The 4th International ConferenceofAsian Allelopathy Society

Tokyo University of Agriculture and Technology

September 8-10, 2018, Tokyo, Japan

4th International Conference of Asian Allelopathy Society

K-1
Insights into the mechanism of proliferation on the special
microbes mediated by phenolic acids in the Radix pseudostellariae
rhizosphere under continuous monoculture regimes
Wenxiong Lin*1,2,3
1Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, College of Life Sciences,

Fujian Agriculture and Forestry University, Fuzhou 350002, P. R. China
2Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops,

Fujian Agriculture and Forestry University, Fuzhou 350002, P. R. China.
3Key Laboratory of Crop Ecology and Molecular Physiology (Fujian Agriculture and Forestry University),

Fujian Province University, Fuzhou 350002, P. R. China.

Radix pseudostellariae L. belonging to the Caryophyllaceae family is a common and popular medicine inChina, which is mainly produced in Fujian Province, Southeast of China. R. pseudostellariae contains
ginseng saponins, polysaccharides, amino acids, flavonoids, and cyclic peptides, which can be used as acure for spleen deficiency, anorexia, and palpitations. The average annual yield of R. pseudostellariae is
about 5000 tons, accounting for more than 22 million dollars per year. However, this medicinal plants
seriously suffer from the replanting disease in the popular monocropping system, in which the biomass andquality of root tuberous roots of the medicinal plants were decreased, indicating that the biomass oftuberous roots was declined by 33.3%, the polysaccharide content and ginseng saponins Rb1 in the
tuberous roots were reduced by 88.08% and 44.33%, respectively. Therefore, it has become a top priority
to elucidate the underlying mechanisms of replanting disease, especially in the case of medicinal plant
production.
In this studies the phenolic acids, as potent allelochemicals, are believed to be associated withreplanting disease and cause microflora shift and structural disorder in the rhizosphere soil of continuously
monocultured R. pseudostellariae. Nine types of phenolic acids were identified both in the monoculutred
rhizosphere soil and in culture medium under sterile conditions. The author also found the phenolic acids
could be transformed each other in the rhizosphere soil. Some special microorganisms utilized andcatalyzed the conversion of an allelochemical vanillic acid into vanillin. Meanwhile, the K. sacchari wasable to produce protocatechuic acid from consumption of vanillin, which then became an active agent in
the allelopathy of understory species in the rhizosphere of R. pseudostellariae. The transcriptome
sequencing was used to reveal the underlying mechanisms on the differential response of pathogenic
bacterium Kosakonia sacchari and beneficial bacterium Bacillus pumilus to the allelopathic interactions ofphenolic acids. Transcriptome analysis showed that vanillin enhanced glycolysis/gluconeogenesis, fatty
acid biosynthesis, pentose phosphate, bacterial chemotaxis, flagellar assembly, and phosphotransferase
system pathway in K. sacchari. However, protocatechuic acid, a metabolite produced by K. sacchari fromvanillin, had negative effects on the citrate cycle and biosynthesis of novobiocin, phenylalanine, tyrosine,and tryptophan in B. pumilus. Concurrently, the protocatechuic acid decreased the biofilm formation of B.
pumilus. These results unveiled the mechanisms how phenolic acids differentially mediate the shifts of
microbial flora in rhizosphere soil, leading to the proliferation of pathogenic bacteria (i.e., K. sacchari) andthe attenuation of beneficial bacteria (i.e., B. pumilus) under the monocropping system of R.
pseudostellariae.
This work was supported by the National Natural and Scientific Foundation of China (Grant NO U1205021, 81573530,
81781260274, 31401950), Central leading local science and technology development special fund project (2017L3010008).

 E-mail: lwx@fafu.edu.cn

4th International Conference of Asian Allelopathy Society

K-2
Using allelopathic approaches for ecological weed
management in field crops
Muhammad Farooq*1,2
1

Department of Crop Sciences, College of Agriculture and Marine Sciences
Sultan Qaboos University, Al-Khoud-123, Muscat, Oman.
2
Department of Agronomy, University of Agriculture,
Faisalabad-38040, Pakistan.

Weeds pose a severe threat to food security for future generations. Chemical
herbicides offer an effective control of weeds. However, continuous andindiscriminate use of herbicides may cause of development of herbicide
resistant weed biotypes and environmental pollution. In this regard, allelopathy
offers attractive and eco-friendly option for ecological weed control.
Allelopathy can be utilized, for weed control, through inclusion of allelopathic
crops in rotation, use of allelopathic mulches, intercropping of allelopathic
crops and use of allelopathic plant extracts. In a series of studies, we noted that
inclusion of sorghum in rotation with maize, wheat and rice caused substantial
reduction in weed infestation in conventional and conservation crop production
systems. Application of sorghum mulch, use of sorghum as intercrop, andexogenous use of sorghum water extract also suppressed the weeds. However,combination of more than one approach was more effective than the single
strategy. In this regard, inclusion of sorghum in rotation together with sorghum
mulching (8 t ha-1) and exogenous application of sorghum water extract (15ha-1) was more effective in suppressing weeds in maize, rice and wheat, which
was then translated in increase in yield and profitability. In conclusion,
integrated use of allelopathic approaches offers cost-effective option for
non-chemical weed management in conventional and conservation maize, rice
and wheat production systems.

Email: farooqcp@gmail.com

4th International Conference of Asian Allelopathy Society

K-3
Allelopathy and allelochemicals between
phytophagous insets and plants
Masanori Morimoto
School of Agriculture, Kindai University, Japan.
The term “allelopathy” is defined as an interactive phenomenon between
different species. This is not only a plant-plant interaction, but also includes aninsect-plant and microbial-plant interaction. Most insect tolerant plants imply
any physical and chemical defense system against their natural enemies. One ofthe physical defense system is having a hairy leaves and/or thorns on their plantsurface. On the others hands, the chemical defense system is production andstorage of the insect toxic secondary metabolites. Especially, wild plants
without protection of pesticides and cultivars closed to its native species maybe
maintain the defense system against their natural enemy. Based on this, many
insect antifeedants were isolated from various plants, Asteraceae, Orobancaceae
and Cyperaceae. And the almost isolated natural products were allelochemicals,since these chemicals existed in/on the plant and the phytophagous insect will
be directly contact with them when they fed to the plant.

 E-mail: masanori@nara.kindai.ac.jp

4th International Conference of Asian Allelopathy Society

K-4
Branched broomrape (Orobanche ramosa L.) control
in tomato (Lycopersicon esculentum Mill.) by trap
and other plant species in rotation
Jamal R. Qasem*
Plant Protection Department, Faculty of Agriculture, University of Jordan,
Amman, Jordan
Forty-four plant species of 13 families were tested as traps reducing Orobancheramosa L. infestation on tomato under glasshouse conditions. Many species
were parasitized but Orobanche shoot number and dry weight were greatly
varied. Trifolium alexandrinum was most infested and Citrullus vulgaris least.
Tomato growth was reduced after many tested species while none prevented
parasite infection. Parasite shoot number was highest on tomato followed
Anethum graveolens, Coriandrum sativum, Cucumis melo var. flexuosus, T.
alexandrinum, Sesamum indicum, and Solanum elaeagnifolium, and lowest after
Brassica oleracea var. italic, Brassica rapa var. rapa, Capsicum annuum,
Cicer arietinum,
Citrullus colocynthis, Cuminum cyminum, Capsicum
frutescens, Cucurbita maxima, Hordeum vulgare, Linum usitatissimum,
Spinacea oleracea cv. Epinard greant and Vigna sinensis. Parasite dryweight was lowest on tomato followed C. frutescens, C. melo, Trigonella
foenum-graecum, Hibiscus sabdariffa and Pimpinella anisum but highest after
Oroganum syriacum and C. colocynthis. Many reported Orobanche traps wereparasitized. Tomato growth was doubled following T. alexandrinum and
Ecballium elaterium. Nonhost species reduced O. ramosa on tomato may be
regarded as parasite traps. The best tomato growth and parasite control wereobtained after L. usitatissimum and V. sinensis followed by C. sativum, E.
elaterium, H. sabdariffa, H. vulgare, S. indicum and Sorghum vulgare.

Keywords: Parasitic weed, Branched broomrape, Orobanche ramosa, Tomato,
Traps species, Catch species, Control, Rotation.
 E-mail: jrqasem@ju.edu.jo4th International Conference of Asian Allelopathy Society

K-5
Protoplast co-culture method with digital image
analysis for bioassay of allelopathy
Sasamoto, H.,*1,2 and Fujii, Y.1
1

Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509,
Japan. 2Research Institute for Integrated Science, Kanagawa University,
Hiratsuka, Kanagawa 259-1293, Japan.

‘Protoplasts Co-culture Method’ was developed to study allelopathic activitiesof plants using tissues and their cultured cells. This method is based on the ideaof co-culturing of callus of a test plant to determine its allelopathic effects on
the growth of another callus. However, quantitative evaluation of experimental
results is difficult using the callus co-culturing method. Therefore, wedeveloped a protoplast co-culture method. The response of cultured protoplastsof a recipient plant can be expressed as numbers of non-spherically enlarged,
divided protoplasts, in a 50 µL of medium in a well of a 96-well culture plate.
For example, inhibitory effects of protoplasts of a test plant, on growth ofrecipient protoplasts of lettuce were determined in a Murashige and Skoog’s
medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine, sucroseand osmoticum mannitol. Protoplasts densities were varied. The results were inagreement with those obtained by the conventional in vitro bioassay method andfield tests. Not only lettuce but also other plants, e.g. rice can be used asrecipient plant species. These results will contribute to clarify the cellularmechanism(s) of allelopathy. Furthermore, we also found an accumulation of ayellow color (carotenoid) after cell wall formation and divisions of lettuce
protoplasts. After scanning of 96-well culture plates, the inhibition of yellow
color accumulation was quantified by the Image J analysis. As lettuce is widelyused as a recipient plant in conventional in vitro bioassay methods, this simple
high-throughput method (DIA-PP method) will make possible to survey effects
of a large numbers of test plants and contribute to the discovery of newallelochemicals.

 E-mail: sasamoto@ynu.ac.jp

4th International Conference of Asian Allelopathy Society

K-6
Control of Panama disease of banana byintercropping with Chinese chive:
Role of allelochemcals
Rensen Zeng, Zhenfang Li, and Yuanyuan Song
College of Crop Science, Fujian Agriculture and Forestry University, Jinshan,
Fuzhou 350002, P.R. China
Panama disease of banana caused by Fusarium oxysporum f.sp. cubense (FOC)
is a devastating soil-borne fungal disease causing extensive failure ofcommercial plantation of banana throughout banana producing areas of theworld. The pathogen is extremely difficult to control due to its long persistence
in soil, and to date no effective control measure has been found. Our study
showed that intercropping banana with Chinese chive (Allium tuberosum
Rottler) could control the Panama disease. Both volatiles and water extracts ofChinese chive suppressed mycelial growth of FOC. Three chemicals including
dimethyl trisulfide (DMT), dipropyl trisulfide (DPT) and 2-methyl-2-pentena(MP) isolated from Chinese chive strongly inhibited mycelial growth and spore
germination of FOC. Different varieties varied with their inhibitory effects.
Volatile compounds emitted from the intact growing roots mimicking naturalenvironment inhibited spore germination of FOC. We identified five volatiles
including 2-methyl-2-pentenal and four organosulfur compounds (dimethyl
trisulfide, dimethyl disulfide, dipropyl disulfide, and dipropyl trisulfide) fromthe leaves and roots of Chinese chive. All these compounds exhibited inhibitory
effects on FOC, but 2-methyl-2-pentenal and dimethyl trisulfide showedstronger inhibition than the other three compounds. Our results show that
antifungal allelochemicals released from Chinese chive help control Panama
disease in banana. We conclude that intercropping banana with Chinese chive
can control Panama disease and increase cropland biodiversity.

Key words: Fusarium wilt；Panama disease of banana；Intercropping；Chinese
chives；Ecological control
 E-mail: rszeng@fafu.edu.cn, rensenzeng@163.com

4th International Conference of Asian Allelopathy Society

K-7
Allelopathic extractions improve growth andyield of wheat
Zahid Ata Cheema,* and Sardar Alam Cheema**
Department of Agronomy, University of Agriculture, Faisalabad, Pakistan.
Allelopathy is an eco-friendly science emerged in the past few decades.
Allelopathic plants suppress or promote the growth of other plants. The
allelopathic plants posses several secondary metabolites which are highly watersoluble. These can easily be extracted by soaking dry herbage in water. We haveconducted series of field experiments at farmers fields and research stations.
Results of the trails have shown considerable improvement in growth of crops.
The water extracts of brinjal, garlic and sorghum (3%) applied at tillering andearing, enhanced the yield of wheat by 10 to 40%. In conclusion use ofalleolpathic extracts at low rates (3%) is highly economical and viable forfarmers. Results of some of the recent experiments will be discussed.

Key words: Allelopathy, Garlic, Brinjal, Sorghum, Wheat.
* E-mail: cheemaza@gmail.com, **E-mail: Sardaralam35@gmail.com

4th International Conference of Asian Allelopathy Society

O-1
Characterization of plant growth-promoting effectof γ-terpinene and β-caryophyllene in lettuce andmaize seedlings
Ebina, S.,*1 Sunohara, Y.,1 Komatsu, S.,2 Yamaguchi, H.,3
Hitachi, K.,3 Tsuchida, K.,3 Yamaguchi, T.,1 and Matsumoto, H.1
1

2

University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Fukui University of Technology, Fukui, Fukui 910-8505, Japan.
3
Fujita Health University, Toyoake, Aichi 470-1192, Japan.

In our previous studies, screening of volatile compounds having plant growthpromoting activity was performed using various plant species, and we foundthat γ-terpinene and β-caryophyllene showed growth-promoting activity inlettuce roots. In this study, the growth-promoting effects of γ-terpinene and βcaryophyllene on root and shoot of lettuce and maize seedlings were
investigated. Root fresh weight increased up to 130% of control at 138 µmol/Land 150% at 518 µmol/L in maize seedlings 3 days after treatment with γterpinene and β-caryophyllene, respectively. On the other hand, β-caryophyllene
reduced the fresh weight of maize hypocotyl by 70% of control 3 days after
treatment, whereas γ-terpinene had no effect on the hypocotyl fresh weight. In
lettuce seedlings, 357 µmol/L of γ-terpinene stimulated root length and third
leaf area up to 120% and 140% of control 14 days after treatment, respectively.
γ-Terpinene (0–357 µmol/L) also increased the number of lateral root in aconcentration-dependent manner in lettuce. Similarly, β-caryophyllene
stimulated root length of lettuce up to 150% of control 14 days after treatment,
but inhibited the third leaf area by 50% of control at 50 µmol/L. Overall, thegrowth-promoting effects of β-caryophyllene were more significant than that of
γ-terpinene. In proteomic analysis, γ-terpinene increased the abundances of 70
proteins
including
IAA-aminoacidhydrolaseandxyloglucan
endotransglucosylase/hydrolase (XTH) in lettuce roots. From these results,
auxin and XTH might be involved in the growth-promoting effect of γ-terpinene
in lettuce roots.

 E-mail: s1721105@s.tsukuba.ac.jp

4th International Conference of Asian Allelopathy Society

O-2
Involvement of ROS and xyloglucan
endotransglycosylase/hydrolase in
S-(+)-carvone-induced growth inhibition in
Arabidopsis thaliana seedlings
Saitoh, R.,* Sunohara, Y., Yamaguchi, T., and Matsumoto, H.
University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
(S)-(+)-2-Methyl-5-(1-methylethenyl)-2-cyclohexenone (S-(+)-carvone) is abioactive volatile terpeniod produced by Aloysia polystachya and it shows
strong growth inhibitory effects on several plant speices. However, its mode ofaction is not yet understood. The present study was conducted to investigate themechanism of growth inhibition of S-(+)-carvone in Arabidopsis thaliana
seedlings. Shoot and root growth of A. thaliana were inhibited by S-(+)-carvone(0-527.6 μmol/L) in a dose-dependent manner. S-(+)-Carvone increased thegeneration of reactive oxygen species (ROS) in shoots and roots at
concentrations >255.6 µmol/L and > 114.2 µmol/L 5 days after treatment,
respectively. This compound also caused lipid peroxidation in shoots andprogramed cell death in roots at concentrations >255.6 µmol/L and >527.6µmol/L 5 days after treatment, respectively. Furthermore, S-(+)-carvoneinduced root growth inhibition was reduced by application of ROS scavenger,
ascorbic acid or 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine. In qRT-PCR
analysis, S-(+)-carvone (255.6 µmol/L) markedly suppressed transcription ofseveral xyloglucan endotransglycosylase/hydrolase (XTH) genes in roots of A.
thaliana 24 hours after treatment. From these results, primary action of S-(+)carvone to inhibit growth of A. thaliana may be caused by ROS overproduction
and subsequent oxidative damage. However, additional studies are required toelucidate the involvement of XTH gene suppression in S-(+)-carvone-induced
plant growth inhibition.

 E-mail: s1821103@s.tsukuba.ac.jp

4th International Conference of Asian Allelopathy Society

O-3
The existence of functional substances released frommycorrhizal fungi for the germination of
Spiranthes sinensis. var. amoena
Chiba, S.,1 Kimura, S.,2 and Tomita-Yokotani, K.2*
1

Faculty of Life and Environmental Sciences, University of Tsukuba,
Tsukuba, Ibaraki 305-8572, Japan.
2
Graduate School of Life and Environmental Sciences, University ofTsukuba, Tsukuba, Ibaraki 305-8572, Japan.

There are many symbiotic species in plants and microbes. It is well known thatmycorrhizae are fungi living inside plants. The seed germination of orchid
plants depends strongly on the existence of mycorrhizae. We have investigated
the relationships between an orchid, Spiranthes sinensis var. amoena, and amycorrhizal fungus, Epulorhiza repens, during their germination process. There
are several steps during seed germination. The first step is the swelling of seeds.
In this study, swelling activity was investigated as the first step of theirgermination.
All of the plant samples were collected from the campus of University ofTsukuba. We have already established a method for calculating the biomass of amycorrhizal fungus growing in Spiranthes sinensis var. amoena in ourlaboratory. Epulorhiza repens was collected by Mr. Fujimori and purified as astrain Epulorhiza repens T338. Epulorhiza repens T338 stored at our laboratory
was used as symbiotic fungus species. The seeds were too small to countindividually, so, the correlation of the wight and their seed number wasinvestigated. After sterilization with sodium chloride, seeds were put on thesurface of medium with and without the fungus. The seed germination wasobserved by microscopes and the rate of germination was calculated.
Our results indicate that some functional substances exist in the medium
released from grown fungi. We will show the results and other recent our study
related to Orchids plants and thier symbiotic fungi.

 E-mail: yokotani.kaori.fn@u.tsukuba.ac.jp

4th International Conference of Asian Allelopathy Society

O-4
Determination of allelopathic potentialities ofBangladeshi plant species
Begum, K.,1* Fujii, Y.,1 and Shammi, M.2
1

United Graduate School of Agricultural Science, Tokyo University ofAgriculture and Technology, Saiwaicho 3-5-8, Fuchu,
Tokyo 183-8509, Japan.
2
Department of Environmental sciences, Jahangirnagar University,
Savar, Dhaka-1342, Bangladesh.

Competitive relationships of crops and weeds are the major problems in thefield culture which reduces gross crop production every year in the South Asian
country, Bangladesh. Excessive use of synthetic fertilizers and herbicides may
create a resistant feature to the weeds and ultimate sickness of soil and farmers’
health. From the perception of Allelopathy, we are piercing natural chemical asa secondary metabolite that might reduce the aggressiveness of native weeds
including invasive plant preserving agricultural sustainability. We manipulated
sandwich (SW) method developed by Fujii et al. (2003). Lettuce growth in SW
was observed to know the influence of leaching the phytochemical from thedifferent parts of the plants as allelochemical from these plant species. We
screened 87% allelopathic plant species among the 215 plant species. Where
37% species shown higher inhibitory (HI), followed by 21% species, moderate
inhibitory (MI) activities under the considerable standard 10 mg in lettuce
radical length.
From this study, we found among 37%, HI species Acacia concinna,
Couroupita guianensis, Piper longum, Aegle marmelos and followed by
Azadirachta indica, Sapindus mukorossi caused strong inhibition on lettuce
radicle growth. Among the screened plant species, Shikakai (Acacia concinna),family Fabaceae, followed by Cannonball tree (Couroupita guianensis),
Lecythidaceae showed a strong allelopathic activity. Advance studies about the
selective plants to identify the potential allelochemical would be considerable.
As a concern of weed management, these allelopathic plants are the significant
potential alternatives relate to eco-friendly crop culture system.
 E-mail: noorkeya.bd@gmail.com

4th International Conference of Asian Allelopathy Society

O-5
Carnosic acid; the Principal Plant Growth Inhibitorin Rosmarinus officinalis Leaves
Appiah, K. S.,1* Amoatey, C. A.,2 Oikawa, Y.,3 and Fujii, Y.1,3
1

Department of Biological Production Science, Tokyo University ofAgriculture and Technology, Tokyo, Japan.
2
Department of Crop Science, University of Ghana, Legon, Ghana.
3
Department of International Environmental and Agricultural Science,
Tokyo University of Agriculture and Technology, Tokyo, Japan.
Weeds are rapidly developing resistance to synthetic herbicides and exploringallelopathic species can help minimize the ecological threat posed by herbicideresistant weeds. In this study, we aimed to evaluate the contribution of somebioactive compounds in rosemary (Rosmarinus officinalis) to its allelopathy
based on the total activity approach. The phytotoxic activity of crude extracts ofrosemary plant parts (leaves, roots, inflorescences, and stems) was tested on
receptor Lactuca sativa. Soils incorporated with rosemary dried leaves werealso tested on test plants. The various parts of rosemary were evaluated for thecontent of some bioactive compounds (caffeic, ferulic, gallic, rosmarinic,
carnosic acid, and chlorogenic acids) by reversed-phase HPLC. The specificactivity and total activity of crude extract and individual compound were
evaluated using lettuce as test plant.
We found that the growth inhibitory effect of the crude extracts of therosemary leaves was the highest among all parts. Dry matter and seed
emergence of lettuce were inhibited by soil amended with rosemary leaf debris.
Carnosic acid was the main compound found in rosemary leaves and also had ahigh specific activity. The inhibitory activity of the rosemary leaf extract could
be explained by the high presence of carnosic acid. Hence, we assume based on
the total activity estimation that carnosic acid among the other tested
compounds is the major allelochemical in rosemary leaves

 E-mail: s166196z@st.go.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

O-6
Effects of Garlic Allelochemical Diallyl Disulfide on
Tomato and Its Mechanism in Alleviating
Continuous Cropping Obstacle
Fang Cheng, and Zhihui Cheng*
Northwest A&F University, Yangling, China.
In recent years, the continuous cropping obstacle has become the restricting
factor for the sustainable development of facility vegetables. In our previous
studies, we found that continuous cropping obstacle of tomato (Solanum
lycopersicum L.) could be alleviated by relay intercropping with garlic (Allium
sativum L.), and the allelopathy of garlic root exudates may play a major role.
Diallyl disulfide (DADS) is one of the main allelochemicals in garlic root
exudates and garlic straw, however, the physiological and molecular mechanismof DADS on tomato are still unclear. The clarification of physiological andmolecular mechanism of DADS-promoting effect on tomato plant and its
alleviating effect on obstacles to continuous cropping of tomato have importantsignificance in the sustainable production of greenhouse tomato.
The objectives of this research were: to study the physiological and biochemical
effects of DADS on tomato seeds germination and seedling growth; toinvestigate the effect of DADS on the expression of tomato root genes, analyse
the sensitive metabolic pathways and functions of major genes which response
to DADS; to test the allelopathic effect of DADS on tomato plant growth undercontinuous cropping conditions and analyze the impact of DADS on tomato
root exudates changes. The results showed: 1. DADS promoted tomato root
growth at low concentrations (0.01-0.62 mM) but inhibited root growth at highconcentrations (6.20-20.67 mM); 2. DADS may activate tomato resistance inthree ways: increasing the contents of nonenzymatic antioxidants, such as GSH;
inducing the activity of enzymatic antioxidants, including SOD, POD, CAT,
PPO and GR; and activating ETI; 3. DADS may change the synthesis of plant
hormones and signal transduction, thereby influencing tomato plant growth andresistance; 4. DADS may change the content of tomato root exudates to reduce
autotoxicity and increase the tomato disease resistance.
 E-mail: chengzh@nwsuaf.edu.cn

4th International Conference of Asian Allelopathy Society

O-7
Identification and allelopathic mechanism of green
garlic volatiles on cucumber seedlings
Fan Yang, and Zhihui Cheng*
Northwest A&F University, Yangling, China.
Plants synthesize and emit a large variety of volatile organic compounds(VOC) to interact with other organisms. Garlic and medicine made of garlicvolatiles are increasingly being used as fungicides in modern agriculture.
However, the component and effect of garlic volatiles in crop plants have beenless studied. Here, green garlic volatiles was chose to explore the questions.
First, it was collected by headspace solid-phase microextraction (HSPM)
method and analyzed by gas chromatography-mass spectrometry (GS-MS). The
results showed that diallyl disulfide (DADS) is the main allelochemicalcompound of green garlic volatiles. Here, we exposed 2 true leaf-stage
cucumber plants cultivated in artificial media to different concentrations of agarlic (0, 6 12 and 18 bulbs) and DADS (0 and 1 mM) for 10 d. On this basis,
the allelopathic mechanism of green garlic volatiles in vivo and DADS on the
cucumber (CCMC) seedlings growth and physiology was further studied. In
summary, they can promote the growth of cucumber seedlings and increase theantioxidant enzyme activities and the accumulation of reactive oxygen species
ultimately.

 E-mail: chengzh@nwsuaf.edu.cn

4th International Conference of Asian Allelopathy Society

O-8
Effect of Momilactone B on Seed Germination andRoot Development of Arabidopsis thaliana andthe Related Mechanisms
Xianhui Lin, Jun Long, Yuanyuan Song, and Rensen Zeng*
Institute of Crop Resistance & Chemical Ecology, Fujian Agriculture andForestry University, Fuzhou 350002, Fujian, China
Momilactones are diterpenoid phytoalexins produced by rice (Oryza sativa L.).
Momilactone B (MB) is the key allelochemical of rice allelopathy and one ofmost phytotoxic natural products. However, the mode of action MB is unknown.
In this study, Arabidopsis thaliana was used as a receiver plant to dissect themechanism of allelopathic effect of MB. A dose-dependent inhibitory effect onseed germination and seedling growth of Arabidopsis were observed. Seedtreatment with MB led to up-regulation of ABA synthesis genes and ABA
signaling pathway genes, as well as strong induction of negative regulation
genes in GA signaling pathway. Furthermore, Arabidopsis thaliana mutant abi4
showed less susceptible to MB. Our results indicate that inhibitory effect of MB
on seed germination results from suppression of GA signal pathway andactivation of ABA signal pathway. Momilactone B inhibited the length of mainroots and the number of lateral roots. Confocal microscopy observation showedthat the length of apical meristem was significantly shorter and the number ofcells was significantly decreased after MB treatment, suggesting that MB
inhibits apical meristem and cell division. Exogenous application of 10 mM
GA3 or 0.1 nM NAA alleviated inhibitory effect of MB on main roots andlateral roots. In conclusion, MB inhibited root growth of Arabidopsis by
inhibiting auxin transportation and reducing auxin accumulation at the root tip,
as well as the activity of root apical meristem and cell elongation. Our results
suggest that MB inhibits Arabidopsis by disturbing hormone signalingtransduction.
Key words: Momilactone B; seed germination; root development; rice
allelopathy
 E-mail: rszeng@fafu.edu.cn

4th International Conference of Asian Allelopathy Society

O-9
Allelopathy in context of resolution at ecosystem
level
Naureen Mustafa,* and Maqsood Anwar
Department of Wildlife Management,
Pir Mehr Ali Shah Arid Agriculture University Rawalpindi,
46300, Pakistan.
The molecular interactions by which the plants affect their neighboring species
either at the species or ecosystem level is attributed to allelopathy consequently
affecting the floral populations and communities. Recent evolvement inassessment of the bioassays made possible the examination of theplant/vegetation interaction at molecular level. In this case we have made thecomparison of allelopathic interactions in two different ecosystems instead of
population/community level of resolution thus hypothesizing that allelopathy is
influencing the ecological communities in a more obvious ways. In this study,
as an initial approach impact of herbivory by wildlife harboring the particular
ecosystem is considered which reflects the allelopathic potential of plant species
as their defense mechanism thus altering or affecting the vegetation in the longrun. This is illustrated by the contrasting examples from Karakoram-West
Tibetan Plateau alpine steppe and Potohar Plateau. The earlier area is considered
as roof of the world and presents a mix of Mountain Sub-Tropical Scrub Forests,
Mountain Dry Temperate Coniferous Forests, Mountain Dry Temperate
Broadleaved Forests and Northern Dry Scrub Forests. However, Potohar Plateau
presents the sub-tropical broad leaved evergreen forest ecosystem. We haveassessed how two exotic species has affected the respective ecosystemproperties by producing the secondary metabolites i.e. Ailanthus spp. andProsopis cineraria. In both cases, the production of metabolites has regulated
the key ecosystem processes thus determine the ecosystem functionality andconsequently the structure of vegetation community in respective areas. Our
study supports the hypothesis that allelopathy is more obvious and operational
at ecosystem level of resolution, instead of species level of resolution.

 E-mail: naureenmustafa5@yahoo.com, naureen_mustafa@yahoo.com

4th International Conference of Asian Allelopathy Society

O-10
Management of charcoal rot of black gramby soil amendment with Ageratum conyzoides
Arshad Javaid,* Saira Banaras, and Amna Shoaib
Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan.
Black gram [Vigna mungo (L.) Hepper] is an important pulse widely used inPakistan. Its yield is significantly reduced by charcoal rot disease caused byMacrophomina phaseolina. The present study was carried out to assess thedisease management potential of dry biomass of an asteraceous weed Ageratum
conyzoides used as soil amendment. Pot soil was made sick with M. phaseolina
inoculum and after seven days, dry biomass of the weed was mixed in pot soil at
0.5, 1, …, 3% (w/w). Treatments included a negative control, a positive control(fungal inoculation alone), and fungal pathogen in combination with different
quantities of dry biomass of A. conyzoides. Seeds of black gram were sown inthese pots at 10 seeds per pot and the crop was harvested at maturity. Data
regarding disease incidence, shoot and root biomass, and grain yield wererecorded and analyzed statistically by applying Tukey’s HSD test at 5% level ofsignificance. Activities of defence related enzymes viz. peroxidase (PO) andphenylalanine ammonia lyase (PAL) were noted at flowering stage. The highest
disease incidence (100%) was recorded in positive control that was gradually
decreased with increase in A. conyzoides dry biomass in the pot soil. A 2% soil
amendment completely controlled the disease. The highest grain yield (5.8 g
pot-1) was recorded in 2.5% soil amendment as compared to 1.43 g pot-1 innegative and 0.132 g pot-1 in positive control treatment. The highest PAL and
PO activities were recorded in positive control. These activities were gradually
decreased with increase in dry biomass of the weed in the soil.

 E-mail: arshad.iags@pu.edu.pk, arshadjpk@yahoo.com

4th International Conference of Asian Allelopathy Society

O-11
Allelopathic water extracts helps improve drought
resistance during reproductive stages in wheat
Ahmad Nawaz,1* Muhammad Nabeel,2 Muhammad Farooq,2Mubshar Hussain,1 and Muhammad Ijaz1
1

College of Agriculture, Bahauddin Zakariya University,
Bahadur Sub-Campus, Layyah, Pakistan.
2
Department of Agronomy, University of Agriculture,
Faisalabad, Pakistan

This experiment was conducted in pots where two wheat cultivars viz.,
Mairaj-2008 (M-2008) and Faisalabad-2008 (FD-2008) were grown in the pots
filled with 10 kg of the soil. The pots were irrigated as per need of the crop until
leaf boot stage at 75% WHC which was followed by imposition of drought
stress at reproductive (booting and anthesis) and grain filling (dough stage)stages in both wheat cultivars. When one week passed away after foliage
application of water extracts on sorghum, brassica, moringa and sunflower, thedrought was imposed at each stage by maintaining the pots at 35% WHC.
Drought stress negatively altered performance of wheat crop in terms ofmorphology and yield; however, the yield of wheat under drought was
improved after the foliage application of allelopathic water extracts. M-2008
performed better than FD-2008 under drought stress which was visible throughimprovement in the performance of this cultivar under terminal drought stress.
Although, application of all the AWEs improved the performance of both
cultivars, the water extract of sorghum was most useful under drought stressthan other extract. Drought stress imposition at booting and anthesis was moreinjurious to crop than when imposed at grain filling. In crux, foliage applied
AWEs applied alone or in combination might be pragmatic option to improve
wheat performance under climate change.

Keywords: drought, wheat, morphology, climate change, water extract
 E-mail: ahmadnawaz2006@gmail.com

4th International Conference of Asian Allelopathy Society

O-12
Combined application of natural plant water
extracts and biochar improves the productivity ofbread wheat
Muhammad Ijaz,* Ahmad Nawaz, Sonia Perveen, Ahmad Sher,and Abdul Sattar,
1

College of Agriculture, Bahauddin Zakariya University,
Bahadur Campus Layyah, Pakistan

Bread wheat (Triticum aestivum L.) is staple of Pakistani people. However, its
yield at farmer field is low as compared with its genetic potential. Integration ofvarious crop and soil management strategies might be an option to enhance
wheat productivity at farmer field. This 2-year experiment was conducted tocheck the influence of combine application of natural plant water extracts andbiochar on the productvity of wheat during the winter season of 2015-16. The
experiment consisted of seven treatment viz. (1) control (2) application ofbiochar (0.18 kg pot-1) alone, (3) application of sorghum water extract (SWE)
alone, (4) application of moringa water extract (MWE) alone, (5) application ofbiochar + SWE, (6) application of biochar + MWE, (7) application of biochar +SWE+MWE. The results revealed that application of both crop water extracts incombination with biochar improved the growth and grain yield of wheat. Use of
MWE in combination with biochar enhanced the grains per spike, 1000-grain
weight and grain yield of wheat by 44, 14, and 24%, respectively than thecontrol treatment. In crux, use of MWE in combination with biochar might be a
viable option to improve the productvity of bread wheat.

Key words: bread wheat, biochar, grain yield, natural water extracts
 E-mail: Muhammad.ijaz@bzu.edu.pk

4th International Conference of Asian Allelopathy Society

O-13
Allelopathic potential of African marigold (Tagetas
erecta) for managing root knot nematode(Meloidogyne incognita) in tomato
Sareer Ud Din, and Muhammad Azam Khan*
Department of Horticulture, Pir Mehr Ali Shah Arid Agriculture University
Rawalpindi, 46300, Pakistan.
Root knot nematode (Meloidogyne incognita) pose a significant threat to crop
production in Pakistan which causes a wide range of losses to agriculturalcommodities such as delayed maturity, toppling, reduced yield & quality, highcosts of production consequently loss of income. This study was carried out tocheck the effect of African marigold (Tagetes erecta) to control the root- knot
nematodes (RKN) infesting tomato (Solanum lycopersicum L.) roots. The
treatments included tomato plants (1,2), marigold plants (0,1,2) and RKN
inoculation (0,1) arranged in various combinations in pots. The results showedthat maximum plant height (79.14cm), number of leaves (28.00), leaf area
(24.46cm2), number of fruit plant-1 (42.66), fruit Size (44.26cm2) of tomato after
45 days of transplantation were observed in the treatment having two plants ofmarigold, one plant of tomato along with the inoculum of RKN as compared toother intercropping treatments and control. Pot soil was analyzed for NPK, EC
and pH before and after transplanting. Significant differences were observed for
NPK. It was observed that intercropping marigold was beneficial in controlling
nematode populations to greater depths by altering soil pH moderately acidic(8.01) with high electrical conductivity (0.14). Fruit quality attributes like
firmness (4.86Nmm-1), TSS (4.9°Brix) and shelf life (8.67days) were best inintercropping treatment comprising two plants of marigold with one plant of
tomato having an inoculum of RKN in a single pot. The results may be
attributed due to release of allelochemicals by Marigold in the surrounding
environment that inhibited the activity of Root knot nematode, which had apositive effect on growth and development of tomato accompanied bysignificant enhancement in yield. It may be concluded that plant parasitic
nematodes may be controlled through intercropping tomato with marigold,
which is a safer alternative to traditional application of agrochemicals fordisease management.
 E-mail: drazam1980@uaar.edu.pk

4th International Conference of Asian Allelopathy Society

O-14
The role of negatively indirect allelopathy in tea
consecutively monoculture ratooning problems
Yasir Arafat,1, 2, 3 Sheng Lin,1, 2, 3* and Wenxiong Lin1, 2, 3*
1

College of life Sciences, Fujian Agriculture and Forestry University, Fuzhou 35002, China.
2
Key Laboratory of Fujian Province for Agroecological process and safety monitoring,
Fujian Agriculture and Forestry University, Fuzhou 35002, China.
3
Institutes of Agroecology, Fujian Agriculture and Forestry University, Fuzhou 35002, China.
Long-term consecutive monoculture of crops leads to decreased growth and yield in following years,
due to allelopathy, the key culprit. In long-term consecutive monoculture tea orchards, plant
performance such as growth, quality and yield decreases over time after the establishment of tea
plants in the same soil for several years. Therefore, addressing allelopathic mechanisms of leaves
litters by falling and pruning every year in long-term monoculture tea orchards are critical to tea
industry. To help characterize the allelopathic mechanism of tea, we carried out a large-scale,pyrosequencing analysis of the tea rhizo-compartments microbiomes in response to the stress ofcatechins, well-known allelochemicals from tea leaves litters. Our main result showed that, pH of tea
orchard was significantly lowered with increasing planting age. Furthermore, long-term tea
cultivation resulted in accumulation of allelochemicals in the rhizosphere including Epigallocatechin
Gallate (EGCG), Epigallocatechin (EGC), Epicatechin gallate (ECG), Catechin (±C), Epicatechin(EC) in the form of leaves litters. Meanwhile, phosphomonoesterase, sucrase and urease activity weresignificantly declined. However, the activities of catalase and polyphenol oxidase were highly
increased in long-term 20-30-years consecutively monocultured tea field. Moreover, distinct
distribution patterns of catechins degrading bacterial taxa were analsed in all threerhizo-compartments （i.e, rhizosphere, rhizoplane and endorhizosphere) of 30-year monoculture tea
mediated strongly by soil pH and catechin containing compounds. Pyrosequencing results identified
Pseudomonas and Burkholderia as the dominant groups at genus level while plant growth promoting
bacteria such as Bacillus, Sphingomonas, and Prevotella were significantly lowered in long term
monoculture degraded tea plantation. The qPCR results in situ also confirmed that the whole bacterial
contents in per gram degraded 30-year tea plants soil was significantly lowered as compared to freshsoil tea plantations. However, Pseudomonas population was significantly increased with increasingtea plantation years. In-vitro study indicated that with increasing the concentration of theseallelochemicals, the growth of catechin degrading bacteria such as Pseudomonas was increased but
reverse was true in the case of the growth promoting bacteria such as Bacillus . Furthermore,invitro-interaction results showed that the Pseudomonas genera are involved in the degradation of
catechins into more phytotoxic allelochemicals such as EC and PCA. The pH of the broth with theaccumulation of PCA was also decreased to 0.36-fold after 72 hours. Therefore it can be concluded
that the shift of beneficial microbes and the accumulation of active allelochemicals may be the causeof acidification, soil sickness and regeneration problem which may attribute to hindrance of plant
growth, development and low yield in 30-year long-term monocultured tea plantation. These results
helped us to clarify the role of leaves litters contamination in long-term continuously ratooned andmonocultured tea orchard that can inevitably lead to soil acidification and fertility decline, in turnresulted in decresed yield and quality of tea.
Keywords: Monoculture; Allelochemicals; Microbiomes; Rhizo-compartments; High-throughput sequencing;
Redundancy analysis (RDA); High performance liquid (HPLC), Chromatography-Electrospray Ionization-Mass
spectrometry (HPLC-ESI-MS), rhizosphere restoration, organic agriculture

E-mail: lwx@fafu.edu.cn, lsjk1958@163.com

4th International Conference of Asian Allelopathy Society

O-15
Identification of antifungal constituents from
Agaricus bisporus (J.E. Lange) Imbach
Akbar, M.,1* Waqas, H. M.,1 and Iqbal, M. S.1
1

Department of Botany, University of Gujrat, Gujrat, 50700, Pakistan.

In agriculture, fungal invasions cause heavy loss and affect food security andfood safety. Chemical pesticides are available to control these pathogens but
these are associated with environmental and public health concerns. In recent
years, there is growing trend towards nature friendly methods of pathogen
control. Use of allelochemicals from mushrooms is a promising fileld. In thepresent study, antifungal activity of an edible mushroom, Agaricus bisporus
was investigated. For this purpose, different organic solvent extracts of A.
bisporus, were evaluated against plant pathogenic fungal species viz.
Macrophomina phaseolina, Alternaria alternata, Fusarium oxysporum,
Drechslera australiensis and Aspergillus flavus. To check the antifungal
activity of A. bisporus, serial dilution method was used. Six levels of treatmentsof each n-hexane, chloroform and ethyl acetate (0, 5, 10, 15, 20 and 25 mg/ml)
were applied against all fungal species. Generally, all organic solvent extracts
reduced the fungal biomass significantly with the increase in concentration but,ethyl acetate fraction exhibited better results and reduced the fungal growth insome species up to 50% like D. australiensis and 44% each in A. alternata andF. oxysporum. From GCMS analysis of ethyl acetate fraction, 10 compounds
were identified. Out of these 10 compounds, only one compound [1,2Benzenedicarboxylic acid, mono (2-ethylhexyl) ester], molecular formula=
C16H22O4 and molecular weight= 278, showed 91.31% peak value. It was
concluded that this compound being in the highest concentration in the ethyl
acetate fraction of A. bisporus mushroom is responsible for its antifungal
activity, recorded in the present study. The present investigation concluded that
A. bisporus has bioactive compounds that can be exploited to develop
ecofriendly fungicides against a number of plant pathogens.

E-mail: makbarpu@gmail.com

4th International Conference of Asian Allelopathy Society

O-16
Change on the allelopathy and rhizospheric soil
microbial diversity by regulating OsMYB57
expression on rice
Changxun Fang,1,2,3 Weisi Chen,1,3 and Wenxiong Lin*1,2,3
1

Fujian Provincial Key Laboratory of Agroecological Processing and Safety
Monitoring, College of Life Sciences, Fujian Agriculture and Forestry
University, Fuzhou 350002, P. R. China
2
Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple
Utilization of Crops, Fujian Agriculture and Forestry University,
Fuzhou 350002, P. R. China.
3
Key Laboratory of Crop Ecology and Molecular Physiology (Fujian
Agriculture and Forestry University), Fujian Province University,
Fuzhou 350002, P. R. China.
Allelochemicals are mainly produced as plant secondary metabolites. MYB
transcription factors are documented that effectively regulate plant secondary
metabolic pathways, which suggests a potential role of MYB in rice allelopathic
agents. The study here showed that transcription factor OsMYB57(LOC_Os02g40530) positively regulates rice allelopathic effect on
barnyardgrass. The gene expression level of O-methyltransferase and cinnamoylcoenzyme A were up-regulated in the OsMYB57 transcriptional enhanced rice,whilst they were down-regulated in the OsMYB57 transcriptional repressed rice,compared to wild-type rice. Enhancement of OsMYB57 expression also led toincreased microbial populations in the rhizosphere of rice. Among the microbes,
Bacillus spp., Pseudomonas spp., and Streptomyces spp. were observed
suppressing the barnyardgrass growth with higher inhibitory rate, and their
populations were highest in the rhizospheric soil of OsMYB57-enhanced rice. A
further study indicated that the phenolic acids, including p-hydroxybenzoic
acid, cinnamic acid, and ferulic acid were able to facilitate the growth of
Bacillus subtilis, Streptomyces and Pseudomonas spp., revealing theinteractions between these microbes and phenolic acids. Our results
demonstrated that transcription factor OsMYB57 functions in modulation of therice allelopathic potential by regulating the genes from phenylpropanoid
pathway. The allelochemicals secreted by rice roots promoted the gathering of
microorganisms, and the joint-interactions between allelochemicals andparticular microbes dominate the allelopathic effect on target barnyardgrass.
 E-mail: lwx@fafu.edu.cn

4th International Conference of Asian Allelopathy Society

O-17
Influence of Trichoderma harzianum in growth,plant defense-related genes and metabolites of Radix
pseudostellariae
Jun, Chen.,1 Linkun, Wu.,1 and Wenxiong, Lin.1
1

College of Life Sciences, Fujian Agriculture and Forestry University,
Fuzhou, 350002, China.

Consecutive monoculture of Radix pseudostellariae in the same field results inserious decrease in both yield and quality. Many Trichoderma species arereported to be antagonistic towards plant pathogen. In this study, we study how
the interaction of plant with Fusarium oxyproum and Trichoderma affect theplant’s growth. A three-year field experiment was used to explore the shifts inthe structure and diversity of rhizosphere Trichoderma communities by DGGE.
Bioassays were performed in the fields, and yield of the plants was evaluated.
The effect of Trichoderma treatment on the expression of R. pseudostellariae
defense-related and pathogenesis-related genes was analyzed by real-time PCR.
In addition, we also analyzed the root samples of R. pseudostellariae treatedwith T. harzianum ZC51, based on metabonomics.
The results demonstrated that extended monoculture significantly declined thediversity of Trichoderm spp. Quantitative PCR analysis showed a significant
decrease in Trichoderma spp, especially in T. harzianum. Furthermore, a strain
of T. harzianum ZC51 antagonistic to F. oxyproum was isolated fromrhizosphere soil. Plant biomass showed that T. harzianum ZC51 performed goodand protected R. pseudostellariae from infection by F. oxysporum, and the yieldof R. pseudostellariae which was treated with T. harzianum ZC51 could reach
77.04% of the newly plant (FP). Moreover, the interaction of plants with T.
harzianum ZC51 affects the level of expression of two defense-related genes,
and three pathogenesis-related genes. In addition, metabonomics results showthat 18 kinds of mutual metabolites with significant differences, including
amino acids, sugars, organic acids, and organic compounds. In conclusion, thisstudy demonstrated that extended monoculture of R. pseudostellariae could
alter the Trichoderma communities in the plant rhizosphere, leading torelatively low level of antagonistic microorganisms. But TR treatment could
enhance the expression of defense-related genes, and the yield of R.
pseudostellariae.
 E-mail: chenjunfafu@163.com

4th International Conference of Asian Allelopathy Society

O-18
Features of microbiomes in the rhizosphere soils of
Achyranthes bidentata tolerant to consecutive
monoculture
Juanying Wang1,2 and Wenxiong Lin1,2,3,*
1

Fujian Provincial Key Laboratory of Agroecological Processing and Safety
Monitoring, College of Life Sciences, Fujian Agriculture and Forestry
University, Fuzhou 350002, PR China. 2 Key Laboratory of Crop Ecology andMolecular Physiology, Fujian Agriculture and Forestry University, Fuzhou
350002, PR China. 3 Key Laboratory for Genetics, Breeding and Multiple
Utilization of Crops, Ministry of Education / College of Crop Science, Fujian
Agriculture and Forestry University, Fuzhou 350002, PR China.
In the long-term practice, it was found that most medicinal plants suffered fromserious replanting diseases. Interestingly, in case of A. bidentata it is opposite to
the aforementioned and a very suitable plant species for continuous
monoculture. Therefore, in this study, the rhizosphere soils which was grown ina monoculture for multiple years were investigated for analyzing therhizosphere microbial community structure by means of high-throughput 16S
rDNA sequencing and denaturing gradient gel electrophoresis (DGGE)
techniques. The results showed that there was significant enhancement in theabundance of some beneficial species such as Pseudomonas spp and Bacillus
spp. The DGGE profiles revealed that the abundance of Pseudomonas
aeruginosa (P. aeruginosa) was distinctly higher in consecutively monocultured
plots than in the newly planted plot. Further, in the exogenous addition
experiment showed that: A strain of P. aeruginosa W39 from our lab was foundto have plant growth promoting activities, such as build-up of antagonistic
effect, production of auxin and increase in biomass of A. bidentata (P<0.5). The
results of the study suggest that one of the chief reasons for A. bidentata
tolerance to continuous cropping may be the increase in the number ofbeneficial bacteria mediated by root exudates in the rhizosphere soil with theincreasing years of continuous cropping.

*E-mail: lwx@fafu.edu.cn

4th International Conference of Asian Allelopathy Society

O-19
Comparative metagenomics analysis of soil
microbial communities under Rehmannia glutinosa
consecutive monoculture
Linkun Wu,1,2 Jun Chen,1,2 Hongmiao Wu,1,2 Juanying Wang,1,2Zhigang Xiao,1,2 Xianjin Qin,3,4 Zhongyi Zhang,3,4 Wenxiong Lin1,3
1

College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, P.R. China. 2Fujian
Provincial Key Laboratory of Agroecological Processing and Safety Monitoring, Fujian Agriculture andForestry University, Fuzhou, Fujian, P.R. China. 3Key Laboratory of Crop Ecology and Molecular Physiology(Fujian Agriculture and Forestry University), Fujian Province University, Fuzhou, Fujian, P.R. China.
4
College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, P.R. China.

Rehmannia glutinosa, a perennial herbaceous plant belonging to the family Scrophulariaceae, ishighly valued in traditional Chinese medicine. However, this plant suffer from severe allelopathic
autotoxicity, also known as replant disease or consecutive monoculture problem in the agricultural
production. Consecutive monoculture of this plant in the same field results in very serious decline inboth quality and yield of tuberous roots, with poor field performance and insufficient resistance. The
root-associated microbiome, referred to as the second genome of the plant, is closely associated withthe soil health and plant performance. In this study, pyrosequencing of 16S rDNA genes andwhole-community shotgun metagenomic sequencing were applied to investigate the shifts in soil
microbial community structure and functional potentials under R. glutinosa consecutive monoculture.
The results from barcoded pyrosequencing of 16S rDNA genes showed consecutive monoculture ofthis plant clearly affected the root-associated bacterial communities in both the rhizosphere andrhizoplane. R. glutinosa monoculture significantly decreased the relative abundances of the phyla
Firmicutes and Actinobacteria and the families Pseudomonadaceae (i.e. Pseudomonas), Bacillaceae
and Micrococcaceae, while increased the relative abundances of the families Sphingomonadaceae and
Xanthomonadaceae. Metagenomics analysis also showed R. glutinosa monoculture significantly
decreased the relative abundances of Pseudomonadaceae and Pseudomonas while increased therelative abundance of Sphingomonadaceae. Moreover, dual culture demonstrated the decline in theabundance of Pseudomonas spp. with antagonistic activity against fungal pathogens undermonoculture. Besides, comparative metagenomics analysis showed that for potentiallyharmful/indicator microorganisms, the percentages of reads categorized to defense mechanisms (i.e.
ABC transporters, efflux transporter, antibiotic resistance) and biological metabolism (i.e. lipid
transport and metabolism, secondary metabolites biosynthesis, transport and catabolism, nucleotide
transport and metabolism, transcription) were significantly higher in the two-year monocultured soil
than in the newly planted soil, but the opposite was true for potentially beneficial microorganisms,
which might disrupt the equilibrium between beneficial and harmful microbes. In conclusion, ourresults provide important insights into the shifts in genomic diversity and functional potentials of
rhizosphere microbiome in response to R. glutinosa consecutive monoculture.
Keywords: Rehmannia glutinosa, replant disease, barcoded pyrosequencing, metagenomics, soil
microbiome, functional potential

 E-mail: lwx@fafu.edu.cn

4th International Conference of Asian Allelopathy Society

O-20
Analysis of fungal community structure variation on
successive rotations of Casuarina equisetifolia as
determined by pyrosequencing
Zeyan Wu,1,2,3,4* Jinfu Liu,2,5 Zhifang Chen,5 Jianjuan Li,5
Siren Lan,5 and Wei Hong5
1

Life Sciences College of Fujian Agriculture and Forestry University,
Fujian350002, China.
2
Cross-Strait Nature Reserve Research Center,Fujian Agriculture and Forestry University, Fuzhou 350002, China.
3
Fujian Provincial Key Laboratory of Agroecological Processing and Safety
Monitoring, School of Life Sciences,
Fujian Agriculture and Forestry University, Fuzhou 350002, China.
4
Key Laboratory of Crop Ecology and Molecular Physiology(Fujian Agriculture and Forestry University),
Fujian Province University, Fuzhou 350002, China.
5
Forestry College of Fujian Agriculture and Forestry University,
Fujian350002, China.

The reproduction of Casuarina equisetifolia are severely compromised under
the successive monoculture regime. In this study, the aim was to analyse theshifts of rhizospheric microbial community structure under the increasing yearsof successive rotations conditions by applying the technology of deep
pyrosequencing. The results demonstrated that there were obvious differencesamong the first rotation Casuarina equisetifolia plantation (FCP), the second
rotation plantation (SCP) and the third rotation plantation (TCP). In FCP, thefungal community showed a significantly higher both the Shannon and Simpson
diversity indexes than in SCP (P<0.05). The relative abundance Fusarium,
Thelephora, Hortaea and Penicillium significantly increased under the
successive rotations. Oppositely, the Tolypocladium and Trichoderma,
conducted a significantly decrease. The results revealed that long-term
successive rotations of C. equisetifolia induced the alternations in therhizospheric soil microbial community structure.
Key words: Casuarina equisetifolia, successive rotation, microbial community
composition, deep pyrosequencing
 E-mail: wuzeyan0977@126.com

4th International Conference of Asian Allelopathy Society

O-21
Allelopathic Water Extracts Helps Improve Drought
Resistance during Reproductive Stages in Wheat
Ahmad Nawaz,1* Muhammad Nabeel,2 Muhammad Farooq,2Mubshar Hussain,1 and Muhammad Ijaz1
1

College of Agriculture, Bahauddin Zakariya University,
Bahadur Sub-Campus, Layyah, Pakistan.
2
Department of Agronomy, University of Agriculture,
Faisalabad, Pakistan.

This experiment was conducted in pots where two wheat cultivars viz.,
Mairaj-2008 (M-2008) and Faisalabad-2008 (FD-2008) were grown in the pots
filled with 10 kg of the soil. The pots were irrigated as per need of the crop until
leaf boot stage at 75% WHC which was followed by imposition of drought
stress at reproductive (booting and anthesis) and grain filling (dough stage)stages in both wheat cultivars. When one week passed away after foliage
application of water extracts on sorghum, brassica, moringa and sunflower, thedrought was imposed at each stage by maintaining the pots at 35% WHC.
Drought stress negatively altered performance of wheat crop in terms ofmorphology and yield; however, the yield of wheat under drought was
improved after the foliage application of allelopathic water extracts. M-2008
performed better than FD-2008 under drought stress which was visible throughimprovement in the performance of this cultivar under terminal drought stress.
Although, application of all the AWEs improved the performance of both
cultivars, the water extract of sorghum was most useful under drought stressthan other extract. Drought stress imposition at booting and anthesis was moreinjurious to crop than when imposed at grain filling. In crux, foliage applied
AWEs applied alone or in combination might be pragmatic option to improve
wheat performance under climate change.

Key words: Drought, wheat, morphology, climate change, water extract
 E-mail: ahmadnawaz2006@gmail.com

4th International Conference of Asian Allelopathy Society

O-22
Influence of different allelopathic weed management
strategies on wheat performance in different
wheat-based cropping systems of Punjab, Pakistan
Mubshar Hussain,1* Muhammad Shahzad,1,2 Khawar Jabran,3and Muhammad Farooq4,5,6,7
1

Department of Agronomy, Bahauddin Zakariya University, Multan, Pakistan.
Department of Agronomy, University College of Agriculture & Environmental Sciences,
The Islamia University of Bahawalpur, Baghdad-ul-Jadeed Campus, Bahawalpur, Pakistan.
3
Department of Plant Protection, Faculty of Agriculture and Natural Sciences,
Duzce University, Turkey
4
Department of Crop Sciences, College of Agricultural and Marine Sciences,
Sultan Qaboos University, Al-Khoud 123, Oman.
5
Department of Agronomy, University of Agriculture, Faisalabad, Pakistan.
6
The UWA Institute of Agriculture, The University of Western Australia, 35 Stirling Highway,
Crawley WA 6009, Australia.
7
College of Food and Agricultural Sciences, King Saud University,
Riyadh 11451, Saudi Arabia.
2

Weed infestation is a major constraint reducing crop productivity all over theworld. In various agro-ecosystems, the weeds are impacted and prevailingcropping systems and the weed control methods. Herbicides are sole method of
weed control in most of the cropping systems; however, the method is facing
problems such as evolution of herbicide resistance in weeds. Moreover, recent
cropping systems are highly simplified which are allowing the best adapted
weed species to proliferate in the field. To solve these issues, crop rotation is a
viable option for preventing weed proliferation in a specified cropping system.
The blending of crop rotation with weed management practices seems highly
effective in this regard. Hence this two-year study was conducted to study theeffect of wheat-based cropping systems viz. fallow-wheat (FW), rice-wheat(RW), cotton-wheat (CW), mungbean-wheat (MW) and sorghum-wheat (SW)on weed infestation, wheat allometry and productivity under divergent weed
management strategies viz. false seedbed, allelopathic water extracts andherbicides application while weed free and weedy check were maintained ascontrols. Wheat growth was substantially low when planted after sorghum due
to its allelopathic affects; nonetheless, mungbean favored the wheat growth.
Chemical weed control decreased weeds infestation which was followed by useof allelopathic extracts; while false seedbed was least effective.
Keywords: Weed managing strategies; Crop allometry; Cropping systems; Wheat performance E-mail: mubashiragr@gmail.com

4th International Conference of Asian Allelopathy Society

O-23
Exogenous of moringa plant residues and aqueous
extract at low and high concentrations on
germination, growth and yield of sunflower
Javaid Iqbal,1 Javeria Irshad,2 Adnan Noor Shah,3and Muhammad Yousaf41

Department of Agronomy, Ghazi University,
Dera Ghazi Khan 32200 Punjab Pakistan.
2South Punjab Institute of Science and Technology,
Dera Ghazi Khan 32200 Punjab Pakistan.
3
Department of Agronomy, Gomal University Dera Ismail Khan KPK, Pakistan.
4
Department of Soil and Environmental Science, Ghazi University,
Dera Ghazi Khan 32200 Punjab Pakistan.
Moringa oleifera belonging to family Moringaceae, is considered as one of theactive allelopathic plant. Allelopathic interactions are considered asconcentration dependent. In this study the allelopathic potential of Moringa
oleifera was studied at both low and high concentration of plant material on
growth and yield of sunflower. A pot trial was conducted in Completely
Randomized Design with four replications at the research area of Ghazi
University, Dera Ghazi Khan Punjab Pakistan. Leaves of Moringa were applied
as soil mixed and surface application at the rate of 0,100,200 and 300gpot-1.
Addition of leaves in the pots increased shoot fresh (39-43%) and dry weights(23-44%), root fresh (44-74%) and dry weights (23-35), head diameter (7-14%),achene’s count (5-21%) and achene’s yield per plant (9%), and 1000 acheneweight (1%) at lower concentration @100-200gpot-1. The improvement in theseparameters was higher @100gpot-1 as compared to 200gpot-1. But at higher rate
@300gpot-1 these parameters were adversely affected by Moringa as compared
to control (-1 to -24%). Mixing of leaves with soil perform better as comparedto surface application (1-14%). It is concluded that Moringa residues havepromotive effect upto a certain concentration but at higher concentration they
have inhibitory effects on growth and yield of sunflower.
Keyword: Morinaga oleifera, Allelopathy, Sunflower, promotive effect,
inhibitory effect E-mail: javaidleghari@hotmail.com

4th International Conference of Asian Allelopathy Society

O-24
Rhizosphere responses to environmental conditionsin the Radix pseudostellariae
rhizosphere under continuous monoculture regimes
Hongmiao Wu,1,2 Xianjin Qin,2,3 Juanying Wang,1,2and Wenxiong Lin1,2,3*
1

Fujian Provincial Key Laboratory of Agroecological Processing and Safety Monitoring,
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China.
2
Key Laboratory of Crop Ecology and Molecular Physiology(Fujian Agriculture and Forestry University), Fujian Province University, Fuzhou 350002, PR China.
3
Key Laboratory for Genetics, Breeding and Multiple Utilization of Crops,
Ministry of Education / College of Crop Science, Fujian Agriculture and Forestry University,
Fuzhou 350002, PR China.

Radix pseudostellariae (Radix Pseudostellariae L.) is one of the most common and highly
demanded Chinese medicines. High-quality Radix pseudostellariae is mainly produced in
ZheRong, Fujian province in southern China, where the soil and climate conditions are themost favorable for its growth. However, consecutively monocultured Radix pseudostellariae
are prone to severe diseases, which may result in reduced biomass, especially of tuberous
products. This phenomenon is known as replanting disease or soil sickness. More than 70 %of medicinal plants, especially tuberous root medicinal plants, are reported to be attacked byvarious replanting diseases. Therefore, replanting disease incidence has resulted in atremendous hurdle to obtain maximum agricultural production of Radix pseudostellariae.
The root exudates of R. pseudostellariae can deteriorate the microbial community in therhizosphere, including hosting specific pathogens at the expense of beneficial
microorganisms. We found the effect of phenolic acids and organic acids were invoked as adriver of the changes seen in the rhizosphere soils. Then we study the effects of an artificiallyapplied root exudates (organic acids, phenolic acids) of Radix pseudostellariae, on Radix
pseudostellariae seedling growth, rhizosphere soil microbial communities, and soil
physicochemical properties. The deep pyrosequencing and qRT-PCR analysis demonstrated
that the treatment of phenolic acids significantly decreased the relative abundance ofTrichoderma, Penicillium, Pseudonocardiales, Xanthomonadales, Streptomycetales,
Micrococcales, Gemmatimonadales. And the organic acids had significantly negative effecton the relative abundance of Pseudonocardiales and Streptomycetales, which significantly
increased the abundance of Fusarium, Xanthomonadales, Micrococcales andGemmatimonadales. The non-invasive micro-test technique analysis indicated that the root
exudates increased the H+ efflux in the pathogenic fungi (Fusarium oxysporum andTalaromyces helicus) and decreased the H+ efflux in the beneficial fungi (Trichoderma
harzianum), which creates an acid environment for the proliferation inhibition of beneficial
bacteria and build-up of specialized plant pathogens. This study explains the reasons why theroot exudates are able to mediate a microflora shift and structure disorder in continuously
monocultured Radix pseudostellariae rhizosphere soil.
Key Words: Radix pseudostellariae; Replanting disease; root exudates; non-invasive micro-test
technique; deep pyrosequencing

E-mail: lwx@fafu.edu.cn

4th International Conference of Asian Allelopathy Society

O-25
Advanced utillization of allelochemicals in Japanesecherry blossom
Tomita-Yokotani, K.,1* Kimura, Y.,2 Kobayashi, H.,3
Kimura, S.,1 and Fujii, Y.4
1

Faculty of Life and Environmental Sciences, University of Tsukuba,
Tsukuba, Ibaraki 305-8572,
2
Faculty of Human life, Jumonji University,
Niiza, Saitama 352-8510,
3College of Agro-Biological Resource Sciences, University of Tsukuba,
Tsukuba, Ibaraki 305-8572, Japan,
4Agriculture and Technology, Internal Environmental and Agricultural Sciences,
Fuchu Campus, Fuchu, Tokyo, 183-8509 Japan.
Japanese cherry blossom, “Sakura”, is a very famous tree in Japan because of
their beautiful flowers. There are many species and varieties in its genus. We
have already reported a part of their allelochemicals in leaves of a species ofJapanese cherry blossom. Many pleople have been focusing on the existence ofallelochemicals in “Sakura” which contribute to human health. Actually, we
identified chemicals in the extract from leaves of a species that showed two
enzyme’s activities related to our health. During our experiments and reporting
our research, the amount of general information related to “Sakura” increased ina field of food or dietary supplements. At this time, we will show some of thefunctional substances in the extracts from leaves of Japanese cherry blossoms
and an increase in the amount of general information related to “Sakura”.
Materials were collected from University of Tsukuba. The leaves were driedand stored at room temperature until starting the extraction. Functional
substances were extracted with water from leaves because we need an easy
method for getting the functional substances. This process is like making tea.
General information related to “Sakura” was found in several newspapers inJapan.
We will show the advanced utilization of allelochemicals and discuss the
future of the allelopathy study.

 E-mail: yokotani.kaori.fn@u.tsukuba.ac.jp

4th International Conference of Asian Allelopathy Society

O-26
Allelopathic and antimicrobial study of Acacia
modesta and Buxux papillosa available in District
Hangu, KPK, Pakistan
Shahida Ayub, Saima Hashim*
Department of Weed Science, The University of Agriculture,
Peshawar-Pakistan
The current study was to asses’ allelopathic activities and antimicrobial
potential of Acacia modesta Wall and Buxus papillosa C. K. Schneider. A
laboratory based studied was made to investigated the allelopathic potential of
A. modesta and B. papillosa leaves and roots on Lettuce seed (Lactuca sativa L.
v. Great lakes 366). Results were significant which showed that 100 g/L water
extraction of leaves of both the plants having high allelopathic potential toinhibit the seedling growth of lettuce seeds. Roots water extractions of both theplants induce seedling growth of tested plant. The in vitro antimicrobial
investigations of crude methanolic extracts of A. modesta (bark and root) and B.
papillosa (leaves, stem and root) plants were analyzed against five pathogenic
bacteria including one Gram positive Bacillus subtilis, four Gram negative i.e.
Escherichia coli, Klebsiella pneumonia, Xanthomonas campestris andAgrobacterium tumefaciens using disc diffusion method. We obtained
significant results from this experiment. It was observed that K. pneumonia was
the most resistant bacterium to all extracts. Present study concluded that theseplants are good sources of antibacterial compounds for the curing of many
pathogenic diseases. These results confirm strong allelopathic and antimicrobial
potential of these both of these candidate species.

 E-mail: saimahashim@aup.edu.pk

4th International Conference of Asian Allelopathy Society

O-27
Remediation of deterioration in microbial structure
in continuous Pinellia ternata cropping soil by crop
rotation
Zhigui Heab，Renjun Maoa，Juan'e Donga, *, Zongsuo Liangac, **，Haihua Zhangac, Lin Liua*a Northwest A & F University, College of Life Sciences, Yangling 712100, Chinab Guilin Tourism University, Institute of Food Science and Biological Engineering,
Guilin 541006, Chinac Zhejiang Sci-Tech University, School of Life Sciences, Hangzhou 310018, China

Pinellia ternata is a traditional Chinese herb that has been shown to sufferfrom continuous cropping, which significantly decreases both yield and quality.
The influence of continuous cropping on microbiome in P. ternata rhizosphereand effects of remediation on microbiota by rotational cropping were assessedby using Illumina high-throughput sequencing technology. Microbial
composition analysis showed that microorganisms belonging to Proteobacteria,
Acidobacteria, and Actinobacteria dominated in all cropping treatments.
Moreover, continuous cropping tended to decrease a-diversities along withcultivation time, whereas rotational cropping tended to increase them.
Differentially abundant analysis showed that microbial structure was importantfor maintaining the health status of P. ternata rhizosphere. Results suggested
that continuous P. ternata cropping may result in simplification and shifts in
microbial structures leading to rhizosphere deterioration. Meanwhile, rotational
cropping between P. ternata and wheat can revert structural changes and thus
remediate the rhizosphere. PCoA and hierarchical analyses generated three post
hoc clusters of sampled soils, which were named as healthy, health-suppressed,and health-depressed soil, respectively. We also found that these three types ofsoils represented different soil physicochemical properties and microbiome
activities. Based on this finding, potential indicator taxa that respond to existingsoil were also identified.
Key words: microbial diversity, community structure, differentiallyabundant taxa, functional features, potential indicators
 E-mail: james1916@126.com

4th International Conference of Asian Allelopathy Society

O-28
Expression difference between allelopathic andnon-allelopathic rice cultivars
Yazhou Liu1, Yiding Sun2*, Jian Fu2, Ru Li1, Hong Lu1, Yiqing Guo1*
1 Yunnan Agricultural University, Kunming, China 650201
2 Biotechnology & Genetic Germplasm Resources Institute, Yunnan Academy ofAgricultural Sciences, Kunming, China 650223
Rice allelopathy means that allelochemicals from rice are secreted around the rhizosphere and suppress the growth ofweeds. The process is regulated/induced by many genes such as signaling transfer from the donor plant to target plant andallelochemicals released from the donor plant. Phenolic compounds were earliest found as one of bioactive chemiclas fromrice. C4H (cinnamic acid 4-hydroxylase) catalyzes a key reaction in the biosynthesis of a large number of phenolic
compounds in higher plants, also is a gene coding for cinnamic acid to p-coumaric acid, which is closely related toallelopathic activity. There are 4 genes encoding C4H in rice. In this study, allelopathic and non-allelopathic rice PI312777
and Lemont were used as materials, the full length sequence of 4 C4H gene from the two rice were cloned and sequencealignment, gene expression was detected by q-PCR after treated for certain time by SA (salicylic acid), JA (jasmonic acid),
UV-B and barnyardgrass, and then a target gene was selected for genetic transformation in order to further understanding themechanism of rice allelopathy regarding phenolic compounds. The results showed that the lengths of amino acids from thetwo rice lines were almost same, and the similarity was about 98.69% with typical domain of P450, and a variety ofcis-acting elements such as transcriptional regulatory factors existed in the promoter of the two rice. The expression of the 4C4H genes in time and pattern was differ under stress condition, in which Os01g0820000 and the Os02g0467000 had similar
expression level after treatment by JA and SA, but Os02g0467600 and Os05g0320700 were up-regulated in PI312777 much
more than that in Lemont. After 1 h treatment by UV-B or 7 days by barnyardgrass, the expression of Os02g0467600 wasincreased in the 2 rice lines, but up-regulated for PI312777 and down-reguilated for Lemont. The expression level of thegenetically transformed rice with Os02g0467600 was increased at 26.41%. These results suggested that C4H-encoding gene
Os02g0467600 plays an important role in rice allelopathy to weed. There are 7 difference loci in the amino acid sequence
between allelopathic and non-allelic rice, and a large number of transcriptional regulatory factors in the promoter sequencewhich may result in difference of allelopatic potential of rice cultivars under stress condition. Further study will be continued
to confirm.

Key words: rice allelopathy; cinnamic acid-4-hydroxylase; Os02g0467600; stress; level of gene expression.

Acknowledgments: The research is funded by the NSFC of China and Fujian Provincial Key Laboratory of Agro-ecological
Processing and Safety Monitoring, College of Life Sciences, Fujian Agriculture and Forestry University (NSFC 31260439,
31760523 and NYST-2017.02).

 E-mail: ichinguo@163.com, yidingsun@hotmail.com

4th International Conference of Asian Allelopathy Society

O-29
Chemical Composition and Behavioral Responses ofEssential Oil Isolated from Eupatorium
adenophorum to Lasioderma serricorne
Lihong Tao, Tingting Cai, Min Ye*
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,
Yunnan Agricultural University, Kunming 650201, China

Essential oil was extracted from the fresh leaves of Eupatorium
adenophorum through hydro-distillation, using a Clevenger type apparatus. The
responses of Lasioderma serricorne to the essential oil were evaluated using a Y
shaped olfactometer. Unsexed adult tobacco beetles were attracted to odor fromthe essential oil when the amounts of essential oil were in the range from 0.1 μg
to 100 μg. When the amount of essential oil went up to 1000 μg, the beetles
were repelled to odor from the essential oil. Beetles did not respond better toodor from the essential oil when the amount of essential oil was 0.01 μg. In thiscase numbers of beetles that moved upwind toward the odor were notsignificantly higher than numbers of that moved upwind toward blank control.
Chemical composition analysis of the essential oil was carried out on a
combined gas chromatograph-mass spectrometer (GC-MS) fitted with an
HP-5MS capillary column at a temperature programme of 40 to 250°C at a rateof 5°Cꞏmin-1 and held for 10 min. The chemical components of the essential oil
were identified by comparing the obtained mass spectra of the analytes withthose of authentic standards from the NIST 08 library with a resemblance
percentage above the ninety percent. The chemical components relative content
was calculated based on the peak area of total ion current chromatogram. The
major components identified in the essential oil include: (-)-δ-Cadinol 18.96%,
β-Bisabolene 7.24%, α-Bisabolol 7.071%, (-)-Bornyl acetate 6.84%, Cadinene
5.78%, (Z)- β-Farnesene 4.66%, β-Cubebene 4.64%, Velleral 4.54%,
β-Caryophyllen 4.21%.
Key words: behavioral responses, Lasioderma serricorne, essential oil,
Eupatorium adenophorum, chemical components

 E-mail: yeminpc@126.com

4th International Conference of Asian Allelopathy Society

O-30
Plant-soil negative feedback in a sanqi production
system mediated by root exudates and soil microbe
Shusheng Zhu*, Yin Yang
State Key Laboratory for Conservation and Utilization of Bio-ResourcesinYunnan,
Yunnan Agricultural University, Kunming 650201, China;
Key Laboratory forAgro-biodiversity and Pest Control of Ministry of Education, Yunnan
Agricultural University, Kunming 650201, China
Replant failure caused by negative plant-soil feedback (NPFS) in agricultural ecosystems is a
critical factor restricting the development of sustainable agriculture. Sanqi ginseng [Panax
notoginseng (Burk.) F. H. Chen], a member of the Araliaceae family, isone of the most important
herbal medicines in China and is in high demand. In China, the commercial cultivation of Sanqi
ginseng has been practiced for more than 200 years. Because ginsenosides have significant effects
against cancer and cardiovascular disease, requirement for these compounds have increased rapidly.
Recently, more than 4 million hectares of Sanqi ginseng have been sown annually in China. However,
Sanqi ginseng plants are often hampered by replant failure, which results in yield reductions and otherdifficulties when re-establishing plants in cultivated fields due to low seed germination, poor seedling
growth, and severe disease. Many factors potentially contribute to this problem, including thedeterioration of soil physicochemical characteristics, nutrient imbalance, soil-borne diseases, andautotoxicity. In the last three decades, researchers and farmers have hypothesized that Sanqi ginseng
replant failure results from the accumulation of pathogens in the soil. Many types of soil-borne
pathogens that cause diseases in Sanqi ginseng have been identified, including fungi, Oomycetes,bacteria, nematodes, and viruses. However, Sanqi ginseng cannot be successfully cultivated again on
the same land even after sterilizing the soil by using fungicides or fumigation. In addition, many
tested fertilizer application and soil modification practices have yielded unsatisfactory results.
Recently, it was hypothesized that the secondary metabolites released by plant residues and root
exudates that accumulated in the soil could be autotoxic to P. notoginseng during replanting.
Autotoxicity, which is defined as a deleterious allelopathic effect among individuals of thes ame
species, has been documented in various crops. For many medicinal plants, suchas Rehmanni
aglutinosa, Pseudostellaria heterophylla and Salvia miltiorrhiza, autotoxicity results in significantly
reduced yields and quality. The autotoxicities in ginseng (P. ginseng) and American ginseng (P.
quinquefolius), which belong to the same genus as Sanqi ginseng (P. notoginseng), were recently
reported as one possible factor that contributes to replant failure. Some phenolic acids in the root
exudates and rhizosphere soils of American ginseng have been identified as potential autotoxic
compounds. However, phenolic acids have frequently been identified in other crops as putative E-mail: shushengzhu79@126.com

4th International Conference of Asian Allelopathy Society
allelopathic substances and have been identified in uncultured soil. Thus, we speculated that otherspecial autotoxic compounds might result in Sanqi ginseng replant failure. Ginsenosides are theprimary biologically active compounds that are produced by Sanqi ginseng plants. At least 20
different ginsenosides occur in Sanqi ginseng, which account for more than 6% of the plant's dry
biomass. Ginsenosides can be released into the rhizosphere soilas root exudates, by leaching andvolatilization, or from the decomposition of plant residues by Sanqi and American ginseng. The root
exudates, root extracts, and extracts from consecutively cultivated soils showed significant
autotoxicity against seedling emergence and growth. Ginsenosides, including R1, Rg1, Re, Rb1, Rb3,
Rg2, and Rd, were identified in the roots and consecutively cultivated soil. The ginsenosides, Rg1, Re,
Rg2, and Rd, were identified in the root exudates. Furthermore, the ginsenosides, R1, Rg1, Re, Rg2,
and Rd, caused autotoxicity against seedling emergence and growth and root cellvigor at aconcentration of 1.0 μg/mL.
We elucidated the response of P. notoginseng to the autotoxic ginsenoside Rg1via transcriptomic
and cellular approaches. Cellular analyses demonstrated that Rg1 inhibited root growth by disrupting
the cell membrane and wall. Transcriptomic analyses confirmed that genes related to the cell
membrane, cell wall decomposition and reactive oxygen species (ROS) metabolism were up-regulatedby Rg1 stress. Further cellular analyses revealed that Rg1 induced ROS (ꞏO2− and H2O2) accumulationin root cells by suppressing ascorbate peroxidase (APX) and the activities of enzymes involved in theascorbate-glutathione (ASC-GSH) cycle. Exogenous antioxidants (ASC and gentiobiose) helped cells
scavenge over-accumulated ROS by promoting superoxide dismutase (SOD) activity and the
ASC-GSH cycle. Collectively, the autotoxin Rg1 caused root celldeath by inducing theover-accumulation of ROS, and the use of exogenous antioxidants could represent a strategy forovercoming autotoxicity.
Ginsenosides are primary components of ginseng and play a multi-purpose ecological role. Likeother saponins, ginsenosides are fungitoxic in plants and act as a host for chemical defenses at highconcentrations. In the rhizosphere, ginsenosides not only exert autotoxic effects on Sanqi ginseng
plants, resulting in poor defense and growth, but also stimulate the growth of soilborne pathogens.
Previous reports have shown that ginsenosides can stimulate the growth of soil-borne P.
quinquefolium and P. notoginseng pathogens, such as Cylindrocarpon destructans, Fusarium solani,
Phytophthora cactorum, and Pythium irregulare. If the autotoxicity of ginsenosides and the infectionof soil-borne pathogens act synergistically in the rhizosphere, a potentially stronger replant failure
effect is expected. Some previous studies have indicated that the interactions between Fusarium
oxysporum and autotoxic cinnamic acid may enhance soil sickness. Thus, additional studies areneeded to evaluate the interactions between autotoxicity and the other factors involved in replantfailure, such as soil-borne pathogens, the deterioration of soil physicochemical properties, and theimbalance of the soil microbial community. These studies could be used to understand themechanisms of replant failure in Sanqi ginseng.

 E-mail: shushengzhu79@126.com

4th International Conference of Asian Allelopathy Society

P-1
A comparison of extraction methods of phenolicacids in cropland soil
Yan Meng Bi, Xiao Lin Jiao, Jun Fei Li, Gei Lin Tian, Xiao Lu,
Xi Mei Zhang, and Wei Wei Gao*
Institute of Medicinal Plant Development, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100193, China
Phenolic acids play important roles in soil ecosystem. It is critical to measure
their level in soil and various methods to extract them were used by researchers.
To compare and evaluate these methods, different solvents included water,
dichloromethane, 1 M and 2 M sodium hydroxide solution (NaOH) were chosen
to extract phenolic acids in wheat (Triticum aestivum L.) and American ginseng(Panax quinquefolium L.) cultivated soil. All sample solutions, with or without
concentrating process, were tested with high performance liquid
chromatography. Nine individual phenolic acids (p-hydroxybenzoic acid,vanillic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, benzoic acid,salicylic acid and cinnamic acid) were detected in 1 M and 2 M NaOH extracts
with concentrating, while only five individual phenolic acids were detectedwithout concentrating. However, the levels of p-hydroxybenzoic acid, vanillin
and p-coumaric acid detected after concentrating were significantly lower. Only
five (American ginseng soil) or four (wheat soil) individual phenolic acids weredetected in water extracts, and none was detected in dichloromethane extracts.
The results indicate NaOH and water recovered different phenolic acids fromsoil, and concentrating might cause certain phenolic acids loss. Besides,
concentrating may make more individual phenolic acids detectable in NaOH
extract.

 E-mail: wwgao411@sina.com

4th International Conference of Asian Allelopathy Society

P-2
Allelopathic potential of Root Endophytic fungal
metabolites of Casuarina equisetifolia
R. Huang, S. K. Jin1, X. Wang, Z. X. Xu, H. M. Li and L. Li*
Ministry of Education Key Laboratory for Ecology of Tropical Islands,
College of Life Sciences,
Hainan Normal University, Haikou 571158, China.
To determine whether endogenous fungi of Casuarina equisetifolia are involved
in allelopathy, their diversity and the composition of their metabolites wasdetermined. By Illumina Miseq high-throughput sequencing platform, a total of
318,972 sequences were obtained from 9 root samples of C. equisetifolia ofdifferent ages, which included 11 phyla, 32 families and 32 genera. Four fungi
were isolated from the roots by the traditional method and identified by biolog
microbial automatic analysis system as Aspergillus aculeatus lzuka, Penicillium
melinii thom, Neosartoryafisccheri (Wehmer) Malloch & Cain BGA, andPenicillium solitum westling BGB. By GC-MS analysis of the fermentation
broth of four endogenous fungi, the pyrogallol, lauric acid, palmitic acid, stearicacid, 2,4-di-tert-butylphenol, 1,2,3,4-butanetriol and globulol and other organic
compounds were identified. Among them, 2, 4-ditert-butyl phenol, 1,2,3, 4butanediol and globulol were also found in soil, root and litters extract, throughthe comparing with previous GC-MS analysis of soil, root and litters extract.
The results implied that root-endogenous fungi are involved in the synthesis ofsensitized substances. The bioassay showed the fermentation broth of the fourroot-endophytic fungi significantly inhibited Thespesia lampas andCalophyllum inophyllum seed germination, A. aculeatus showed the strongest
inhibitory effect on seed germination.

 E-mail: lei-li@126.com

4th International Conference of Asian Allelopathy Society

P-3
The role of low molecular weight organic acidaccumulation on microbial deterioration in theRadix pseudostellariae rhizosphere under continuous
monoculture regimes
Manhong Lin,1,2 and Wenxiong Lin1,2,3,*
1

Fujian Provincial Key Laboratory of Agroecological Processing and Safety
Monitoring, College of Crop Sciences,
Fujian Agriculture and Forestry University, Fuzhou 350002, PR China.
2
Key Laboratory of Crop Ecology and Molecular Physiology,
Fujian Agriculture and Forestry University, Fuzhou 350002, PR China.
3
Key Laboratory for Genetics, Breeding and Multiple Utilization of Crops,
Ministry of Education / College of Crop Science,
Fujian Agriculture and Forestry University, Fuzhou 350002, PR China.

A two-year field trial of consecutive monoculture of R. pseudostellariae wasconducted to further understand the underlying mechanism why continuous
monoculture regimes significantly decline the biomass and quality of Radix
pseudostellariae. Quantitative PCR was used to analyse the total bacteria and fungi inthe monocultured rhizosphere soil and the expression of the biocontrol-related genes
in bacteria. And HPLC analysis was used to detect the organic acids and toxins.
HPLC analysis indicated that continuous monoculture soil was rich in small
molecular weight organic acids which had cumulative effects over time in therhizosphere soil under monoculture regimes. The qPCR analysis demonstrated theaccumulated organic acids concerned in rhizosphere soil led to the higher ratios offungi/bacteria over time. Further analysis suggested that the mixture of organic acids
at the ratio and dosage close to actual situation detected in the rhizosphere soil could
significantly promote the growth of the pathogenic fungi, such as Talaromyces helices,
Fusarium oxysporum and Fusarium moniliforme concerned, and the chemotactic
response, the expression of chemotaxis-related gene (cheA) and biofilm formation of
the specific pathogenic bacterium, Kosakonia sacchari, revise is true in the case of
Bacillus megaterium and Bacillus pumilus concerned. At the same time, the present
result also found that the mixed organic acids stimulate the production of toxins(3A-DON and 15A-DON) as well as H2O2 generations in the specific pathogenic
fungi. Furthermore, the organic acids had negative effects on the expression of thebiocontrol-related genes, and hence resulted in attenuated antagonistic activities of the
PGPR to suppress the mycelial growth of the pathogenic fungi at the dosage close toactual situation in the monoculture rhizosphere soil. This result unveils themechanisms how the accumulated organic acids differentially mediate deterioration ofsoil microbial composition and structure in monocropping system.
 E-mail: wenxiong181@163.com

4th International Conference of Asian Allelopathy Society

P-4
Hijacking underground common mycorrhizal
networks for interplant defense communication
Yuanyuan Song, and Rensen Zeng*
Institute of Crop Resistance & Chemical Ecology,
Fujian Agriculture and Forestry University,
Fuzhou 350002, Fujian, China
Majority of terrestrial plants form mycorrhizal symbiosis, which plays a vitalrole in plant nutrition and stress resistance. Common mycorrhizal networks(CMNs) link multiple plants together in ecosystems. Our study shows that
CMNs mediate plant-plant communication between healthy plants andenemy-challenged tomato plants. After establishment of CMNs with thearbuscular mycorrhizal fungus Glomus mosseae between tomato plants,
inoculation of ‘donor’ plants with pathogen or herbivore insect led to increases
in resistance and activities of the putative defensive enzymes, as well as
induction of defense-related genes in healthy neighboring ‘receiver’ plants,suggesting that CMNs may function as a plant-plant underground talking
conduit for systemic defense. However, use of a JA biosynthesis defective
mutant spr2 as ‘donor’ plants resulted in no induction of defense responses andno change in insect resistance in ‘receiver’ plants, suggesting that JA signaling
is required for CMNs-mediated interplant communication. Our results indicatethat plants are able to hijack CMNs for herbivore-induced defence signaltransfer and interplant defense communication.

Key words: Common mycorrhizal networks; Plant communication; Induced
defense E-mail: rszeng@fafu.edu.cn

4th International Conference of Asian Allelopathy Society

P-5
Changes in Amonia oxidizing community duringconsecutively monocultured Pseudostalleria
heterophylla
Khan, M. U.*, Wu, L., Amjad, H., and Lin, W.
Fujian Agriculture and Forestry University,
Fujian, Fuzhou 35000, China.
Monoculture problems occur and prevail in many crops. Psedustaleria
heterophylla is one of the important medicinal crop in China, which is effectedby the monoculture diseases and is becoming a daunting task to comprehend.
Previous research have pointed out that soil physico-chemical (such as
production of more phenolic acids) and microbial imbalance could lead to thisproblem. Here we explored the population abundance and diversity of ammonia
oxidizing community consisting of ammonia oxidizing archaea (AOA) andammonia oxidizing bacteria (AOB). We also quantified the other nitrogen cycle
genes in the rhizosphere soil. The gene abundance of nar, nifH, nirK and nosZ
were significantly lower in all treatments than the newly planted plants. The
DGGE and qPCR was employed to analyze the community structure andabundance of AOA and AOB. Both molecular techniques showed that amoA
gene from AOA and AOB sharply decreased in 2 year and 3 year monoculture
rhizosphere soils. In contrast, the application of the bio fertilizer improved thesoil rhizosphere conditions and triggered the process of reshaping the Ammonia
oxidizers in the soil close to the newly planted rhizosphere soil. However, it wasmore in favor of AOB as compared to AOA. We concluded that using bio
oraganic fertilizer could reduce the monoculture disease problems by partiallyreverting the ammonia oxidizing population close to newly planted ammonia
oxidizers population.

 E-mail: umar.khan018@yahoo.com

4th International Conference of Asian Allelopathy Society

P-6
Identification of a putative chemical floral filter inSisyrinchium rosulatum
Nakatani, S.,* Saruta, Y., and Inagaki, H.
Shizuoka University, Shizuoka city, Shizuoka 422-8529, Japan.
Most angiosperms attract insect pollinators. Entomophily influences floral
shape and pollination syndrome—floral traits that have evolved in response todifferent pollination needs and type of pollinators. For example, flowers visitedby hoverflies are flat and have a simple shape. In contrast, flowers visited bybees have a complex shape and are deep. Flowers of annual blue-eyed grass(Sisyrinchium rosulatum) have a flat and simple shape, characteristic of theentomophily by hoverflies. However, in our study using the quadrat method, we
could not observe any S. rosulatum flower-visiting hoverflies or bees during
field studies conducted in 2017 and 2018. Furthermore, we could not observe
any flower-visiting hoverflies (Syrphinae) for S. rosulatum in the test
experiments performed in a breeding box, even though the hoverflies frequently
visited another flower, wood sorrel (Oxalis corniculata). Based on the results ofthe preference tests conducted using a T-shaped pipe, we found that Syrphinae
significantly avoided feeding on S. rosulatum (p=0.0004) and instead chose O.
corniculata (p=0.0081). Recently, it has been suggested that flowers emit
specific chemicals not only to attract select pollinators but also to repel otherinconsequential insects. Although studies on floral scents and their repellent
actions have been conducted mostly in sexually deceptive orchids and corpse
flowers, our results on grasses indicate that S. rosulatum, a common weed,
repels hoverflies through chemicals. Alternatively, our data on the genetics offlower color revealed a recessive phenotype in the field. The presence ofinterspecific hybrids between S. rosulatum and the blue-eyed grass (S.
iridifolium) further indicates outcrossing, and that self-pollination does notoccur in S. rosulatum. The pollinators, specifically of S. rosulatum, are yet to beclarified.

 E-mail: inagaki.hidehiro@shizuoka.ac.jp

4th International Conference of Asian Allelopathy Society

P-7
Screening of weed extracts for allelopathic activity
on nodulation in legumes
Suzuki, H.,* Tominaga, A., Naruse, K., and Inagaki, H.
Shizuoka University, Shizuoka city, Shizuoka 422-8529, Japan.
It is well known that allelopathy of weeds not only influence the germinationand growth of other plants but also affect soil microorganisms. Allepathic
activities of some weeds are known to influence the action of rhizobia
coexisting with leguminous plants. Some substances having negative
allelopathic activity on root nodule bacteria may possibly be used for the
suppression of harmful legume weeds, such as Kudzu (Pueraria montana).
Contrarily, substances having positive allelopathic activity on root nodule
bacteria are expected to enhance the productivity of crops, such as soybean.
Therefore, we screened the weed extracts that affect the formation of legumeroot nodule using soybean, which is the main crop, and Lotus japonicas, which
is a model plant, as the test subjects. After investigating 21 weed extracts, theextracts of red clover (Trifolium pratense) and broomsedge bluestem(Andropogon virginicus) were found to suppress the development of Lotus
japonicas root nodules. However, it was not clear whether Andropogonvirginicus root extracts promoted, rather than inhibited, the formation ofsoybean root nodules.

 E-mail: inagaki.hidehiro@shizuoka.ac.jp

4th International Conference of Asian Allelopathy Society

P-8
Evaluation of allelopathic activities of
56 aquatic plants on seed germination andseedling growth of upland weeds
Hirano, R., and Inagaki, H.*
Shizuoka University, Shizuoka city, Shizuoka 422-8529, Japan.
Allelopathy is an important factor affecting the establishment of native plant
species. Studies on allelopathic activity have mainly focused on the interactionsof plants occurring in the same habitat area, such as on the effects of aquaticplants on aquatic weeds and the effects of terrestrial plants on upland weeds. It
is believed that plants growing in the same environment develop resistanceagainst the allelopathic substances of each other. In contrast, it is suggested thatplants growing in different environments (such as in aquatic and upland areas)
may not develop resistance to allelochemicals produced by each other, which
may heighten the inhibitory effects upon encounter. Therefore, we evaluated theallelopathic activities of the extracts of 56 aquatic plants and the water in which
the aquatic plants were cultured, on seed germination and seedling growth of
upland weeds. All tested aquatic plants displayed inhibitory activities. The
extracts of aquatic plants tended to strongly inhibit the elongation of the roots ofupland weeds and the culture water tended to strongly inhibit the germination of
upland weeds. Germination of lettuce, used as a control, was remarkably
inhibited in the culture water of Daphnia lucifera, water wisteria (Hygrophiladifformis), pearl grass (Micranthemum micranthemoides), and Mayaca
fluviatilis. Germination of white clover, a leguminous weed, was remarkably
inhibited by the extracts of Hygrophila polysperma. Similarly, Lolium
multiflorum, a Poaceae weed, was remarkably inhibited by the extracts of
Eleocharis vivipara. Moreover, the extracts of red Ludwigia (Ludwigia repens),
Hygrophila polysperma, some Commelinaceae species from Pantanal (Brazil),
and water chestnut (Trapa natans) remarkably inhibited root length elongationin both white clover and Lolium multiflorum.

 E-mail: inagaki.hidehiro@shizuoka.ac.jp

4th International Conference of Asian Allelopathy Society

P-9
Allelopathic effect of pruned branches of kiwifruit
trees on weeds
Iwamoto, Y.,*1 Yoto, D.,*1 Nishikawa, K.,1 Naruse, K.,1
Okada, S.,2 Kato-Noguchi, H.,2, Nakamura, D.,3 and Inagaki, H.1
1

Shizuoka University, Shizuoka city, Shizuoka 422-8529, Japan.
2
Kagawa University, Miki town, Kagawa 761-0795, Japan.
3
Nihon Organic Inc., Shizuoka city, Shizuoka 420-0922, Japan.

Large amounts of residue are discharged from agricultural production andprocessing. In this study, the growth inhibitory activity of residual fruits andvegetable extracts, such as from orange, grapefruit, apple, kiwifruit, pineapple,
tomato, radish, Chinese cabbage, cabbage, green onion, lettuce, spinach, parsley,
onion, bean sprouts, carrot, burdock, eggplant, cucumber, bitter gourd, green
peppers, perilla, asparagus, garlic, ginger, okra, taro, yam, potato, sweet potato,
and wasabi, was evaluated. Results indicate a strong growth inhibitory activity
particularly of kiwifruits. Furthermore, this activity was not only limited to thekiwifruit but also the stems and leaves of kiwifruit plants. This prompted us tofocus on pruning the branches of kiwifruit, which presents as a major residue
problem. So we evaluated the weed control effect of pruned branch tips of
kiwifruit in the field. In the field experiments, we validated the suppression of
weeds by placing the tips of pruned kiwifruit branches in the field. Moreover,
we suppose that the weed control effect is not only due to the covering effectbut also due to the effect of allelopathy. This is because the weed suppression
effect increased by puncturing the chip treatment compared with covering thechip treatment. Currently, we are focused on identifying the allelochemicals inpruned kiwifruit branches and developing weed control material by pelletizing
pruned branches.

 E-mail: inagaki.hidehiro@shizuoka.ac.jp

4th International Conference of Asian Allelopathy Society

P-10
Screening of weed extracts for induction of systemicacquired resistance to anthracnose.
Usui, Y.,*1 Tominaga, A.,1 Kato, K.,2 and Inagaki, H.1
1

2

Shizuoka University, Shizuoka city, Shizuoka 422-8529, Japan.
Agri Open Innovation Practical and Applied Research Center, Numazu city,
Shizuoka 410-0321, Japan.

Recently, systemic acquired resistance (SAR) against microbial pathogens—theintroduction of broad-spectrum and long-lasting immunity in non-infected
tissues via induction of the plant’s own defense mechanism—has gained
considerable attention in crop production. While several reports havedocumented the antifungal and insecticidal activities of natural products, studies
on the natural product-induced SAR itself are limited. In the present study, we
screened leaf extracts from 203 weed species for ability to induce SAR byperforming a bioassay using cucumber plants and the pathogen Colletotrichum
lagenarium. Many of the leaf extracts examined showed antifungal properties
against anthracnose when used to treat the leaves of cucumber. The weed
extracts of 48 species showed an inhibition rate of 80%. Furthermore, theextracts of several weeds inhibited the anthracnose infection in the cucumber
leaves even in the untreated upper leaves. For example, a high inhibition rate of>60% was obtained in the upper leaves with the extracts from the following 11
weed species: Boehmeria nipononivea, Persicaria scabra, Dianthus armeria,
Ranunculus japonicus, Ranunculus sceleratus, Sedum oryzifolium, Sedum
sarmentosum, Trifolium pratense, Pueraria lobata, Cuscuta pentagona, andGnaphalium purpureum. Such inhibition of the anthracnose infection on theupper leaves of the plants could have two possible explanations. First, theantifungal ingredient in the extracts could have been transported from the lower
to the upper leaves, in which case the active ingredient of the weed extracts
could be a low molecular weight compound. This could be a potential precursor
for the development of a new fungicide. Second, the weed extracts inoculated in
the cucumber plants could have led to SAR induction. In order to confirm thelatter hypothesis, we have been determining the expression of the SAR marker
gene in the model plant Arabidopsis thaliana.
 E-mail: inagaki.hidehiro@shizuoka.ac.jp

4th International Conference of Asian Allelopathy Society

P-11
Synthesis of Molecular Probes of Cis-Cinnamic Acid
Analog with Antigravitropic Activity
Furusawa, Y.,2 Sun, J.,2 Iwata, T.,1 and Shindo, M. 1*
1

Institute for Materials Chemistry and Engineering, Kyushu University, Kasuga,
Fukuoka 816-8580, Japan.
2Interdisciplinary Graduate School of Engineering Sciences, Kyushu University,
Kasuga, Fukuoka 816-8580, Japan.
Cis-cinnamic acid (cis-CA) was reported by Hiradateand Fujii to be a biologically active substance derived
from an allelochmical produced by Spiraea thunbergii
Sieb. Cis-CA shows significant inhibitory activity
toward plant growth.1,2 Through structure-activity relationship (SAR) study
about this molecule, we found that analog 1 has strong antigravitropic activity
toward lettuce roots.3 In this study, we investigated design and synthesis ofmolecular probes of 1 in order to reveal its biological mechanism.
Design of molecular probes 5 is based on our early SAR study about 1.
Bromide 2 and boronic acid 3 were used as starting materials, and sequential
transformations including Suzuki coupling, HWE reaction and Mitsunobu
reaction provided primary amines 4. Finally, fluorescent groups were connectedto the amino group. Totally, 12 fluorescent probes 5 were prepared. Preliminary
fluorescent imaging experiments using lettuce roots indicated that the root cap
was especially stained. Synthesis of photo-affinity probes will also be shown.

1) S. Hiradate, Y. Fujii, at al., J. Chem. Ecol. 2005, 31, 591. 2) S. Hiradate, Y. Fujii,
Phytochemistry 2004, 65, 731. 3) manuscript in preparation.

 E-mail: shindo@cm.kyushu-u.ac.jp

4th International Conference of Asian Allelopathy Society

P-12
Characterization of growth-promoting activity ofvolatile substances released from Crocus sativus
Fujimaru, Y.,* Sunohara, Y., Yamaguchi, T., and Matsumoto, H.
University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Plant growth-promoting activities of volatile compounds from 13 plant species
were examined using lettuce seedlings (Lactuca sativa L.cv. Legacy) at thegermination stage by a dishpack method to find new natural compounds that
could be sources of lead compouds for environmentally safer novel plant
growth regulators, and the characterization of the promising compound was
examined to understand the mechanism for the promoting activity. The volatilecompounds from Crocus sativus L. showed the strongest promotive effect on
lettuce root growth, followed by those from Zanthoxylum piperitum (L.) DC.,
Mentha spicata L. and Foeniculum vulgare Mill.. Gas chromatography-mass
spectrometry analysis identified seven volatile compounds including safranal(2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde),isohorone(3,5,5trimethyl-2-cyclohexene-1-one) and limonene (1-methyl-4-(1-methylethenyl)cyclohexene) from C. sativus, three volatile compounds, sylvestrene (1-methyl5-(1-methylethenyl)-cyclohexene), neryl acetate ([(2Z)-3,7-dimethylocta-2,6dienyl] acetate) and beta-myrcene (7-methyl-3-methylideneocta-1,6-diene) fromZ. piperitum and eight volatile compounds including limonene, L-fencone(1,3,3-trimethylbicyclo[2.2.1]heptan-2-one) and anethol (1-methoxy-4-[(1E)prop-1-en-1-yl]benzene) from F. vulgare. Among these volatile compounds,
safranal showed the highest growth-promoting activity on lettuce roots,
followed by anethol, limonene and beta-myrcene. Safranal (19 µmol/L)
stimulated lettuce root length up to 162% of control 3 days after treatment. In
maize (Zea mays L. cv. Honey Bantam), safranal also tended to promote
primary root elongation and lateral root formation at 0.9 µmol/L 3 days after
treatment.

 E-mail: s1821144@s.tsukuba.ac.jp

4th International Conference of Asian Allelopathy Society

P-13
In Vitro Bioassay of Allelopathy in
Caffeine-containing Coffee Cells using Protoplast
Co-Culture Method with Digital Image Analysis
Ogita, S.,1* and Sasamoto, H.2
1

Prefectural University of Hiroshima, Shoubara,
Hiroshima, 727-0023, Japan.
2
Tokyo University of Agriculture and Technology, Fucyu,
Tokyo 183-8509, Japan.
Caffeine (1,3,7-trimethylxanthine), a purine alkaloid, is known as anallelochemical in such as coffee and tea plants. To unveil the cellularmechanism(s) of allelopathy, the protoplast co-culture method for assay ofallelopathy was developed in a 50 µL liquid medium using a 96 well culture
plate. Inhibitory effects of caffeine and its metabolites on the growth ofrecipient lettuce protoplasts were reported using this method. In the present
study, a unique embryogenic cell (EC) line of Coffea canephora (robusta
coffee) which accumulates caffeine during subcultures was investigated.
Protoplasts were isolated from four-week cultured EC using Cellulase R10 in
0.6 M mannitol, which condition was selected from 24 combinations of six
kinds of cell wall degrading enzymes. Numbers of non-spherically enlarged or
divided protoplasts in a Murashige and Skoog’s medium containing 2,4dichlorophenoxyacetic acid and benzyladenine, sucrose and mannitol, werecounted under an inverted microscope. Protoplasts densities varied from 6 x
10^3 to 5 x 10^5 / mL. The growth of recipient lettuce protoplasts were
described as % of control. Coffee protoplasts stimulated the cell wall formationof recipient lettuce protoplasts, but strongly inhibited cell divisions depending
protoplast densities of coffee. Furthermore, effects of coffee protoplasts onyellow color accumulation of lettuce protoplasts were quantitated by the Image
J analysis of a scanned digital image of a 96-well culture plate (DIA-PP
method). Differences and causes of the allelopathic activities were discussed
comparing with other plant species.

 E-mail: ogita@pu-hiroshima.ac.jp

4th International Conference of Asian Allelopathy Society

P-14
Isolation and identification of allelopathic
substances from Lippia canescens
Tomita, M., 1* Shigemori, H.,1 and Yamada, K.1
1

Graduate School of Life and Environmental Sciences, University of Tsukuba,
Ibaraki 305-8572, Japan.

Recently, the cultivation of ground-cover plants received much attention as a
new method of weed management. Because using ground-cover plants for weed
management is a weed control method that does not use herbicides, it canmarkedly contribute to organic and environmental preservation agriculture.
These plants can deprive weeds of sunlight and grow into dominant spices.
Some of these plants have a competitive advantage not only because they coverweeds, but also because they exhibit an allelopathic potential. The objective of
this study was to evaluate the allelochemicals produced by ground-cover plants.
To assess the allelopathic activity, the sandwich method was used. Lippia
canescens, which exhibited significant plant growth-inhibitory activity, wasselected as a plant material. The candidate allelochemicals were purified froman acetone extract by bioassay-guided column chromatography and HPLC,
based on the inhibitory activity against the growth of monocot (lettuce) anddicot (timothy) seedlings. The growth-inhibitory compounds were identified by
ESI-MS and 1H and 13C NMR spectra analysis. From L. canescens extract,
cornoside, arenarioside and acteoside were isolated as potential allelochemicals.
Whether these compounds are present at sufficient levels to exhibit anallelopathic potential is also discussed.
This study may contribute to the further utilization of ground-coverplants.

 E-mail: s1721157@s.tsukuba.ac.jp

4th International Conference of Asian Allelopathy Society

P-15
Assessment of allelopathic potential ofsome crop residues on wheat and some associatedweeds (chard and wild oat)
Abdelkhalek, R., 1* Ismail, T.,2 Karetum, A.,2 Shams, M.,1
Adam, F.,2 and Fujii ,Y.3
1
2

Weed Research Central Laboratory, Agricultural Research Center, Egypt.
Department of Pesticide Chemistry and Toxicology, Agriculture Faculty,
Kafrelsheikh University, Egypt.
3 Department of International and Environmental Agriculture Science,
Tokyo University of Agriculture and Technology, Japan.

In Egypt a huge amount of crop residues remained in fields and are usually
burnt by farmers. In this study we attempted to utilize some agriculture wastes
such as rice straw, sorghum, sunflower and wild mint under both laboratory andgreenhouse conditions to determine their allelopathic potential. In laboratory
bioassay, sandwich method with lettuce seed has been used. The results showedthat all allelopathic plants significantly reduced lettuce radical length. The
highest inhibitory effect at 50 mg was observed by sunflower (82%) followedby sorghum (73%), rice straw (56%) and finally by wild mint (57%).
Greenhouse experiment was carried out to evaluate the potency of using thesecrop residues in combating weeds under environmental conditions. Test plantpowders were used at two levels 0.5 and 1.5 % as soil incorporation against twoof its obnoxious weeds, chard and wild oat, and wheat. Germination percentage,
chlorophyll content, root and shoot elongation, fresh and dry weight were
evaluated after 30 days from sowing. Regarding sunflower residues, it achived
the highest inhibitory activity against wheat and its weeds. Sunflower residues
significantly inhibited germination process by (90%, 40% and 56%) andreduced root growth by (96.7%, 81.1and 81%) in chard, wild oat and wheat,
respectively comparing to control at 1.5% powder. Sorghum residues caused
also high inhibitory activity against the test weeds while, its inhibitory effectagainst wheat was not significant. As for rice straw and wild mint, both caused
subsential suppression in root growth of chard (93.8% and 97.1%, respectively).
It was concluded that it is possible to incorporate the tested crop residue withsoil to control weeds instead of herbicides under field conditions.

 E-mail: tamermadeh3000@yahoo.com

4th International Conference of Asian Allelopathy Society

P-16
Investigation of Allelochemicals Activity in Cassava
Leaf and Callus for Genetic Transformation
Sanjaya, B. R. L.,1,2 Ara, M. T.,2 and Ogita, S.2
1

Jember University, Center for Development Advance Sciences andTechnology (CDAST), Jember, 68121, East-Java, Indonesia.
2
Graduation School of Comprehensive Scientific Research,Prefectural University of Hiroshima, Shobara, Japan.

Cassava (Manihot esculenta) is a perennial woody shrub in Euphorbiaceae
family that most important sources of food energy and industrial raw materials
in tropical and subtropical regions. Cassava is the third most important sourceof calories and the sixth most important crop (Burns et al., 2010,
doi:10.3390/su2113572). Cassava can produce some phenolic compounds
which contributed to the antioxidant and antimicrobial activity. For example,
ascorbic acid and β-carotene are known as an allelochemicals in cassava. We
currently focus on the research to explore morphological and physiologicalresponses of Asian (mainly Indonesian) cassava under drought stress. The
present study was aimed to investigate allelochemicals activity of two typicalcassava varieties from Okinawa Japan. At first, morphological andphysiological characteristics of these local cassava varieties were evaluated.
Allelochemicals activity was tested on seeds germination of Lactuca sativa,
using sandwich method. Then, antimicrobial activity of cassava leaf and cassava
callus was tested in bacteria species Escherichia coli JM109, Agrobacterium
tumefaciens LBA4404 and A. tumefaciens EHA101 strains by measuring the
growth inhibition zone and/or microplate reader based bio-assay. Differences
and causes of the allelochemicals activities were discussed. The minimum zoneof inhibitory between A. tumefaciens LBA4404 and EHA101 indicated as
effective vehicle for plant transformation. Finally, a callus culture system wasdeveloped using petiole and stem explants on Murashige and Skoog medium
with various concentrations of phytohormons (3 M 2, 4-D + 3 M BA, 10 and
50 M picloram). The infection time and bacterial density could have someeffects on the transformation efficiency.

 E-mail: bellarhea78@gmail.com

4th International Conference of Asian Allelopathy Society

P-17
Screening for Potential Allelopathic Activity of
Medicinal Plant in Phnom Kulen National Park,
Cambodia
Yourk, S.,1,2* Chan, S.,2 Khou, E.,3 and Fujii, Y.1
1

Department of International Environmental and Agricultural Science, Tokyo University ofAgriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
2
General Secretariat of National Council for Sustainable Development, Morodok Techcho(Lot 503) Tonle Bassac, Chamkarmorn, Phnom Penh, Cambodia
3
National Authority for Preah Vihear, Thamacheat village, Sraem Commune,
Choam Khasant District, Preah Vihear Province, Cambodia

Recently, the term of allelopathy is wisely used to enhance sustainable agriculture,
particularly for weed control. Medicinal plant, known as herbicide, contains many
pharmacological activities are commonly used with many prescribed drugs; however
their several functions including defense against insects, fungi, diseases andherbivorous mammals, are reported allelopathic properties which influent tosurrounding plants for both positively and negatively (Ahn K. 2017; Abbiw 1990; Rice
1984). This present study aims to screen potential allelopathic activity of medicinal
plants in Phnom Kulen National Park, where known the richness of medicinal plants
and cultural values in Cambodia. To achieve this objective, 113 medicinal plant
species from 57 families were screened by using sandwich method (Fujii et al. 2004).
Lettuce seeds (Lactuca sativa L. Great Lakes No. 366) was observed as a test plantmaterial in bioassay to determine the influent of root exudates.
As a result to allelopathic activity exhibited through plant leachates, the radicle
elongation percentage of lettuce seeding were in range 4 to 118.8% and 3.1 to 98.8%
for 10 mg and 50 mg sample treatment. We observed that radicle growth inhibition>90% occurred in 34 species, 70–90% in 32 species, 50–70% in 27 species, 30–50%
in 13 species, and 4–30% in 7 species. Additionally, 32 plant species show thesignificant inhibition on lettuce radicle as evaluated by using standard deviation
variance (SDV). We also found that Iris Parllida (4%) showed the strongest inhibitory
activity among 113 test plants. Following by Peliosanthes tesa (8.2%), Suregada
multiflora (21.3%), Ervatamia microphylla (22.4%), Allophyllus serrulatus (23.3%),
Eupatorium odoratum (24.1%), and Stephania rotunda (28.7%). This is the firstmassive screening allelopathic activities of medicinal plants in Cambodia. This result
could be used as a benchmark for future research on isolation and identification
allelochemicals among plant species in Cambodia.

 E-mail: thearith.yourk@gmail.com

4th International Conference of Asian Allelopathy Society

P-18
Evaluation of allelopathic activity ofHypericum oliganthum-Toward practical use as cover plants for weed management
and conservation of Hypericum oliganthum1

Kato, T.,1 Yoshioka, T.,2 and Fujii, Y.1
Tokyo university of Agriculture and Technology, Japan.
2
Fukui prefectural university, Japan.

Hypericum oliganthum is distributed to the western part of Kanto, Japan and Korea, living at
paddy levees or riverbeds. This plant is designated endangered species rank IB. According tosurvey by the Ministry of Environment at 2007, average decreasing rate was 61%, so it’surgent to conserve this species. Although this plant is endangered species, propagation of thisplat is not difficult (Yoshioka, 2015). Hypericum oliganthum is perennial, creeping plant.
These characteristics indicate that there is a possibility to be useful as cover plants for weed
suppression at paddy levees. In addition, its flowers are beautiful. If Hypericum oliganthum
can be useful as cover plants, not only conservation of this, but also reduction of farmer’sburden and beautification of paddy levees will be expected, so it will get easier to get
consensus of people to conserve Hypericum oliganthum. On the other hand, allelopathy is
one of factors related to weed suppression. However, allelopathic activity of Hypericum
oliganthum is not reported, so allelopathic activity of Hypericum oliganthum was evaluated.
Three methods: Sandwich method, plant box method and rhizosphere method wereconducted. Allelopathic activity through leaching from dried leaves, stems, amd roots ofHypericum oliganthum were evaluated by sandwich method. By plant box method,
allelopathic activity through root exudates was evaluated. Allelopathic activity of chemicals
existing in rhizosphere soil was evaluated by rhizosphere soil method. As a result, inhibition
percentage of radicle growth of lettuces seeds (Species: Legacy) were as follows. Sandwich
method: 2h dried leaves (10 mg) 20.2 (%), dried leaves (50 mg) 50.6 (%), dried stems (10mg) 6.5 (%), dried stems (50 mg) 46.7 (%), dried roots (10 mg) 6.4 (%), dried roots (50 mg)
39.5 (%), Plant box method: 66 (%), Rhizosphere method: dried rhizosphere soil (3.0 g) 41.3(%). Dried leaf leachates and root exudates had high allelopathic activity.
Leaves of Hypericum oliganthum are dropped and accumulated on the surface of soil in
winter, so chemicals related to weed growth inhibition may be leached and accumulated on
the surface.
In addition to shading light by creeping, dried leaf leachates and root exudates from
Hypericum oliganthum may contribute to weed suppression. So, possibilities for Hypericum
oliganthum to be useful as coverplants became higher than before.

 E-mail: taitai.kato@gmail.com

4th International Conference of Asian Allelopathy Society

P-19
Possible involvement of nitric oxide in promotion ofrice seed germination under low temperature by
inoculation of Bacillus pumilus TUAT1 strain
Xiao, L., Yamada, T.,* Yokoyama, T., and Fujii, Y.
Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi,
Tokyo 183-8509, Japan.
In direct sowing culture of rice in cold areas, high seed germination rate in soil
and vigorous growth of the seedling under low temperature are important traits.
Bacillus pumilus TUAT1 strain is one of plant growth-promoting rhizobacteria.
In rice seedings inoculated with TUAT1, production of nitric oxide (NO),which is a signal molecule in allelopathic interactions, was increased and it
resulted in a promotion of crown root formation. In this study, the effects of
inoculation with TUAT1 to seed germination in soil and seedling growth of rice
under low temperature (15℃) were confirmed. The relationship between
increase of NO production and promotion of germination in rice seeds was also
investigated.
The seeds of japonica rice ‘Hitomebore’, ‘Fukuhibiki’, ‘TAT-26’ and‘LTAT-29’ were sown on granular soil and cultivated at 15ºC. When the seeds
treated with TUAT1 spore suspension (107 CFU/mL), higher germination rate
was observed in all varieties. In ‘Fukuhibiki’ and ‘LTAT-29’, growth amount of
seedlings was significantly increased. These results showed that inoculation of
TUAT1 promotes seed germination and seedling growth of rice under low
temperature.
When the seeds were treated together with NO scavenger (100 µM cPTIO)
and TUAT1, the promotion effect of TUAT1 on seed germination in soil was
dissapeared. The NO production level in the seed of ‘Hitomebore’ estimatedusing DAF-FM DA was higher after treated with TUAT1 than control seeds.
These results suggest that possible involvement of the increase in NO
production level to the promotion of germination under low temperature in riceseeds treated with TUAT1.

 E-mail: teyamada@cc.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-20
Enumeration of soil microorganism which degradeL-3,4-dihydroxyphenylalanine, an allelochemical of
Mucuna prurience var. utilis
Hareyama, Y., Fujii, Y., and Tarao, M.
Tokyo University of Agriculture and Techonology, Fuchu,
Tokyo 183-8509, Japan.
Since study dealing with allelopathy have been conducted mainly in vitro. Thus,
the effect of allelopathy on soil microorganism, such as allelochemicaldegrader, remain unclear. We hypothesize that the number of allelochemicaldegrader increases when allelochemical donor plants are grown in soil. Mucuna
prurience var. utilis, a tropical legume, is grown for edible bean, cover crop andgreen manure in south America or southeast Asia, M. prurience has been known
to have allelopathic effect. Experiments in vitro suggest that L-3,4dihydroxyphenylaranin (L-DOPA) is a putative allelochemical and inhibits
growth of plants. In this study we measured the number of L-DOPA-degrading
microorganism in several soils by using MPN method and the amount of LDOPA in soil in which M. prurience was grown. More than 10⁵ MPN g⁻ ¹ of Lthere was no significant difference in the number of L-DOPA-degrading
microorganisms between M. prurience grown soil and no-growth soil. The
amount of L-DOPA in M. prurience grown soil was lower than detection limit(<2.48 µg g-1). These results suggested that L-DOPA released from M.
prurience may be degraded by soil microorganisms because they are at highdensity in soil or the amount of L-DOPA released from Mucuna prurience var.
utilis is very small. Further studies would be needed to verify these hypotheses.

 E-mail: s186788y@st.go.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-21
Phytotoxic effect of soil-incorporated dried leaf
residue of Euphorbia golondrina L.C. Wheeler
on tomato growth and yield
Ndam, L. M.,1* Mih, A. M.,1 Tening, A. S.,2 Fongod, A. G. M.,2
Fujii, Y.,3 and Nkegua, T.4
1

University of Buea, Dept. of Botany and Plant Physiology, Cameroon
2
University of Buea, Dept. of Agronomy, Cameroon
3
Tokyo University of Agriculture and Technology, Japan
4
ABA Home Health Care Inc., United States

Invasive alien plant species play a major role in agricultural and natural
ecosystems by determining vegetation pattern, plant dominance, and yield loss.
Euphorbia golondrina, an annual native to Central America, is now reported in
Cameroon where it is a nuisance in farming systems. This study aimed toevaluate the phytotoxic effects of E. golondrina soil-incorporated dried leaf
residue on growth and yield of tomato in the Greenhouse of the University Buea,
Cameroon. Incorporation of the leaf residue to the soil showed chlorosis,
necrotic lesions and wilting of tomato under treatments 10 g and 17 g at 2 WAP.
The number of leaves, leaf area, dry shoot and root weight of tomato grown inthe soil-leaf mixtures were also significantly reduced. The inhibition
percentages due to the addition of the three concentrations of E. golondrina
dried leaf residue on the dry shoot weight at 4 WAP were 42.1 g, 49.6 g and75.9 g for tomato respectively. Similarly, declines in the dry root weight ranged
from 42.9 to 57.1% under amended soil treatments. Further, the dry fruit yieldand shoot weight of tomato under the treatments 7 g, 10 g and 17 g reducedwith increase in leaf residue concentrations. The reductions in the fruit yield andfresh head weight caused by treatments 10 g and 17 g were 54.2 and 71.4%
respectively. The mineral contents in the leaves of tomato showed significant
differences in the uptake of N, Mg, Na, Cu and Fe by the tomato plants.
However, the P content was relatively constant in the leaves of tomato at 4 and
12 WAP. At 12 WAP, the Fe content in soils with treatments 10 g and 17 g wasmarkedly enhanced in comparison with the other nutrients. Hence, E.
golondrina is phytotoxic to tomato and warrants phytochemical analysis.
 E-mail: nlmonah77 gmail.com

4th International Conference of Asian Allelopathy Society

P-22
Endophytic Fungi Isolates from an Allelophatic
Plant: The Case of Vicia villosa RothTaheri, P.,* Okazaki, S., Ndam, L. M., and Fujii, Y.
Department of International Environmental and Agricultural Science,
United Graduate School of Agriculture, Faculty of Agriculture,
Tokyo University of Agriculture and Technology, Tokyo, Japan
Plant-microbes symbiotic such as mycorrhizal fungi and some endophytes play a vitalrole in ecosystem services and function by enhancing nutrient uptake and growth, and/or deter antagonistic microbes attack.
Fungal endophytes are fungi that spend either full or a considerable part of their life
inside living plant tissues without causing any visible harm on the host. Secondary
metabolites produced by fungal endophytes are useful resources for natural products
which have a wide range of applications in medicine and agriculture. Hairy vetch(Vicia villosa Roth.), an allelophatic legume native to some part of Europe andwestern Asia such as Japan contains the secondary metabolite cyanamide responsible
for allelophatic potential and serve as the central ingredient in nitrogen fertilisers.
Previous studies have elucidated the cover crop attributes and secondary metabolites
from V. villosa vegetative parts but none have determined the endophytic profile.
Hence, this study was aimed to document the endophytic mycoflora diversity of V.
villosa with the view of exploitation in biotechnological applications.
Random samples, from asymptomatic surface sterilized tissues (root, stem, leaf) fromV. villosa were used for fungal assemblage and diversity investigation following
comparative morphological descriptors with the aid of a light microscope. Forty-three
endophytic fungi were isolated from V. villosa. The isolates were characterized andgrouped according to culture characteristics and colony growth morphology.
Thirty-six morphotypes, equivalent to 83.7% were assigned to ascomycotina.
Twenty-seven isolates, equivalent to 62.8 % were found in the leaf tissues.
Among the isolated endophytic fungi from leaves, 15 and 1 isolates were confined toroot and stem tissues, respectively. Further, penicillium spp. were the most dominant
endophytic fungi in all tissues.
This study provides a base line data on the fungal association of V. villosa for usage as
an eco-friendly source of natural metabolites in the medical and agriculturalindustries.

 E-mail: parisa.taheri00@gmail.com

4th International Conference of Asian Allelopathy Society

P-23
A Perspective on Allelopathic Potential of Some
Turkish Plant Species by Sandwich Method
Tugba, G. I. O.,* and Fujii, Y.
Tokyo University of Agriculture and Technology, International
Environmental and Agricultural Sciences, Fuchu, Tokyo 183-8509 Japan
Turkey is one of significant country in the world from the stand point of plant
genetic resources and plant diversity. Although Turkey’s flora presents potential
for allelopathy researches, there is a lack of studies that screen the large number
of Turkish plant species by bioassasy for allelopathic activity. That is why, themain objective of current research is to screen allelopathic activity of somecrops, herbs, spices, medicinal and aromatic plants from Turkey and to
determine strong allelopathic species for next studies. In the present study, 165
dry plant samples supplied from herbalists in different cities in Turkey and wereassayed by the Sandwich Method for their allelopathic activity. The Sandwich
Method is a bioassay method which is reliable, effective, less time-consuming,
and widely used in determining allelopathic effects of dry plant materials underlaboratory conditions. Lettuce seeds (Lactuca sativa L.) was used as a test plantmaterial in the bioassay. Inhibitory activity of dry plant samples against lettuce
seedling elongation was evaluated, by comparing the radicle lengths of treatedseeds with that of control seeds.
Top ten plant species, which have had maximum inhibition activity on theradicle lengths of treated seeds, were Punica granatum L. (Lythraceae), Alcea
rosea L. (Malvaceae), Lavendula stoechas L. (Lamiaceae), Silybum marianum(L.) Gaertn. (Asteraceae), Nigella sativa L. (Ranunculaceae), Camilla sinensis(L.) O. Kuntze. (Theaceae), Pinus brutia Tenore (Pinaceae), Liquidambar
orientalis L. (Altingiaceae), Malva sylvestris L. (Malvaceae), and Citrus
sinensis (L.) Osbeck. (Rutaceae).
This is the first comprehensive report on screening of over hunder number of
Turkish plant species by bioassasy for their allelopathic activity. Further studies
would be applied to determine allelochemicals which cause allelopathic activity
in regarding plants. Presented data could be utilized as benchmark information
to develop natural herbicides for weed control.
 E-mail: tugbagonca_ozkan@yahoo.com

4th International Conference of Asian Allelopathy Society

P-24
A perspective on allelopathic activity of someTurkish plant species by Dish Pack Method
Tugba, G. I. O.,* and Fujii, Y.
Tokyo University of Agriculture and Technology, International
Environmental and Agricultural Sciences, Fuchu, Tokyo 183-8509 Japan
Although Turkey’s flora with its existing diversity present potential forallelopathy researches, there is a lack of studies that screen the large number of
Turkish plant species by bioassasy for allelopathic activity. That is why, themain objective of current research is to screen allelopathic activity of somecrops, herbs, spices, medicinal and aromatic plants from Turkey and to
determine strong allelopathic species for next studies. In the present study, 79
dry plant samples supplied from herbalists in different cities in Turkey and wereassayed by the Dish Pack Method for volatile allelopathic activity. The Dish
Pack Method is a quick bioassay method which is widely used in screening forthe presence of volatile allelochemicals from plant materials under laboratory
conditions. Lettuce seeds (Lactuca sativa L.) was used as a test plant material in
the bioassay, because of its high sensitivity to inhibition by allelochemicals andreliability for germination. Inhibitory activity of dry plant samples against
lettuce seedling elongation was evaluated, by comparing the radicle lengths oftreated seeds with that of control seeds.
Top ten plant species, which have had maximum inhibition activity on theradicle lengths of treated seeds, were Centaurium erythraea Rafn
(Gentianaceae), Hibiscus rosa-sinensis L. (Malveceae), Salvia officinalis L.
(Lamiaceae), Lavendula stoechas L. (Lamiaceae), Valeriana officinalis
L.(Caprifoliaceae), Melissa officinalis L. (Lamiaceae), Prunus mahaleb L.
(Rosaceae), Hypericum perforatum L. (Hypericaceae), Ceratonia siliqua L.
(Fabaceae), and Rosmarinus officinalis L. (Lamiaceae). This is the firstcomprehensive report on screening of large number of Turkish plant species bybioassasy for volatile allelopathic activity. Further studies would be applied todetermine allelochemicals which cause allelopathic activity in regarding plants.
Presented data could provide not only a pathway to identify new naturalchemicals that would serve sustainable agriculture, but also it could be utilized
as benchmark information to develop natural herbicides for weed control.
*E-mail: tugbagonca_ozkan@yahoo.com

4th International Conference of Asian Allelopathy Society

P-25
Effect of Mechanical Weeding on the Allelopathic
Potential of Weed
Biramahire, B.,* and Fujii, Y.
Tokyo University of Agriculture and Technology, International
Environmental and Agricultural Sciences, Fuchu, Tokyo 183-8509 JapanThe allelopathic contribution on both vegetation growth inhibition and species
richness depression by trampling and cutting of an herbaceous plant community
will be evaluated. In fact, plant wounding by either biotic or abiotic agentsinduces an emission of allelochemicals, mainly volatile, such as leaf aldehydes,
leaf alcohol and ethylene, from wounded planted. Therefore, in the no-till
system, the vegetation will be trampled and removed using a grass roller and agrass trimmer, respectively, as mechanical plant wounding agents. Four
replications of five treatment and control (undisturbed) plots were randomly
designed in the field experiment located in Tokyo University of Agriculture andTechnology. The five treatments include vegetation cutting and four different
trampling intensities: 25, 75, 200 and 500 Passes. Species richness will berecorded before and after treatments. Plant coverages and heights of thecandidate plants species will be recorded one day before, as well as, 15; 30; and75 days after treatment to investigate the species traits, resilience and resistance.
The soil hardness tester will show the difference between soil hardness incontrol and treated plots. Solid Phase Microextraction (SPME) and Gas
Chromatography-Mass Spectrum (GC-MS) will identify leaf aldehydes. While
ethylene will be detected by Gas Tech System. Moreover, a bio-assay will be
conducted to check the effects of aldehydes and ethylene on the growth oflettuce and the candidate species in the laboratory. The rhizosphere soil method
will reveal the increase of soil allelopathy, due to increased root exudation ofallelochemical after plant wounding. Eventually, based on the study findings,
we intend to suggest grass rolling as a new eco-friendly weeding method.

 E-mail: biramahirebienvenu@gmail.com

4th International Conference of Asian Allelopathy Society

P-26
Efect of relay living mulches with maiz
(Zea mays L.) on weed communities under reduced
soil-tillage
Angelo, C.,1,2* Domingos, D.,2 Fernando, C. J.,2 Gabriel, B.,2and Fujii, Y.1
1

Tokyo University of Agriculture and Technology, International Environmental andAgricultural Sciences, Fuchu, Tokyo 183-8509 Japan2
Mozambique Research Institute of Agriculture, Sussundenga road 216, Mozambique
The Shift from conventional to reduced soil-tillage lead to the development of high weed
infestation pushing farmers to intensively use herbicides. However, one of the sustainable
alternatives to reduce the demand for herbicides is the use of living mulches. Field
experiment was conducted in the rainy season 2017/2018 at Sussundenga research station,
Manica province (33o15’E 190 20’S and 635m at sea level) to investigate the effect of relaymaize with brachiaria (Brachiaria decumbensis), velvet bean (Mucuna prurens), cowpea(Vigna unguiculata L.), soybean (Glycine max) and sunhemp (Crotalaria juncea) comparedwith sole maize on weed composition and diversity; maize yield. In addition, living
mulch soil coverage (%) and height were recorded to determine multiplied dominance ratio(MDR), as well as aboveground biomass. Data were recorded at 35 and 83 days after planting(DAP) and the end of grain filling. The MDR of living mulches showed a tendency toincrease markedly during the maize growth and better performance was observed undervelvet bean (94.5m3m-2) and brachiaria (68.5m3m-2). From the results of analysis of weed
functional groups, a significant decrease was observed in annual monocotyledonous grasses
under brachiaria andperennial
dicotyledonous
broadleaved
undercowpea.
Furthermore, brachiaria, velvet bean and soybean reduced weed density by 58.5%, 46.3% and43.6% more than under sole maize respectively. On another hand, the Leguminous living
mulches showed a tendency to boost the weed aboveground biomass production opposing theweed density pattern. In addition, weed diversity among treatments did not vary during themaize growth. The introduction of a different type of living mulches had no significant effecton maize grain and stover yield. However in terms of total plot biomass produced (maize
stover yield + living mulches aboveground biomass) under brachiaria there was an increase
by up to 11174.4 to 13957.8 kg/ha more than sole maize and within living mulches, except
under mucuna which outperformed sole maize, intercropped sunhemp, soybean and cowpea
by up to 5450.8 to 8234.2 kg/ha. Thus, under reduced soil-tillage, relaying maizewith brachiaria and velvet bean could be an appropriate strategy to suppress weed, reduce
heavy use of herbicides and biomass production. However further studies on the effect ofsowing date and densities are critical issues to improve maize productivity.

E-mail: angelocumbane@gmail.com

4th International Conference of Asian Allelopathy Society

P-27
Presence and variations of l-canavanine in hairy
vetch (Vicia villosa) during seeds maturation stages
Mardani, H.,1* Motobayashi, T.,2 and Fujii, Y.1
1

Department of International Environmental and Agricultural Sciences,
Tokyo University of Agriculture and Technology, Fuchu, Japan
2
Field Science Centre for Education and Research,
Tokyo University of Agriculture and Technology, Fuchu, Japanl-canavanine is a nonprotein amino acid with allelopathic potential that has
recently been found in several leguminous species. Due to its unique structure
and its potential in many domains such as medicine and agriculture,l-canavanine has drawn much attention. Recently, l-canavanine has beenrecognised as precursor for cyanamide biosynthesis in young seedlings of hairy
vetch (Vicia villosa). However, quantification and qualification of thiscompound in mature plant and during seed maturation stages has not beenreported. Therefore, we assessed the allelopathic activity and quantified thel-canavanine in different parts of the mature plants of hairy vetch by using
GC-MS. Sandwich method of immature seeds showed stronger inhibitory
activity in comparison to the other parts of the plant. EC50 of l-canavanine wasestimated as 5.5 µmol/ml. We found no l-canavanine in the leaf, stem, and rootof hairy vetch. However, it was found that l-canavanine is biosynthesised inhigh concentration in immature seed during the globular (6µg/100mg) andincreases throughout the cotyledon, early maturation and fully maturation stages
in the seed (400 ng/100mg). These results were consistent with the bioassay of
aqueous ethanol extract of hairy vetch immature seeds. We think thatincorporation of hairy vetch into the soil during the seed maturation stage
maybe increase its weed controlling potential in the field condition.

 E-mail: fw3115@go.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-28
Contents of l-Canavanine and Free Amino Acids inGerminated Seeds and Protoplasts of Etiolated
Seedlings of Hairy vetch (Vicia villosa).
Mardani, H.,* Sasamoto, H., and Fujii, Y.
Department of International Environmental and Agricultural Sciences,
Tokyo University of Agriculture and Technology, Fucyu,
Tokyo 183-8509, Japan.
l-Canavanine is known as a precursor for biosynthesis of cyanamide, the main
allelochemical in leaves of hairy vetch (V. villosa var. Kantaro). In the
protoplast co-culture bioassay method of allelopathy, epicotyl protoplasts of
etiolated seedlings and l-canavanine were both strongly inhibitory, while
cyanamide was not so at low concentrations. In this report, we quantified thel-canavanine and free amino acids (FAAs) concentrations in tissues ofgerminated seeds and protoplasts of etiolated seedlings by using GC-MS. Majortransport and storage compounds (FAAs), ASN and GLU rapidly decreased inepicotyl tissue after days of germination, while other FAAs increased
significantly. Similarly, the l-canavanine content decreased, however, itsconcentration (20 mg/g fresh weight) at the day 7 was yet significantly higher
than ASN (10 x) and GLU (7 x). l-Canavanine content in epicotyls protoplasts
was estimated to be 0.23 µmoles /10^5 protoplasts and lower in root protoplasts.
These concentrations agreed with our results for pure l-canavanine in the
protoplast co-culture. Thus, in the V. villosa, l-canavanine may act as anallelopathic compound as well as transport-storage compounds similar to thoseof major storage FAAs. In general, it is possible that the V. villosa at early
growth stage utilizes l-canavanine as its main allelopathic compound to combat
neighboring species.

 E-mail: fw3115@go.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-29
Evaluation of allelopathic ground cover plants andisolation of azetidine 2-carboxylic acid asallelochemical from big blue lilyturf
Maeda, H., Okumura, K., Nakamura, R.,
Nomura, T., and Fujii, Y.
Tokyo University of Agriculture and Technology (TUAT),
3-8-1 Harumi-cho, Fuchu-shi, Tokyo 183-8538, JapanFor the selection of effective weed control, seven ground cover plants, creeping
thyme (Thymus serphyllum), moss phlox (Phlox subulata), carpetweed (Phyla
canescens), trailing ice plant (Mesembryanthemum spectabile), mondo grass(Ophiopogon japonicus), pennyroyal (Mentha pulegium), big blue lilyturf(Liriope platyphylla) were compared by field experiment for four years, andallelopathic activity of each plant was examined. In field experiment, dryweight of weed, cover degree, MDR, weed inhibitory rate and species of weeds
were examined. Dry weights of weed at creeping thyme, carpetweed, mondo
grass and big blue lilyturf were lower. Weed inhibitory rate and cover degree ofbig blue lilyturf were more than 90 % from 2nd year to 4th year. MDR of big
blue lilyturf was the highest. The number of emerged weed species of big blue
lilyturf was the least. Allelopathic activity was evaluated by sandwich method
and plant box method. Mondo grass and big blue lilyturf inhibited the growth oflettuce more than 90 % by sandwich method and plant box method. Trailing ice
plant inhibited growth of lettuce about 90 % by sandwich method.
Big blue lilyturf is a Japanese traditional perennial ground cover plant andshowed best results both in field experiment and allelopathic activity.
Allelochemicals of big blue lilyturf were evaluated by total activity. It was
found that azetidine 2-carboxylic acid (AZC) is considered to be a main
allelochemical of big blue lilyturf.

 E-mail: s152768t@st.go.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-30
Nitrogen mineralization and microbial biomass
dynamics in different soils following amendmentwith contrasting plant resources
Omari, R. A.,* and Fujii, Y.
Tokyo University of Agriculture and Technology, International
Environmental and Agricultural Sciences, Fuchu, Tokyo 183-8509 JapanThe need to evaluate common underutilized organic residues (OR) as soil
amendments in small-holder agro-ecosystems is necessary. This study was
conducted to evaluate the nitrogen (N) mineralization, soil pH and microbial
biomass dynamics of contrasting ORs in different soils. Six ORs (Leucaena
leucocephala, Centrosema pubescens, Gliricidia sepium, Pueraria phaseoloides,
Azadirachta indica, and Theobroma cacao), each at 24 mg g-1 dry soil wasadded to three soils and incubated for 120 days. Inorganic N contents variedamong the ORs, soil types and incubation days. Regardless of soil type,
Gliricidia had the highest net inorganic N while the lowest was in Theobroma.
Nitrogen mineralization rate of 1.4 to 1.5 mg N kg-1 soil day-1 was respectively
observed for Legon and Techiman soils, and was twice higher compared to
Nyankpala. However, Nyankpala soil released higher cumulative mineralized N
than Techiman and Legon, implying high N mineralization efficiency in theformer. Consistent soil pH and microbial biomass C (MBC) increase wererespectively observed for Theobroma and Pueraria treatments in all soils.
Inherent soil properties accounted for shifts in MBC and soil organic C while
OR quality indices controlled pH and N mineralization. Thus, we conclude thatsoil properties and OR type are important for optimal utilization of organic
resources.

Email: talk2jafakingonline@gmail.com

4th International Conference of Asian Allelopathy Society

P-31
Insect antifeedant activities of sesquiterpene lactones
from Asteraceae glandular trichomes
Tsunaki, K., Morimoto, M., and Matsuda, K.
School of Agriculture, Kindai University, Japan.
Yacon (Smallanthus sonchifolius (Poepp. & Endl.) H. Rob) and Helianthus
strumosus L. are perennial Asteraceae, and their roots are foodstuff. Since bothplant species have a high tolerance to pests, they can cultivate with minimal use
of pesticides. They possess a large number of glandular trichomes on leaves,which are likely implicated in resistance to herbivores. Thus, the leaves wererinsed using dichloromethane (DCM) to obtain the rinsate, and plant residues
were subsequently extracted by DCM to obtain the DCM extract and these two
fractions were tested on the common cutworms (Spodoptera litura F.). It was
found that the rinsate showed a higher insect antifeedant activity against thecommon cutworms than for DCM extract. The active principle of the glandular
trichomes were isolated by silica gel flash chromatography using a hexane andethyl acetate solvent system, and were identified as the sesquiterpene lactone,
Uvedalin(5-Acetoxy-4-epoxyangeloyloxy-6-methylcarboxygermacrenolide)and its oxidative congener, Enhydrin (5-Acetoxy-10-epoxy-4-epoxyangeloyloxy-6-methylcarboxy germacrenolide), from yacon, and Erioflorin, from H.
strumosu. Both of Uvedalin and Enhydrin showed strong insect antifeedantactivities with ED50 values of 18 and 9.0 nmol/cm2, respectively, whereas
Erioflorin had a moderate activity with ED50 value of 7.32 µmol/cm2 against thecommon cutworms. Thus, it is suggested that the substituents of these
germacrenolide contribute, at least in part, to the insect antifeedant activity andthat Uvedalin and Enhydrin serve as chemical defense weapons in yacon toward
phytophagous insects.

 E-mail: 1833670014v@nara.kindai.ac.jp

4th International Conference of Asian Allelopathy Society

P-32
Weed control using herbal medicine extraction
residue as natural mulch
Oyama, K.,1, 2* Morimoto, M.,2 Horimoto, S.,3 and Matsuda, K.2
1

Miyazaki University, Faculty of Agriculture, 1-1, Gakuen Kibanadai-nishi,
Miyazaki 889-2192, Japan.
2
Kindai University, School of Agriculture, 3327-204, Nakamachi, Nara city,
Nara, 631-8505, Japan.
3
Kyoto Institute of Technology, Saga Ippongichou, Ukyo-ku, Kyoto city,
Kyoto, 616-8354, Japan.
Although herbal medicine extraction residues are organic wastes, some of them areused to make compost for agricultural use. Therefore, we have evaluated the effects ofthem made from the cinnamon bark, ginger and magnolia bark on weed control in thefield as well as identified components responsible for the weed control.
The field test on weed control using the residue resulting from extraction of the
cinnamon (Cinnamomum cassia) bark with 70% EtOH extraction (RCB) was
performed on June 8th, 2016. Additionally, field tests of the other two residues
resulting from extraction of ginger (Zingiber officinale) and magnolia bark (Mongolia
obovate and M. officinalis) with 70% EtOH extraction (RG, RMB) was performed on
May 8th, 2017. These field tests were carried out in Kyoto Institute of Technology,
Kyoto, Japan. These residues were provided by Nippon Funmatsu Yakuhin, Co., Ltd.
Mulch was prepared with thicknesses of 0, 1, 2, and 3 cm for RCB or 0 and 3 cm for
RG and RMB on the soil surface. Weed species and their masses were measured at 3
months after the treatment.
The MeOH extracts were prepared from these residues by MeOH extraction,
respectively. Phytotoxic compounds were isolated using silica gel flash column
chromatography and identified by spectroscopic data. Phytotoxicity tests were
performed using lettuce (Lactuca sativa cv great lakes) and Italian ryegrass (Lolium
multiflorum cv wasefudo) seedlings.
The RCB mulch completely controlled various annual weeds, while did not affect
the growth of some perennial weeds. Only t-cinnamic aldehyde was isolated andidentified as a phytotoxic substance in the RCB MeOH extract. The RG mulch was
muddy after a rain, as the results it was unsuitable for mulch. The RMB mulch also
completely controlled various annual weeds. And then magnolol was isolated andidentified as a phytotoxic substance from the RMB MeOH extract.

 E-mail: aa18001@student.miyazaki-u.ac.jp

4th International Conference of Asian Allelopathy Society

P-33
Evaluation of putative allelochemicals, cinnamic
acid and an anthocyanin, in Spiraea thunbergii and
S. cantoniensis using in vitro bioassay method ofallelopathy, the protoplast co-culture
with digital image analysis
Suzuki, S.,1* Wasano, N.,1 Kimura, M.,1 Yasuda, R.,1
Nakagawa, W.,1 Sasamoto, Y.,2 Fujii, Y ,1 and Sasamoto, H.1,3
1

Tokyo University of Agriculture and Technology, Fucyu,
Tokyo 183-8509, Japan.
2
National Institute for Agro-Environmental Sciences, Tsukuba,
Ibaraki 305-8604, Japan.
3
Kanagawa University, Research Institute of Integrated Science, Hiratsuka,
Kanagawa 259-1293, Japan.
Strong inhibitory allelopathic activities of Spiraea thunbergii and its putative
allelochemical, cis-cinnamic acid, and moderate activity of S. cantoniensis werereported by conventional bioassay methods. To know the cellular mechanism(s) of allelopathy, we introduced another bioassay method of allelopathy,
protoplast co-culture method with digital image analysis (DIA-PP method),which quantitate the effects of protoplasts of test plants on the growth ofrecipient lettuce protoplasts, co-cultured in a 50 μL medium in a 96-well culture
plate. Green leaves of aseptically grown seedlings of both Spiraea species, andpurple leaves of S. cantoniensis, which anthycyanin-synthesizing gene wasintroduced, were investigated. Enzymatic conditions for protoplast isolation
were optimized for each material. Effects of cis-, trans-cinnamic acid, andcyanidin glucoside were also quantitated. Numbers of non-spherically enlarged(cell wall formation stage) or divided protoplasts (cell devision stage) werecounted under an inverted microscope, and Image J analysis of scanned digital
image of yellow color accumulation in a 96-well plate was performed. Growthof lettuce protoplasts was described as % of control. Green leaves of S.
thunbergii and purple leaves of S. cantoniensis and cyanidin glucoside strongly
inhibited lettuce growth at cell division stage. The results were compared withthose investigated using the same DIA-PP method.
 E-mail: ssakae@cc.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-34
Phytochemical screening and allelopathic activity of
ethanolic extract and solvent fractions ofVernonia Amygdalina Del. leaf
Hien, N.,T.,H.,1* Danh, L. T.,2 and Fujii, Y.3
1

Department of Biological Production Science, Tokyo University of Agriculture
and Technology, Tokyo 183-8509, Japan
2
College of Agriculture and Applied Biology, Can Tho University, Vietnam
3
Department of International Environmental and Agricultural Science,
Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan

Vernonia amygdalina Del. is a native African plant of the Asteraceae family
that has been commonly known as bitter leaf due to its bitter taste. The possible
medicinal use of V. amygdalina by wild chimpanzees was first described since
1989 and subsequent researches has unveiled that V. amygdalina contains awide variety of bioactive compounds. However, there has not been any detailed
research on allelopathic activity of this species. In 2017, the allelopathic
potentiality of this plant was primarily evaluated by sandwich and dish packmethods at Fujii laboratory (TUAT). V. amygdalina showed significant strong
inhibitory effect on lettuce radicle and hypocotyl elongation, compared to otherlocal botanical species in Mekong Delta Vietnam. Therefore, the aim of thisstudy was to continue investigating the phytochemical component and inhibitive
activity of the extracts of V. amygdalina leaf on lettuce seeds. Dried leaves wereextracted with 95% ethanol and then seperated by column chromatographyusing various solvents: hexane, ethyl acetate, n-butanol and water. The
phytochemical analysis revealed the presence of secondary metabolites in crude
extract, e.g. flavonoids, saponis, tannins, phenolics, steroids, alkaloids,
terpenoids and cardiac glycosides. The ethyl acetate fraction showed the highest
total phenolic content (74.83 mg QEA/g extract) and the highest flavonoid(63.62±1.11 mg RU/g extract), while the highest total tannin content wasachieved in water fraction (1.77±0.02 %). The strongest inhibitory activity on
the growth of lettuce roots was found in n-butanol fraction by 58.41%, yethypocotyl elongation was not statistically different among separated fractions.
At the concentration of 0.5 mg/mL, lettuce radicle and hypocotyl elongation
were lowest by 73.26 and 78.29%, respectively. These results suggest that V.
amygdalina can be an allelopathic candidate for further studies.
 E-mail: nthhien59@gmail.com

4th International Conference of Asian Allelopathy Society

P-35
Species-specific allelopathic potential of
Vietnamese tea (Camellia sinensis)
Pham, V. T. T.,* Mishyna, M., and Fujii, Y.
Tokyo University of Agriculture and Technology
The bio-herbicides isolated from plants are applied for weed control byreplacing synthetic herbicides or can be discovered as lead chemicals forsynthesis of a new herbicide. It was reported that tea plant (Camellia sinensis)
has a significant allelopathic effect from tea extract and residues. However,each kind of weeds or crops has different sensitivity to inhibitory compounds
extracted from allelopathic plants. In this study, we evaluated allelopathic
potentials of aqueous extracts of green and oolong tea from Vietnam on growthof twelve crop and weed species. For the evaluation of plant germination, 1 mlof each extract (50, 75 and 100 mg/ml) was added to a sheet of filter paper (ϕ42.5 mm) in a 50 mm Petri dish, and then ten seeds of each tested species wereplaced on the filter paper, and incubated in dark condition at 22°C for n-days.
For the evaluation of plant growth: 700 µl of the crude extracts of tea leaves atconcentrations of: 1, 2, 4, 6, 8, and 10 mg/ml were added to the filter paper.
Then five lettuce seedlings were put on the filter paper, and incubated in dark at
22oC for 3 days. The length of radicle and hypocotyls/coleoptiles of lettuce
seedlings were measured on the third day.
The resulst showed seed germination of all the tested species was inhibitedby green and oolong tea extract at concentration 100 mg/ml, but inhibitory
effect induced by extract was not observed on the germination at both ricecultivars. Based on EC50 value, green tea extract significantly inhibited thegrowth of the weeds such as: timothy, white clove and red clover by possible
presence of high caffeine content. By contrast, extract had a slight effect on thegrowth of rice, carrot and hairy vetch at the tested concentrations (4, 6, 8, 10
mg/ml). The observed differences in the response (germination, radicle andhypocotyls/coleoptiles elongation) were possibly due to the difference in seedsize and seed coat permeability which are responsible for caffeine uptake. Ourfindings revealed that extract from green tea significantly inhibited the weed
species than oolong tea, suggesting a new weed control potential.

 E-mail: phamvanbq@gmail.com

4th International Conference of Asian Allelopathy Society

P-36
Evaluation on the allelopathic potentials of
Malaysian plants, and identification ofgoniothalamin as a potent allelochemical from
Goniothalamus andersonii J. Sinclair
Ismil, R., 1* Hirai, N.,1 and Fujii, Y.2
1

Kyoto University, Graduate School of Agriculture, Sakyo-ku,
Kyoto 606-8502, Japan.
2
Tokyo University of Agriculture and Technology, Graduate School of
Agriculture, 3-5-8, Saiwai-cho, Fuchu-shi, Tokyo 183-8509 Japan.
Evaluation on the allelopathic potentials of 145 plants was conducted byusing the sandwich method. The bark of Goniothalamus andersonii
exhibited the greatest inhibition rate of 81% on the radicle growth of lettuce
seedlings at 10 mg dry weight. Goniothalamin was identified as a potent
allelochemical from the bark by bioassay-guided purification. This
compound showed the EC50 values 50 and 125 μmol L-1 for both radicle andhypocotyl growth of lettuce seedlings, respectively. Soil incorporation withthe bark powder of G. andersonii was conducted to assess the plant growth
inhibitory activity of this plant against lettuce (Lactuca sativa L.), cucumber(Cucumis sativus L.), white clover (Trifolium repens L.) and perennial
ryegrass (Lolium perenne L.). Various concentrations of the bark powder
incorporated into soil have resulted in the reduction on the growth rate, freshand dry weight of those tested plants parallel with the increasing
concentration of bark powder treated. At the highest concentration of 2%,
the growth of perennial ryegrass was strongly inhibited after 14 days ofsowing (95%). The strong detrimental effect on this invasive perennial
ryegrass in particular indicates that the bark of G. andersonii could be very
beneficial as a bio-herbicide for practical weed management.

E-mail: raihan.ismil@gmail.com

4th International Conference of Asian Allelopathy Society

P-37
Identification of allelopathic substances of desert
plant Sea-buckthorn
Bao, L.,1* Aniya1, Bai, M.,2 Adouqinfu,2 and Fujii, Y.1*1

Tokyo University of Agriculture and Technology, Graduate School of
Agriculture, Fuchu Campus, 3-5-8, Saiwai-cho, Fuchu,
Tokyo 183-8509 Japan.
2
Inner Mongolia University for the Nationalities, Pharmaceutical Institute
Mengniu, No. 536 Huolinhe Street, Horqin District, Tongliao City,
Inner Mongolia Autonomous Region, China.
Leaves, fruits and other parts of 22 plants from desert plants of Inner Mongolian
Autonomous Region were examined by the “Sandwich Method” which is
widely used to assay allelopathy of plant parts. The results of the test are as
follows: Hippophae rhamnoides (Sea Buckthorn) 96%, Artemisia sieversiana
94%, Salsola collina 92%, Artemisia annua 87%, Platycodon grandiflorus 85%,
Salix cheilophila 83%, Agriophyllum squarrosum 83% (in order from strong toweak). Strong activity was observed in most of the species. As a result of thebioassay by the dishpack method, which tests the allelopathy of plant volatile
substances, strong activity was observed in the order of Artemisia annua (Sweet
annie) 85%, Sabina vulgaris 55%, Artemisia ordosia (strong to weak) 26%.
The strongest Sea Buckthorn fruit inhibited the growth of the recipient plantlettuce by 96% at 10 mg-dry weight/10 mL agar. Sarcosine and asparagine weredetected as candidates for potential allelochemical as a result of GC-MS
analysis of water extract of Sea Buckthorn fruit.
In the test of volatile substances of Sweet annie, the growth of therecipient plant lettuce was inhibited and 1, 8-cineole was detected as the major
compound by GC-MS analysis.

 E-mail: bao18747378714@163.com

4th International Conference of Asian Allelopathy SocietyR

P-38
The possible role of allelopathy in invasive
succession of Papaver dubium L.
Izumi, M., Mardani, H. and Fujii, Y.
Tokyo University of Agriculture and Technology, Faculty of Agriculture,
Fuchu, Tokyo, Japan.
Papaver dubium L. is an annual alien species in Japan that was first time foundin Setagaya, Tokyo in 1961. Since then, it has been widely spread across Japan,
especially in central and southern areas. Allelopathy has high contribution toinvasive properties. Therefore, it is essential to know their ecologicalcharacteristics, especially allelopathy.
In this experiment, in order to determine the possible role of allelopathy in the
spread of P. dubium in Japanese agroecosystem, Sandwich method (SW), Plant
Box method (PB) and soil incorporation test were adapted and its inhibitory
activity on elongation and dry weight of lettuce seedlings were measured.
Moreover, fresh leaf and root were extracted with aqueous methanol todetermine the EC50 on lettuce radicle growth. HPLC was used to analyse thechemical composition in the aqueous methanol extract of the P. dubium.
P. dubium showed high inhibitory activity by SW (Leaf 50mg: 82%, Root
50mg: 56%), PB (63%) and the soil incorporation test (EC50: 1.7% (w/w) ). The
extracts of fresh leaf showed inhibitory activity by increasing concentrations(EC50: 30mg/mL). The HPLC analysis revealed that the fresh leaf and the root of
P. dubium contain high concentration of berberine (Leaf: 13.2µg/mg, Root:
13.3µg/mg). Pure compound of berberine showed inhibitory activity byincreasing concentrations (EC50: 25µg/mL). The total activity of the berberine
was estimated 0.53. Based on the present results, It is possible that berberine
play some roles in invasive succession of Papaver dubium L.

 E-mail: s154075w@st.go.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-39
Allelopathic and antimicrobial study ofAcacia modesta and Buxux papillosa available
in District Hangu, KPK, Pakistan
Shahida Ayub, and Saima Hashim*
Department of Weed Science, The University of Agriculture,
Peshawar-Pakistan
The current study was to asses’ allelopathic activities and antimicrobial
potential of Acacia modesta Wall and Buxus papillosa C. K. Schneider. A
laboratory based studied was made to investigated the allelopathic potential of
A. modesta and B. papillosa leaves and roots on Lettuce seed (Lactuca sativa L.
v. Great lakes 366). Results were significant which showed that 100 g/L water
extraction of leaves of both the plants having high allelopathic potential toinhibit the seedling growth of lettuce seeds. Roots water extractions of both theplants induce seedling growth of tested plant. The in vitro antimicrobial
investigations of crude methanolic extracts of A. modesta (bark and root) and B.
papillosa (leaves, stem and root) plants were analyzed against five pathogenic
bacteria including one Gram positive Bacillus subtilis, four Gram negative i.e.
Escherichia coli, Klebsiella pneumonia, Xanthomonas campestris andAgrobacterium tumefaciens using disc diffusion method. We obtained
significant results from this experiment. It was observed that K. pneumonia was
the most resistant bacterium to all extracts. Present study concluded that theseplants are good sources of antibacterial compounds for the curing of many
pathogenic diseases. These results confirm strong allelopathic and antimicrobial
potential of these both of these candidate species.

E-mail: saimahashim@aup.edu.pk

4th International Conference of Asian Allelopathy Society

P-40
How to promote the growth of young Japanese Peartrees with the cyanamide and establish the system todetermine when we should use the cyanamide
112

Toya, T., * Oshida, M., Tomiyama, M., Fujii, Y.

3

1

Chiba Prefectural Agriculture and Forestry Research Center.
2
Nippon Carbide Industries Co., INC.
3
Tokyo University of Agriculture and Technology.

Cultivation of Japanese pear, which is a major fruit in Japan, has been
expanding in East Asia. Japanese pear is necessary to replant in every 30 years,
but the growth of replanted trees were usually suffered from soil sickness
syndrome. Cyanamide, a putative allelochemical from hairy vetch, hasanti-microbial, insecticidal and herbicidal activity, and also used practically topromote the flowering of grape.
In order to establish the method for culturing trees of Japanese Pear ‘Kosui’
growing up in a short period by applying the cyanamide, which is the chemical
used for stabilizing the flowering, we carried out the following verification test
and system development.
・In case the cyanamide was sprayed on the nursey trees in orchard field, thesprouting rates of leaf bud at a year and two were 94.0% and 96.5% respectively,
both of which were significantly greater than no-treatment trees (controls). The
number of shoots and total elongation of shoots were greater than the controls at
three years.
・In case the cyanamide was sprayed on an aging nursery in the replanted
orchard field, both sprouting rates of leaf bud and the number of shoots weresignificantly greater than controls, as an aforementioned example. Therefore,
we consider that cyanamide could be used not only for stabilizing the flowering,
but also for facilitating the early bearing of replanted orchard field.
・We developed a system which calculates the appropriate timing for spraying
the cyanamide by downloading the temperature data by AMeDAS(Japan
Meteorological Agency official website) into a personal computer automatically,
and then calculates the DVI (developmental indexes) according to the growthforecast model.
*E-mail: t.ty5@pref.chiba.lg.jp

4th International Conference of Asian Allelopathy Society

P-41
Identification of allelopathic compound of fenugreek
and its allelopathic potential against some weeds
under laboratory and greenhouse conditions.
Ismail, T.,1* Abdelkhalek, R.,2 and Fujii, Y.3
1

Department of Pesticide, Agriculture Faculty., Kafrelsheikh University, Egypt.
2
Weed Research Central Laboratory, Agricultural Research Center, Egypt.
3
Department of International and Environmental Agriculture Science, Tokyo
University of Agriculture and Technology, Japan.

Fenugreek is one of the promising medicinal herbs. It has potent allelopathic
activity, and the activity is confirmed through laboratory and greenhouse bioassay
in this study. laboratory bioassay tests have been carried out by sandwich method
and plant box method with lettuce seeds. As for sandwich method the inhibition
percentage of lettuce radicle by fenugreek leaves (91%) was greater than its seeds(60%). Regarding the result of plant box method showed that the fenugreek root
exudates have allelopathic effect which able to suppress lettuce seedling growth by
86.1%. To identify fenugreek allelochemicals, dried leaves were extracted by
distilled water then partitioned with different solvents. Chloroform layer showed
the highest biological activity. Isolation step has been performed using C18 and
HLB columns, inhibitory activity was found in 80% methanol fraction. Further
cleanup has been done using Preparative- HPLC. The EC50 values of this fraction
have been evaluated against some plant species namely: lettuce (4.8 ppm), Alfalfa(19.4 ppm), white clover (28.8 ppm), Verdolaga (31.7 ppm), Centipede grass(37.75 ppm), Timothy (3.5 ppm). This fraction has been identified by GC-MS
instrument as dihydroactinidiloide compound. Regarding greenhouse experiment,
three levels of fenugreek leave powder (0.5, 1 and 1.5 %) incorporated with soil.
Two obnoxious weeds, wild oat and dandelion have been used in this test. The
results showed that both of 1 and 1.5% of fenugreek leaves powder caused totally
inhibition on germination process of dandelion. While wild oat, fenugreek leaves
caused significantly decrease in root and shoot length. Knowledge of fenugreek
allelopathy will offer possibilities for ecological management of weeds under field
conditions.
 E-mail: tamer.ismail@agr.kfs.edu.eg

4th International Conference of Asian Allelopathy Society

P-42
Evaluation of the allelopathic activity of Sudaneseplants for weed control in Sorghum bicolor
Elmadni, H.S.M.,1,2 Mishyna, M.,1,3 and Fujii, Y.1
1

Tokyo University of Agriculture and Technology, Tokyo, Fuchu, 1838509, Japan.
2
Ministry of Agriculture and Forest, Khartoum, 461, Sudan3
Тel Hai College, Upper Galilee 1220800, Israel

Sudan is a major Sorghum producing country; however, its production has been
hindered by the presence of serious invasive weeds (Azhari, 2000; Badi et al.,1990). Intensive use of chemicals to control weed species has led to increase weed
resistance to pesticides (Michael et al, 2005). One of the effective alternative ways
in weed control is allelopathy, i.e. using of natural chemicals from plants tominimize the dependency on synthetic herbicides. Therefore, our research aims toassess the allelopathic potential of fifty-nine plants originated from Sudan and to
identify allelochemicals and their specific activity. Allelopathic potential ofsoluble and volatile compounds was evaluated using Lactuca sativa as receptor
plant according to (Fujii et al., 1994; Fujii et al., 2000). Identification of potential
allelochemicals was done using GC-MS and HPLC; the contribution of anindividual compounds into the total inhibitory activity was determined by (Golisz
et al., 2007) and (Mishyna et al., 2015). The top list of Sudanese plants with thestrongest allelopathic activity through exudates includes Lawsonia inermis (leaves,
powder), Hibiscus sabdariffa (fruit), Adansonia digitata (fruit), and Balanites
aegyptiaca (leaves). The highest suppression of the hypocotyl growth of lettuce
seedlings by volatiles compounds was observed for Terminalia brownie (wood),
Euphorbia hirta (leaves), Diospyros mespiliformis (leaves), Adansonia digitata(leaves), Corchorus olitorius (leaves), and Hibiscus sabdariffa (fruits). Moreover,
1-decyne was identified as the main volatile compound naturally released fromdried A. digitata leaves and responsible for the allelopathic activity expressed byvolatiles from baobab leaves against lettuce growth. The allelochemical lawsone
has been identified in Lawsonia inermis leaves by HPLC and mainly contributed
to the total allelopathic activity of soluble exudates from Lawsonia inermis leaves.
Therefore, plant species from Sudan showed a high allelopathic potential and canbe used for future organic herbicide development.

 E-mail halaaahmed79@yahoo.com

4th International Conference of Asian Allelopathy Society

P-43
L‐DOPA in Vicia faba sprouts andgene that produce L‐DOPA
Mochizuki, A.*, Taku, K., Sasaki, N., and Fujii, Y.
Tokyo University of Agriculture and Technology, Japan

L-DOPA plays two roles of treatment of Parkinson's disease and allelopathy.
Parkinson's disease is commonly treated by ingestion of L-DOPA. In plants,
L-DOPA has allelopathic activity to inhibit radicle growth, and the use of
weed control has attracted attention. L-DOPA is made from tyrosine bytyrosine hydroxylase and tyrosine hydroxylase is classified in 14-3-3 protein
family in eukaryotes. However, genes that produce L-DOPA in plants havenot been elucidated. If it is clarified, it is expected to contribute to thedevelopment of medical and agricultural aspects. In this study, thecorrelation between the content of L-DOPA and the expression level of
L-DOPA genes in sprout, the purpose of cloning 14-3-3d sequence of Vicia
faba produced as L-DOPA genes The experiment was carried out.Previous
studies have obtained data suggesting a change in L-DOPA content in Vicia
faba sprouts and have experimented with the hypothesis that theexpression of L-DOPA genes also fluctuates. In this study, I measured theamount of L-DOPA contained in the Vicia faba sprout HPLC. As a result, it
was found that the content of L-DOPA increased about 2-fold from 7th day
to 14th day after seeding. Next, BLAST search was performed based on thesequence of broad bean 14-3-3 protein to obtain the 14‐3‐3 amino acidsequence and cDNA sequence of Vicia faba, then I called it vf14‐3‐3dsequence. Primers for cloning and expression were designed based on theinformation of this base sequence. We succeeded in amplifying the DNA
fragment by PCR with the ORF primer using hydroxylase cDNA and genomic
DNA for vf14‐3‐3d.

 E-mail:s1796677r@st.go.tuat.ac.jp

4th International Conference of Asian Allelopathy Society

P-44
Evaluation of 91 Chinese medicinal plant species forallelopathic potential
Aniya,1* Fuerdeng,2 Nomura, Y.,1 and Fujii, Y.1
1

Tokyo university of Agriculture and Technology, Japan.
Xinjiang Institute of Environmental Protection., China.

2

In plant kingdom allelopathy is an essential ecological factor as well as phytotoxic in
medicinal plants (Samanta et al., 2011). Medicinal plants contain a relatively large number of
secondary metabolites and allelopathic activities have been frequently found in those plants(Fujji et al., 1990). This study focused on Chinese medicinal plants, which are mainly
distributed in northeastern and southeastern of China. The objectives of this study were: (1)Screening of allelopathic activity of medicinal plants in China. (2) Evaluation of allelopathic
activity of plants candidates for weed control. (3) Identification of allelochemical(s) frompotential allelopathic plant species.
For the materials, 91 species medicinal plants were collected from Xinjiang Institute of
Ecology and Geography and Beijing Hospital of Traditional Chinese Medicine. Plant
materials were screened using the Sandwich method (Fujii et al., 1994) and Dish Pack
method (Fujii et al., 2000) to evaluate their allelopathic effect on the growth of lettuce(Lactuca sativa). Phytotoxic activity experiment was used to determine allelopathic activity
on crops and weed species. Cotton swab method was used to determine allelopathic activityof volatile allelochemical (Mishyna et al., 2015). Identification and quantification of potential
volatile allelochemical(s) were done by GC-MS.
As the results, fruits of Illicium verum showed significant inhibitory acitivity in both
Sandwich and Dish-pack method. The fruits of the Chinese star anise (Illicium verum)
contain SA to the extent of 17.14% on dry wt. basis and this is now the main source forcommercial production of SA (Jagat Chandra Borah, 2015). According to the EC50 result, it
was concluded that radicle growth inhibition of lettuce caused by crude extract of star anise
could be explained by contribution of biological activity of shikimic acid. GC-MS analysis of
Illicium verum (0.5g) showed anethole could be the major volatile compound in this plant bycomparing with database. The result of cotton swab method showed the strong growth
inhibition of authentic anethole and star anise powder on lettuce respectively. The result
indicated that anethole has a strong inhibitory activity and has a big contribution inallelopathic activity of star anise. It also indicated that EC50 of anethole in head space of staranise powder is almost similar to that of authentic anethole, so anethole could explain as
principle inhibitory volatile in head space of star anise.
In conclusion, fruits of Illicium verum showed significant inhibitory activity in bothsandwich and dish-pack method. Two major compounds, shikimic acid and anethole in staranise have showed strong inhibitory activities on crops and weed species. They could be theputative allelochemicals in Illicium verum and could be utilized in weed management andsustainable agriculture.

 E-mail: any2014@foxmail.com

4th International Conference of Asian Allelopathy Society

P-45
CREST project and future dream for research-Elucidation of rhizosphere chemical world for the
regulation of crop robustnessSugiyama, A.,1 Fujii, Y.,*2 Sakurai, N.,3 Kobayashi, M.,1 Aoki, Y.,4
Onodera, T. 5
1

2

3

Kyoto University, Tokyo university of Agriculture and Technology, Kazusa DNA
4
5
Research Institute, Tohoku University, Kyusyu University, Japan.

Crops such as soybean and tomato can only be grown to about half of theirpotentially achievable yield due to various problems related to soils. In thisresearch, we develop a multimodal sensor device to diagnose minerals andmetabolites in the rhizosphere, which has strong influence on the crop growth.
We identify biomarkers for robustness by integrating data from sensors andomics and analyzing in multimodal deep learning. We verify biomarkers underfluctuating environment to achieve crop robustness for the increase of
production.
For allelopathic research, we are starting to use hairy vetch as green manure
plant for soybean growth. Yield of soybean after hairy vetch usually 10 to 20 %higher than control and quality of bean also increase. Metabolomic analysis incombination with other factors will explain the important factor in the soil.

 E-mail: yfujii@cc.tuat.ac.jp