling the BSR. Two different experiments were carried out. In the first experiment, rubber
wood blocks of size 3 x 3 x 6

cm were used to grow the G. boninense inoculum for 1 month. The blocks were theninoculated to one primary root of 5 months old mycorrhizal and control oil palm seedlings. In the second experiment,
the soil collected from rhizosphere of Infected palm by G. boninense was used as media to grow the three months old
mycorrhizal and control oil palm seedlings. The first experiment showed that both mycorrhizal and control seedlings
were infected by G. boninense.

However, in the control seedling, the length of primary root that rot by the pathogen

was longer than that of mycorrhizal. In the second experiment, no infection of G. boninense were observed in
mycorrhizal and control seedlings.

Introduction

associated with agricultural crops is the arbuscular

Basal stem rot (BSR) caused by Ganoderma species ismycorrhiza fungi (AMF). Infection by AMF has been

the most serious disease of oil palm. Infection by theshown to stimulate the growth of many plant species

fungi causes significant loss in yield, often resulting in(Smith and Smith, 2011; Zhang et al., 2010), increase

the palm’s death as the disease progressed. The BSR

nutrient uptake especially phosphorus (Rini, 2004; Smithaffects the root and basal stem portion of the palm.

et al., 2011), improve the soil structure (through external

Infection by the fungus begins in the roots and move intohyphae that extends into the soil) for better aeration andthe stem causing a dry rot, which eventually lead to thewater percolation, and improve plant physiologicaldeath of the palm.

Infection of living palm occurs

processes such as photosynthesis rate and water relation

through contact of healthy palm root with the infected(Lu et al., 2007; Rini et al., 2000; Ruiz-Lozano androot mass or bole tissue which serve as the inoculum

Azcon, 2010). Arbuscular mycorrhiza also has beensource (Turner, 1981, Paterson, 2007). Generally, theproposed as an alternative for the management of soil

first visible symptom of infected field palm is theborne pathogen. AM fungi has been proven to impair

presence of excessive spear leaves, while the foliage

the development of soil borne pest and pathogens andappears pale green when compare to that of healthy palm.

consequently inhibit or reduce disease severity (AmerProgressive yellowing, desiccation and mottling of theand Abou-El-Seoud, 2008; Jung et al., 2012; Tsvetkov etlower fronds, followed by necrosis is the characteristical., 2014). Therefore, this study was conducted tofeature of the disease of young palms. In older palms,

evaluate the ability of arbuscular mycorrhiza fungi inthe typical symptoms are skirting of the lower fronds,

controlling the BSR.

production of multiple unopened spears and overall

Material and Methodpaleness of the canopy (Fee, 2011; Gurmit, 1991).

The first experiment. A single factor experiment

Mycorrhizal fungi are ubiquitous and form symbiotic

arranged in a completely randomized design was used

relationship with the roots of majority terrestrial plants

with ten replications per treatment. The treatment was

including oil palm (Sieverding, 1991; Smith and Read,

inoculated with (+M) and without (-M) AM fungi. The

2008). The mutual symbiosis benefits both the host and

Ganoderma boninense inoculum was prepared on rubber

the fungus.

wood blocks measuring 3 x 3 x 6 cm by inoculating the

The largest group, which predominantly- 75 -

block with five 1 cm2 plugs from 7-10 days old G.

root that rot due to G. boninense (Table 1). The length ofboninense culture grown on malt extract agar with one

primary root that rot in nonmycorrhizal seedlings wasplug on each side of the block (done in the air laminar

11.1 cm compared to only 7.4 cm in mycorrhizalflow). The blocks were incubated at room temperature

seedling. Length of primary root that rot as a resulto

27 ± 1 C for ten weeks. A small hole (± 3 cm depth) was

Ganoderma infection was significantly reduced when

made on the top of the blocks using an electric drill.

root was earlier precolonized by AMF. This indicate

Five month old mycorrhizal (inoculated with inoculumthat the spread of Ganoderma infection within primarycontaining

mosseae,root of mycorrhizal seedlings was slower compared to

Scutellospora callospora, and Acaulospora laevis) andthat of nonmycorrhizal control. In the present study, thenonmycorrhizal seedlings were inoculated with theselength of rotten root can be estimated 1.23 cm and 1.68

blocks using single root inoculation technique. One of

cm per month. The spread of Ganoderma infection in thethe primary root of the seedling was washed with tap

present study was faster compared to Arifin and Idris

water, the root tip was excised and the cut end of the root(1990) who found only 1 cm/month, especially for

was inserted into the hole of the inoculum block. The

nonmycorrhizal seedlings (1,68 cm/month). This fasterinoculated seedlings were then put inside a polybag andspeed could be due to the different size and substrates

filled up with the soil (mineral soil:sand = 2 : 1) andused to grow Ganoderma inoculum.

water daily. Six month after inoculation, the seedlings

showed that utilization of different subtrates as source ofwere removed from the polybags.

The inoculated

Ganoderma inoculum resulted in different growth rate ofprimary roots were then carefully separated from thethe Ganoderma mycelia within the primary roots of oilbole of the seedlings. The length of inoculated root that

palm.

mix

speciesof

GlomusIdris (1999)

rot due to G. boninense and AMF root infection were
measured. Total phenolic content in the roots wasanalyzed following the method of Anderson and Ingram

Table 1. Length of primary roots infected byG.boninense after 6 months of inoculation(1993). Data obtained were subjected to t-test analysis.

+Mycorrhiza

Non-mycorrhiza

7.4

11.1

The second experiment. The treatment design used was
Length(cm)
P valuea factorial design 4 x 2 with 5 replication arranged incompletely randomized design.

The first factor was<0.05

application of AMF i.e. without AMF (control, m0),
inoculation with AMF Glomus sp. (m1), Entrophospora
sp. (m2) and mixture of Glomus sp. and Entrophospora

Total phenolic content in the roots was also significantly

sp. (m3). The second factor was Ganoderma i.e. withouthigher in mycorrhizal seedlings.

Ganoderma (planting media was sterile soil) and with

24.43% and 21.75% in mycorrhizal and nonmycorrhizal

Ganoderma (planting media was rhizosphere soil

seedlings respectively (Table 2.)

The values werecollected from Ganoderma infected palm + fruiting bodyor sporophore of Ganoderma). The one month old oilpalm seedlings were inoculated with AMF according toTable 2. Total phenolic content in mycorrhizal andnon mycorrhizal root after 6 months of G. boninense
inoculation

treatment and the seedling were kept in green house for 2+Mycorrhiza

months after which the seedlings were then transferred tobigger polybag according to Ganoderma treatments. The
seedlings were then kept for another 5 months in green
house. At the end of experiment, data on fresh and dry

Total phenolic in
the roots (%)
P value

Non-mycorrhiza

24.43

21.75
<0.05

weight of shoot and root and Ganoderma infection wererecorded.

Plant phenolic are the most widespread classes ofResult and Discussions

secondary metabolites and are known to be involved in

The first experiment. Percentage of AMF colonization

plant microbe interactions. One of the biological

6 month after Ganoderma inoculation was in rangefunctions of phenol is its antimicrobial activity which

42.7—49.0%. Presence of AMF in the root of oil palm

play an important role in the plant defence mechanism

significantly reduce the length of the inoculated primary(Morandi, 1996). Result from this study show that total- 76 -

phenolic content in the roots of mycorrhizal seedlings isTable 4. Dry weight of shoot and root of 8 months old oil

higher that that of nonmycorrhizal ones, suggesting that

palm seedling treated with AMF and Ganoderma.

phenolic compounds could be implicated in disease
resistance, resulting in slower rate of Ganoderma spread

Treatment

within the primary root of seedlings. Devi and Reddy

Dry Weight (g)
Shoot

Root

13.5 b

3.3 a

Glomus sp. (G)19.3 a4.2 a

Entrophospora sp. (E)

17.6 a3.8 aincrease in free and total phenolic contents in

G+E

19.0 a4.0 a(2002) reported that AMF significantly increase theControlquantity of phenolics compound in roots and shoots ofgroundnut. In (1998), Rabie believed that a significant
preinoculation of G. mosseae in faba bean contributed to

LSD 5%

3.5

0.9

increased resistance of the plant to chocolate spot

Sterile Soil16.7 a3.2 bdisease.

Ganoderma Infected18.1 a

4.4 a

2.5

0.6

Soil
The second experiment.

LSD 5%

Data obtained from analysis of variance showed that
there were no interaction between AMF factor withIn this study, AMF gave the beneficial effects on oil palm

Ganoderma factor for all data recorded. Moreover,seedling growth as indicated by fresh and dry weight of

results showed that seedlings growth were significantly

shoot and root. The enhancing in growth could be due toenhanced by AMF treatment. All AMF inoculated

the increase in uptake of nutrient especially phosphorus

seedlings had better shoot and root fresh weight and dry

as mycorrhiza hyphae that developed in the soil canweight compared to control one. However, no differences

absorb nutrients directly from the soil matrix (Neumannwere observed within AMF treatment. All AMF treatedand George, 2010; Rini, 2005) and improve in plantseedlings whether single (Glomus sp. or Enthrophosporawater relation such as increase in water uptake andsp.) or their combination statistically had the same shootphotosynthesis rate (Ruiz-Lozano and Azcon, 2010;

and root fresh weight and dry weight. For Ganoderma

Doubkova et al., 2013).

treatment, no effect were detected in shoot fresh and dry

In this study, AMF treatment gave a better impact onweight.

Contrary to shoot, Ganoderma treatment

plant growth. However, its significance in reducing or

increase root fresh and dry weight. Oil palm seedling

control Ganoderma infection cannot be examined. All

planted in Ganoderma infected soil had higher root fresh

seedling planted in Ganoderma infected soil mix with its

weight and root dry weight (Table 3 and Table 4).

fruiting body had no Ganoderma infection in their root
(Table 5).

Table 3. Fresh weight of shoot and root of 8 months old
oil palm seedling treated with AMF and Ganoderma.

Table 5. Root infection by AMF and Ganoderma as aresult of AMF and Ganoderma treatments

Treatment

Fresh Weight (g)
Shoot

Root

Control

43.3 b09.7 b

Glomus sp. (G)66.4 a13.1 aControl0

Entrophospora sp. (E)64.0 a13.9 aGlomus sp. (G)0

G+E

66.7 a13.5 aEntrophospora sp. (E)0

LSD 5%

9.3

2.8

G+E

0

Sterile Soil

56.2 a10.0 b

LSD 5%-

Ganoderma Infected

64.0 a15.0 aSterile Soil0

Ganoderma Infected

0

6.5

2.0

Treatment

Soil
LSD 5%

Root infection (%) by
Ganoderma

Soil
LSD 5%- 77 --

Contrary to the first study, Ganoderma inoculum
prepared in rubber wood block successfully infect bothmycorrhizal and nonmycorrhizal seedling. Base on thisresult, it can be suggested that type of inoculum affectthe success of Ganoderma to infect the root of oil palm
seedling. Using rubber wood block to grow theGanoderma inoculum confirmed the statement ofTurner (1981) that Ganoderma pathogen is a facultative
parasite.

It is capable of living saprophytically on

rotting stumps and roots. When a suitable host like oilpalm root becomes available, the pathogen will colonize
it and establishes a parasitic relationship. Using infectedsoil mix with the Ganoderma fruiting bodies as
Ganoderma inoculum failed to cause infection.

This

result suggest that Ganoderma spores that exist in thesoil and spores within fruiting body, within theconstraints of this study, is not capable in infecting oilpalm seedling root.

Conclusion
Base on the results from the study, the following
conclusions could be made: (1) arbuscular mycorrhiza
fungi improved the oil palm seedling growth andincrease the seedling tolerance to Ganoderma infection(2) Spores of Ganoderma from the infected soil and
Ganoderma fruiting bodies were failed to caused disease
infection, contrary to the inoculum prepared on rubber
wood block that successfully infect the seedling root.

Acknowledgement
The author would like to thank to staff of Labortorium
Produksi Perkebunan, Faculty of Agriculture, The
University of Lampung for their help in conducting thisresearch.

Reference1)Amer MA and Abou-El-Seoud II (2008) Mycorrhizal
fungi and Trichoderma harzianum as biocontrol agentsfor suppression of Rhizoctonia solani damping-off
disease of tomato. Commun. Agric. Appl. Bio. Sci.,73(2):217-232.
2)Anderson JM and Ingram JSI (1993) Tropical soil
biology and fertility. A handbook of methods.
Wallingford:Cab International.
3)Ariffin D and Idris AS (1990) Artificial inoculation ofoil palm seedlings with Ganoderma boninense. Paper
presented at Joint meeting agronomy/Breeding/
Pathology, pp.3, Palm Oil Research Institute of Malaysia.
4)Devi MC and Reddy MN (2002) Phenolics acidmetabolism of groundnut (Arachis hypogaea L.) plants
inoculated with VAM fungus and Rhizobium. Plant
Growth Regulation,87:151-156.
5)Doubkova P, Vlasakova E and Sukova R (2013).
Arbuscular mycorrhizal symbiosis alleviates drought
stress imposed on Knautia arvensis plants in serpentine
soil. Plant and Soil,370:149-161.
6)Fee C G (2011) Management of Ganoderma diseases

in oil palm plantations. The Planter,87(1022):325-339.
7)Gurmit S (1991) Ganoderma: The scourge of oil palms
in the coastal areas. The Planter,67:421-444.
8)Idris AS (1999) Basal stem rot (BSR) of oil palm(Elaeis guineensis Jacq.) in Malaysia:factors associatedwith variation in disease severity. Ph.D. Thesis, Wye
College, University of London.
9)Jung SC, Martinez-Medina A, Lopez-Raez JA and Pozo
MJ (2012) Mycorrhizal-induced resistance and primingof plant defenses. J. Chem. Ecol.,38: 651-664.
10)Lu J, Liu M, Mao Y and Shen L (2007) Effects ofvesicular-arbuscular mycorrhi-zae on the drought
resistance of wild jujube (Zizyphys spinosus Hu)
seedlings. Front. Agric. China,1(4):468-471.
11)Morandi D (1996) Occurrence of phytoalexins andphenolic compounds in endomycorrhizal interactions, andtheir potential role in biological control. Plant andSoil,185:241-251.
12)Neumann E and George E (2010). Nutrient uptake:the arbuscular mycorrhiza fungal symbiosis as a plant
nutrient acquisition strategy. In Arbuscular Mycorrhizas:
Physiology and Function. Edited by H Koltai and Y
Kapulnik, pp.137-167. New York, Springer.
13)Paterson RRM (2007) Ganoderma disease of oil palm
– A white rot perspective necessary for intergrated
control. Crop Protection,26:1369-1376.
14)Rabie GH (1998) Induction of fungal disease
resistance in Vica faba by dual inoculation with
Rhizobium leguminosorum and vesicular arbuscular
mycorrhiza fungi. Mycopathologia,141:159-166.
15)Rini MV (2004) Influence of arbuscular mycorrhiza
infection on growth, P uptake, and root morphology ofoil palm seedlings (Elaeis guineensis Jacq.). Jurnal
Tanah Tropika, 18:145—154.
16)Rini MV (2005) Effect of arbuscular mycorrhiza
inoculum density on growth, root infection, and nutrient
uptake of oil palm seedlings. Jurnal Tanah Tropika(journal of Tropical Soils),10(1):39-44.
17)Rini MV, Jamal T, Idris ZA and Azizah H (2000)
Effect of arbuscular mycorrhiza fungi colonization on
growth and physiological responses of grafted cocoa
under field condition. Malaysian Journal of Soil Science,
4: 67—78.
18)Ruiz-Lozano JM and Aroca R (2010) Host responses
to osmotic stresses: stomatal behavior and water use
efficiency of arbuscular mycorrhizal plants. In Arbuscular
Mycorrhizas: Physiology and Function. Edited by H
Koltai and Y Kapulnik, pp 239-256.
New York,
Springer.
19)Sieverding E (1991)
Vesicular Arbuscular
Mycorrhizae Management in Tropical Agroecosystem.
Deutsche Gesellschaft fur Technische Zusammenarbeit(GTZ) Gmbh, Eschborn.
20)Smith SE and Read DJ (2008) Mycorrhizal Symbiosis,rd
3 edition. Elsevier, New York.
21)Smith FA and Smith SE (2011) What is thesignificance of the arbuscular mycorrhizal colonisation ofmany economically important crop plants? Plant Soil,348:
63—79.
22)Smith SE, Jakobsen I, Gronlund M and Smith F A(2011) Roles of arbuscular mycorrhizas in plant
phosphorus nutrition: Interactions between pathways of- 78 -

phosphorus uptake in arbuscular mycorrhizal roots haveimportant
implications
forunderstandingandmanipulating plant phosphorus acquisition. Plant
Physiol,156:1050–1057.
23)Tsvetkov I, Dzhambazova T, Kondakova V and
Batcharova R (2014) Mycorrhizal fungi Glomus spp. andTrichoderma spp. in viticulture (Review). Bulgarian J.
of Agric. Sci.,20(4):849-855.
24)Turner PD (1981) Oil palm diseases and disorder.
Kuala Lumpur, Oxford University Press.
25)Zhang Y, Zhong CL, Chen Y, Jiang QB, Wu C andPinyopusarerk K (2010) Improving drought tolerance ofCasuarina equisetifolia seedlings by arbuscular
mycorrhizas under glasshouse conditions. New Forests,
40: 261-271.

- 79 -

- 80 -

OS IV-3
Potency of watery extract compost plus Beauveria sp. after storage for controlling
planthopper and rice bug
PURNOMO，Radix SUHARJO, Ainin NISWATI, Yuyun FITRIANA, and Siti JARLINA(Faculty of Agriculture, University of Lampung）

SUMMARY
Watery Extract Compost (WEC) is a liquid fertilizer that has been reported can be used as a biological control of plantpests and diseases. One of the entomopathogen which was widely used is Beauveria sp. This research was aimed toinvestigate survival and virulence of Beauveria sp. that was added into WEC after storage to planthopper and rice bug.
The compost used in this study was made using Trichoderma sp. as a starter. The fungus Beauveria sp. used in thisstudy was obtained from corn rhizosfer. The fungus was added into WEC before storage. The treatment were WEC
suspension which were keep for 1, 2, 3, 4, 5 and 6 months. Each of experimental unit consist of 20 planthopper or ricebug, with ratio 1 : 1 for male and female. The result showed that after 6 months storage, the fungus Beauveria sp. which
was added into WEC was still had capability to infect and cause mortality of planthopper and rice bug. After 6 months
storage, the mortality of planthopper caused by WEC-Beauveria application reach > 50%. Meanwhile, the mortality ofrice bug reach 45%.

Introduction

The purpose of the reaerch were to study ability to

Soil quality degradation as well as pest and plantsurvival and virulence of entomopathogen Beauveria sp.

diseases are two important issues in crop productivity infungi which is added to the WEC after storage against theIndonesia. Therefore, several effort have been done to findplanthopper and rice bug. Rice planthopper and rice bug

environmentally friendly technology. The combination ofare an important pest of rice plants.

wateryextracts

ofcompostedmaterials andMaterial and Method

entomopathogenic fungi are one of alternative for solvedthat problem in which it need to be developed.

The experiment were conducted at February until

Watery Extract Compost (WEC) or commonly call

October 2017. Compost were made by rice straw mixed

compost tea are fermented watery extracts of composted

with 10% cow dung with starter was Trichoderma sp

materials that are used for improving plant nutrition andfungi. Preparation and production of WEC werecrop yield including antimicrobial activities (Dionne et al.,performed at Laboratory of Biotechnology, Faculty of2012). Compost tea as a liquid fertilizer were made by

Agriculture, University of Lampung.

mixing compost and water for 2-8 days by adding

biopesticidematerials that can increase the microbial population in itapplication for rice planthopper and rice bug were held at(Scheuerell, 2004). Variuous number of stake holder

Crop Protection Laboratory, Trimurjo, Central Lampung.

using biofertilizers and biocontrol agents (biopesticide) in

Beauveria sp. fungi used were collection of Laboratory ofan effort to increase crop productivity are a very good step

Biotechnology that were isolated from corn rhizosphere.

in terms of ecological aspects related to the many bad

Beauveria sp. fungi with density of 109 ml-1 wateryconsequences of using chemical fertilizers continuously

compost were storaged before application with duration of(Swastika, 2007) and using chemical pesticides without

1, 2, 3, 4, 5 and 6 month in room temperature.

(WEC

+

Beauveria

Rearing andsp.)suspension

The experiment were arranged with Completely

good control (Untung, 2001). Application of WEC, thatwere liquid biofertilizer integrated with entomopathogen

Randomized Design.

Beauveria sp. fungi, were reported by (Purnomo et al.,experiment, one for rice planthopper and one for rice bug.

2017) increased paddy field productivity. The problem is

Six replications were used for every treatment. Every

how long entomopathogen Beauveria sp. fungi canexperimental unit were given with 20 individual (10 males

survive during storage in WEC without lose its

and 10 females) of rice planthopper and rice bug,capability?.

respectively.

- 81 -

There were two packages ofObservation were conducted on rice

Table 2. Percentage of mortality of rice bug (%) by
application of WEC plus Beauveria sp with different

planthopper and rice bug mortality attacked by Beauveria
sp. Infection and emerged of planthopper nymph.

storage duration.

Result and Discussions

Beauveria sp.

The results showed that compost tea is a good

Treatments

medium for the growth of insect pathogenic fungi. It was
showed by after stored 6 months in compost tea it still
keep continued high capability to kill insects. Table 1
showed that Beauveria sp. had ability to kill rice
planthopper more that 50% after storage 6 months incompost tea with room temperature. This high virulence
was seen at 14 days after application. At 7 days after
application, the ability to kill 50% or more is only forstorage of 4 months or less.

7 days after

14 days after

application

application

Control00

1 month

68.3

85.00

2 months

62.5

78.33

3 months

51.7

70.83

4 months

41.7

55.83

5 months

42.5

56.67

6 months

33.3

45.00

The symptom of infected planthopper and rice bug
Table 2 showed that storage of Beauveria sp. for 5

by Beuveria sp. could be seen two days after application.

month in compost tea was be able to kill more than 50%

Four to five days after application, almost the wholebody of insect had already covered by white muscardine

rice bug tested after 14 days application. At the 7 days

(Fig. 1)after application, 50% mortality of rice bug that killed by
Beauveria sp. which storage less that 3 months.

A

Entomopathogen fungi that have virulence more thanB

50% commonly assumed as a potential source forbioinsecticides. Therefore, some researcher worked manyeffort to increase the virulence of entomopathogen fungi bymanipulation of medium for fungi growth (Nuryanti et al.
2012; Fan et al. 2007; Herlinda et al. 2006).
Beside Beauveria sp. after storage in tea compost had
Fig. 1 Rice bug (A), rice planthopper (B) infected byhigh virulence infected adult of rice planthopper and rice

Beauveria sp.

bug, it also could suppress nymph of rice planthopper thatemerge from the egg.

Table 1. Percentage of mortality of rice planthopper (%)
by application of WEC plus Beauveria sp. with different
storage duration.

Fig. 2 showed that practicallystorage of Beauveria sp. in tea compost for 6 months were
not able to suppress emergence nymph of rice planthopper.
However, the storage 5 month or less, Beauveria sp. could
suppress better the emergence nymph of rice planthopper.

Beauveria sp.
Treatments

7 days after

14 days after

application

application

Control00

1 month

71.7

82.50

2 months

67.5

80.00

3 months

58.3

70.83

4 months

60.0

68.33

5 months

41.7

50.83

6 months

40.8

52.50
Fig. 2 Population of nymph of rice planthopper emerging
after Beauveria sp application.

- 82 -

Conclusion

3) Herlinda S, Utama MD, Pujiastuti Y and Suwandi

WEC or compost tea can be a good host for insect(2006) Kerapatan dan viabilitas spora Beuveria

pathogenic fungi Beauveria sp. Application of compost

bassiana akibat sub-kultur dan pengayaan media,

tea has contain Beauveria sp. is able to suppress theserta virulensinya terhadap larva Plutella xylostella

population of rice planthopper and rice bug, even though(Linn.). Jurnal Hama dan Penyakit Tumbuhan Tropika,

the compost tea has been stored for 5 months at room

6(2): 70-78.
4) Nuryanti NSP, Wibowo L and Azis A (2012)

temperature.
It is needed to continous research focused on WEC

Pemanfaatan beberapa jenis bahan nutrisi pada media

as a liquid biofertilizer which combined with biocontrol

perbanyakan untuk meningkatkan virulensi Beuveria

agent for controling plant pest and diseases, for example

bassiana terhadap walang sangit. Jurnal Hama dan

research of microbial content in compost tea.

Penyakit Tumbuhan Tropika, 12(1): 64-70.
5) Purnomo, Suharjo R, Niswati A, Solihatin U, Fitriana
Y and Indriyati (2017) Aplikasi Compost Tea dan

Acknowledgement
We thank the staff at Crop Protection Laboratory,

jamur Beauveria Bassiana menekan perkembangan

Trimurjo, Central Lampung for permitting this study andhama dan penyakit serta meningkatkan pertumbuhan

supporting the green house for experiment. Our study

dan produksi tanaman padi. Prosiding Seminar

was supported by Product Applied Research Grant

Nasional dan Rapat Tahunan BKS Pertanian Barat di(Penelitian Produk Terapan), Ministry of Research

Universitas Bangka dan Belitung.

Pangkalpinang,

20 Juni 2017.

Technology and Higher Education Republic of Indonesia

6) Scheuerell SJ (2004) Compost Tea production

with contract number 1638/UN26.21/KU/2017.

practices, microbial properties, and plant disease

Referencessuppression. Paper at I International Conference on1) Dionne A, Tweddell RJ, Antoun H and Avis TJ

Soil and Compost Eco-Biology September 15th – 17 th
2004, León – Spain.

(2012) Effect of non-aerated compost teas on
damping-off pathogens of tomato. Canadian Journal

7) Swastika DKS,

Wargiono J,

SoejitnoS

andHasanuddin A (2007) Analisis kebijakan peningkatan

of Plant Pathology, 34(1): 51- 57.
2) Fan Y, Fang W, Guo S, Pei X, Zhang Y, Xiao Y, Li

produksi padi melalui efisiensi pemanfaatan lahan

D, Jin K, Bidochka MJ, and Pei Y (2007) Increased

sawah di Indonesia. Analisis Kebijakan Pertanian, 5Insect Virulence in(1): 36-52.

Beauveria bassiana Strains

Overexpressing an Engineered Chitinase. Applied and8) Untung K ( 2001).
Terpadu.

Environmental Microbiology, 73(1): 295 - 302.

Yogyakarta.

- 83 -

GadjahPengantar Pengelolaan Hama
Mada

University

Press.

- 84 -

OS IV-4
Low pH-tolerant mutant of Trichoderma spp. induced by EMS,
gamma rays and UV irradiation
Yuyun FITRIANA, Radix SUHARJO, Maria Viva RINI, Kuswanta Futas HIDAYAT(Faculty of Agriculture, Lampung University）

SUMMARY
Basal stem rot caused by Ganoderma boninense is one of the most important problems causing severe economic losses
in many oil palm industries, including in Indonesia. Recently, Trichoderma spp. has been widely used as biologicalcontrol agent of G. boninense, however, this fungus was negatively affected by low pH condition. In Indonesia, oil palm
commonly cultivated in marginal soil such us ultisol and peat soil with low pH (acid). In this study, three mutagens(EMS, gamma rays and UV irradiation) were used to generate low pH-tolerant mutant of Trichoderma spp. to increase
its survival and effectiveness when it is applied in the previous mentioned field circumstance. Four Trichoderma isolates
were used as the wild type. The mutant isolates were able to grow and produce spores in the pH 2-Potato Dextrose
medium but not for the wild type. Only 1 out of 109 potentially low pH-tolerant mutant of Trichoderma showed bettercolony growth, sporulation, viability and antagonist to G. boninense than the wild type. These results showed that EMS,
gamma rays and UV irradiation can be used to improve low-pH tolerant of Trichoderma spp.. However, the mutants
must be carefully evaluated for any possibility of its negative impacts.

Introduction

resistant to low pH. The mutation techniques used were

Basal stem rot caused by Ganoderma boninense is one ofGamma rays irradiation, Ultraviolet irradiation (UV), andthe most important problems causing severe economic

Ethyl Methane Sulfonate (EMS).

losses in many oil palm industries, including in Indonesia.
Initially, this fungi attacked the roots of oil palm. Over

Material and Method

time, it attacks continue to grow toward the base of the

Fungalstem (Rini, 2003). At the beginning of the attack, theTrichoderma spp. (T1, T2, T3 and T4) that had been

symptoms cannot be seen in the plant canopy. However,screened was cultured in Petri dishes (90 mm diameter)when the symptoms have appeared in the plant canopy

containing Potato’s dextrose agar (PDA) for 2 weeks.

(unopened shoots, pale green leaves, and old leaves begin

Conidial suspensions were prepared by scraping theto droop, fruiting bodies appear at the base of the stem),

conidia/mycelia into sterile 0.1% Tween 80 and thenthe fungal attack on the root and base of the stem isfiltering the mixture through sterile cloth (0.2 mm insevere. Almost 50% of the base of the stem is damaged

mesh size) to provide a suspension of conidia.

preparation.

Four

isolatesofwild-type(Gurmit, 1991). Therefore, for controlling the Ganoderma

Gamma rays and UV irradiation. Conidia of theis more difficult, because early symptoms is occur within

wild-type isolates were irradiated with gamma rays at Badan

the ground that are difficult to detect.

Tenaga Nuklir Nasional (BATAN), Serpong. UVTrichoderma is a fungi that can act as a biological agent

irradiation and EMS was done at Biotechnology

and can increase plant growth (Tsvetkov et al., 2014). In

Laboratory, Faculty of Agriculture, Lampung University.

Indonesia, most of oil palm is grown on marginal land

Twenty mL of the conidial suspension was injected onsuch as Ultisol and peat soil with low soil pH. In addition,

membrane filter and then was placed in each replicate plastic

intensive use of chemical fertilizers can further decrease

Petri dish (60 mm diameter). The dishes were irradiated at asoil pH (Firmansyah and Sumarni, 2013) and decrease therange of doses 0, 30, 100, 300, 1000 and 3000 Gy

population and microbial effectiveness in soils, including(Gamma ray). For UV irradiation, wave length that was

beneficial microbes for plants (Rousk et al., 2009)used was 245 nm with 10, 20 30, 40 and 50 minutes

including Trichoderma.

irradiation. All dishes were incubated at 20°C for 24 hours

In this study, four isolates were used as a material forthen moved into 3 mL Potato Dextrose (PD) liquidmutation activity to obtain mutant Trichoderma that were

medium.

- 85 -EMS solution. Ten mL of the conidial suspension was

Gamma rays irradiation. A total of 54 isolate mutants ofcentrifused for 10 minutes at 3000 rpm. The pellet was

low pH (pH 2) obtained in this irradiation. Ten mutants ofsoaked on 1.5 and 2% of EMS suspension for 30 and 50

T1 have larger colonies diameter than wild-type and 9 theminutes. Then, it was recentrifuged for 10 minutes atother putative mutants had smaller colonies diameter than3000 rpm. The pellet was incubated for 10 minutes at 0°C.

wild-type. The colonies diameter of the mutant resuted

Ten mL of Phosphate buffer salin steril was added to thefrom T1 ranges from 4.6 to 7.4 cm. From the isolate T2, 6

pellet then it was centrifuged for 10 minutes at 3000 rpm.

mutants had larger colonies diameter than wild-type,This step was replicated twice. The pellet was incubatedwhile the other 12 isolates have smaller colonies diameterat 20°C selama 24 jam.

than wild-type. The diameter of mutant isolates from T2

Inoculation of Trichoderma isolates that have been

ranges from 4.68 to 8.00 cm. T3 isolate, produced 1

treated on medium with low pH conditions. Each ofmutant isolate, and has colonies diameter larger than wild

treated isolates grown on 4 mL of PD Broth (liquid

type (6.61 cm). For T4 isolates, 14 mutant isolates had

medium) at pH 2. Twenty mL of the incubated conidial

larger colonies diameter than wild-type and 2 isolates had

suspension were entered into each medium. Observationcolonies diameter smaller than wild-type. The diameter ofwere made daily for 7 days on the growth of fungus, themutant isolates from T4 ranged from 3.73 to 7.50 cm (Fig.

rate of growth and the formation of conidia. The

1).

wild-type were also included as a control.
The

growthofcolonies

diameter,

Isolated mutants of T1

sporulation,

viabilitity dan antagonism of Trichoderma Mutant
Suspects to Ganoderma sp. This procedurs wereconducted to know whether the mutants have the sameability as its wild type. Measurement methods of growth,
sporulation, viability and ability of mutant antagonists
Isolated mutants of T2

were performed according to the same measurement
method used in the screening stage to obtain selected
Trichoderma fungi. Isolate to be tested previously moved
from liquid medium to solid PDA media.

Isolated mutants of T3

Result and Discussions
Isolates of the Trichoderma Putative Mutants. A totalof 109 mutant suspects that capable of growing at pH 2
were obtained in this study. The mutant suspectedisolates were obtained from gamma ray irradiation (54Isolated mutants of T4

isolates), UV irradiation (8 isolates) and EMS (47isolates). The first step of determining the mutant wasseen from the growing ability and spore production ofeach isolate that has been treated on each type of media
compared to the wild type. If the treated isolate has adifferent appearance than the wild type, the isolate wasFig.1 Colonies diameter of low pH mutant irridiateddetermined as putative mutant. On the medium of pH 2,from gamma rays irradiation

the putative mutant isolates resulting from gamma ray
irradiation were able to form spores (marked with green

UV irradiation. There were 8 isolates of low pH mutantscolony color) but not for wild type. Wild type was still(pH 2), consisting of 6 isolates of T2 and 2 isolates of T3.

able to grow, however it did not produce spores (marked

Two mutants of T2 resulted smaller colonies diameterwith colony color that remains white). This also occured

than wild-type and 6 isolates mutants larger thanin UV irradiation.

wild-type. While 2 mutants of T3 had larger colonies

The growth of colony diameter of Low pH putative

diameter than wild-type (Fig. 2).

mutants.

- 86 -

higher sporulation than wild-type (> 6.88 x 108 spore/ml),
while the other 6 mutants had sporulation lower thanwild-type. T3 isolate produced 3 mutants that had
sporulation smaller than wild-type (> 3.38 x 108 spore/ml),
while the other 9 mutants were larger than wild-type. T4
isolate produced only 2 mutants that had higher
sporulation than wild-type (> 6.25 x 108 spore/ml), whileFig.2 Colonies diameter of low pH mutant irridiated fromthe other 10 mutants were smaller than wild-type (Fig.5).

UV irradiation
Isolated mutants of T1

EMS solution. Total mutants isolates of low pH of 47
isolates consisted of 12 mutants of T1, 12 mutants of T2,
11 mutants of T3 and 12 mutants of T4. At 2 days after
inoculation, 2 mutants of T1 produced larger colonies
growth than wild-type meanwhile 10 mutants of T2

Isolated mutants of T2

produced lower colony diameter than wild-type. Six
mutant of T2 had same colonies diameter as wild-type
and the other 6 mutants is lower. Five mutants of T3 hadsame colonies diameter as wild-type but the other 6
mutants were smaller. Only 1 mutant of T4 had larger
colonies diameter than wild-type, the other 11 mutants

Isolated mutants of T3

were smaller.
Sporulation.
Gamma rays irradiation. Of all the isolates obtained, 2mutants of T1 produced spore higher than wild-type andIsolated mutants of T4

the other 16 mutants have spores lower than wild-type.
Spores production of mutants T1 had range from 2.25 to
16.88 x 108 spores/ml. One mutant of T2 had spore
production higher than wild-type, while 17 mutants had
lower spore production than wild-type. Spores production
from mutants of T2 ranged from 1.38 to 18.75 x 108

Fig.3 Sporulation of low pH mutant irridiated fromspores/ml. The mutant isolates derived from T3 isolates

gamma rays irradiation.

are currently still in the process of analysis. All of themutants of T4 had lower spore production than wild-type.
Spores production of mutants T4 ranged from 2.19 to
13.75 x 108 spores/ml (Fig. 3).
UV irradiation. Sporulation of 5 mutants of T2 were
smaller than wild-type (15.63 x 108 spore/ml) and only 1
mutants equal to wild-type. While 2 mutants of T3, 1
mutant was larger than wild-type (8.13 x 108 spores/ml),
but 1 mutant had smaller than wild type (Fig.4).
EMS solution. The highest sporulation produced by T2
isolate (6.88 x 108 spores/ml) and the lowest was T3
8

isolate (3.38 x 10 spores/ml). Three mutants of T1

Fig.4 Produksi spora isolat terduga mutan pH rendah
hasil iradiasi UV

8produced spore higher than wild-type (> 3.75 x 10

spores/ml), while the other 8 mutants have lower spore
than wild-type. T2 isolate produced 6 mutants that have

- 87 -

Spore viability.

to 93.48%. From T2, 5 mutants had better antagonism

Gamma rays irradiation. Seven mutants of T1 produced

capability than wild-type and 13 mutants had lower

spore viability higher than wild-type and the other 12

antagonism capability than wild-type. The percentage ofmutants had lower spore viability than wild-type. Spore's

inhibition produced by mutants of T2 ranged from 54.78

viability ranged between 94.17-100%. From T2, 15

to 96.09%. From T3, the mutant had smaller antagonism

mutants produced higher spore viability than wild-type,capability than wild-type. The resulting percentage iswhile the other 3 isolates had lower spore viability than

82.17%. From T4, 4 mutants had better antagonism

wild-type. The spore’s viability of mutants T2 ranged

capability than wild-type and 12 mutants had smallerfrom 95.45-100%. For unexpected isolates of mutants

antagonism capability than wild-type. The resulting

derived from T3, currently is still in a process of analysis.

percentage ranged from 73.91-94.35%.

From T4, 9 mutants of T4 had higher spore viability thanwild-type, while the other 7 isolates had lower spore
viability than wild-type. The spore’s viability were
between 82.32-100%.
UV irradiation. Five mutants of T2 have spore viability
lower than wild-type (<100%) while 1 mutant had spore
viability same as wild type. Two mutants of T3 had spore
viability higher than wild-type (Fig. 5).

Fig.5 Spore viability of low pH mutant irridiated from
UV irradiation
EMS solution. Immersion of EMS solution resulted 2
mutants of T1 that lower than wild-type (<98.52%); 1
mutant was equal to wild-type and 8 mutants were higher
spore viability than wild-type. Two mutants of T2 werelower than wild-type while 8 mutants were higher thanwild-type. From T3, 2 mutants had lower spore viability
than wild-type and 10 mutants were as same as wild-type.

Fig.6 Spore viability of low pH mutant iridiated fromThree mutants of T4 had spore viability lower thanEMS

wild-type and 9 mutants were higher than wild-type (Fig.
6).

UV irradiation. Six mutants of T2 were not better
inhibition to wild-type (92.1%) meanwhile 2 mutants of

The Antagonism of Low pH Mutants to Ganoderma
Gamma rays irradiation. Of the 54 low pH mutants (pHT3 were better inhibition than wild-type (86.61%) (Fig.
7).

2) obtained, 5 mutants of T1 had better antagonism
capability than wild-type, 1 mutant had same antagonism
capability as wild-type and 13 mutants had lower
antagonism capability than wild-type. The percentage ofinhibition produced by mutants of T1 ranged from 62.17- 88 -

In this research, we used 3 mutation agents (mutagen)namely gamma ray irradiation, ultraviolet irradiation andEthyl Methane Sulfonate (EMS). These three mutagens
have generally been widely used to generate mutants(Darwis, 2006; Kava et al., 1995). Treatment with thesethree mutagens will lead to changes in various chemical
and molecular bonds of reproductive cells frommicroorganisms (Darwis, 2006).
Piri et al. (2011) mentioned that gamma ray irradiation
Fig.7 Antagonism to G. boninense of Low pH obtained

has been widely used to produce mutants from plants andfrom UV irradiation

microorganisms. Gamma rays irradiation have also beensuccessfully used to produce resistance fungicide of

EMS solution. Four wild-type produced various mutants.

Isaria fumosorosea and thermotolerant mutant of

Mutants of T1 and T2 were not able to had better

Metarhizium anisopliae s.l (Shinohara et al., 2013;

antagonism of wild-type (90.7% and 92.1%, respectively).

Fitriana et al., 2014). UV irradiation and EMS have also

Whereas 5 mutants of T3 had better antagonism ability

been reported successfully used to create mutants fromthan wild-type (86.61%) and the other 7 mutants weremicroorganisms (Pelczar and Chan, 1986). Radha et al.

smaller than wild-type. Four mutants of T4 were better(2012) reported that the use of EMS can produce

antagonism than wild-type but the other 8 mutants wereAspergillus niger mutants capable of producing proteases

smaller antagonism than wild-type (Fig.8).

1.5 times higher than wild-type. UV Irradiation was also
reported to be able to make mutant of Aspergillus niger
capable of producing proteases 2 times higher.
From this research, in total, we obtained 109 mutants
isolates from three mutagens (UV irradiation, gamma ray
irradiation and EMS) that were low pH resistant. All of
the mutant isolates have different performance withwild-type. After its treated with mutagen, almost all
mutant isolates have better performance than wild-typewhen it were grown on low pH medium. The results
showed that some mutant isolates had different colonies
colors compared with wild-type. However, there are also
isolates that do not change colonies color either on PDB
media or after being transferred back using PDA media.
The ability of mutant isolates to grow, sporulation, spore
viability and antagonism to Ganoderma sp. were also
various. There were some mutant isolates that had
performance lower than wild-type, some of they are
similar to wild-type and some else had better performance
than wild-type.
Najafi and Pezehki (2013) reported that mutations in
microorganisms can affect or not affect the nature ofthese living things. The other research reported that
thermotolerant resistant of Metarhizium anisopliae
obtained from gamma-ray irradiation had lower virulencewith wild-type, but others had higher virulence thanFig.8 Antagonism to G. boninense of Low pH mutants

wild-type (Fitriana, 2015).

from EMS solution- 89 -

Mikrobiologi 1. Universitas Indonesia Press. Jakarta.

Conclusion

9) Piri I, Mehdi Babayan M, Tavassoli A and Javaheri M.

The mutant isolates were able to grow and produce

2011. The use of gamma 4 irradiation in agriculture.

spores in the pH 2-Potato Dextrose medium but not for

African Journal of Microbiology Research, 5:5806-5811.

the wild type. Only 1 out of 109 potentially low10) Radha S, Babu RH, Sridevi A, Prasad NBL and

pH-tolerant mutant of Trichoderma consistently showed

Narasimha G (2012) Development of mutant fungal

betterandstrains of Aspergillus niger for enhanced production ofantagonist to G. boninense than the wild type. These

acid protease in submerged and solid state fermentation.

results showed that EMS, gamma rays and UVEuropean Journal of Experimental Biology, 2 (5):colonygrowth,sporulation,

viability

irradiation can be used to improve low-pH tolerant of1517-1528.

Trichoderma spp.

11) Rini MV (2003) Arbuscular Mycorrhiza association
decrease Rate of Spread of Ganoderma boninense

Acknowledgement
This

work

was

made

Infection within Oil Palm Root. Dalam Prosiding
possiblethroughvarious

Simposium

Nasional

dan

Kongres

PeragiVII.

facilitations. We thank to BPDP 2015 and Financial

Perhimpunan Agronomi Indonesia. Hlm. 324-327.

Department. We also thank our former students12) Rousk J, Brookes PC and Baath E (2009)(Fransiska Dina and Fransiskus Ellyando Sinaga) for

Contrasting soil pH effects on fungal and bacterial

valuable assistance during research.

growth suggest functional redundancy in carbon
mineralization. Applied Enviromental and Microbiology,

Reference
1) Darwis D (2006) Sterilisasi produk kesehatan (HealthCare Products) dengan radiasi berkas elektron. Proseding
Pertemuan dan Presentasi Ilmiah Teknologi Ahelerator
dan Aplikasinya Edisi khusus: 78-86.

dua

Varietas

BawangMerahand3) Fitriana Y, Satoh K, Narumi I and Saito T (2014)
gamma-rayirradiations

followingionizing

radiation-induced

Trichoderma

spp.

in

Viticulture(Review).

Bulagarian Journal of Agricultural Science, 20(4):Jurnal Hortikultura, 23(4):358- 364.
andfumosorosea

Batcharova R (2014) Mycorrhizal Fungi Glomus spp.

(Allium

ascalonicum L). pada Entisols-Brebes Jawa Tengah.

Ion-beamT (2013) Enhanced fungicide resistance in Isaria

14) Tsvetkov I, Dzhambazova T, Kondakova V and

Pupuk N terhadap pH Tanah N-Total Tanah, dan Serapan
Hasil13) Shinohara S, Fitriana Y, Satoh K, Narumi I and Saito

mutagenesis. FEMS Microbiology Letters, 349(1): 54-60.

2) Firmansyah I dan Sumarni N (2013) Pengaruh Dosis
N

75(6): 1589-1596.

849-855.

inducethermotolerant mutants in the entomopathogenic fungus
Metarhizium anisopliae s.l. Biocontrol Science andTechnology, 24(9):1052-1061.
4) Fitriana Y, Shinohara S, Satoh K, Narumi I and Saito T(2015) Benomyl-resistant Beauveria bassiana mutants
induced by ion beams. Applied Entomology and Zoology,
50(1):123-129.
5) Gurmit S (1991) Ganoderma: The Scourge of Oil
Palmsin the Coastal Areas. The Planter, 67:421-444.
6) Kava-Cordeiro V, Luna-Alves-Lima EA and Azevedo
JL (1995) Survival and mutant production induced bymutagenic agents in Metarhizium anisopliae. Sci. Agric.,
52:548-554.
7) Najafi MBH and Pezehki P (2013) Bacterial mutation;
types, mechanisms and mutant detection methods: areview. European Scientific Journal, 4:628-638.
8) Pelczar MJ and Chan ECS (1986). Dasar-Dasar- 90 -

OS V-1
Management of rice and wheat blast pathogen in Bangladesh
Manzur-E-Mohsina FERDOUS，Mohammad Mahmood HASAN , Adeeba RAIHAN, Lutfur
RAHMAN, Abu Shamim Mohammad NAHIYAN(Advanced Seed Research and Biotech Center, ACI Ltd., Dhaka, Bangladesh）

SUMMARY
Rice is a primary source of dietary energy for more than half of the world population. However, one of the mostdevastating fungal diseases ‘rice blast’ (caused by Pyricularia oryzae) hampers 60-100% yield. In this study, 11 popular
rice (Oryza sativa Indica) accessions from Bangladesh were screened for blast resistance through artificial inoculation
with mixed and single isolates. Disease responses varied among the accessions. Only BR15, BR16 and BRRI dhan 33
showed moderate resistance, with most germplasm susceptible. These accessions were also screened for candidate
genes providing horizontal resistance. The gene specific microsatellite markers identified the Pi 35t gene in the BR 15
and 16, which is complementary to the disease scale after artificial inoculation and can be further used to develop blast
resistant variety through marker-assisted breeding. On the other hand, wheat blast also occurred in Bangladesh recently.
This incident is also first time in Asia and very alarming. Resistance breeding is the smart alternative but complex due
to combination of several genes. On the basis of above circumstances, an experiment was planned to use biocontrol
agent (Trichoderma sp.) on 1 mega variety of wheat in Bangladesh BARIGOM 28 and the ASRBC developed 8 mutant
lines against wheat blast.

Introduction

in many rice growing countries over the last decade i.e.

Rice (Oryza sativa L.) is one of the most important

20-100% yield losses have been reported in India, Japan

leading food crops of the world which is consumed by

and Kenya (Khush and Jena, 2009; Sharma et al., 2012;

50% of the world population (Fairhurst and Dobermann;

Kihoro et al., 2013) depending on the rice variety grown.

2002). It is also the most important to millions of small

Leaf blast lesions decrease the net photosynthesis rates,

farmers and landless workers. In Bangladesh, Rice has awhich causes more damage than the visible portions oflong cultivation history and it is grown all over the

diseased leaves (Bastiaans, 1991). Neck blast iscountry year round except in the south-eastern hilly areas

considered(Shelly et al., 2016). It is the staple food for about 156trans-boundary disease (Zhu et al., 2005) which causes

million people of the country. Although the agro climatic

extreme and permanent damage (Biswas, 2017). In

condition is suitable, the national average rice yield is

Bangladesh, 11,000 MT crop losses have been reported

much lower (2.94 t/ha) than the other rice-growing

by neck blast on an area of 3000 ha (approx.) at several

countries (BBS, 2012). The estimated amount ofnorthern and southern parts of the country (Hossain,

imported rice is 100,000 MT based on the local market2017) resulted from favorable condition such as adverse

demand for 2016/17 marketing year which will beweather, high rainfall, prolonged dew and late planting

increased to 125,000 for 2017/18 marketing year (Lagosetc (Lagos and Hossain, 2017). High relative humidity

and Hossain, 2017). The population increases at a rate ofplays the most important role in sporulation, germination

2 million per year and concurrently the total cultivable

and release of blast conidia (Ou, 1985; Koutroubas,

land is decreasing at a rate of more than 1% per year due2009). Severe outbreak of wheat blast has been taken

to urbanization. Moreover, biotic and abiotic constraints

place last February 2016 in Bangladesh (Callaway, 2016),

create adverse condition, which results serious yieldthreatening the food security of major wheat producing

reduction.

neighbouring countries (Islam et al., 2016). In case of

Among biotic stresses, rice blast is a very prominent, andrice, 267 blast occurring races have already been

widespread disease, which caused by fungal pathogen

identified in Bangladesh, which can become a serious

Pyricularia oryzae and reduces crop yield significantly

threat for 86 rice growing counties including Bangladesh- 91 -

themostdestructive

phase

of

this

in near future. Therefore, attempts should be made to

growth chambers and later vegetative stages in a green

increase the yield per unit area of rice in a sustainable

house. Gene specific microsatellite markers were used tomanner for the food and nutritional security of thisdetect genes, which may be providing horizontal

highly populated country. So, the identification and use

resistance against blast.

of resistant or tolerant cultivars will be the mosteconomic and environment friendly way to manage thisResult and Discussions

blast disease.

The pathogen P. oryzae was successfully isolated withthe filter paper method and then and cultured on water

Material and Method

agar

Isolation and characterization of the pathogen
Pyricularia oryzae
The suspected causal agent for neck blast disease,
Pyricularia oryzae was collected from blast affected
plants of rice field. Infected leaves and nodes were cut
into small pieces and placed on wet filter papers insterilized Petri dishes to keep them moisturized. The
Petri dishes were covered and kept under light at 24°C.
After 24 hours of incubation period, the tissues were
examined under stereo microscope to check for thefungal sporulations. The spores were then transferred towater agar media with an inoculation loop and incubatedfor 3 days at 24°C. Single isolates were identified usingmorphological characteristics, microscopic studies andfinal confirmation by ITS sequences. The Petri dishes of

medium.

Microscopic

characterizationandmolecular analysis (Fig 1) were done for final
confirmation. Disease symptom was appeared on
susceptive plant leaves after inoculation, on the other
hand, no visible symptom was found on healthy anddisease free plants. Only BR15, BR16 and BRRI dhan33
showed moderate resistance among 13 accessions usedin this experiment (Fig. 2 and 3). However, BRRI
dhan28 and BRRI dhan29 showed severe susceptibility.
The result was evaluated and determined for both single
and mixed strain inoculations by scores of disease rating
scale provided by IRRI in 1996. The presence of target
gene in selected germplasm has been identified bymolecular assay. The screened 13 accessions showedhorizontal resistance against blast. The gene specificmicrosatellite markers identified the Pi35t gene in the
BR15 and BR16 (Fig. 1).

purified cultures were stored at 4°C for further use.

Disease responses varied between the greenhouse andPreparation of inoculums
The inoculums were prepared from pre-inoculated plates

growth chamber as well as between seedling andof single and mixed strains culture. The spores were

germplasm screening against rice blast in Bangladesh,suspended in distilled water and the final concentration

though huge numbers of rice genotype were screened

1x106

and found resistant against blast (Ahmed et al., 1985;

was

adjustedtospores/ml

by

using

vegetative stages. Very few reports are available on ricehaemocytometer.

Islam et al., 2001) but identification of involved genes

Artificial inoculation of P. oryzae
Seeds of the rice accessions were sown in several earthen

has been less practiced to maintain and utilize the known

pots and placed on the benches of the greenhouse. After

BRRI dhan33 showed moderate resistance, and most

two weeks, the seedlings were inoculated with aqueous

germplasm was found susceptible. Mohanta et al. (2003)

suspension containing 1x106 spores/ml of virulent

reported to have 3 highly resistant, 12 resistant and 16

isolates of single and mixed strains of P. oryzae. Freshmoderately susceptible in screening trails between 28

water was sprinkled on plants three times every day

restroed lines and 4 standard checks.

between 11:00 and 16:00 hours to ease the disease

The gene specific microsatellite markers identified thedevelopment. The greenhouse temperature and humidity

Pi35t gene in the BR15 and BR16 in this study, which iswere maintained at 28°C and 90-100% respectively.

complementary to the disease scale after artificial

Screening of rice germplasm against rice blast disease
The seedlings were scored through ‘Standard evaluation
system of rice on 0-9 scale (IRRI 1996)’ based on visualobservation of the symptom at 10, 20 and 30 days aftergene pool. In this experiment, only BR15, BR16 andinoculation. In a similar experiment, Khan et al. (2014)
identified blast resistant genes Pish, Pi9 and Pita fromresistantvarieties

Bawaibhog.

inoculation. The experiment screened seedlings in- 92 -

chinigura,BRRI

dhan50andBiswasJK(2017)

http://www.daily-sun.com/

printversion/details/221224/Rice-Blast-Disease-andSome-Related-Issues
Callaway E (2016). Devastating wheat fungus appears inConclusion

Asia for first time. Nature, 532:421-2.
Fairhurst TH and Dobermann A (2002). Rice in the global
food supply. Better Crops Int., Vol 16:3-6.

aHossain T (2017). Grain and Feed Update, Bangladesh,b

2017. Global Agricultural Information Network (GAIN)
Report. USDA Foreign Agricultural Service. Report No.
BG7006.
Lagos JE and Hossain T (2017) Grain and Feed Annual of
Bangladesh, 2016. Global Agricultural Information
Network (GAIN) Report. USDA Foreign Agricultural
Service. Report No. BG6005.
IRRI (1996) Standard evaluation system for rice. 4th ed.
IRRI, Manila, Phillipine.
Islam MT, Croll D, Gladieux P, Soanes DM, Persoons A,
Bhattacharjee P, Hossain MS, Gupta DR, Rahman MM,
Mahboob MG, Cook N, Salam MU, Surovy M.Z.,

Effective evaluation leads breeders to choose advanced

Sancho VB, Maciel JLN, NhaniJúnior A, Castroagudín

germplasm as parental lines for future work and to use as

VL, Reges JTA, Ceresini PC, Ravel S, Kellner R,

a gene pool in order to improve blast resistance in rice.

Fournier E, Tharreau D, Lebrun MH, McDonald BA,

Host resistance form BR15 and BR16 are being used todevelopblast

resistantrice

varieties

Stitt T, Swan D, Talbot NJ, Saunders DGO, Win J andthroughKamoun S (2016). Emergence of wheat blast in

marker-assisted breeding.

Bangladesh was caused by a South American lineage of
Magnaporthe oryzae. BMC Biology, 14:84

Acknowledgement

Islam KMH, Islam MK, Rahman AJMM, Jalaluddin M.,
Special thanks to Dr. FH Ansarey, Managing Director

Mia MAT. (2001) Screening of some rice varieties andand CEO, ACI Agribusinesses, ACI Ltd. for his

induced mutants against blast (Pyricularia grisea),continuous support for conducting the research.

Bangladesh J. Plant Pathol. 17 49–53.

Reference

Khan JA, Jamil FF, Cheema AA, and Gill MA (2001).

Ahmed HU, Haque MA, Shahjahan AKM, Miah SA

Screening of rice germplasm against blast disease caused(1985). Blast resistance in rice germplasm in Bangladesh,by Pyricularia oryzae In: Proc. National Conf. of Plant

Bangladesh J. Plant Pathol., 1:73–74.

Pathology, held at NARC. Islamabad. Oct 1-3. pp. 86-9.

Bastiaans L (1991) Ratio between virtual and visual

Khush GS, and Jena KK (2009). “Current status andlesion size as a measure to describe reduction in leaf

future prospects for research on blast resistance in ricephotosynthesis of rice due to leaf blast. Phytopathol., 81:(Oryza sativa L.),”in Advances in Genetics, Genomics

611-615.

and Control of Rice Blast Disease, eds GL Wang and B

BBS (Bangladesh Bureau of Statistics) (2012) Statistical

Valent (Dordrecht: Springer), 1–10.

Year Book of Bangladesh. Ministry of Planning,

Kihoro J, Bosco NJ, Murage H, Ateka E and Makihara D

Government of the People’s Republic of Bangladesh:(2013). Investigating the impact of rice blast disease on

33–36.

the livelihood of the local farmers in greater Mwea region- 93 -

of Kenya. Springer Plus, 2:308.
Koutroubas SD, Katsantonis D, Ntanos DA, Lupotto E(2009). Blast disease influence on agronomic and quality
traits of rice varieties under Mediterranean conditions
Turk J Agric For., 33 487-494c.
Mohanta BK, Aslam MR, Kabir ME, Anam MK,. AlamMK and Habib MA (2003). Performance of different
genotypes/cultivars to blast disease of rice in Boro and T.
Aman crop in Bangladesh. Asian. J. Plant Sci., 2(7):575-7.
Ou SH (1985) Rice Diseases, 2nd ed. Commonwealth
Mycological Institute, Kew, Surrey, England.
Sharma TR, Rai AK, Gupta SK, Vijayan J, Devanna BN,
and Ray S (2012). Rice blast management throughhost-plant

resistance:

retrospect

andprospects.

Agric.Res., 1, 37–52.
Shelley IJ, Takahashi-Nosaka M, Kano-Nakata M, HaqueMS and Inukai Y (2016) Rice Cultivation in Bangladesh:
Present Scenario, Problems, and Prospects. J Intl Cooper
Agric Dev., 14: 20–29.
Zhu YY, Fang H, Wang YY, Fan JX, Yang SS, Mew TW,
Mundt CC (2005) Panicle blast and canopy moisture inrice cultivar mixtures. Phytopathol 95: 433-438.

- 94 -

OS V-2
Control of eggplant and tomato bacterial wilt by grafting in Indonesia
Triwidodo ARWIYANTO, YMS MARYUDANI, Kin Lay LWIN, Bellarminus TRIMAN, Sri
SULANDARI, Suryanti SURYANTI, Putri LAESHITA(Faculty of Agriculture, University Of Gadjah Mada)

SUMMARY
Bacterial wilt disease caused by Ralstonia solanacearum, reduce production of Solanaceous crops such as potato,
tomato, tobacco, and eggplant. The disease was difficult to control by available means. Grafting with resistant rootstock
is an alternative control which has been practiced in many countries. In Indonesia, grafting with Solanum torvum
suppressed eggplant and tomato bacterial wilt in the greenhouse experiments. All grafted plants as well as S. torvum did
not exhibit wilt symptoms until one month after inoculation. However, non grafted eggplant wilted within the first week
after inoculation and at 30 days after inoculation the disease index reached 96.4. The non grafted tomato completely
wilted at 30 days after inoculation. Local cultivars of tomato grafted with Eg-203 showed lowest disease index in theexperimental field infested with R. solanacearum. The grafted plants showed better growth and better fruit production
compared with non grafted plants.When the local cultivars were grafted with H-7996, the disease index were higher
compared with those grafted with Eg-203, and produced more fruit compared with non grafted plants. Two local tomato
cultivars when tested as rootstocks, the grafted plants exhibited lower disease index both in the vinyl polybags and in
the field experiments.

Introduction

the seeded media was watered daily with ground water.

Bacterial wilt caused by Ralstonia solanacearum is one

The seedlings then watered with a half strength ofof the important diseases in Solanaceous crops such as

Hoagland’s solution until ready for grafting. Two

potato, tomato, tobacco, and eggplant. The diseasemethods of grafting were performed. The first methods

destroyed cigar tobacco plantation in north Sumatra

was cleft grafting as described elsewhere (Oda, 1999)(Arwiyanto and Hartana, 2001), and along with root knot

and tube grafting was performed according Arwiyanto etnemathode devastated keretek tobacco in Temanggung,al., 2015b. A challenge bacterial strain of Ralstonia

Central Java (Dalmadiyo, 1991). Although the figure ofsolanacearum Phylotype 1, race 1, biovar 3 was used inlosses has never been investigated, the disease also

the greenhouse experiments. In the field experiments the

reduce significantly the production of potato, eggplant,

artificial inoculation with bacterial strain were notchilli pepper, and tomato. The disease is difficult to

conducted, instead its used natural infection of thecontrol due to complexity of infection entrance to root

disease. In the second experiments, a local tomato

and due to genetic variability of the pathogen. Vegetable

cultivar (Martha) was used as scion. The rootstocks usedgrafting has been practiced since long time ago in Japan

were Solanum torvum (T), H-7996 (H), Eg-203 (E),to control soilborne plant pathogen including R.

Amelia (A) and Mawar (M). Thus, TM was denoted tosolanacearum (Lee et al., 2010). The methods then has

Martha grafted with Solanum torvum, HM those which

been developed and adopted elsewhere in the world.

grafted with H-7996, and so on.

Here we reported the use of Solanum torvum, two local

Result and Discussions

varieties of open pollinated tomato, H-7996, and Eg-203

The first experiments with the used of solely Solanum

as rootstocks to control bacterial wilt.

torvum as a rootstock has been reported elsewhere

Material and Method(Arwiyanto et al., 2015a). All grafted plants as well as S.

Sowing media was a mixture of cocopeat and rice hull

torvum did not exhibit wilt symptoms until one monthcharcoal (1:1, v/v). Solanum torvum was seeded 3 weeks

after inoculation. However, non grafted eggplant wilted

before scions. Tomato rootstocks were seeded two days

within the first week after inoculation and at 30 days

before the scion. At the first day until cotyledone emerge,

after inoculation the disease index reached 96.4. The non- 95 -

grafted tomato completely wilted at 30 days after

About ten weeks after transplanting, infection of

inoculation (table 1).

bacterial wilt were observed on non grafted plants. At 12

Table 1. Average disease index of plants after inoculation

weeks after transplanting the disease progres on nonwith R. solanacearum

grafted plants increase then significantly the disease

Treatments

intensity bcome higher. At the same time, tomato grafted

Solanum torvum

Average disease index atdays after inoculatiom
7
14
21
28
0*
0
0
0a**
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a
0
0
0
0a

‘Kalenda’ eggplant

29

59

79

96b

solanacearum as showed in the table 2. There was‘Sanmarino’

31

78

100

100b

infection of bacterial wilt when artificially inoculated

R1S1
R1S2
R1S3
R1S4
R2S1
R2S2
R2S3
R2S4
R3S1
R3S2
R3S3
R3S4

with Mawar (MM) and H-7996 (HM) were infected in
low intensity and kept at this point until the end ofexperiment. While, the non grafted plants reachedmaximum bacterial wilt intensity in the field. All grafted
plants showed lower disease intensity compare with nongrafted plants (Table 2). The lower disease intensity inthe field experiments indicated that there was low
population of the bacterial wilt pathogen. While in thevinyl polybag experiments, the disease intensity was
higher because of artificial inoculation with highly
virulent of Ralstonia solanacearum. In the field
experiments, tomato grafted with rootstock Eg-203 (EM)and with Solanum torvum (TM) exhibited no disease
infection. Again, the resistance of S. torvum againts
bacterial wilt was proven. The resistance of Eg-203 wasbroken when challenged with dense population of R.

with dense population of highly virulent of R.

tomatosolanacearum, although the figure was small.

*) means of five replications

Table 2. Disease intensity of grafted tomato plants**) number followed by the same letter were notTreatment

significantly different (P=0.05) with Duncan’s Multiple

Disease Intensity (%)
Field

Vinyl Polybag

Range Test.

HM

0,95 a2,67 aSource : Arwiyanto et al., 2015a.

EM

0a8,67 aSeveral years later, grafting experiments were performed

AM

1,66 a5,67 aby tube grafting methods. In field experiment, due to low

MM1,64 a10,00 apopulation of R. solanacearum in soil, the disease

TM

0a

-

progres was slow (Figure 1).

M

13,86 b50,67 b

Number followed by the same letter in the same column
were not significantly different (P=0.05) with Duncan’sMultiple Range Test.
The best yield was obtained when the tomato plants weregrafted with Mawar (MM) which produced 2.7 kg/plantfollowed by those grafted with H-7996 (2.6 kg/plant)
then Amelia (2.5 kg/plant) (Table 3). Tomato plants
grafted with tomato rootstocks (HM, AM, MM) showed
better performance on fruit production compare withthose grafted with Eg-203 or S. torvum. On the contrary,
although there was no infection of bacterial wilt,
Figure 1. Bacterial wilt disease development of grafted

production of tomato grafted with S. torvum showed thetomato- 96 -

lowest production (1.1 kg/plant), even compared withthose non grafted plants (2.0 kg/plant). It is assumed that
incompatibility might happen so that the supply ofnutrients from the soil was not in maximum capacity.
Table 3. Fruit production of grafted tomato
MarketTreat-

Fruit

able

ment

number

fruit

HM

33.7d

33.4d

NumberProductionof rotten(kg/plant)

fruit
0.3a

2.6c

EM

25.5b

25.3b

0.1a

2.0b

AM

30.5cd30.3cd

0.2a

2.5c

MM

32.3d

32.0d

0.3a

2.7c

TM

15.1a

14.9a

0.2a

1.1a

M

26.8bc26.6bc0.2a2.0b

Number followed by the same letter in the same column
were not significantly different (P=0.05) with Duncan’sMultiple Range Test.

Conclusion
Bacterial wilt caused by Ralstonia solanacearum can becontrolled by grafting with the use of local tomato
rootstock. Due to the variability of the pathogen, it isnecessary to conduct the same experiment in manydifferent area.

Acknowledgement
This research was financed by the grant from Ministry of
Researh, Technology and Higher Education, Republic ofIndonesia. The first author thanks to Gifu University forgiving oportunity to present this paper by grants fortravel and accomodation.

Reference
1) Arwiyanto, T, Lwin, KL, Maryudani, YMS, andPurwantoro, A (2015)a Evaluation of Local Solanum
torvum as a Rootstock to Control of Ralstonia
solanacearum in Indonesia. Acta Horticultura 1086:
101 -106.
2) Arwiyanto, T, Nurcahyanti, SD, Indradewa, D and
Widada, J (2015)b Grafting local commercial tomato
cultivars with H-7996 and Eg-203 to suppress bacterial
wilt (Ralstonia solanacearum) in Indonesia. Acta
Horticultura 1069:173-178.
2) Arwiyanto, T and Hartana I (2001) Field experimenton biological control of tobacco bacterial wilt disease(Ralstonia solanacearum). Mediagama 3:7-14. In
Indonesian.
3) Lee JM, Kubota C, Tsao SJ, Bie Z, Hoyos Echevarriae
P, Morraf L, Oda M (2010). Current status of vegetable
grafting: Diffusion, grafting techniques, automation.
Scientia Horticulturae 127: 93–105.
4) Oda, M. 1999. Grafting of vegetables to improvegreenhouse production in Japan.
http://www.agnet.org/linrary/article/eb 480.html- 97 -

- 98 -

OS V-3
Detection and characterization of Asia soybean rust in Bangladesh
Md. Motaher Hossain1，Naoki Yamanaka2(1Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh, 2Japan International Research Center
for Agricultural Sciences, Japan）

SUMMARY
Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most serious diseases of soybean. During
April-May 2016, as soybean crops were nearing maturity, the disease was found throughout the region of southwestern
Bangladesh, which includes Lakshimpur, Noakhali and Bhola District, where approximately 99% of the soybean crop of
Bangladesh is produced. A total of 23 soybean fields in Noakhali, Lakshmipur and Bhola districts of Bangladesh weresurveyed for the presence of ASR during April-May in 2016. Disease was detected in all those fields where nearly allplants showed symptoms. Affected crops were mostly in growth stages R4 to R6, except for a few fields that had been
visited later and were in a late R7 stage. Leaf lesions were reddish brown, irregularly shaped, and were more abundanton the abaxial surface. Under the dissecting microscope, uredinia were observed as erumpent pustules with a conspicuous
central pore. Masses of urediniospores were expelled through the pore and covered the pustules. Urediniospores were
hyaline to pale yellow-brown, sub globose to ovoid and an average size of 17 to 36 × 14 to 25 μm μm. ASR susceptible
soybean cultivar BRS154 and a local soybean cultivar BARI Soybean-5 inoculated with the spore suspension of three
ASR isolates, each from each of three districts yielded symptoms and sign equivalent to those observed in the field. These
results confirmed the presence of P. pachyrhizi in Lakshimpur, Noakhali and Bhola District, Bangladesh. Inoculation ofa range of soybean genotypes was used for diversity and virulence analyses.

Introduction

South America and the USA over the course of the last

Bangladesh is one of the countries with huge consumption

hundred years. The disease is responsible for prematureof soybean. However, the local production of soybean isdefoliation and early maturation of pod leading to large

much lower than the national demand. As a result, large

yield losses. ASR caused by P. pachyrhizi can reduceamounts of soybean and soybean products are imported

soybean yields up to 67% (Kumudini et al., 2008).

from other countries. Projections indicate that soybean

Several major resistance genes Rpp1, Rpp2, Rpp3, Rpp4,

production in Bangladesh has made spectacular progress in

Rpp5 and Rpp6 have been identified. However, thethe last few years (Shurtleff and Aoyagi, 2013). However,durability of the disease resistance genes cannot be

the actual yield is very often lower than most of thepredicted because of our lack of understanding aboutsoybean growing countries.

fundamental aspects of the fungus biology. In 2016, a

To achieve the desirable level of productivity, proper crop

survey was conducted to detect the presence of soybean

management actions should be undertaken. Until somerust disease in the hot spots of soybean cultivation inyears ago, diseases that imposed important economic

Bangladesh. Soybean rust collected from various regions

losses to Bangladeshi soybean growers were regarded as ofwere studied for their morphological and pathologicalinsignificant importance. With the global spread of thecharacterization. Characterization of local soybean rust

Asian soybean rust (ASR), caused by the fungus

will provide useful information towards designing of better

Phakopsora pachyrhizi Sydow & Sydow, the local soybean

disease management programs in the infected areas.

production system is facing a new challenge that requires

Material and Method

a new set of management skills from the soybean

A total of 23 soybean fields in Noakhali, Lakshmipur andresearchers and growers. The challenges are enormous

Bhola districts of Bangladesh were surveyed for thesince no soybean growing region is free from thepresence of ASR during April-May in 2016 (Table 1,

occurrence of the ASR and no resistant variety has yet been

Figure 1). In a single locality, soybean field was randomly

released to soybean growers. The disease was first reported

selected. Depending on the size of the field, 5 to 10 sectionsin Japan and initially limited to tropical and subtropicalof 10-20 ft row length were scouted. Sites were chosenareas of Asia and Australia, but it has spread to Africa,

throughout the field and the canopy were examined for- 99 -

symptoms of the disease. Using a hand lens, the upper andmoistened filter paper. The lower end was covered withthe lower leaf surface were looked for chlorosis or black

triple thickness of moistened filter papers. Frozen

pinpoint spots and sporulation. Ten to twenty leaves wereurediniospores of each ASR population were thawed incollected, placed them in a sealed plastic bag and brought

water bath at 39OC for 1 min and suspended in 1 mL of

to the laboratory. Each leaf let was examined under

distilled water containing 0.04% polyoxyethylene sorbitanstereomicroscope for the presence of uredinia andmonolaurate (Tween 20). Inoculation was done byurediniospores. In each filed, urediniospores werespreading spore suspension homogenously on the lower

collected in 50 ml cryogenic tubes by leaf agitation. The

surface of the leaves using a piece of filter paper.

cryogenic tubes were kept in a styrene foam box while

Inculcated leaves were kept in the growth chamber in dark

completing the collection of the day. The spores wereovernight and then cultured at 21°C under a 12-h lightbrought to the laboratory 2 days after collection and air-period of approximately 3,000 lux provided by fluorescentdried overnight at room temperature. On the following day,lamps. Sterilized distilled water was pipetted to theOspores were stored at -80 C.

moistened filter paper every 2-3 days. Following each

In order to recover the cultures of P. pachyrhizi, sporesinoculation time, aliquots (20 µl) of urediniospores

from each location were multiplied on detached leaves ofsuspension were incubated overnight on a slide glass

an ASR susceptible soybean cultivar BRS184 according tocontaining 0.5% water agar for assessment of germination

Hossain et al., (2015). Pathogenicity of three ASR isolates,of spores. Two weeks after inoculation, each leaflet was

ASR-1, ASR-18 and ASR-22 collected from Lakshimpur,

evaluated for SL and NoU per lesion. A maximum of 30

Noakhali and Bhola, respectively was performed on

lesions from each leaf let was examined undersusceptible Brazilian cultivar BRS154 and local soybean

stereomicroscope to score the SL and NoU. Finally,

cultivar BARI Soybean-5. When plants reached V3 to V4

average NoU and SL were calculated from three

growth stage (approximate 3 weeks after sowing), they

replications.

were inoculated. The optimal spore concentration used forResult and Discussion

4

inoculation of soybean plants was 5 × 10 urediniospores

Asian soybean rust, caused by P. pachyrhizi has been

per mL. Plants were first sprayed with distilled water

known to occur in the Indian Subcontinent since 1970.

containing

polyoxyethylenesorbitan

More recently, it was reported from Nigeria in 2001, Brazilmonolaurate (Tween 20) using an atomizer. After air-and Paraguay in 2002 and United States in 2004.

drying for 15 min, plant leaves were inoculated by spraying

Aerobiological models suggests that urediniospores of thethem with freshly prepared spore suspension until run-off.

pathogen are responsible for widespread dissemination of

Plants were allowed to air

the soybean rust. In our study, ASR was observed in all the

dry before being placed in a humid chamber maintaining

23 fields in Noakhali, Lakshimpur and Bhola districts

high humidity and dark conditions overnight and thensurveyed (Fig. 1; Table 1). In each field, all plants showedtransferred to a growth chamber with the same conditions

suspected rust symptoms. Infected plants were atas described previously (Yamanaka et al., 2010). Twophysiological maturity but had not senesced. The crops

weeks after inoculation, three infected leaflets were

were mostly in growth stages of R4 to R6, except in Bholadetached from each inoculated plant, and the abaxial side

where plants were in a R5-R7 stage. This may be due towas examined microscopically for examining leaf

two week delay from the original starting date of survey insymptoms and determining sporulation level (SL) and the

Bhola 2. Leaf lesions were reddish brown, irregularly

numbers of uredinia (NoU) per lesion in a maximum of 30

shaped, and were more abundant on the abaxial surface.

lesions in total, 10 lesions from each leaflet. Additionally,

Infection was most found in middle and lower part of thea set of standard soybean genotypes were used to test theplant canopy. Disease severity index ranged between 3.25

pathogenicity of ASR populations. Seeds of these varietiesand 3.60, where the lowest and highest were in Noakhali

sown in a plastic tray containing vermiculite. One week

and Bhola, respectively (Table 1). Typical erumpent

after, three plants in each genotype were transplanted topustules with a conspicuous central pore on infected leaves

pots filled with field soil. When the plants reached V3-V4

were readily apparent under the dissecting microscope.

stages, three leaflets were derived from the first trifoliate

Masses of urediniospores were expelled through the poreleaf of three independent plants. Leaflets were laid abaxial

and covered the pustules. Urediniospores were hyaline tosurface up in the trays placing the lower end on thepale yellow-brown, sub globose to ovoid and an average

0.04%

v/v

- 100 -

Bangladesh on soybean host differentials (data were notshown). Several distinct pathotypes were identified. This
Table 1 Number sites, soybean growth stage anddisease severity during survey of soybean rust in
2016
Location(District)

No. ofsites

Lakshimpur
Noakhali
Bhola

10
8
5

Plant
growth
stage
R4-R6
R4-R6
R5-R7

Mean
diseaseseverity
3.40
3.25
3.60

indicates the existence of pathogenic variation in the ASR
populations in Bangladesh. Most R genes display
specificity with regard to their effectiveness against
Fig.1 Locations of soybean fields where soybean
rust were surveyed in Bangladesh in 2016

particular pathogen race. However, some genes in crops

size of 17 to 36 × 14 to 25 μm. After germination, spore

the Avr genes and confer resistance to a wide array of

produced a long germ tube. Inoculation of susceptible

pathotypes (Ellis et al. 2014). Our study has proven thesoybean cultivar BRS154 with three ASR isolates resulted

efficacy of two major genes against diverse pathotypes ofin development of the expected tan-colored lesions and the

ASR fungus in Bangladesh, as they conferred complete

production of many uredinia with abundant sporulation.

resistance against all tested populations. These genes may

The NoU in BRS154 ranged from 2.76 to 3.93, while thehave valuable use as a source of resistance to rust isolates

SL were 2.96 to 3.00 (Fig. 2). Inoculation of local soybean

incultivar, BARI Soybean-5 with the same ASR populations

characterization of ASR pathogen in Bangladesh.

gave also susceptible infection and induced an abundant

Conclusion

have been referred as “broad spectrum” as they recognize

uredinia and uredioniospore. The NoU in BARI Soybean5 ranged from 2.71 to 3.54, while the SL were 2.94 to 3.00.
These results confirmed the presence of highly virulent P.
pachyrhizi in Lakshimpur, Noakhali and Bhola District,
Bangladesh.
Soybean rust pathogen has high evolutionary potential.
Continuous monitoring for the presence and changes invirulence are essential in order to guide the developmentof soybean varieties with durable host resistance. In this

This

is

the

first

comprehensive

In conclusion, our study successfully detected andcharacterized the ASR pathogen in the soybean hot spotsof Bangladesh. The pathogen is highly virulent andvariable which infected all cultivable varieties in the field.
Identification of most indispensable major genes against
the existing ASR pathotypes will help prioritize thedeployment R gene(s) in soybean varieties throughbreeding program. Future studies are needed to analyze
virulence diversity of ASR pathogen in consecutive years
and efficacy of gene-pyramiding lines to confer adequateand durable resistance to soybean cultivars in Bangladesh.

Acknowledgement

3This study was partly financially supported by Ministry of
Education, Bangladesh (No.-37.20.0000.004.033.013.
2015) and partly by the JIRCAS research project‘Development of technologies for the control of migratory
plant pests and transboundary diseases’

2

Reference

BRS154

5

Mean NoU and SL levels/lesion

Bangladesh.

BARI Soybean-5

4

1
0
NoU
ASR-1

SL

NoU
ASR-18

SL

NoU

SL

ASR-22

Fig.2 Mean values of number of uredinia per lesion(NoU) and sporulation level (SL) against three
ASR isolates in BRS154 and BARI Soybean-5
study, we apprised ASR pathogenic diversity in1) Hossain MM, Akamatsu H, Morishita M, Moria T,
Yamaoka Y, Suenaga K, Soares, RM. Bogadoe, AN,
Ivancovich AJG and Yamanaka N (2015) Molecularmapping of Asian soybean rust resistance in soybean
landraces PI 594767A, PI 587905 and PI 416764. Plant
Pathology, 64:147–56.
2) Kumudini S, Godoy CV, Board JE, Omielan J, andTollenaar M (2008) Mechanisms involved in soybean rustinduced yield reduction. Crop Science, 48:2334–42.
3) Shurtleff W and Aoyagi A (2013) History of soy flour,
grits and flakes (510 CE to 2013): Extensively annotated- 101 -

bibliography and sourcebook, Soyinfo Center, Lafayette,
CA, USA4)Yamanaka N, Yamaoka Y, Kato M, Lemos NG,
Passianotto ALL, Santos JVM, Benitez ER, Abdelnoor RV,
Soares RM and Suenaga K (2010) Development ofclassification criteria for resistance to soybean rust anddifferences in virulence among Japanese and Brazilian rust
populations. Trop Plant Pathol, 35:153–162.
5) Yorinori JT (2008) Soybean germplasms with resistanceand tolerance to Asian soybean rust and screening methods.
In: Kudo H, Suenaga K, Soares RM, Toledo A, eds. Facing
the challenge of soybean rust in South America. Tsukuba,
Japan: JIRCAS Working Report No. 58, 70–87.

- 102 -

OS V-4

In vitro selection of sugarcane (Saccharum officinarum L)
for Fusarium-pokah bung (Pb) resistance
NANDARIYAH and Endang YUNIASTUTI(Study Program of Agrotechnology, Faculty of Agriculture, Sebelas Maret University (UNS),
Surakarta Indonesia, E-mail: nandar.suroso@yahoo.com)

SUMMARY
The aim of the research was to obtain mutants of sugarcane callus resistant against Fusarium-Pb after treated with
Dioxane as mutagen then infected by Fusarium filtrate. The research was conducted at the Laboratory of Tissue
Culture and consisted of five steps: (1). Growing callus of three clones: Ps-59, Ps-56 and F-154, (2). Treated in
Dioxane 2 cc/l in MS medium for 21 days, (3) Treated with Fusarium filtrate: 20% and 50% concentrations.
Fusarium-Pb was prepared before by isolated of single spore, (4). Selection of resistant callus against Fusarium-Pb
filtrate, (5) Aclimatization of planlet. This experiment using a Randomized Complete Block Design (RCBD) withtreated of three sugarcane clones and each of which was repeated three times. Variables observed were number ofgrowing callus, weight and percentage of survival callus and number of planlet. Data analysis with variance andcovariance. The result showed that Dioxane concentration of 20 % give higher number of survival callus andplanlets growth than 50% after treated with Fusarium-Pb Filtrat. Sugarcane Clone Ps-56 showed the highest resistantagainst Fusarium-Pb filtrate than Ps-59 and F-154 clones.

Introduction
Sugarcane is an important agricultural commodity as
one of the nine main needs of Indonesia people. The
needs of sugar always increase but sugar production has
not fulfilled the whole national demand because
narrower sugarcane plantation and utilization of
seedling contaminated of diseases. One of the diseaseattack sugarcane is “Pokah bung” (Pb) caused ofFusarium moniliformae Sheldon. The disease has 3stage of symptoms namely Pb1, Pb2 and Pb3. Pb1 isusually identified by chlorotic symptom appearing fromyoung leaves of sugarcane. In the further development
the Pb1 can be the Pb2 that shows brownish red lines
extending into deeper cavities and bent condition. The
last stage is the Pb3 that is the death symptom ofsugarcane because the pathogen has attacked thegrowing point (Pratiwi et al, 2013).
Pratiwi et al, (2013) said that countermeasures pokahattack bung common is the use of fungicides. However,the use of this fungicide had detrimental effects on theenvironment, among others, the destruction of predatory
animals and have negative effects on the health offoodstuffs. Utilization of resistant varieties is the mosteffective method. One way that can be applied is theuse of resistant varieties and high quality. In theassembly of improved varieties of crops needed becauseof the high genetic diversity of the population that areavailable to do the selection of superior properties as
desired.
Resistant varieties of sugarcane are obtained throughconventional breeding programs through crossbreeding

and selection has produced several varieties that areresistant to pokahbung (Yunita, 2009). Breeding
methods in vitro by using somaclonal variation
combined with the mutations used to produce plants
resistant to pokah-bung. Plant tissue regeneration
results in in vitro culture is likely to have a tolerant
phenotype selection condition. This in vitro method formore efficient mengeuntukan with conditions that canbe made homogeneous, does not require space andmaterials in large quantities, and the effectiveness of thehigh selection.

Material and Method
The research was conducted in Tissue Culture
Laboratory of Biology Faculty University of Gadjah
Mada consisted of five steps: (1). Research to knowwhat clone gave the highest growing ability of callus in
Murashige and Skoog (MS) medium (Nandariyah,1990). Research arranged in Randomize Complete
Block Design with treated of three sugarcane clones:
Ps-59, Ps-56 and F-154 and each of which was repeatedthree times. (2). Increasing sugarcane variability bytreating of callus in Dioxane 2 cc/l as mutagen mixed in
MS medium for 21 days. Every clone used of 40
samples compared with control (medium without
dioxane). (3) Treating of callus to know the survival
callus against Fusarium-Pb by droped of 20% and 50%
concentrations of Fusarium-Pb filtrate. The aim of theresearch was to know resistance of sugarcane callus
against Fusarium pb filtrate. (4) Selection of resistantcallus against Fusarium-Pb filtrate, by calculated- 103 -

number of survival callus after treatment in FusariumPb filtrate. Resistant callus appearing white and clean
performed then subculture in MS medium with ad
kinetin plant growth regulator to induce planlet growth.
(5) After 2 months age planlets then transplanted insand medium contains humus. Variables observed were
number of growing callus, weight and percentage ofsurvival callus and number of growing planlets.
Calculated and analysed data with variance andcovariance analysis.

Sourchof
Variati
on
Block

Degre
es of
freedo
m
9

Clone

2

Media

1

Result and Discussions
Number of growing callus

Clone x
Media
Error

Average number of growing callus after one month
showed not significant different among three clones(Table 1).
Table 1. Variance analysis of callus growing ability
F tableSourch Degre Sum of Mean Fcal
es of Square Squar c
of
5% 1%
e
Variati freedo
m
on
Block
9
15082, 1675, 12,7 2,4 3,6
66
85
6
3
0
Clone
2
45,60
22,80 0,17 3,5 6,0
NS
5
1
Error
18
2363,7 131,3
3
2
Noted: NS = Not significant different at 5% level
Table 1 showed the growing ability of calli of threetested clones, were not different from one to the other.
Similarly for number of callus grow from in vitro
method, were not different too. This is good forvariability of the sugarcane by used mutagen dioxane.
Purnamaningsih (2006) in her research to induced
callus of Musa paradisiaca showed that the best
medium formulation for induction of callus formation
was MS + 2,4-D 2 mg/l + casein hidrolisat 3 mg/l,
while the best medium formulation for callus
regeneration was MS +BA 3 mg/l + thidiazuron 0,1
mg/l.
Increasing variability of callus was done by ad dioxaneof 2cc/l media MS compared with media without
dioxane. Dioxane is a kind of chemical mutagen thathas effected against genetically characters change like
Ethyl methane sulfonate (EMS) (Avivi, 2014).

Number of growing callus after 3 weeks
subculture in media added dioxane

Sum
of
Squar
e
2932,
00
281,0
3
24,07

Mean
Squa
re

Fcalc

265,7
8
140,5
2
24,07

12,48
6,60*
*
1,13N
S

2

Ftable
5%

1%

3,3
9
4,2
4
3,3
9

5,5
7
7,7
7
5,5
7

193,6
96,83 4,54*
3
45
958,6
21,30
0
Noted: * = Significant different at 5% level** = Significant different at 1% level
NS= Not Significant different at 5% level
Table 2 showed that there was significant different
between three clones of number of callus grow after
three weeks of subculture on dioxane was supplemented
to media MS and were significant different ininteractions between clone and media. Although media
MS added dioxane showed were not different on thenumber of callus life.The use of mutagen to increase
variation somaclonal is in line with the effort to done by
Lestari et. Al (2006). Avivi (2014) in his research toincrease variability sugarcane callus for water lodging
resistance used EMS of 0,3%. Fig 1. Showed Ps 59
callus after 3 weeks subculture on media without
dioxane.

Fig. 1. Ps 59 Callus after 3 weeks subculture on media
without dioxane.

Weight of three clone callus in media added
dioxane
The ultization of dioxane also able to raising weight ofthree clones on subculture in media added dioxane(Table 3).

Sub culture callus then following test for variability by
growing callus in medium with dioxane 2 cc/l. As
comparison used callus growing in medium without
dioxane (Table 2).
Table 2. Analysis of variance number of growing callus
after 3 weeks sub culture

Table 3. Average weight of three clone callus in media
is added dioxane after three weeks of subculture- 104 -

Degrees Sum of Mean
SourchSquareSquareofof
Variation freedomBlock90,31530 0,035000
Clone
20,15460 0,077300
Media
1
0,32269 0,322690
Clone x 2
0,00227 0,001135
Media
Error
45
0,85590 0,019000
Noted: * = Significant different at 5% level** = Significant different at 1% level

NS= Not Significant different at 5% level
FtableFcalc

Covarianceof Surviving Calli of Three
5% 1%
Clones After added By Filtrats at 20% and
50% Concentration

4,06519*
3,39 the5,57
From
analysis of covariance obtained (Table 4)
16,96589** turns
4,24 dioxane
7,77 use significantly affect the amount of
0,05967NS callus
3,39 life
5,57
and the analysis proved that the amount ofcallus life is not significantly different from each otherand there is no real interaction between clones andmedia.

Table 4. Analysis of Covariance of Surviving calli of three clones after added by filtrats at 20% and 50%
concentration
Sourch of
Variation

Degreesoffreedom

Sum of Product
Xx

xy

Yy

Sum
59
801,4
86,6
38,73
Block
9
97,73
9,43
4,4
Clone
2
152,1
1,35
0,23
Media
1
0,6
0,8
1,066
Clone x Media
2
1,9
-0,25
0,0334Error
45 549,0667
75,26 33,0003
Clone + error
47 701,1667
76,617 33,2333
Difference fortesting
adjustedclone means
media + error
46 549,6667 76,0677 34,0669
Difference fortesting
adjustedMedia means
Clone + Media+ Error47 550,9667 75,0172 33,0337
Difference fortesting
adjustedMedia means
Noted : NS= Not Significant different at 5% levelFig 2. Describe the F154 callus growth after treatment
fitrat the concentration of 50% with the overall browncolor whereas the callus nearly. Fig.3 callus Ps 59 withall parts brown color after the treatment of the filtrateconcentration of 20% and fig.4 showed F 154 on a
callus growth medium without treatment dioxane
filtrate shows the growth of plantlets.
Use of filtrate concentration of 20% and 50% realdecrease the amount of callus life. The higher
concentration of the filtrate further reduced the numberof resistant callus. By using the filtrate of Fusarium-PB
as the selection agent can be obtained subklon resistant.

- 105 -

Degreesoffreedomy adjusted for x
Sum ofMean
SquareSquareF calc

F
table

44
46

22,6826
24,86140,51552
45

2,178823,54021,0894

2,1133NS

3,39

0,8576

0,8576

1,6636NS

4,24

0,06860,1331NS

3,39

46

22,81982

0,1372

Fig 2. F154 callus after treatment fitrat theconcentration of 50%

ReferenceFig 3. Ps 59 after treatment filtrat the concentration
20%

Fig. 4. F 154 on a callus growth medium without
treatment dioxane
Jumjunidang et al. (2005) on his research showed thatconcentration of filtrate toxin fusarium culture at 60 and
40% could be used as a selection medium for in vitro
screening techniques for Musa resistance to Foc. The
higher concentration of filtrate toxin of Foc culture andfusaric acid, the faster disease incubation periode
appeared. There was recovery of attacked plant due tofusaric acid and filtrate toxin of culture Foc treatment.
This filtrate can be used as an early testing medium forresistance to fusarium wilt of banana.

Conclusion
The results of analysis showed that the growing abilityof calli of three tested clones, were not different from
one to the other. The 2 cc/l dioxane concentration could
not increase the growing ability of the calli after thetreatment with 20% and 50% of filtrate concentrations.
The higher the filtrate concentration the higher thenumber of calli became dead. The addition of 20% and
50% filtrate really influence the plantlet growth.

1) Avivi S, Sigit S, and Muhammad Afif (2014)
Selection of resistance puddle on the callus ofsugarcane(Saccharum
officinarum)
after
perlakuann EMS (Methanol Ethyl Sulfate).
Proceedings of the National Seminar, Faculty ofAgriculture, UNS 24 April 2014.
2) Birtha Niken Pratiwi, Liliek Sulistyowati, Anton
Muhibuddin and Ari Kristini (2013) Test control
pokahbung disease (Fusarium moniliformae) insugarcane (Saccharum officinarum) using
Trichoderma sp. inidigenous in vitro and in vivo.
Journal HPT 1 (3): 119-129.
3) Jumjunidang, N Nasir, Riska, H Hand (2005) In
vitro screening techniques for resistance of Musato fusarium wilt disease by using filtrate toxin
from Fusarium oxysporum f.sp. cubense culture. J.
Hort. 15 (2): 135-139.
4) Khrisnamurthi, M and J. Tlaskal (1974) Fiji
Disease Resistance Saccharum officinarum var.
Pindar Sub Clone. Tissue Culture Proc. ISSCT
15th Cong; 130-137.
5) H Lestari, Endang G, I Mariska, I Roostika and M
Kosmiatin (2006) Mutative induction and in-vitro
using fusaric acid selection to resist against
fusarium wilt disease in Banana cv "Ambon
Green".News Biology 8 (1).
6) Nandariyah (1990) Sugarcane (Saccharum
officinarum L.) for resistance against Fusarium. Pb,
selection through tissue culture. Thesis. Study
Program of Agronomy, Gadjah Mada University.
7) Ragapadmi Purnamaningsih (2006) Induction of
callus and regeneration optimization of four
varieties of rice through in vitro culture.
AgroBiogen Journal 2 (2): 74-80.
8) Vasil, I. K, WR Scowcroft, and KJ Frey (1982)
Plant Improvement and Somatic Cell Genetics.
Acad Press. New York.
9) Yunita R (2009) Utilization of somaclonalvariation and selection in vitro in assembly plants
tolerant to abiotic stresses. Journal of Agricultural
Research 28 (4).

Acknowledgement
The authors are grateful to the Sebelas Maret University
and the research team, so this research can be done well.

- 106 -

Poster Session Abstracts- 107 -

- 108 -P-1Fumonisin production recovery in a Fusarium fujikuroi strain by complementation of FUM21,
FUM6 and FUM7 genes
1
Sharmin SULTANA , Hironori KOBAYASHI1, Ryuou YAMAGUCHI1, Masafumi SHIMIZU1,
Koji KAGEYAMA2, Haruhisa SUGA3(1 Faculty of Applied Biological Science, Gifu University, Gifu, Japan; 2 River Basin Research Center, Gifu University;
3Life Science Research Center, Gifu University)

SUMMARY
A part of Fusarium fujikuroi are known to produce a carcinogenic mycotoxin, fumonisin. Sixteen clustered genes
associated with fumonisin biosynthesis (FUM cluster) are revealed in the F. fujikuroi genome. A FUM cluster was
detected not only in fumonisin producers but also in non-producers (the strains do not produce detectable level offumonisin). Therefore, fumonisin production of non-producers is still ambiguous and genetic elucidation of fumonisin
non-production is required for accurate assessment of fumonisin producibility in F. fujikuroi. The causative mutationsof fumonisin non-production was investigated with a non-producer Gfc0801001 (G1) in this study. FUM21 is one of thecause of fumonisin non-production in G1. However, a complementation by FUM21 from a fumonisin producer
Gfc0825009 (G9) failed recovery of fumonisin production in G1 and it suggested that G1 has an additional mutation(s)in the FUM cluster. In order to identify the additional mutation(s), simultaneous complementation by FUM21 andFUM6/FUM7 of G9 was conducted in G1. Fumonisin production was recovered in the transformants and these indicatethat additional mutation(s) causing fumonisin non-production in G1 is present in FUM6/FUM7 region.

Introduction

production but also for obtaining important information

Mycotoxin contamination poses threat to food chainsto prevent mycotoxin contamination by inhibition target

including crop cultivations, storages and distributions.

key gene function. In previous study, genetic mapping

Fusarium fujikuroi, an important member of Fusarium

using the crossing progenies between a fumonisinfujikuroi species complex, is the causal agent of riceproducing strain G9 and a non-producing strain G1bakanae disease. A part of F. fujikuroi produces

indicated that fumonisin non-production in G1 attributes

fumonisin that is associated with human esophageal

to the FUM cluster (北嶋 2012). PCR results indicatedcancer, leukoencephalomalacia in horse and pulmonarythat G1 retain entire FUM cluster. Though FUM21

edema in swine and its contamination in grains (Marasas

expression was detected in G1 by RT-PCR after culturing

2001; Marasas 1996; Harrison et al 1990). Fusarium

by fumonisin induction medium, lack of remaining threeverticillioides and Fusarium proliferatum are well known

FUM gene expressions suggested dysfunction of FUM21

for fumonisin production. F. fujikuroi and F. proliferatum

causes fumonisin non-production in G1. However,are sibling species which can occasionally undergo

complementation by FUM21 of G9 failed to recoversexual reproduction and produce interspecific hybrid

fumonisin production in G1 and further complementation

progeny but distinguished by molecular marker andwith FUM6, FUM7, FUM8, FUM3, FUM10, FUM11,

chemotaxonomic criteria (Leslie et al 2004; Leslie et al

FUM2 and FUM13 recovered fumonisin production in2007). They are considered as potential fumonisin

G1. These results suggested additional causative

producers on rice (Cruz et al 2013). Not all F. fujikuroi

mutation(s) to FUM21 is present in the FUM cluster inproduce fumonisin. Information of the genes affecting

G1. Objective of this study is clarification of the

fumonisin production can be a target for chemicalcausative mutation(s) of fumonisin non-production indevelopment to inhibit this mycotoxin production.

G1.

The aim of this study is elucidation of the genetic
mutations critical for fumonisin production in F. fujikuroi.

Material and Method

This is not only for specifying key genes for fumonisin- 109 -

Confirmed transcription of some FUM gene of G1






and G9 by RT-PCR.

Transcription of Histone H3, FUM21, FUM1, FUM6,

Plasmids were prepared by cloning the objective

FUM8 and FUM10 in G1 and G9 were confirmed bygene from G9 and point mutation of plasmids.

RT-PCR. Amplified DNA with expected size of all genes

FfT67FUMKOD-1

tested were detected in G9 but only it was detected forand

FfT21FUMKOD-2,transformants of G1 those carried FUM6/FUM7

HistoneH3, FUM21 and FUM1 were in G1 (Fig. 2A andand FUM21 of G9 respectively were used for

2B). Lack of transcription of multiple FUM genes

furtherwassuggested dysfunction of the transcription factor FUM21

performed according to Suga et al (2016) andin G1 because fumonisin production recovery was failed

created twenty transformants.

by complementation of FUM21 in G1 (小林 2013) (Fig.

Fumonisin production recovery of transformants

3).

transformation.

Transformation

was detected by ELISA by culturing in corn
medium for 10 days in 25º C and extracting
fumonisin with 75% methanol. Then, fumonisins inthree of each transformant series including previous
studies ( 小 林2013) were quantified by

LC-MS/MS analyses.

Result and Discussions
Sequence comparison of FUM genes between G1 and
G9
The distribution and the direction of each FUM gene in

Fig. 2 RT-PCR result of (A) Gfc0825009, (B)G1 are the same as in G9. High sequence homology

Gfc0801001(97.3-99.8 %) was observed in FUM genes between G1

and

(C)

FfT21FUMKOD(#2).

RT-PCR product was subjected to 2 % agarose gel

and G9 (data not shown). One substitution and 11 amino

electrophoresis. (+) means that expected size ofacid deletion at C-terminal were found in FUM21. These

RT-PCR product was detected, (-) not detected.

mutations suggested dysfunction of FUM21 in G1 (Fig.
1)

FUM gene complementation by transformation
Fumonisinproduction

recoverywasfailedbycomplementation of only FUM21 but it was succeededby complementation of FUM21 and FUM6/ FUM7/
FUM8/ FUM10/ FUM11/ FUM2 FUM13 (小林 2013,
FfDTFUM21_6_13 in Fig. 3). In order to identify whichof g.888G>C (p.D261H) or g.2551G>T (p.G678*) (Fig.
1) is the causative mutation of fumonisin non-productionin G1, complementation of FUM21 with either of thesepoint mutation was conducted in G1.The plasmid thatcarry a point mutation at 888th (pDT21G888C-1) and
2551th (pDT21G2551T-2) nucleotide in FUM21 of G9
was created and transformed into FfT67831011213-30 (atransformant of G1 carrying FUM6/ FUM7/ FUM8/
FUM10/ FUM11/ FUM2/ FUM13 regions of G9) (Fig. 3).
Fig. 1 Comparison of the amino acid sequences of

None

FUM21 of fumonisin producing strain (upper)

production

Gfc0825009(FfDTFUM21G2551T_6_13 in Fig. 3) while fiveandGfc0801001 (lower).

fumonisin

non-producing

of

the

transformants
inoffumonisin

pDT21G2551T-2

transformants recovered fumonisin production in case ofpDT21G888C-1

RT-PCR of FUM genes

case

recovered(FfDTFUM21G88C_6_13

Fig.

3).

These results suggested that g.2551G>T (p.G678*)- 110 -

rather than g888G>C (p.D261H) is one of the cause oftransformant of G1 FfT21FUMKOD (#2) in thatfumonisin non-production in G1 (Fig. 1). We sequenced

functioning of integrated FUM21 was confirmed by

the terminal portion of FUM21 of additional threetranscription of FUM6, FUM8 and FUM10 in G1 (Fig.

fumonisin non-producing strains (GL-24, Gfc0625008

2C).

and Gfc1034001) and three fumonisin producing strainstransformant series were also created by transformation(Gfc0821004,

revealof FfT67FUMKOD (#1) with plasmid carrying FUM21

specificity of g.2551G>T substitution in fumonisinof G9 (FfDT21T67KOD-1 in Fig. 3). Fumonisin

non-producing strains (data not shown) and observed

production was recovered in both transformant series

that(Fig. 3). Multiple mutation(s) causing non-production ofall

Gfc0009063

fumonisin

and41-79)

non-producing

tostrains

carriedFUM21/

FUM6/

FUM7

complementary

secondary metabolite in the biosynthetic gene cluster

g.2551G>T (p.G678*) in FUM21 as G1.
In order to identify the additional mutation(s) in

were reported as aflatoxin non-production in Aspergillus

FUM6/ FUM7/ FUM8/ FUM3/ FUM10/ FUM11/

oryzae(Tominaga

FUM2/ FUM13, simultaneous complementation of

non-production in F. proliferatum (Malonek et al 2005).

FUM21 and FUM6/FUM7 regions was conducted in G1.

These results suggested that mutations causing fumonisin

Previous studies showed that fumonisin production

non-production in G1 were distributed in FUM21, FUM6

recovery was failed by independent complementation ofand FUM7.

FUM21 and FUM6/FUM7 regions in G1 (Fig. 3).

The plasmid carrying FUM6/FUM7 region of G9
wastransformed

into

a

FUM21

Conclusion

complementary- 111 -

et

al

2006)

and

gibberellin

B. A. (2004). Interfertility of two mating populationsin the Gibberella fujikuroi species complex.
European Journal of Plant Pathology 110: 611-618.
6.

Leslie J. F., Anderson L. L., Bowden R. L. and Lee
Y-W. (2007). Inter- and intra-specific genetic
variation in Fusarium. International Journal of Food
Microbiology 119: 25-32.

7.

Malonek S., Rojas M. C., Hedden P., Hopkins P. andTudzynski B. (2005). Restoration of gibberellin
production in Fusarium proliferatum by functional
complementation of enzymatic blocks. Applied andEnvironmental Microbiology 71: 6014-6025.

8.

Marasas W. F. O. (1996). Fumonisins: history,
worldwide

occurrence

and

the

impact.

In:

Fumonisins in food (Jackson L. S. D., Vries J. W.
and Bullerman L. B. eds): 1-17. Plenum Press.

Acknowledgement
A grant from Ministry of Agriculture, Forestry and9.

Marasas W. F. O. (2001). Discovery and occurrenceofFisheries of Japan (Research project for improving foodtheahistoricalperspective.

Environmental Health Perspectives 109: 239-243.

safety and animal health).

10. Suga

Reference

fumonisins:

H.,

Kageyama

K.,

ShimizuM.

andHyakumachi M. (2016). A natural mutation
1.

Cruz A., Marín P., González-Jaén M. T., Aguilar K.

involving both pathogenicity and perithecium

G. I. and Cumagun C. J. R. (2013). Phylogenetic

formation in the Fusarium graminearum species

analysis, fumonisin production and pathogenicity ofcomplex. G3 6: 3883-3892.

Fusarium fujikuroi strains isolated from rice in thePhilippines. Journal of Science of Food and

11. Tominaga M., Lee Y-H. Hayashi R., Suzuki Y.,
Yamada O., Sakamoto K., Gotoh K. and Akita O.

Agriculture 93: 3032–3039.

(2006). Molecular analysis of an inactive aflatoxin
2.

Harrison L. R., Colvin B. M., Green J. T., Newman

biosynthesis gene cluster in Aspergillus oryzae RIB

L. E. and Cole J. R. (1990). Pulmonary edema andstrains. Applied and Environmental Microbiologyhydrothorax in swine produced by fumonisin B1, a

72: 484-490.

toxic metabolite of Fusarium moniliforme. Journalof Veterinary Diagnostic Investigation 2: 217-221.
3.

北嶋美葉 (2012). Fusarium fujikuroi におけるフモニシン産生遺伝子の発現解析. 岐阜大学大学
院応用生物科学研究科 修士論文: 1-43.

4.

小林弘典 (2013). 遺伝子導入による Fusarium
fujikuroi のフモニシン産生能の回復. 岐阜大学
応用生物科学部 学位論文: 1-22.

5.

Leslie J. F., Zeller K. A., Wohler M. and Summerell- 112 -

P-2
Genetic mapping of chromosome No.1 region associated with pathogenicity
in Fusarium head blight pathogen
1
Rina OKUMURA , Maho IKAWA1, Yuki HIRATA1, Masafumi SHIMIZU1, Koji KAGEYAMA2,
Haruhisa SUGA3(1Faculty of Applied Biological Sciences, 2River Basin Research Center, 3Life Science Resarch Center,
Gifu University）

SUMMARY
Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens causing Fusarium
head blight (FHB) in wheat and barley worldwide. A natural pathogenicity mutant (Fusarium asiaticum strain 0233007)
was found in a sample of the Fg complex collected in Japan. The mutant strain did not induce symptoms in wheat
spikes beyond the point of inoculation, and did not form perithecia. No segregation of phenotypic deficiencies occurredin the progenies of a cross between the mutant and a fully pathogenic wild-type strain, which suggested that a single
genetic locus controlled both traits. The locus was mapped to ca. 200 kbp region in the chromosome No.1 by using
sequence-tagged markers. This suggested the gene involving pathogenicity and perithecium formation is present in thisregion.

Introduction

susceptibility to FHB. Each plant was inoculated about

In previous studies, several F. asiaticum strains that102 conidia added to the wounded lower spikelet. The

show non-pathogenicity to wheat and non-perithecium

disease symptoms were evaluated 14 days after

formation have been discovered in Japanese collection.

inoculation. Pathogenicity was scored as pathogenic or

One of the strains, 0225022 lacks ca. 2 kbp on

non-pathogenic based on whether the symptoms werechromosome No.2 and loss of FGSG02810 gene in that

confined in the initially inoculated spikelet, as forregion causes lost of both traits (Suga et al 2016). On the0233007, or whether they spread to the neighboringother hand, another F. asiaticum strain 023307 withflorets, as for 0407011 (Fig.1).

non-pathogenicity and non-perithecium formation has no

Perithecium formation ― Perithecium formation wasdeletion on chromosome No.2, and introduction of

assayed on carrot agar plates (Klittich and Leslie 1998)

wild-type FGSG02810 gene by transformation failed

as described above, expect that the mycelial plug ofrecovery of both traits in the strain 0233007. Therefore,

progeny was placed in the center of the plate.

the causative mutation in this strain is expected to be

Perithecium formation was observed until 3 weeks after

different from that of 0225022. In previous studies, itaerial mycelium knockdown.

was estimated that the causative genomic region in thisDNA extraction and PCR―Genomic DNA for PCR was

strain is approximately 3,480 kbp (between VNHK909

extacted from 3-day-old mycelium cultured on potato

and VHNK917 markers) on chromosome No.1 (井川dextrose broth (Suga et al 2008). The final DNA pllet

2016). Further mapping was performed in this study.

was dissolved in 200 µl of water.

Material and Method

Mapping―For setting markers at arbitrary positions, theStrain ― The wild-type strain F. graminearum s. str.

National Center for Biotechnology Information (NCBI)

0407011,

perithecium

website was used. A gene with more than one intron

formation, and the mutant-type strain F. asiaticum

showingaround in the target region was selected and PCR-RFLP

0233007,

andmarker was developed. Data obtained with PCR-RFLP

non-perithecium formation, were used (Suga et al 2008).

markers were processed using JoinMap®4 to detect a

Forty progenies obtained by their crossing (⑩67P7101-

significant⑩67P7147 in 井川(2016)) were also used.

perithecium formation; the markers were processed using

Pathogenicity

pathogenicity

showing

test ― The

andnon-pathogenicity

pathogenicity

test

was

association

betweenlogarithm of odds (LOD) scores.

performed using wheat cultibar Apogee, which has high- 113 -

pathogenicity

andResult and Discussions

Mapping―Nineteen progenies were analyzed with 27

Pathogenicity test―In 0407011, whole spike whitenedmarkers. Six markers (VNHK909 ～ VNHK917) inafter 14 days, whereas in 0233007, symptom waschoromosome No.1 showed high LOD values (井川observed only in the spike injected with the spore

2016). Therefore, PCR-RFLP markers (PRHS857/Hae ,suspension or only in the adjacent spike 14 days after the

PRHS859/XhoI,

inoculation (Fig.1). Twenty two progenies showed

PRHS865/RsaI, PRHS867/RsaI, PRHS869/Hpa ) was

pathogenicity,

set to be approximately equal positions between the

and

18

progenies

showednon-pathogenicity.

PRHS861/Hinc ,PRHS863/AluI,

markers. Among these, the PRHS859/XhoI marker
showed the highest LOD value (LOD=11.95). Then,additional PCR-RFLP markers were developed for the
middle region between PRHS859/XhoI and adjacent
PCR-RFLP markers and obtained LOD value. This task
was repeated and finally, four markers (PRHS892/NsiI,

04070110233007
Fig. 1 Result of pathogenicity test of the parent

PRHS859/XhoI, PRHS894/MseI, PRHS896/HhaI) withperfect match between marker type and phenotype in thestrains

40 progenies were obtained (Fig.3). According to thePerithecium formation―A parent strain 0407011 and 22

information of chromosome No.1 of F. graminearum s.

progenies showed perithecium formation. A parent strainstr.

0233007 and 18 progenies showed non-perithecium

HG970332.1, total length 11,760,950 bp), there are 82

formation (Fig.2). Perithecia formation ability andgenes in ca. 200 kbp from PRHS890/MseI to PRHS

pathogenic

886/HaeIII

strains

werecompletely

identical

inprogenies.

strainPH-1

onadjacent

NCBI

to(accession

PRHS892/NsiI

numberand

PRHS896/HhaI, respectively.

04070110233007
Fig. 2 Result of perithecium formartion assay of theparent strains

Fig. 3 Mapping of non-pathogenicity and non-perithecium formation in the mutant strain 0233007
The result of a part of progenies is indicated above. The logarithm of odds (LOD) value obtained by analyzing 40
progenies and the position in chromosome No.1 of each maker were indicated below. White and black circles on lines
indicate the 0233007 and 0407011-type allele, respectively; white and black circles next to the progeny’s nameindicate mutant phenotype (non-pathogenic and non-perithecium formation) and wild-type phenotype (pathogenic
and perithecium formation), respectively. The marker within the vertical dotted lines showed a perfect match between
allele-type and phenotype in the 40 progenies.

- 114 -

Conclusion
It was presumed that a gene involving pathogenicity andprithecium formation is present in ca. 200 kbp inchromosome No.1 by linkage analysis using 40
progenies. There are 82 genes in this region. In the future,gene complematation analysis would be able to identify
the gene from them.

Acknowledgement
This work was supported by JSPS KAKENHI Grant
Number JP16K07612.

Reference
1) 井川 真帆 (2016). ムギ類赤かび病菌における病原力関連ゲノム領域のマッピング. 岐阜大学応用生
物科学部 学位論文: 1-21.
2) Klittich C. J. R. and Leslie J. F. (1988). Nirtate
reduction mutants of Fusarium moniliforme (Gibberella
fujikuroi). Genetics 118: 417-423.
3) Suga H., Karugia G. W., Ward T., Gale L. R.,
Tomimura K., Nakajima T., Miyasaka A., Koizumi S.,
Kageyama K. and Hyakumachi M. (2008). Molecularcharacterization of the Fusarium graminearum species
complex in Japan. Phytopathology 98: 159-166.
4) Suga H., Kageyama K., Shimizu M. and Hyakumachi
M (2016). A natural mutation involving both
pathogenicity and perithecium formation in the Fusarium
graminearum species complex. G3: Genes, Genomes,
Genetics 6: 3883-3892.

- 115 -

- 116 -P-3
Isolation of plant probiotic Bacillus spp. from tea rhizosphere
Nusrat Ahsan1, Tomoki Nishioka1, Haruhisa Suga2, Hiroyuki Koyama1,
Masafumi Shimizu1(1 Faculty of Applied Biological Sciences, Gifu University; 2 Life Science Research Center, Gifu University)

Abstract
The aim of this study was to isolate and characterize rhizosphere spore-forming bacteria with plant probiotic activities.
Plant probiotic is a general term for microbes which exert health promoting and nutritional benefits on plants.
In this study, a total of 80 isolates of heat-tolerant spore-forming bacteria were isolated from rhizosphere soil of organicallycultivated tea plants. Phylogenetic analysis based on 16S rRNA gene sequences showed that 78 of them belonged to genus
Bacillus and remaining 2 isolates belonged to genus Lysinibacillus. In order to evaluate the plant probiotic potential, the bacteria
were screened in vitro for the siderophore production, indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1carboxylate (ACC) deaminase production, phosphate solubilisation, and antagonistic effect against Sclerotinia sclerotiorum. The
results showed that all isolates produced IAA, ranged from 0.2 to 12.7 μg/ml, in the presence of L-tryptophan. The ability tosolubilize tricalcium phosphate was displayed by 67 isolates of Bacillus spp. and 1 isolate of Lysinibacillus sp. (GUCS32).
Seventy two isolates of Bacillus spp. and the isolate GUCS32 of Lysinibacillus exhibited the ability to produce siderophore.
Additionally, 71 isolates of Bacillus spp. and Lysinibacillus isolate GUCS32 were found to have antagonistic activity against S.
sclerotiorum. On the other hands, none of the isolates have an ability to produce ACC deaminase.
For further in planta experiments, seven Bacillus isolates (GUCS 17, GUCS 18, GUCS 22, GUCS 42, GUCS 58, GUCS
70, and GUCS 78) and Lysinibacillus isolate GUCS32 were randomly selected. The plant growth promotion study showed thatthe inoculation of these isolates significantly increased shoot dry weight of spinach seedlings under climate chamber conditions.
Furthermore, the inoculation of soil with the isolates GUCS 22, GUCS 32, GUCS 42, GUCS 58, and GUCS 78 reduced theseverity of cabbage damping-off caused by S. sclerotiorum. The highest reduction (63%) was recorded for the isolate GUCS42
which was most closely related to the type strain of Bacillus cereus.
In order to understand the importance of chemotaxis in the growth promoting and biocontrol behaviours of the selectedbacterial isolates, their chemotactic activity towards oxalic acid, which was known to be produced by spinach and S. sclerotiorum,
was investigated by a capillary assay. However, none of the isolates exhibited chemotaxis towards oxalic acid.
This study demonstrated that a selected isolate GUCS42 could be used for the development of a novel plant probiotic
product having growth promoting and disease suppressive effects.

- 117 -

- 118 -P-4
Microbial basis of Fusarium wilt suppression by Allium-cultivated soils
Tomoki NISHIOKA1, Malek MARIAN1, Haruhisa SUGA2, Masafumi SHIMIZU1(1The United Graduate school of Agricultural Science, Gifu University;
2Life Science Research Center, Gifu University)

Abstract
Crop rotation and intercropping with Allium plants has been reported to suppress the incidence of Fusarium wilt (FW) in various
crops caused by Fusarium oxysporum. However, the mechanisms responsible for FW suppression in Allium-cultivated soil have not been
fully elucidated. In this study, we revealed the underlying mechanism of the FW suppressiveness of Allium-cultivated soil using culturedependent and independent approaches.
The Allium-cultivated soils exhibited an inhibitory effect on cucumber FW (CFW) caused by F. oxysporum f. sp. cucumerinum(Focu). However, the suppressive effects of cultivated soils were drastically eliminated by soil pasteurization at more than 60C. These
results suggest that gram-negative bacteria play a key role in the FW suppression by Allium-cultivated soils. Furthermore, thecharacterization and comparison of rhizobacterial communities by 16S rRNA amplicon sequencing revealed that four gram-negative genera,
Burkholderia, Pseudomonas, Flavobacterium, and Chryseobacterium, were the predominant groups that preferentially accumulated in therhizospheres of Allium plants. The isolates of Burkholderia, Pseudomonas, and Flavobacterium recovered from rhizosphere soils of Allium
plants tended to exhibit suppressive effects against CFW. Therefore, we speculate that the accumulation of these antagonistic gram-negative
bacteria plays a key role in FW suppression by Allium-cultivated soil.
Several Burkholderia and Pseudomonas isolates recovered from Allium rhizosphere soils exhibited antagonistic activity toward Focu onagar medium. In comparison with non-antagonistic Burkholderia and Pseudomonas isolates, antagonistic isolates showed significantly
higher suppressive effects on CFW, suggesting that the CFW suppressiveness of these antagonistic isolates may be attributed to the
production of antifungal compounds.

Acknowledgments
This work was supported by a Grant-in-Aid for Young Scientists (B) (grant number 24780317) and for JSPS Fellows (grant number
953381) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

- 119 -

- 120 -P-5
Isolation of novel deoxynivalenol-degrading microorganisms from Poaceae planted soils
Hiroyuki MORIMURA, Sotaro CHIBA, Daigo TAKEMOTO, Kazuhito KAWAKITA, Ikuo SATO
(Graduate School of Bioagricultural Sciences School of Agricultural Sciences, Nagoya University)

SUMMARY
The mycotoxin deoxynivalenol (DON) is produced by several plant pathogen Fusarium species and is the result of
Fusarium Head Blight (FHB) which is known as worldwide serious problems in cereal crops. A biological control for the
degradation of DON using DON-degrading microorganisms is seem as the one of remarked method in livestock stage,
although the species of DON-degrading microbial have been isolated limitedly and known slightly where they habit. In
this study, we isolated some DON-degrading microbes from Poaceae (Oryza sativa, Eleusine indica, Avena sativa) planted
soils. Collected samples were inoculated into mineral salt medium with 100 µg mL-1 DON and inoculated media were
cultured several times. The concentration of DON in the media were monitored by using HPLC. The media culture
decreasing DON was selected and plated onto 3-fold-diluted R2A gellan gum. Finally, we succeeded in isolate three
microbes degrading DON from the randomly selected microbial grown on the gellan gum media.

Introduction

which are able to degrade DON from Poaceae (Oryza

The several plant pathogen Fusarium species infect a lot

sativa, Eleusine indica, Avena sativa) planted soils.

of cereal crops and cause Fusarium Head Blight (FHB).

Material and Method

FHB is known as the one of most hazardous plant disease

Fig. 1 describes a detail process of enrichment culture. To

around the world and accumulates several mycotoxin

isolate DON-degrading microbe, a variety of samples were

deoxynivalenol (DON), and results in huge economic

collected from natural environment. The Poaceae plants

losses. For example, the wheat and barley losses in U.S.A

including these leaves, stems, spikelets and these planted

and Canada during the 1990s are estimated in about $3

soils, were mainly collected for isolating, because there isbillion and $220 million respectively (10). DON, apossibility to capture DON-degrading microbes for thesecondary metabolite produced by the pathogens is notreason that the many microbes have already been isolated

only essential to promote the infection spread against hostfrom wheat or rice planting environments. For enrichment

plants, but also poisonous toward eukaryotes (1, 6, 7, 9).

of the microbes, approximately 0.03-0.04g of the collected

The main toxicity of DON is to inhibit the protein synthesis

each samples was suspended in 600-800µL mineral saltcaused by binding the ribosome in the cell both humans

medium (MM) (4, 8) containing 100 µg mL-1 DON as aand crops (1, 6, 7, 9). Ingesting DON during long periodcarbon source. And the cultures were incubated in a shaker

results in serious symptoms included vomiting, diarrhea,

with the condition of shaking 150rpm and temperature 28

leukopenia, hemorrhage (6). These reasons force us tofor 7 days. After that, 6-8µL of the cultures was added into

protect our cereal livestock from the pathogen producing

600-800µL same medium and the process was repeated 2-

DON by using sustainable and effective methods. A

4 times.

biological

DON-degrading

In order to determine whether the microorganism

microorganisms and these microbe’s enzymes are drawing

degraded DON in the cultures or not, the cultures filteredattention as a way to solve the contamination problem inthrough 0.45-µm membrane were analyzed by HPLC.

managementusing

food supply chain. Our laboratory has 16 strains, of DON
degradingbacteria

including4

Devosiaspp.,

Sphingomonas sp., Marmoricola sp. and 10 Nocardioides
spp. that have been isolated from a variety of environment
samples (3, 4, 5, 8) although the information about DONdegrading microbial is limited. Therefore, we triedexperiment with isolating new promising DON-degrading
microorganisms. As a result, we isolated 3 microorganisms

Fig. 1 Enrichment culture of DON-degrading microorganism.

- 121 -

The culture samples with decreasing DON wereDON

selected and inoculated on 3-fold-diluted R2A gellan

IMS

gum (4). And then, the colonies grown on the media
were randomly selected and inoculated to MM with 100µg mL-1 DON. The inoculated MM were analyzed by
HPLC to determine DON decrease after 7 days
incubation as described previously (8).

Result and Discussions
Table 1. Microbes of isolated from Poaceae planted soils.
StrainIsolation soil
Location
Date
IMS
Oryza sativa
Mie
13.06.2017
KOM Eleusine indica
Saitama
07.10.2017
HEM
Avena sativa
Hokkaido
07.09.2017

KOM

culture among 212 samples collected from Poaceae
planted environments including 77 leaves, 37 stems, 68
spikelets and 30 soils. We isolated 3 DON-degrading
microbial strains from the 3 out of the 8 cultures
originated from soils planted Oryza sativa, Eleusine
indica and Avena sativa named IMS, HEM and KOM,
respectively. (Table 1). Figure 2 showed the HPLC

Absorbance (mAu)

There were 8 cultures that decreased DON in theHEMelution profiles of DON and its DON metabolites
expected. The existence of DON was identified by thepeak at 4.0 min in MM containing 100 µg mL-1 DON.
The profiles of both microorganisms named IMS andMM with

KOM showed same peak at 9.1 min, but the profile of

100 µg mL

HEM showed unique peak at 8.5 min. The HEM also

DON

-1

demonstrated peaks at 3.6 min around DON peak.
And common peaks were indicated at 1.8 and 2.4 minin all treatments, but the peaks increased in DONdegrading microbial treatments, so this result suggests
unidentified compounds and the components of MM be
mingled. These above peaks illustrated there arepossible to be metabolites of microbes although further
analyses are needed.
In fact, 5 samples of enrichment cultures other the 3
DON-degrading microbes have could decompose DON.

Retention time (min)This result implies more DON-degrading microbes will

Fig. 2 DON-degrading by microorganisms derived frombe unveiled from Poaceae plants. But, these 5 samples

Poaceae planted soils. This figure shows HPLC profiles ofwere not isolated what microbe had the capacity of

DON and DON metabolites in MM containing 100 µg mL

degrading DON. As a reason for that there is possible-1

DON with IMS (first panel) and with KOM (second panel) andthat a few of DON-degrading microbial increased on thewith HEM (third panel) and with neither (forth panel) from top

media because of multiplying more slowly than otherto bottom. The white arrows indicate unidentified compounds

microbes in the MM, or the solid media were bad

except for only MM. The black arrow indicates unidentified

condition for the microbial colonization. In addition,

compounds or the components of MM. The bar shows the peakanother mycotoxin degradation by two bacteria wasof DON.

already reported (2), so it is necessary to think about the- 122 -

degradation of consecutive action of several bacteria. Thus,
the unknown microbes in the samples should be carefullysought.
Our research group has 16 strains bacteria containing 4
Devosia spp. Sphingomonas sp. Marmoricola sp. 10
Nocardioides spp. which are isolated from wheat or rice
plants and aquatic environment (3, 4, 5, 8). The
Marmoricola sp. strain MIM116 derived from wheat heads
has been reported already as it is able to remove DON on
the wheat kernel (4). The report suggested it be capable todecrease DON in harvested wheat grain affected by FHB
with applying other DON-degrading bacteria. Moreover,Sphingomonas sp. strain KSM1 derived from lake water
has the ability for both DON and another mycotoxin
nivalenol (NIV) to assimilate these mycotoxins (5). This
study indicated DON-degrading bacteria exist in a wide
variety of environments and unknown will be newly
discovered in the future.
Finally, we should continue exploring new unidentified
DON-degrading microorganisms. And so, we consider
hereafter microorganism using method establishing assustainable and effective way.

Conclusion
We present the isolation of novel 3 DON-degrading
microorganisms from Poaceae including Oryza sativa,

3) Ikunaga Y, Sato I, Grond S, et al., 2011. Nocardioides
sp. strain WSN05-2, isolated from a wheat field, degrades
deoxynivalenol, producing the novel intermediate 3-epideoxynivalenol. Applied Microbiology and Biotechnology
89, 419-427.
4) Ito M, Sato I, Koitabashi M, Yoshida S, Imai M,
Tsushima S, 2012. A novel actinomycete derived from
wheat heads degrades deoxynivalenol in the grain of wheat
and barley affected by Fusarium head blight. Applied
Microbiology and Biotechnology 96, 1059-1070.
5) Ito, M., Sato, I., Ishizaka, M., Yoshida, S., Koitabashi,M., and Tsushima, S. 2013. Bacterial Cytochrome P450
System Catabolizing the Fusarium Toxin Deoxynivalenol.
Applied and Environmental Microbiology 79:1619-1628.
6) Kazan K, Gardiner Dm, Manners Jm, 2012. On the trail
of a cereal killer: recent advances in Fusarium
graminearum pathogenomics and host resistance.
Molecular Plant Pathology 13, 399-413.
7) Pestka Jj, 2010. Deoxynivalenol: mechanisms of action,
human exposure, and toxicological relevance. Archives of
Toxicology 84, 663-679.
8) Sato I, Ito M, Ishizaka M, et al., 2012. Thirteen novel
deoxynivalenol-degrading bacteria are classified within
two genera with distinct degradation mechanisms. Fems
Microbiology Letters 327, 110-117.
9) Vanhoutte I, Audenaert K, De Gelder L, 2016.
Biodegradation of Mycotoxins: Tales from Known andUnexplored Worlds. Frontiers in Microbiology 7, 20.
10) Windels Ce, 2000. Economic and social impacts ofFusarium head blight: Changing farms and ruralcommunities in the Northern Great Plains. Phytopathology
90, 17-21.

Eleusine indica, Avena sativa, planted soils. This result
implies more DON-degrading microbes will be unveiled
from Poaceae plants. Furthermore, it is possible to exist
some DON metabolites by the microorganisms. However,
the information of DON-degrading microorganisms islimited. Therefore, we ought to run the experiment withsearching DON-degrading microorganism from a large
variety of environments and analyzing the compounds
produced by the microorganisms.

Acknowledgement
This work was supported by JSPS KAKENHI Grant
Number 17K15233. We are grateful to Prof. Seiya
Tsushima (Tokyo Agricultural University, Japan), Dr.
Motoo Koitabashi (NARO, Japan), Dr. Shigenobu Yoshida(NARO, Japan) and Dr. Michihiro Ito (Ryukyu University,
Japan) for valuable suggestions and for technical supports.

Reference
1) Audenaert K, Vanheule A, Hofte M, Haesaert G, 2014.
Deoxynivalenol: A Major Player in the Multifaceted
Response of Fusarium to Its Environment. Toxins 6, 1-19.
2) Heinl S, Hartinger D, Thamhesl M, et al., 2010.
Degradation of fumonisin B-1 by the consecutive action of
two bacterial enzymes. Journal of Biotechnology 145, 120129.

- 123 -

- 124 -P-6
Plant growth-promoting traits of rhizospheric Flavobacterium and Chryseobacterium
Fumiya MIZUTANI1, Tomoki NISHIOKA2, Haruhisa SUGA3, Koji KAGEYAMA4, Masafumi
SHIMIZU1(1Faculty of Applied Biological Sciences, Gifu University, 2The United Graduate school of Agricultural Science, Gifu
University, 3Life Science Research Center, Gifu University, 4River Bacin Research Center, Gifu University)

Introduction
Recent advances in molecular techniques to study microbiota has led to considerable progress in understanding therhizosphere bacterial communities. The phylum Bacteroidetes is one of the abundant bacterial lineages in the rhizospheresof various plants. Among them, the family Flavobacteriaceae, especially genera Flavobacterium and Chryseobacteium,
were found to be the most abundant bacteria in the rhizospheres of a wide variety of plants. Accordingly, we hypothesizedthat Flavobacterium and Chryseobacteium species might play important roles in the health and development of host plants.
However, it was difficult to analyze the function of Flavobacterium and Chryseobacteium species because there was nosuitable medium for the isolation of these bacteria. Recently, we developed the optimized medium for the isolation of
Flavobacterium and Chryseobacterium species from rhizosphere soil (PSR2A-C/T medium, Nishioka et al., 2016). In thepresent study, we isolated rhizospheric Flavobacterium and Chryseobacterium species with PSR2A-C/T medium, andevaluated their plant growth-promoting traits.

Materials and Methods
Bacteria were isolated from rhizosphere soil of pot-grown Welsh onion (Allium fistulosum L.) and onion (A. cepa
L.) by dilution plating on PSR2A-C/T medium. Yellow or orange colored colonies were purified and then identified by
16S rRNA gene sequence analysis to ascertain their taxonomic positions. Subsequently, bacterial isolates were evaluatedin vitro for their plant growth-promoting traits like production of indole acetic acid (IAA), siderophore, 1aminocyclopropane-1-carboxylate (ACC) deaminase, hydrogen cyanide (HCN), and phosphate solubilization.

Results and Discussion
In this study, six isolates of Flavobacterium and 37 isolates of Chryseobacterium were successfully obtained fromthe rhizosphere soils of Welsh onion and onion. By phylogenetic analysis based on 16S rRNA gene sequences, all
Flavobacterium isolates were grouped into one clade containing F. anhuience which was originally isolated from fieldsoil. On the other hands, Chryseobacterium isolates were fell into five distinct clades containing various species that wereisolated from soils, freshwater lake and waste water. Results of in vitro assays showed that all of the isolates did not haveabilities to solubilize insoluble phosphate, produce siderophore, ACC deaminase and HCN but produced IAA in liquidmedium, ranged from 0.2 to 5 μg/ml. IAA-producing rhizobateria are known to directly promote plant growth. Therefore,
this study may underline the importance of rhizospheric Flavobacterium and Chryseobacterium species in thedevelopment of plants under natural environments.

- 125 -

- 126 -P-7
Establishment of global Phytophthora database for quarantine control
Ayaka Hieno1, Mingzhu Li2, Kayoko Otsubo1, Haruhisa Suga3 and Koji Kageyama1(1River Basin Research Center, Gifu University; 2College of Life Science, Shaanxi Normal University; 3Life Science
Research Center, Gifu University)

Abstract
Plant pathogenic Phytophthora species cause destructive
diseases in agriculture and forest worldwide. For
effective quarantine control of Phytophthora species,
alternative detection methods based on accurate databaseare highly required. For development of quarantine
control, there is a problem that the type isolates of some
Phytophthora species remain unclear and complicated,for example, two different isolates are enlisted as type of
one species. Therefore, we established an accurate list of
type strains based on new species description papers, andreliable web databases, such as Phytophthora database(http://www.phytophthoradb.org/), and Q-bank(http://www.q-bank.eu/Fungi/) (Fig. 1). According to thelist, we collected DNA sequences of 8 genomic regions,rDNA-ITS, rDNA-LSU, β-tubulin, elongation factor 1α,
cox1, cox2, cox spacer, and Ypt1 from public database. In

N

P

3

2

1

TobaccoPeriwinkle
4
5

3

2

1

Ivy

CBS-KNAW, WPC and ATCC for analyzing sequences.

N

researchers and the public bioresource institutes such asPtype strain, we collected the ex-type isolates from the3case that there is no available sequence information in

Tomato1Hydrangea
2

Fig. 1 Coverage of culture, DNA and sequence
data of type strain

The coverage of the sequence data reached 75.7~98.5%
in 8 genomic regions (Fig. 1).
Based on the collected DNA sequences, we selected Ypt1
gene for designing genus specific PCR primer, one
primer pair was selected from 22 pair of designed
primers by checking of specificity. The selected primer
pair was able to detect 104 out of the tested 105 species.
Only one species, Ph. aquimorbida, was not able to be
detected. We confirmed no amplification of 35 species ofPythium, 15 species of Phytopythium, 1 species ofFig. 2 Phytophthora genus specific detection from
inoculated (ivy, tomato and hydrangea) andnatural infected (periwinkle and tobacco) plants

Aphanomyces and 6 species of soil-borne pathogens.
Only 4 minor species of Pythium and 1 species of

Acknowledgements

Phytopythium were showed unspecific detection.

hedraiandra, and natural infected plants; P. nicotianae to

Phytophthora isolates used in this study were kindly
provided from Dr. Treena Burgess, CPSM, Australia,
Prof. Michael D. Coffey, WPC culture collection, USA,
Dr. Everett Hansen, Oregon Univ., USA, Dr. Chuanxue
Hong, Hampton Roads AREC, USA, Dr. Thomas Jung,
Mendel Univ., Czech Republic, Dr. Paul W. Tooley,
USDA ARS, USA, Dr. Bevan S. Weir, ICMP culturecollection, New Zealand.

periwinkle and tobacco (Fig. 2). The PCR-based

Reference

Extracted DNA from inoculated and naturally infected
plants were subjected to PCR detection using the primer
pair. Detection of the target DNA were successful in all
DNA extracted from inoculated plants; tomato with P.
nicotianae, ivy with P. citrophthora, hydrangea with P.

detection method will contribute to establish an effective
quarantine system for Phytophthora species.

Erwin, D.C. and Ribero, O.K. (1996) Phytophthora
Diseases Worldwide. APS Press.

- 127 -

- 128 -P-8Morphological and molecular identification of causal agent ofcocoa pod rot disease in Indonesia
1,23
Masanto , Ayaka Hieno , Arif Wibowo2, Siti Subandiyah2, Masafumi Shimizu4, Haruhisa Suga5,
Koji Kageyama3(1Faculty of Agriculture, University of Gadjah Mada; 2UGSAS, Gifu University; 3River Basin Research Center, Gifu
University; 4Faculty of Applied Biological Science, Gifu University; 5Life Science Research Center, Gifu University)

Abstract
Cocoa pod rot disease is considered as one of the mostimportant problems in Indonesia as the third cocoa
producing country in the world. In this study, 55 isolates
infecting cocoa pod rot were collected from 23 cocoa
growing provinces in Indonesia. The pathogen wasbrecovered from the adjacent of the infected and healthy
part of symptomatic cocoa pod on semi-selective agar
medium for oomycetes. Identification was conducted byaobserving the morphological characteristics (hyphae,

Fig. 1 Characteristics of morphological featuresof pathogenic agents isolated from symptomatic
cocoa pod rot in cacao growing areas ofIndonesia (a) appearance of hyphae, sporangia andchlamydospores on V8 agar medium, (b) globose(top) ovoid- (bottom), (c) obpyriform-, (d)ellipsoidal- (e) distorted-shaped sporangia.

sporangium and chlamydospores) under the microscope;
while the molecular assay was carried out by multiplex
PCR with species-specific primers. The results showedthat the collected isolates produced clumsy, coenocytic,
and hyaline hyphae, pappillate sporangium (with rangeof length about 32.82-76.18 µm, range of breadth

c

a

d

e

b

between 21.61-41.60 µm, l/b ratio around 1.16-2.14, as
well as length of pedicel and pappillate ranging between
1.92-9.38 µm and 1.93-8.05 µm, respectively); andchlamydospores (with range of 18.30-48.87 µm incdiameter). These features were recognized as thecharacteristics

ofPhytophthorapalmivora.

Then,molecular analysis confirmed that all isolates werepositively detected with the PCR. The study on genetic
diversity of these isolates is still in progress.

Fig. 2 Performance of DNA band from all isolates
after molecular assay using multiplex PCR
method with universal (18S-69F and 18S-111R)
and specific (GUPal6fw and GUPal8rv) primers(a) isolates no. 1 – 21, (b) isolates no. 22 – 42, (c)isolates no. 43 – 55 with (+) and (-) control wereP0633 and SDW, respectively.

Table 1 Morphological features of collected isolates
Features

Remarks

Sporangium Type

Distorted, ellipsoidal,
globose, obpyriform, ovoid

Length (L) (µm)

32.82 – 76.18

Breadth (B) (µm)

21.61 – 41.60

Acknowledgements

L/B ratio

1.16 – 2.14

This research was conducted under collaboration ofFaculty of Agriculture, University of Gadjah Mada,
Yogyakarta (Indonesia) and River Basin Research Center,
Gifu University, Gifu (Japan) within “6-months
Sandwich Program in UGSAS-GU”.

Length of pedicel (µm)

1.92 – 9.38

Length of pappillate (µm)
Semi-pappillate

1.93 – 2.43

Full-pappillate

2.77 – 8.05

Chlamydospores
Shape

Spherical

Production site

Intercalary and terminal

Diameter (µm)

18.30–48.87

References
Erwin, D.C. and Ribero, O.K. (1996) Phytophthora
Diseases Worldwide. APS Press
Faostat (2017) Cocoa bean producing countries in theworld. www.fao.org/faostat- 129 -

- 130 -P-9
Biocontrol of tomato bacterial wilt using Ralstonia and Mitsuaria species
Malek MARIAN1, Tomoki NISHIOKA1, Hiroyuki KOYAMA1, Haruhisa SUGA2, Masafumi SHIMIZU1(1The United Graduate School of Agricultural Science, Gifu University; 2Life Science Research Center, Gifu University)

Abstract
Bacterial wilt caused by the soil-borne pathogen Ralstonia solanacearum is one of the most destructive bacterial diseases of solanaceous
species worldwide. The management of bacterial wilt is difficult owing to the viability, adaptability, and genetic diversity of the
responsible pathogen. The current countermeasures used against bacterial wilt include biological controls, chemical controls, and cultural
practices. However, chemical controls using soil fumigants are potentially harmful to the environment and have not been efficient ineradicating R. solanacearum. Moreover, cultural practices through commercially grafted seedlings is expensive and labor intensive andnew virulent races of the pathogen might overcome the resistance, resulting in colonization and migration of the pathogen into susceptible
scions and causing wilt symptoms. Thus, other alternative methods for controlling bacterial wilt are required. The biological control
method of using beneficial microbes has been proposed as an effective, safe, and sustainable approach. Additionally, beneficial bacteria
which are capable of colonizing the rhizosphere may provide the first line of defense against the attack of various soil-borne pathogens
including R. solanacearum. Thus, the aim of this study was to isolate rhizobacteria and evaluate their biocontrol potential against tomato
bacterial wilt.
A total of 442 bacteria were isolated from the rhizosphere soil of tomato (Solanum lycopersicum cv. Ohgata-Fukuju), Chinese chive(Allium tuberosum Rottler ex Spreng., cv. Super green belt), and Welsh onion (Allium fistulosum L., cv. Kujo-hoso), which were grown
at Gifu University (Yanagido, Gifu city, Gifu prefecture, Japan). Of these isolates, 276 exhibited in vitro antibacterial activity against R.
solanacearum. By analyzing a partial sequence of 16S rRNA gene, these antibacterial isolates were assigned to 24 genera, including
Burkholderia, Pseudomonas, Acinetobacter, Arthrobacter, Achromobacter, Ralstonia, and Mitsuaria. The suppressive effect of theselected antibacterial isolates against tomato bacterial wilt was further examined using the tomato seedling bioassay. In the first trial, 15of 276 isolates showed >50% reduction in disease severity. Therefore, the disease suppressive effect of these 15 isolates which belonged
to the genera Ralstonia and Mitsuaria were further evaluated in a second trial of tomato seedling bioassay. All of the tested isolates
exhibited strong suppressive effects (ranging from 68.5% to 95.9% reduction in disease severity). Because none of the isolates belonging
to genus Mitsuaria has been reported as a biocontrol agent against R. solanacearum, we evaluated the biocontrol efficacy of all of our
Mitsuaria isolates, against tomato bacterial wilt in the first pot experiment. As a result, an isolate TWR114 was selected as final candidate
for the second pot experiment based on its strong biocontrol effect (60% reduction in disease incidence). Furthermore, an isolate TCR112
was selected as final candidate from eight Ralstonia isolates for the second pot experiment because it showed the highest suppressive
effect in both trials of seedling bioassay. In the second pot experiment, the final candidate isolates TCR112 and TWR114 significantly
reduced the area under disease incidence progress curve by 63% and 48%, respectively. Furthermore, in field experiment, the wilt
incidence at 50 days after transplanting was significantly reduced by 57% and 86% in plots drench-treated with TCR112 and TWR114
at weekly intervals, respectively. The isolates effectively reduced the pathogen population in the rhizosphere and above ground stem
(crown) of pot grown tomato plants. Monitoring the population dynamics of the biocontrol isolates revealed that both isolates have stablerhizosphere and endophytic colonization capacities. The findings from the current study demonstrate that the isolates TCR112 andTWR114 are promising biocontrol agents against tomato bacterial wilt.

- 131 -

- 132 -P-10
Comprehensive evaluation of the resistance of root-stock-used Cucumis melo stock to
Meloidogyne incongnita
Wanxue BAO(Life Science Research Center, Gifu University)

SUMMARY
In this study, using Meloidogyne Incongnita separated from local pathogenic tomato plants in Guangxi of China as
pathogen, I inoculated the pathogen to 9 shares of homozygous root-stock-used muskmelon to evaluate their resistanceto root-knot nematode by measuring growth index of experimental rootstocks after inoculation. The result showed theresistance rank of 9 shares of root-stock-used muskmelons, which from top to bottom indicated that T05, TKH1, T06,
T03, T07T, T02, T04, T03T, and T07.
night it should not be under 15 . The soil temperature

Introduction
Root-knot nematode are common diseases whichshould not be under 5 . When tomato seedlings were

influence production and quality of melon in Guangxi,

infected for 40 days, I dig the roots and collected root

China. Cultivating by grafting seedling is an effective

nematode.

way to solve this problem, but selecting root-stocks with

Preparation of Meloidogyne incongnita egg liquid:

resistance of root-knot nematode is faced with huge

Cleaning and cutting pathogentic into pieces, and putting

difficulty now. Nowadays, although Yunnan Cucurbita

them into distilled water mixed with NaHClO as volume

ficifolia Bouché is widely used in melon grafting

ratio 1:1, stirring fast for 1min. Putting the mixture onto

cultivation due to its high grafting efficiency, but it has

3 sifters which from top to bottom were 50 holes, 350

been proved that its resistance to root-knot nematode isholes and 500 holes. Watering the back of 500 holes

poor. Therefore, selecting rootstocks with high resistancesifter and collecting liquid in the beaker. Putting distilled

to root-knot nematode is an effective way to increasewater in to the beaker to adjust the concentration to 1000

melon production and economic benefits of farmers.

egg per ml.

In this study, using Meloidogyne incongnita separated

Inoculation: The method was same to Expanding

from local pathogenic tomato plants in Guangxi of China

propagation. Putting 6 ml distilled water to seedlings’

as pathogen, I inoculated the pathogen to 9 shares ofroot as the control group(CK).

homozygous root-stock-used muskmelon to evaluate

By counting root numbers method, disease level of

their resistance to root-knot nematode by measuringroot-stock nematode was set to 0 to 5(table 1). Disease

growthafter

index(DI)=∑(disease level × plant numbers of thisinoculation. The evaluated method was new discovered

level)/(plant numbers × the highest disease level)×100%.

and the result may be more comprehensive and reliable.

Resistance evaluation criterion was set to 6 levels by

The result showed the resistance rank of 9 shares ofcalculating DI.

index

of

experimentalrootstocks

root-stock-used muskmelons. The conclusion can not

Besides calculating DI, measuring growth characters

only provide reference to root-stock breeding, but also

such as plant height, stem diameter, fresh weight of overguide to complete criterion to evaluate disease resistanceground part, fresh weight of underground part, dryof melon.

weight of over ground part, dry weight of underground

Material and Method

part, and root surface area. Then I calculated relative

Expanding propagation of Meloidogyne incongnita:

growth of infected rootstocks. Relative growth=(growth

Growing seedlings of susceptible tomato Y4 before 20

character number of tested group-growth characterdays of inoculation, then transplant them to nutrition pot

number of CK)/(growth character number of CK). And I

15 days later. I chose seedling substrate mixed with sandmeasured and calculated resistance characters such as

as volume ratio 1:1. I poured Meloidogyne incongnita

nodules

egg liquid near tomato root. 6ml per seedling.

coefficient, and disease index.

The

concentration of liquid was 1000 egg per ml. The
temperature in daylight should not be over 28 , and at

numberindex,

egg

index,

propagation

Nodules number index=nodule number of one
seedling/root fresh weight- 133 -

Egg index=egg number of one seedling/root fresh
weightPropagation coefficient=egg number of one seedling/
infected egg number
Table 1 Classification criteria of root-knot nematode
Disease level

Disease symptom0

No root knot

1

1-2 root knots

2

3-10 root knots

311-30 root knots

431-100 root-knots

5

More than 100 root knots

Table 2 Evaluation criteria of resistance of root-knot
Resistance level

Evaluation criteria（I） Immunity

DI=0（HR）High resistance

0.0≤DI≤10.0（R）Resistance

10.1≤DI≤30.0（MR）Medium resistance

30.1≤DI≤50.0（S）Susceptible

50.1≤DI≤75.0（HS）High susceptibleDI≥75.1

Result and Discussion

Fig 1 Root scan photos of infected muskmelon underFounder Z2400 scanner(From top to bottom, and eachline’s right to left are T02, T03T, T04, T05, T06, T07,
T07-T, and TKH1).
These photos elucidated that when were inoculated
by Meloidogyne incongnita, root-knot formed on
experimented muskmelon. The size and numbers ofroot-knots varied among different varieties. The results
verified that experimented pathogen had intensive
pathogenicity muskmelons could be infected by thepathogen. The quantity of root-knots on each root washuge, and some small root-knot taking group amounts
into consideration, it results to difficulties for counting
root-knot numbers, and the deviation may be generated
during the counting process. Hence, it is necessary tochoose an applicable way to evaluate resistance on
root-knot nematode of root-stock-used plants.

- 134 -

Table 3 Relative growth rate of root-stock-usedmuskmelon
NO.

H

D

FW of

FW of

DW of

DW of

Root

OP

UP

OP

UP

SA

taken as measurable indexes of evaluating resistanceon Meloidogyne incongnita of root-stock-usedmuskmelons.
Table 4 Change of resistance index ofroot-stock-used muskmelon after been infected
Meloidogyne incongnita

T02-37.15-25.96-27.95-25.88-34.76-20.00-40.90T03-22.89-28.42-26.05-46.80-16.48-40.80-47.74

Nodules

Egg number

Propagation

DiseaseT04-19.62-30.08-39.45-45.05-37.65-45.56-63.92

number(/g·FW)

coefficient

index

T05

-13.92-25.64-13.55-37.94-19.26-41.98-60.84(/g·FW)

T06-14.02-29.89-26.50-49.10-23.20-46.28-62.17T02

56.62

41419.24

87.60

70.0

T07

-14.61-31.22-16.57-43.34-34.21-41.67-59.00T03

9.7924065.91

40.40

48.0

T03T

-23.30-35.67-27.68-40.87-28.39-39.87-63.67T04

18.34

17455.10

32.55

54.0

T07T

-18.55-31.82-19.96-43.28-25.31-44.15-63.68T05

27.73

23722.01

41.63

58.0

TKH1

-24.05-20.07-27.15-50.68-23.29-49.07-69.12T06

33.27

29576.29

48.79

60.0

CV(%)

34.93

15.52

30.36

17.36

27.18

41.04

15.14

T07

54.22

49166.83

80.76

76.0

H means plant height, D means stem diameter, FW

T03T

37.97

37169.31

60.57

64.0

means fresh weight, DW means dry weight, OP means

T07T

60.65

56029.86

104.02

72.0

NO.

(%)

over-ground part of plant, UP means under-ground part

TKH1

73.56

65600.08

123.2186.0

of plant. The following tables was as the same. SA

CV(%)

51.28

42.51

46.01

18.11

means surface area.

After inoculation, the resistance index have changed todifferent degree. Among 9 experimented materials, theRelative growth rate revealed that after been

nodules number of per gram fresh weight and diseaseinoculated by Meloidogyne incongnita, the strength ofindex of T03 was the lowest, the egg number of per gram

activity that kept its own growth potential. It changed
more slightly, the negative impact whichroot-stock-used muskmelon got was slighter, and theresistance or tolerance may be stronger. It(Table 3)
showed that the growth indexes of inoculated groupdeclined, the relative growth rate of inoculated groups
to control groups was minus number, indicating that
Meloidogyne incongnita restrained experimented
root-stock-used muskmelons, and different rootstocks
showed diverse descent degrees. T05 and T03 showed
the least descent degrees of 9 root-stock-usedmuskmelons on relative growth rate. These tworootstocks had resistance or tolerance to the pathogen.
Relative growth rate of T04 and TKH1 was lower thanothers. It may be the sigh that Meloidogyne incongnita
had strong suppression of these two root-stocks, theresistance or tolerance to the pathogen was theweakest among the experimented root-stock-usedmuskmelons. Counting CV(coefficient of variation) of
these relative growth rates, the result showed that CVof dry weight of underground part and plant height
was higher, respectively were 41.04% and 34.93%.
The result suggested that after inoculation, the growthindex that plant height and dry weight of undergroundwere affected severely by the pathogen, and could

fresh weight of T05 was the lowest, and the propagation
coefficient of T04 was the lowest. However, these four
resistance indexes of TKH1 was the highest. The CV of
nodules number and egg number of per gram fresh
weight and propagation coefficient were higher, counting
as 51.28%, 42.51% and 46.01%. By contrast, the CV of
disease index was the lowest, indicated 18.11%. We
could get conclusion that if the resistance was evaluatedonly by disease index, the consequence might be there
was no difference of resistance among these rootstocks.
But by calculating resistance indexes, the otherness
appeared although the disease index seemed similar.
Therefore, it may be circumscribed if only taking diseaseindex into consideration when we evaluate resistance of

Meloidogyne incongnita.

- 135 -

NO.

Table 5 Subordinate function value of 5 indexes

per gram of fresh weight and reproduction coefficient,of root-knot nematode resistance of root-stock-usedmuskmelon

the resistance of the rootstocks to southern root-knot

H

DW

NN

EN

PC

Mean

nematode is comprehensively evaluated, and T05
Rrootstocks are found to be the most resistant to thenematode, followed by TKH1, T06, T03, T07T, T02, T04,

of rootT02

0.56950.43320.68230.56820.7032

0.5682

6

T03T and T07 respectively.

T03

0.5056

0.58740.6506

0.63140.67880.6117

4

AcknowledgementT04

0.41240.60020.65840.55090.66740.53997

T05

0.59560.52010.81310.73410.73190.6652

1

T06

0.44010.52790.74740.72530.76300.6141

3

T07

0.32610.65010.51000.47080.54720.4367

9T03T

0.44360.50000.59160.56090.58170.53908

T07T

0.36990.50000.63770.68520.59500.57205TKH1

0.5547

0.6666

0.68380.66170.5826

0.6164

2

Doing subordinate function analysis of 5 indexes
which had huge CV of inoculated root-stock-usedmuskmelons, as plant height(H), dry weight of root(DWof root), nodules number(NN), eggs number(EN) andpropagation coefficient(PC). By calculating the meanvalue of these 5 indexes of each rootstock, we got therank. T05 had the highest mean value, as 0.6652, among
all the experimented rootstocks. It proved that T05 hadstrongresistanceor

tolerancetoMeloidogyne
incongnita. Where as T07 had the lowest mean value,
as 0.4367, suggesting that T07 had the weakest
resistance of Meloidogyne incongnita. The resistancerank from top to bottom was T05, TKH1, T06, T03,
T07T, T02, T04, T03T and T07.

This study was guided by professor Wenjin Yu in
Guangxi University. It is honored to work with professor
Yu and our group members.

Reference
1) Boiteux L S and Charchar J M. (1996) Cenetic
resistance to root-knot nematode (Meloidogyne
javanica) in eggplant (Solanum melongena). Plant
Breeding, 115: 198-200.
2) Wenming C. (2016) Comprehensive evaluation ofresistance on Fusarium wilt and root-knot nematodeof root-stock-used cucurbit. Nanning, China,
Guangxi University.
3) Ke L, Yu D, Dexi S, Yingchun Z, Zhijun Z, Junpu L.
(2016) Selection of rootstocks with resistance on
southern root-knot nematode. Chinese Melon
Vegetables, 04:15-18.
4) Chengjing L. (2010) Resistance on temperaturestress and root-knot nematode of graftingwatermelon. Fuzhou, China, Fujian Agriculture andForestry University.
5) Jones J T, Haegeman A, Danchin E G, Gaur H S,
Helder J, Jones M G, Kikuchi T, Manzanilla-Lopez
R, Palomares-Rius J E, Wesemael W M, Perry R N.
(2013) Top 10 plant-parasitic nematodes in mocularplant pathology. Molecular Plant Pathology,
14:946-961.

Conclusion
In the experiment, it is discovered that after ninespecimens of rootstock-used melons are inoculated withsouthern root-knot nematodes, significant changes
happen to their growth indexes. Compared with thecontrol group injected with clear water, rootstock height,
stem thickness, aboveground and underground dry andfresh weights decline to different extent, which
demonstrates that the growth of rootstock specimens are
impacted by the southern root-knot nematodes, and somedifferences in resistance indexes of the strains. It isempirically proven that there would be great errors inmerelydetermining

diseasegradesofroot-knot

nematode based on number of root-knots by humanvisual observations and counting. After calculating
membership functions of indexes of tested rootstocks
with higher coefficient of variation after grafting,including stem length, underground dry weight, numberof root-knots per gram of fresh weight, number of eggs

- 136 -

P-11
Population genetics analysis of Phytopyhtium helicoides in Japan
Auliana AFANDI1, Emi MURAYAMA2, Ayaka HIENO2, Haruhisa SUGA3,
Koji KAGEYAMA2(1United Graduate School of Agricultural Science, Gifu University; 2River Basin Research Center, Gifu University;
3Life Science Research Center, Gifu University)

Abstractby their geographical origin. While Shizuoka populations

Phytopythium is a recently established genus containing

were divided into two major clusters. One cluster wereorganisms formerly classified as Pythium clade K species.

shared with the isolates from Iriomote-Jima which have

The species belong to this clade were morphologicallysimilar asexual reproductive behaviour. Another werelook like Pythium but genetically closer to Phytophthorashared with several isolates from different prefecture but(deCock et al., 2015). Phytopythium helicoides was firsthas same host plants (Fig.1). The results suggest thatisolated on 1930 by Drechsler from Dahlia in USA. This

geographical origin will play important role on thehigh temperature pathogen were not popular until

population structure of P. helicoides.

recently. In Japan, it was first isolated on the miniature

Table 1. The analysis of molecular variance(AMOVA) of P. helicoides population
Est.Var** %Fst***
Sourcedf*
Among Pops
14
0.453
19
0.187
Among Indv
139
0.744
31
Within indv
154
1.221
50
Total
307
2.418
100
*degree of freedom, **estimated variance, ***fixationindex

rose in Gifu in 1996 (Kageyama et al., 2002). The
pathogen is able to produce large number of zoospores as
secondary inoculum source. Thus, it can rapidly spreadon the hydroponic farming system.
Understanding the genetic variability on a pathogen
could help in developing effective disease management
strategy. The observation on the microevolution and2

population structure are necessary to predict theadaptation and migration ability of a pathogen. The
pathogen that has high genetic diversity and highmobility

will

perform

betteradaptation

on

the

environmental change. Study on the population genetics
can be done by several methods such as RAPD, RFLP,
AFLP, SNP, and microsatellites. The microsatellites are
the most convenience due to their codominant,multiallelic, highly polymorphism and require only small
amount of DNA for PCR analysis (Yin-Ling et al., 2009).

●Oita ▲Rebun Island ♦ Saga ♦ Shiga ○Shizuoka ● Tochigi ▼ Toyama ∆ Yamanashi ▼In this study, microsatellite markers containing 4 lociunknown ● Aichi ■ Fukui ■ Gifu ▲ Hokkaido ▲ Iriomote-Jima ▼Kagawa ▼ Mie ●(EM-AGC1, EM-AGGCA, EM-GGA1 and EM-TCA1)

Fig. 1 Phylogenetic Analysis on P. helicoides
population using Neighbor Joining algorithmwith 37 alleles were used for the population genetics
analysis.
One hundred fifty five isolates from 15 prefectures inJapan and four overseas isolates were applied to
GenAlEx software in Microsoft Excel. The overseas
isolates were originated from South Africa, USA,
Netherland and Spain. The Hardy- Weinberg Equilibrium
were significantly differed from the equilibrium for all
loci, indicating a low number of population or
occurrence of genetic drift. The analysis of molecularvariance showed the significant genetic differentiation
between populations (19%) (Table 1). The phylogenetic
analysis using neighbor joining algorithm revealed fourmain clusters. Gifu and Aichi population were clustered

Acknowledgement
We thank Mr. Mikihiko Suzuki, Dr. Hideki Watanabe,
and Dr. Noriyuki Miyake for providing the isolates usedin this study.

References
1) de Cock AWAM, Lodhi, AM, Rintoul TL, Bala K,
Robideau GP, Abad ZG, Coffey MD, Shahzad S,
Levesque CA (2005) Phytopythium: molecularphylogeny and systematics. Persoonia 34:25-39.
2)Kageyama K, Aoyagi T, Sunouchi R, Fukui H (2002)
Root rot of miniature roses caused by Pythium helicoides.
Journal of General Plant Pathology, 68:15-20.
3)Yin-Ling, Zhou W, Motohashi K, Suga H, Fukui H,
Kageyama K (2009) Development of microsatellite
markers for Pythium helicoides. FEMS Microbiology
Letter, 293:85-91.

- 137 -

- 138 -P-12
Study of a transcriptional regulator of plant pathogenic genes in a soft rot disease causing
bacterium, Dickeya dadantii
Dina Istiqomah1 and Naoto Ogawa1, 2(1The United Graduate School of Agricultural Science, Gifu University; 2Faculty of Agriculture,
Shizuoka University)
Abstract
The soft rot pathogenic bacteria, Dickeya dadantii, causes soft-rot-disease in plants of various host range including
economically important crops. The virulence of D. dadantii is known with its ability to synthesize and secrete pectinase(Pel) enzymes which act as plant cell wall degrading enzymes. PelA, PelD and PelE are among the most important Pelsfor virulence. Among the pel genes, pelD expression is most strongly affected by pectin derivatives, which makes it a key
element in the induction of D. dadantii virulence.
The pathogenicity level of pathogen may be affected by involvement of several regulatory protein. Previous study
revealed that a gene of LacI transcriptional repressor family, ltR, was involved in the expression of virulent factors andpathogenicity of D. dadantii in negative manner. To study the function of ltR, we conducted experiments including cloningof the gene, expression of the protein and then His-tag purification of the protein. By electrophoretic mobility shift assay(EMSA) using the purified protein, we analyzed the binding of LtR protein with promoter regions of related virulence
genes of D. dadantii.
EMSA showed that LtR protein could bind to each of the promoter regions of the pelD, pelA, and pelE. This result
indicated that LtR protein has a potential to be a transcriptional regulator for the three genes. The affinity of the bindingby LtR seemed to be different among the three promoters, suggesting different manner in the regulation by LtR for thepromoters. In vivo analysis of the strain using Reverse-Transcriptase PCR is being conducted to analyze the expressionof the virulence genes.

Keywords: Dickeya dadantii, soft rot disease, transcriptional regulator, virulence gene.

- 139 -

- 140 -P-13
Identification of freshness marker of stored soybean sprouts
Syukri, D.1, Thammawong, M.1, Kuroki, S.2, Tsuta, M.3, Yoshida, M.4, Nakano, K.1(1The United Graduate School of Agricultural Science, Gifu University; 2 Faculty of Agriculture, Kobe University; 3Food Research Institute, National
Agriculture and Food Research Organization, 4Kanagawa Agricultural Technology Center)

Abstract
The objective of this study was to discover a freshness marker of fruits and vegetables from small reactive carbonyl compound which is well known
as a second product of lipid peroxidation. Self-cultivated soybean sprout was used as a sample material, and was stored various temperatures. The
sprout was sampled periodically, and was divided into cotyledon and hypocotyl. At the same time, the respiratory CO2 production was also measured
by a flow-through method using a gas chromatography. The carbonyl compounds were extracted from each part by homogenizing with polar-semipolar mixture solvent. Then, they were derivatized with specific carbonyl compounds labeling agent. High performance liquid chromatography/
electrospray ionization tandem mass spectrometry with multiplexed multiple reactions monitoring was used for the comprehensive detection ofcarbonyl compounds. A large variety of carbonyl compounds were observed in MRM chromatogram from both cotyledon and hypocotyl part ofstored soybean sprouts. Several m/z values were selected as candidates of the freshness maker of soybean sprout by means of principle component
analysis-discriminant analysis (PCA-DA) by relating with the accumulation CO2 production as a reference freshness indicator. The structure
elucidation of these freshness marker candidates will be conducted in future study.

Keywords: freshness evaluation, lipid peroxidation, senescence, soybean sprout- 141 -

- 142 -P-14
Studies on acetaldehyde tolerance system in the budding yeast using myo-inositol
Annisyia Zarina Putri1, Mizuho Inagaki1,2, Masaya Shimada1,2, Takashi Hayakawa1,2, Tomoyuki Nakagawa1,2(1 The United Graduate School of Agricultural Science, Gifu University; 2 Faculty of Applied Biological Sciences, Gifu University)

Abstract
Acetaldehyde is a toxic compound that up to now is still problem during fermentation in yeast. In
recent years many study have been carried out for improving the fermentation capability especially using yeast
Saccharomyces cerevisiae. According to Furukawa et al. (2004), in S. cerevisiae, the intracellular myo-inositol content was found
to affect strain tolerance against ethanol stress. In S. cerevisiae, myo-inositol is produced from glucose 6-phosphate via thereactions catalyzed by the INO1-encoded inositol-3-phosphate synthase and INM1-encoded inositol monophosphatase, and thenparticipated in the biosynthesis of phosphatidylinositol. In the present study, decreasing inositol by simultaneous deletion of INM1
and INM2 enhanced ethanol tolerance. It was shown that, under high ethanol condition (12-20%) with limited inositol in themedium, yeast cells leak intracellular metabolites such as nucleotides, phosphate and potassium. Under high ethanol
concentration, addition of inositol to medium improves ethanol tolerance (Chi et al. 1999). From this background, we want
to try to do the experiment for acetaldehyde tolerance in yeast Saccharomyces cerevisiae using myo-inostiol to improve
fermentation capability.

Keywords: Acetaldehyde tolerance, Saccharomyces cerevisiae, myo-inositol, INO1

- 143 -

- 144 -P-15
Identification of bioaerosols from environmental samples in the AIST, Tsukuba, Japan
Panyapon Pumkaeo, Wenhao Lu, Youki Endou, Tomohiro Mizuno,
Junko Takahashi, Hitoshi Iwahashi(The United Graduate School of Agricultural Science, Gifu University;
Faculty of Applied Biological Sciences, Gifu University)

Abstract
The Bioaerosols are the atmosphere particles, mists or dust of µm range, associated with metabolically active or inactive viable
particles. They contain living organism’s included microorganisms such as viruses, bacteria, and fungi also plant material as well as pollen. Next
Generation Sequencing (NGS) is a novel method of DNA sequencing that quickly and efficiently read the underlying sequence of an organism
by means of massively parallel sequencing. The aim of this study is identifying organisms which contained in environmental samples by using
NGS
This study monitored the environmental sample (Bioaerosols) from November 2013 to January 2015 for 50 days using air samples werecollected at AIST, Tsukuba, Japan. Samples were bio-analyzed using a next-generation sequencing method. In this study, we used two NGS
platform, GS FLX+ (Roche 454 sequencing) and Illumina Misep. The sample was detected plants, eukaryotes, and bacteria. The sample wasdivided into two subgroup subgroups according to the size of its bioaerosols, large subgroup contains bioaerosols whose diameter is bigger than
3.3μm, and small subgroup contains those smaller than 3.3μm.
Most of the plant cells were included in large subgroups; while back filter also contained much plant-derived debris. Samples whose size
ranges from 0.43μm to 3.3μm showed least plant DNA. Regarding the relevance of plant DNA quantities and season, samples collected inspring showed thehighest expression,next was autumn,followed by summer, and winter samples appeared the least DNA expression. Bacteria
did not show a correlation between DNA expressions with the collected data. Components of large subgroups showed no obvious differencecompared with small subgroups. Medicago papillosa is the dominant species in AIST, Tsukuba. Rare, endangered species such as wild rice
were traced. The rbcL universal primer showed high effective for NGS of the environmental sample than other primers that used in this study.
Keywords: Bioareosols, Next-Generation Sequencing, Bacteria, Plant- 145 -

- 146 -P-16
The effect of persimmon (Diospyros kaki) on the prevention of sarcopenia
Nayla Majeda Alfarafisa, Tomio Yabe
(The United Graduate School of Agricultural Science, Gifu University)

Abstract
Sarcopenia is an age-related syndrome that characterized by progressive loss of mass, strength, and function of skeletal muscle.
The loss of skeletal muscle fibers resulted from imbalance between the protein synthesis and degradation, called atrophy, is acommon feature in sarcopenia. Until now, the underlying mechanisms and pathophysiology of sarcopenia remains unclear. Thereis now increasing evidence which suggests that muscle wasting has a strong relationships with increased levels of circulating
inflammatory components in elder people. Some proinflammatory cytokines could enhance the proteolysis and decreasing protein
synthesis. Persimmon (Diospyros kaki), kaki fruit, is a popular fruit and well distributed, especially among Asian countries.
Persimmon is enriched with several bioactive compounds that have beneficial effects on human health. For example, flavonoid
is one of the most dominant phytochemical that can be found on persimmon peel and fruit. Recent study showed that flavonoid
had a positive effect on the prevention of skeletal muscle atrophy in vitro, hypothetically via TNF-α counteracted mechanism.In
this study, the effect of extracts from persimmon on the progression of skeletal muscle atrophy in vitrowill be investigated. TNFα level, the expression of anabolic targets (Akt, mTOR, p70S6k, and 4E-BP1), and catabolic targets (FoxO1, FoxO3a, MAFbx,
MuRF1) would be checked after extracts treatment to evaluate the effect of persimmon fruit to the muscle protein turnover andatrophy development. In addition, the effects of extracts on the production of pro-inflammatory will also be investigated,
persimmon bioactive compoundswill be expected to prevent the progression of atrophy not only by balanced protein turnover but
also by suppress the production of inflammatory cytokines. After treatment, the levels of inflammatory cytokines (NF- κβ, I-κβ,p50, p65, and IL-6) to evaluate the anti-inflammatory effects of persimmon. A better understanding of cellular mechanism of
sarcopenia is required to prevent the progression of sarcopenia based on research evidence.
Keywords: Sarcopenia, Atrophy, Inflammatory Response, Flavonoid, Diospyros kaki

- 147 -

- 148 -P-17Transcriptional biomarkers for managing pulse crop production in acid soil region
Raj kishan Agrahari1, Hiroyuki Koyama2(1The United Graduate School of Agricultural Science, Gifu University;
2

Faculty of Applied Biological Sciences, Gifu University)

Abstract
Crop Production decreased by excessive soil minerals and salts, such as sodium chloride, heavy metals, and Aluminum(Al).These stress induces specific damage and stress-responses in the shoots, which can be evaluated by Molecular level.
Al specific responses in shoots was analyzed by Sawaki et al.(2016) and they found shoot specific Al responsive gene bothin Arabidopsis thaliana and Nicotiana tabacum out of these it is found that ALS3 and PGIP1 are shoot specific Al
responsive gene. ALS3 is ABC transporter that reported in Arabidopsis and it is regulated by STOP1 (sensitive to proton
rhizotoxicity1) proteins for Al specific response but its molecular mechanism and Signal Transduction is still not clear,similarly gene PGIP1 (polygalacturonase inhibiting protein) is a cell wall pectin degrading enzymes such as those produced
by fungal pathogens (Nguema-Ona et al. 2013) but it is reported under low pH (acid soil) expressed in shoot (Sawaki et al.
2016). The existence of microarray data for Al stresses in Arabidopsis provides the opportunity to identify and characterize
key conserved genes downstream of Al stress responses. Some of these genes might be eventually used as biomarkers forthe evaluation of stress responses in crop. They could also expand our knowledge about the genetic mechanisms underlying
the responses to Al stresses in non-model crops. So from this scenario my research area focus on understanding themolecular mechanism of the genes in shoot under Al stress and application of the gene signaling in crops as a biomarker.

Al3+

H

?
UDP‐Glucose?
Al tolerance

Proton Tolerance genes.

NucleusFig.1: Model representing Research of Interest- 149 -

Cell Wall- 150 -P-18
The accumulation of carotenoid in mango during fruit maturation
W. Yungyuen1,2, T.T. Vo3, G. Ma2, L.C. Zhang2, P. Jitareerat3, A. Uthairatanakij3, M. Kato2(1 The United Graduate School of Agricultural Science, Gifu University; 2 Graduate School of Agriculture, Shizuoka University; 3 King
Mongkut's University of Technology Thonburi)

Abstract
Among tropical fruits, mango is one of the most popular fruits worldwide because of its tasty and attractive color. Carotenoids
are considered as an important bioactive compound in mango fruits responsible for bright yellow pigment, which determine quality andappearance of fruits. In addition, carotenoids accumulated in mango fruits can act as bioactive antioxidants, promoting benefits to humanhealth. To date, the accumulation of carotenoids in mango fruits during the maturation process is still unclear. Therefore, the objective of
this research was to investigate the changes in carotenoid content and composition during fruit maturation in the three mango varieties fromThailand. The highest carotenoids level was observed in ‘Kaituk’, follow by, ‘Namdokmai No.4’, and ‘Namdokmai Srithong’ in both peel
and pulp. The accumulations of six carotenoids were found, including all-trans-violaxanthin, 9-cis-violaxanthin, lutein, -cryptoxanthin, carotene, and -carotene. Those carotenoids were highly accumulated in the peel, but their contents were lower in the pulp. Among them,–carotene was observed as a major carotenoid accumulated in the peel and pulp of three mango varieties. During mango fruits maturation,
the carotenoids were accumulated at low level in the green stage fruits. Then, the carotenoid content was significantly increased in thetransition stage from green to yellow at 81 days after anthesis, reaching a maximum in the full ripening stage at 98 days after anthesis in thethree mango varieties. These results showed different levels of carotenoid accumulated in the three mango varieties, which is usefulmaterials for elucidating the regulation of carotenoid metabolism during mango fruit maturation in the future research.

Keywords: carotenoid metabolism, -carotene, mango peel, mango pulp

- 151 -

- 152 -P-19
Augmented nuclease resistance and gene silencing with 3’-end modified small interfering
RNAs and dendrimer based drug delivery
Akash Chandela1, Yoshihito Ueno1,2(1United Graduate School of Agricultural Science, Gifu University; 2Course of Applied Life Science,
Faculty of Applied Biological Sciences, Gifu University)

Abstract
RNA interference (RNAi) is a naturally occurring mechanism where non-coding double stranded RNA regulates thesequence specific posttranscriptional gene silencing. With thousands of druggable target genes, this tool provides a
revolution in gene regulation and treating numerous epigenetic disorders. Two major obstacles in this phenomenon are
instability of natural RNA (nuclease degradation) and inefficient drug delivery. Therefore, this study is aimed atsynthesizing stable siRNA with modified 3’-end and designing branched siRNA for dendrimer based delivery.
siRNA is a key moiety in this pathway that contains two signature characteristics: a two nucleotide 3’-end overhang anda phosphorylated 5’-end. The functional strand, referred as guide strand, interacts directly with the multi domain AGO
proteins to form RNA induced silencing complex (RISC). The 3’-end is recognized by PAZ domain housing ahydrophobic pocket, whereas the 5’-phosphorylated end is recognized by MID domain. This RISC recognizes target
messenger RNAs with sequences complementary to the guide strand, cleaves them and regulate gene expression.
Therefore, we have modified the base at 3’-end with trifluoroethyl and trichloroethyl analogs. These analogs weresynthesized by β-selective introduction at 1-position of D-ribofuranose via an acetal linkage. They have been found to
exhibit better nuclease resistance and comparable gene silencing to the native. Binding assay have shown lower
dissociation energy of these analogs complex with PAZ domain. Hence, we hypothesize upon these findings that it’s the
optimal interaction which drives this mechanism forward.
Lately, cholesterol conjugated passenger strand with analog modified antisense are also being analyzed as previous
literatures have reviewed this combination as more productive to increase interaction with PAZ domain. Furthermore,
we have synthesized branched siRNA using a trebler synthon as solid support. This trebler synthon have beensynthesized using pentaerythritol.
Thus, our current study postulates that modification with hydrophobic analogs at 3’-end of guide strand yields better
nuclease resistance and gene silencing. And, we look forward to analyze the dendrimer based approach for drugdelivery with these tripod siRNA.

- 153 -

- 154 -P-20
Protein-based functional analysis of renin and (pro)renin receptor genes in hypertensive
and diabetic Bangladeshi population: Pursuing the environment-induced molecular
traitsJobaida Akther1, A. H. M. Nurun Nabi2, Tsutomu Nakagawa3, Fumiaki Suzuki3, Akio
Ebihara3(1United Graduate School of Agricultural Science, Gifu University; 2Department of Biochemistry and Molecular
Biology, University of Dhaka; 3Faculty of Applied Biological Sciences, Gifu University)

Abstract
Both renin and (pro)renin receptor [(P)RR] proteins as well as their genetic variations have been found to be
associated with the risk of hypertension and diabetes; and no extensive study has been performed so far in
Bangladeshi population regarding renin and (pro)renin receptor. In addition, environmental factors, which aresometimes imposed to Bangladeshi workers, can make a large influence on the plasma levels of renin and (P)RR.
Their effects can be observed as environmental-induced molecular traits. Thus, this study aims to develop thefollowing methods1. Better ELISA for measuring plasma renin concentration (PRC) and soluble (P)RR [s(P)RR]
2. Storage of proteins in solid state using pullulan (a polysaccharide polymer) and their activity check1. Angiontensin-I ELISA (AI-ELISA) for measuring PRC
Recombinant ovine angiotensinogen (oANG) was prepared using E.coli cells according to the method describedby Yamashita et al. (2016). The rate of angiotensin-I (AI) generation was determined by AI-ELISA (Suzuki F etal. 1990). Prorenin produced in mammalian cell was treated with trypsin to get renin, and its concentration wasdetermined from trypsin treatment time vs renin activity plot. Again, unknown concentration of renin wasdetermined from renin activity vs renin concentration plot. This assay can be utilized to measure PRC as abiomarker.
2. Formation of oANG tablets using pullulan
Pullulan is a polysaccharide produced from starch by the fungus Aureobasidium pullulans and has been used for
encapsulation of biomolecules and solidifying enzymes. Pullulan solution was mixed with oANG protein to maketablets. The effect of pullulan on oANG has been examined by measuring renin activity varying storage periods.
Keywords: oANG, AI-ELISA, PRC, Pullulan- 155 -

- 156 -P-21
Proposals for countermeasures to reduce risk of hydraulic fracturing
adjacent to culvert – A case study
Duy Quan Tran1, Shinichi Nishimura2, Masateru Senge2, Tatsuro Nishiyama2, Fumitoshi
Imaizumi3(1 The United Graduate School of Agricultural Science, Gifu University; 2 Faculty of Applied Biological
Sciences, Gifu University; 3 Faculty of Agricultural, Shizuoka University)

Abstract
Hydraulic fracturing is the propagation and development of cracks under the effect of water pressure when the
stress in the dam is lower than the water pressure. Hydraulic fracturing has been also considered as one of possible
causes leading to concentrated leakage and failure of many fill dams especially at the first reservoir filling. Sherard(1972) pointed out that hydraulic fracturing was the most likely cause in seven of eleven dam failures in Oklahoma
and all three of the Mississippi dam failures that location of the failures occurred directly adjacent to culverts.
Ngambi et al. (1997), according to results of in-situ observations combined with numerical analyses concluded
that hydraulic fracturing is the most likely cause of concentrated leakage and of failure of many low dams along
the outlet conduit especially after heavy rain.
From such experiences, in this study, a case study of dam failure is introduced. The case study is called KE 2/20
REC dam - an agricultural dam in Central Vietnam that was broken just under one-year in operation. Previous
research revealed that the dam failure was related to hydraulic fracturing mechanism (Tran et el. 2017). Whereby,
there were stress decreases at both sides of the pipe culvert by arching action that was induced by effects of culvert
shape and a steep excavation slope. Based on these findings, in this study, the authors focused on proposal of two
countermeasures to prevent risk of hydraulic fracturing adjacent to the culvert of the case study dam. These
countermeasures are combinations created by changing the culvert shape and either shifting the position of the
excavation slope 5.0 meters away from the former position or replacing the fill soil between the culvert and the
excavation slope with a concrete block. Effectiveness of the countermeasures are then verified by numericalmodels using finite element method (FEM). The results showed that around the proposal culvert, normal stress isalways higher than water pressure, and hence, there is no possibility of hydraulic fracturing occurred adjacent tothe culvert.

Keywords: Dam failure, Hydraulic fracturing, countermeasure, Finite Element Method (FEM)- 157 -

- 158 -P-22
Droughts hotspot distribution by long term assessment the Standardized Precipitation Index (SPI)in Indonesia
Yudhi Pramudya, Takeo Onishi(Faculty of Applied Biological Sciences, Gifu University)

Abstract
Differences in weather and climate conditions in Indonesia influenced by the ITCZ (Inter Tropical Convergence Zone) on
January to March and Asian Monsoon in November to December. As a result, this area is frequent experiencing anomalies or climate
irregularities primarily associated with the natural phenomenon of La-Nina and El-Nino, which can cause a disaster nature like a drought
disaster. Rainfall data used to calculate the value of meteorological drought index in Indonesia using SPI method (Standardized Precipitation
Index) to identify drought events means rainfall deficit during peak three months wet season period (SPI-3 during December-February 2000
to 2015). APHRODITE (1961-2007) and CHIRPS (1981-2015) provides estimated rainfall data that can be used to calculate the value of
meteorological drought index in a region. Drought index ranged from -2.51 to 2.67 indicating the criteria very dry to very wet, indicates thedominant drought hotspot (extreme climate condition) occurred in the peak of wet season (December–February) has wider extent andhigher severity respectively in the West Borneo, Banten and Near Jakarta in 2005, Central Java, Southwest Celebes, Ambon, Maluku, andPapua in 2006, Belitung and South part of West Borneo in 2010, West Sumatera, West Borneo, West Sulawesi 2013, and West Sumaterain 2014. The index also describes dry region in wet months caused flood disaster in Dec-Feb 2012-2013. Spatial analysis information ofdrought hotspot distribution important to described as one of the efforts to help warning a farmer not to be failed in harvesting, also ananticipation and evaluation for future conditions, so it can be known patterns, severity and frequency of occurrence of drought disaster.
From the analysis results predicted areas prone to droughts and floods, then compared with topographical aspects and characteristics of theregion (eg agricultural areas, urban areas, etc.) to hydrological planning to face possible disasters (such as drainage, surface water storageetc.). Areas that often experience drought hotspot of the mapping are West Sumatera, West Celebes and Central Java.

Keywords: Drought, Index, SPI-3, Climate, Rainfall- 159 -

- 160 -P-23
The role of floral volatiles for attracting pollinators and reproductive isolation in Mimulus species
Muhammad Arifin, Tomoko Okamoto(Faculty of Applied Biological Sciences, Gifu University)

Abstract
Flowering plants have a wide variety of signals to attract pollinators. Visual signals, including flower size, color and shape havegreat influence for preference of pollinator. In addition, flower also emit a large array of volatile compounds, assuming a varietyof roles, e.g. attracting effective pollinators and avoiding inappropriate flower visitors. Although many studies have been made tounderstand plant-pollinator interaction, most of them either focused on visual and olfactory cues. Previous study revealed thatvisual floral diversity in genus Mimulus (family Phrymaceae) correlated with the type of pollinator-animals, leading to reproductive
isolation between related species. However, the effect of floral volatiles in Mimulus to attract different of pollinators is unknown.
In this study, I try to reveal the ecological and evolutionary role of floral volatiles of Mimulus species growing in Japan. This study
is aimed to (1) clarify the type of pollinator in Mimulus flowers, (2) analyze volatile compounds emitted from Mimulus flowers
and (3) identify the most important volatile compounds to attract specific pollinator animals. After achieving this study, I canunderstand more clearly about the roles of floral volatiles in plant-pollinator interaction and reproductive isolation in Mimulus
species in Japan. The sample of all Mimulus species will be collected from various area in Japan. Observations of flower visitors
will be recorded for a total of 120 hours in several populations to reveal pollinators. Floral volatiles will be collected by head-space
sampling in wild. Floral volatiles will be analyzed with a gas chromatography mass spectrometry. Behavioral test using flower
volatiles and pollinator animals will be conducted to identify the most important volatiles compounds for pollinator attraction in
Mimulus.

Keywords: floral scent, plant-pollinator interaction, pollination, chemical ecology, reproductive isolation- 161 -

- 162 -The United Graduated School of Agricultural Science, Gifu University
1-1 Yanagido, Gifu 501-1193, Japan
Tel: +81-58-293-2984 (or, 2985)
E-mail: renno@gifu-u.ac.jp
Home page: http://www.ugsas.gifu-u.ac.jp/eng/ISBN978-4-909365-02-6
Supporting foundation: Gifu Convention and Visitors Bureau, Japan