M i n i s t r y o f E d u c at i o n , C u l t u re , S p o r t s , S c i e n c e an d Te c h n o l o g y ( M E X T )F o r u m f o r N u c le ar C o o p e r at i o n i n A s i a ( F N C A ) B i o f e r t i l i ze r Pr o j e c t

I s sue No. 9
March, 2 011

Overview of FNCA Bio ferti liz er Project 2010
Tadashi Yokoyama, Tokyo University of Agriculture and Technology (TUAT)
Shotaro Ando, National Agriculture and Food Research Organizatio n (NARO)
Establishment of environmental friendly sustainable

would like to introduce the FNCA 2010 workshop

agriculture and reduction of agrochemical input areon Biofertilizer Project, which was held in Manila,

required in Asia. This FNCA Biofertilizer Project

the Philippines, Nov. 8-12, 2010, joingly with theaims to reduce the amount of chemical fertilizer

workshop on Mutation Breeding Project. A total of

input without decreasing yield of crops, by using

54 experts and staffs from Bangladesh, China,function of beneficial microorganisms in biofertilizer,

Indonesia,

which increase availability of plant nutrients fromThailand, Vietnam, and RCA/IAEA participated in

soil.

this workshop.

We carried out the first phase of Biofertilizer ProjectAt the workshop on Biofertilizer Project, summaryfrom 2001 and the Biofertilizer Manual, which canreport was presented by each participationg country

be downloaded from the website of the FNCA, wasand five topics were discussed. The first topic waspublished as one of the outcomes of the project. In“application of radiation sterilization of carriers” andthe second phase of this project from 2007, we areit was reported that autoclave-sterilization of carriersdevoting our efforts to three objectives as follows.

showed a

The first one is development of multi-functional

Azospirillum, whereas gamma-irradiation did not.

biofertilizer, which consists of multiple inoculants

The second topic was “ development of FNCA

with promoting plant growth or inhibiting plant

Manual for Quality Standard of Biofertilizer” anddiseases. The second one is improvement of

future plan was concluded that FNCA Biofertilizer

inoculants by radiation-based microbial mutation

Quality Standard Manual would be edited based on

breeding in order to keep high quality of inoculants

the Quality Standard manual of biofertilizer ofunder tropical conditions. The third one is

Thailand. The third topic was “ development ofdissemination of radiation sterilization method ofmulti-functional biofertilizer” and different kinds ofcarrier using Co-60 to improve quality of carrier forworks on the development of multi-functional

biofertilizer.

biofertilizers that have multi-effects on the growth,

In this news letter, we introduce activities of thenutrition uptake of plant as well as reduction of plant

FNCA Biofertilizer Project in 2010. At first, we

disease were reported. Then, the forth topic,
1

Japan,

Malaysia,

The Philippines,

deleterious effect onsurvival ofF o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t“commercial application of biofertilizer”, and theThen, they visited Bio-N test field and got an

fifth topic, “ application of biofertilizer with plant

explanation that the farmer used Bio-N to reduce

growth promoter of oligo-chitosan” were discussed.

usage of chemical fertilizer and got a good result for

The special presentation was also given by Dr.

yield of corn.

Mercedes Umali Garcia, University of Philippine,

On November 12th, FNCA 2010 Meeting on

Los Banos (UPLB) on development and promotion

Technology Transfer from Research to Commercialof Bio-N, biofertilizer with Azospirillum, and its

Application was held. Approach for technology

great success story.

transfer in several countries, current status andParticipants visited the Bio-N laboratory inchallenges that nuclear research institutions faced in

the National Institute of Molecular Biology andtransferring research results from the laboratory to

Biotechnology

the market place were discussed.

(BIOTECH)

of

UPLB.

They

observed and discussed about process flow on Bio-N
production, especially on carrier preparation.

Participants of workshops on Mutation Breeding and Biofertilizer Project

Carrier Production of Bio-N,

A scene of Biofertilizer Project Wroskhop

Biofertilizer in the Philippines2F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

T he Effe ct of Gamma-Ste riliz ation of Biofe rtiliz e r ’s Carrie ron the De nsity of Rhiz obia l Ino culants
Kouhei Tejima, Issay Narumi, Japan Atomic Energy Agency (JAEA)
Nitrogen fixation through the legume-Rhizobium
symbiosis is important for legume production. To

Following the sterilization of the carrier materials by
50 kGy of gamma-irradiation or autoclaving at 121°C

utilize symbiotic nitrogen fixation for agricultural
production more effectively, it is necessary to develop
advanced rhizobial biofertilizers that keep the amountfor 40 min, water suspension of Bradyrhizobium
japonicum strain USDA110 was inoculated.
Additionally, biofertilizers that were consisted of theof viable inoculants for a definite period of time. The
inoculants survival in the biofertilizer could be

inoculants and one of the non-sterilized carriers wereprepared as controls. The initial density of inoculants

affected by the physical and chemical properties ofcarrier materials and by the competition with native
microbes in the carrier. Gamma-irradiation is expectedin the biofertilizer was adjusted to 6.6×10 7 cells g–1.
The biofertilizer was packed in polyethylene bags andstored for 6 months in 4°C or 30°C. After storage,

to sterilize the carrier materials without changes inphysical and chemical properties. In an effort toviable inoculants in the biofertilizer were enumerated
by the dilution plate method. The number ofdemonstrate the effect of gamma-sterilization, thesurvivals of inoculants were monitored to assess theshelf life of biofertilizer.

inoculants in biofertilizers made from non-sterilized
carrier was monitored by PCR that amplifies the
nodD-nodY region of B. japonicum.

As carrier materials, three kinds of Japanese typicalsoil (cultivated topsoil of Andosol, Gray Paddy soil

The inoculants density after 6-month storage wasgreater than the initial density in biofertilizers madeand forest subsoil of the Kanto Loam Formation) andJapanese peat soil “Keto-tsuchi”) were prepared. To
make soil-based artificial carriers, each Japanesefrom sterilized carriers, while that was significantly
decreased in biofertilizers made from non-sterilized
carriers (Fig. 2). Gamma-sterilization was superior totypical soil was mixed with charcoal powder (3:1). In
order to optimize gamma-ray dose to sterilize soils,

autoclave-sterilization in enhancing the survival of
inoculants. Because of the stability of supply, thethe survival rate of native microbes in the preparedsoils were determined. As a result, 50 kGy wasdetermined to be suitable to sterilize soils (Fig. 1).

high sterilization effect with lower radiation doses,
and the high performance in maintaining the
inoculants density, “ Keto-tsuchi” was selected as
those most suitable for a biofertilizer’s carrier.

Fig. 1
Surv ival rates of nativ e microbes in the different kinds ofsoil after gamma-irradiation. Surviv al microbes were notdetected after 50 kGy of irradiation.

Fig. 2
Surv ival of rhizobial inoculants in biof ertilizer stored at
4°C (left) and 30°C (right). Error bars indicate ±SE
around means.

3F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

Country New s

Biofertilizer Act ivit ies in Ban gla desh
Md. Said ul Islam, Ban gladesh At omic Energy Commission ( BAEC)
Biofertilizer is becoming very popular in Bangladesh◆Status of biofertilizer development in Bangladesh◆compared to chemical fertilizer for its easy availability

1. Rhizobial biofertilizer for pulse and oil seedand ecofriendly nature to the environment. The following

legume (lentil, chickpea, mungbean, cowpea,

organizations are involved in biofertilizer activities in theblack gram, groundnut and soybean): Intensively

country: Bangladesh Institute of Nuclear Agriculture,studied and best developed in Bangladesh and is a

National Institutemature technology.

AgriculturalofBiotechnology,

Bangladesh

Bangladesh

Agricultural

University,

2. Azolla for wet land boro rice: Mature technology

Research Institute etc.

(not in practice).
3. Phosphorus solubilizing bacteria (PSB): On-farmevaluation going on.
4. Azospirillun / Azotobacter / blue green algae:
Evaluating at field condition.
5. Mycorrhiza: Evaluating at pot and field condition.

A biof ertilizer demonstration f ield of soybean
treated with and without inoculum

A rice field of Dhamkuri village,
Nowgaon District

Sample collection in poly bag

Collected Sample in Laboratory

Sample preparation

Weighing of rice root

B acteria in semi solid medium4F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

Study on H igh E ffect iv e Pho sp hate-s olub iliz ing a ndMult ifun ctio na l B io fertilizers in C h ina
Fan Bingquan, Chinese Academy of Agricultural S ciences (CAAS )1. Recent research of biofertilizer in our group

2. Development of multifuctional Biofertilizers in China

In order toeffects of P-solubilizing

Some multifunctional biofertilizers are developed.

biofertilizers on plant yield, our studies are focusing on

The test strains are (1) Klebsiella pneunoniaee; (2)the optimum combination of P-solubilizing strains with

Penicillium oxalicum P8; (3) Bacillus mucilaginosus;

various soils and crop plants in China. The test crops(4) Trichederma longbrachiatum; (5) Trichoderma

are soybean, paddy rice, wheat and corn, the test soils

onigii; (6) Trichoderma citrinoviride; (7) Bacillus

are Chao soil, Drab soil, ALBISOL, Black Soil, andmucilaginosus; (8) B. megaterium (Ba5); (9) B.

Dark Brown Chernozemic Soil. Phosphate-solubilizing

megaterium (AT CC14581); (10) B. circulans. Test

strains are P8, Pn1, A6, P21，P24-B1，P40-2，P36-1，

crops are soybean, corn and rice. Test soils are Chao

P40-1，P41-3A，P31，10243，P2-1A，10245，11107,

soil and Dark Brown Chernozemic soil. We have

RN5, Pantoea ananatis and Bacillus subtilis. Thirty

gotten 10 kinds of high effective multifunctional

optimum

biofertilizers (Table 2).

increasecombinations ofphosphate-solubilizingstrains with 5 types of soils and 4 crop species in North

The

results showed that the

combination ofChina and North-Eastern China are listed in Table 1.

Azotobacter chroococcumm, Penicillium oxalicum P8,

The result showed that there exists significant

Bacillus mucilaginosus with rice plant in Dark Brown

suitability among P-solubilizing microbes with crops

Chernozemic soil achieved a high biomass yield. The

and soils. Strain P36-1 is suitable for wheat on Chao

combination of P. oxalicum P8, T. longbrachiatum, T.

soil; strain P21 is suitable for wheat on Drab soil;

citrinoviride, and T. konigii with soybean in Chao soil

strain P36-1 and P40-1 are suitable for corn on Chao

got a highest biomass yield. The combination of P.

soil; strain P2-1A is suitable for corn on Drab soil;

oxalicum P8, T. longbrachiatum, T. citrinoviride, andstrain 10245 and P41-3A are suitable for corn on

T. onigii with soybean in Dark Brown Chernozemic

ALBISOL; strain P40-2 is suitable for corn on Blacksoil was the best one. The combination of K.

soil; strain P30-1 is suitable for corn on Dark Brown

pneunoniaee, P. oxalicum P8, B. mucilaginosus , T.

Chernozemic soil; strain P2-1A+RN5 is suitable forlongbrachiatum, T. citrinoviride, and T. konigii withsoybean on Black soil; and strain 11107+RN5 iscorn in Chao soil had a marked effect on cornsuitable for soybean on Drab soil; Pantoea ananatis isbiomass.

suitable for soybean on Dark Brown Chernozemic

Interesting topics of biofertilizer in China are (1) tosoil; and Bacillus subtilis is suitable for Paddy rice on

develop P-solubilizing biofertilizer; (2) to develop

Dark Brown Chernozemic soil.

nitrogen-tolerant N-fixing biofertilizer; (3) to develop
plant growth-promoting biofertilizer; (4) to develop
antagonistic

biofertilizer; andsilicate-dissolving biofertilizer.
5

(5)

to

developF o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

Table 1. Optimum combination of high effe ctive P-solubilizing microbe with various soils and crops

Table 2. Effects of high effective multifuctional biofertilizers in different soils and crops6F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

M utant Induction of M ine ral Phosp hate Solubil iz ing Panto ea
b y Ra diatio nYoung-Ke un Lee , Kore a Atomi c Ene rgy Re se arch Insti tute (KAERI)
Three mineral phosphate solubilizing bacteria (PSB)

However, high P solubilization potential of P2 and P3

were isolated from common bean rhizosphere and dry

indicated the possible involvement of other P

field soil. 16S rDNA analysis indicated that the isolate

solubilizing mechanisms. Mutant clone libraries forP2 and P3 are closely related to Pantoea dispersa whilephosphate solubilizing strains were created by gamma

isolate P4 is Pantoea terrea. Isolates P2 and P3

radiation induced mutagenesis and screened for

exhibited stronger mineral phosphate solubilizing

modified MPS activity. Mutant P2-M1 recorded the(MPS) activity than that of isolate P4. Unlike P2 and P3,

highest P-solubilizing potential among any other wild

prolonged incubation of P4 in Pikovskaya (PVK) andor mutant clones by releasing 504.21 µgml-1 of

National Botanical Research Institute’s Phosphate

phosphorous i.e. 35% higher than its wild type by the(NBRIP) liquid growth media exhibited poor P

end of day 5. A comparative evaluation of T CP

solubilization as well as increase in pH towards neutral.

solubilization by wild type isolates of Pantoea and their

Production of gluconic acid through direct oxidation

mutants, led to select three MPS mutant clones such as

pathway of Glucose (DOPG) by these P solubilizing

P2-M1, P3-M2 and P3-M4 with a potential to release >isolates was confirmed by reverse phase HPLC.

471.67 µgml-1 of phosphorous from TCP. We propose

Specific activity of Glucose dehydrogenase andthat mutants like P2-M1, P3-M2 and P3-M4 withgluconic acid production by isolate P4 was significantly

enhanced MPS activity could be deployed forhigher than that of P2 and P3.

biofertilization.

Fig. 1. Scanning electron micrographs of Pantoea strains (a) P2, (b) P3 and (c) P4.

Pantoea dispersa strain P2

Pantoea dispersa strain P3

Pantoea terrea strain P4

Fig. 2. HPLC detection of gluconic acid production by Pantoea isolates

7

F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

Bright Outlook for Biofertilizer in Malays ia
Khairuddin Abdul Rahim, Malaysian Nuclear Agency (Nuclear Malaysia)
Looking at the increasing entries of innovative
biofertilizer and bioorganic fertilizer products athave been pressed out of them, previously an almost
unwanted and problematic organic mountains, and theinnovation expositions and agricultural exhibitions in
the country for the past few years, one would feel thebreath of fresh air in terms of the acceptability of suchoften foul-smelling fermenting palm oil mill effluent(POME) in vast, space-consuming ponds, are now
being treasured. They are now the bases for new

products for the Malaysian agricultural industry. It isalso heartening to note that the giant plantation

high-value products, including as carriers andingredients in bioorganic fertilizer formulations. The

industries of oil palm and other commodity crops likerubber and rice are taking biofertilizer and bioorganic
fertilizer seriously, although they have been using

plantations are now scrambling to build their own
institu biofertilizer and bioorganic fertilizer plants.
The use of nuclear technology, particularly in the formchemical fertilizers for increased crop productivity
since the sixties. Chemical fertilizers and otherof gamma irradiation sterilization of media andsubstrates for biofertilizer products has been adopted

agrochemicals have been ingrained in the country’s
agricultural programs, and this include in theprovision of fertilizer subsidies for smallholders andby several companies and enterprises. The factors ofsterilization time for bulk samples in pre-packed bags
or containers and energy cost make gamma sterilizationindividual farmers of selected industrial crops.
With increasing awareness on food and environmental

attractive. However, the factors of logistics, including
escalating transportation cost have to be considered,safety from injudicious use of chemical fertilizers andother agrochemical inputs, notwithstanding theincreasing prices of petroleum-based fertilizers andtoo. The available gamma irradiation facilities in
Malaysia may not be able to cope with increasing
demand for sterilization services in the future.

other chemicals, the time has come for the bioorganics
and the functional indigenous microorganisms to

On the success of from laboratory to market, we arepleased to note that Nuclear Malaysia has undertaken

come into the picture, with their own roles in crop
productivity, environmental safety and generalsocietal wellbeing. The platforms of Greento supply fresh cultures of one of her biofertilizer
microorganism strains for an agreed period at anagreed fee, to Malaysian Agri Hi-Tech, an enterprisingTechnology and Waste-to-Wealth (W2W) are now
available for the advancement of biofertilizers andand modern agricultural bioproduct company, which
also utilizes Nuclear Malaysia’s gamma irradiation

other natural products through new industrial
initiatives. Agricultural wastes from large oil palm
plantations, in the forms of empty fruit bunches (EFB)

facility as part of their production. This shows there ishope for R&D to Technology Transfer
Commercialization in the biofertilizer arena.

after the oils and oleo chemicals

Best wishes to all!

8

F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

Dev elop ment o f Mu lti-funct io na l Bio fertilizer in Th a iland
S ompong Meunchang and Achara Nuntagij, Department of Agriculture (DOA)
The increased crop yields are widely observed by the
biological N2 –fixing, by the biological P and K
solubilization, and by the biological antagonistic
associations, even thought by increasing capability ofplant nutritional adsorption. These multimode andnatural functions of microorganisms are able topromote plant growth and yield production.
The multi-functional biofertilizer might be developed
from some microbes that have multi-mechanism.
Some N2-fixing bacteria can increase soil N utilization,
increase solubilization efficiency of fixed soil P or
phosphate rock P, and increase efficiency of use of
fixed clay soil K or potassium rock. The productmight be produced from single strain, multi-strains or
multi-genus co-inoculation.
General procedure of developing multi-functional
biofertilizer production is as follows.

3) Proper carriers should be discovered such as peatsoil, charcoal, compost and other materials. They
might be sterilized by autoclave or irradiation.
4) The distribution or commercialization is also
should be approved.
5) The utilization on each planting system should bealso considered after farmers’ acceptance.
In Thailand, multifunctional biofertilizer withco-inoculants of Azospirillum and Bradyrhizobium forleguminous plants are developing. At first, to selecteffective strains, we screened Azospirillum isolates
respond with Bradyrhizobium specific for vegetablesoybean. We tried pot experiment inoculated withliquid culture of each isolates, and compared
Azospirillum inoculation, Bradyrhizobium inoculation,and co-inoculation (Fig. 1 and 2). To confirm theeffectiveness of co-inoculation, we repeated in field
experiments. Finally, we designed small scale
production in 100 g bag with sterilized carriers by
autoclaving or grammar irradiation and estimated that
inoculants could survive for one year.

1) Selection or screening of effective microbes. We
must approve the special ability in laboratory, in pot
or green house experiment and in field trial.
2) In case of co-inoculation, we have to ensure theimpact of co-inoculation, as if strains do not inhibit ordamage each other. Not only loss of population but
also the point of activity should be investigated, too.

Fig. 1 Diff erent growth of vegetable soy bean af ter inoculation
multif unctional biof ertilizer; T1 control,
T2 NPK, T3 Bradyrhizobium+PK, T4 Azospirillum+PK T5
Bradyrhizobium+ Azospirillum+PK

9Fig. 2 Nodulation of vegetable soy bean af ter inoculation
multif unctional biof ertilizer; T1 control, T2
NPK, T3 Bradyrhizobium+PK, T4 Azospirillum+PK T5
Bradyrhizobium+ Azospirillum+PK

F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

FNC A Biofe rtiliz e r Proje ct
Biofe rtiliz e r Re se arch and De ve lopme nt in Vie tna m
Pham Van Toan , Mi nistry of Agriculture and Rural De velopment (M ARD)
◆Interesting Topic of Biofertilizer◆

6. Biofertilizer containing microorganisms, which are

1. Biofertilizer containing N-Fixing microorganisms

antagonistic to root diseasse pathogens

2. Biofertilizer containing P. Solubilizing microorganisms

7. Organic fertilizer containing benifit microbes like

3. Biofertilizer consist of K. Solubilizing microorganisms

N-fixing, P,K-Solubilizing and PGPR

4. Biofertilizer containing PGPR microorganisms

8. Biofertilizer containing microbes, which improve

5. Biofertilizer containing Mycorhiza

soil properties like water holding capacity

Screening of microorganisms using in biofe rtilizer production

Production of organic biofertilizer

Application of biofertilizer
10

F o r um fo r N uc le a r C o o p e r a tio n in A s ia ( F N C A ) Bio fe r ti liz e r P r o j e c t

Topic of Japan & Thailand

Massage fr om Thai Researcher Lives in JapanKun layakorn Pron gju nthuek, Department of Agricu lture (DOA)
My name is Kunlayakorn Prongjunthuek, I come fromThailand. I work at Soil Microbiology Research Group,
Soil Science Division, Department of Agriculture. My
research field is about PGPR bio-fertilizer (PGPR =Plant Growth Promoting Rhizo Bacteria). I am
participate in MEXT Nuclear Researchers Exchange
Program Fellows in 2010.
My research theme is Indonesia, Japan and Thailand
Azospirillum sp. phylogenetic relation and Mutation
breeding of Azospirillum sp. using ion-beam facility interm of long self life in carrier. In screening I studied for
IAA production ability and DNA isolation. After that I
choose some and send to JAEA for irradiation. My
research is in the level of intensity of radiation used in
the screening for the mutation of microbes. This now isin the summary.
I used to dream that I could be here – Japan the land ofcivilization, cultures and unique language which people
in the world dream of visiting this wonderful land oncein their lives. Now I have been here for one year working
as an exchange researcher in the Tokyo University ofAgriculture and Technology (TUAT), Fuchu, Tokyo.
This university is a famous university for agriculture andengineering.

As soon as I stepped on this place, I saw an enormous
farm, greenhouse experiments that are lined up, a great
number of trees along the path, and high buildings
surrounding with various trees. I could feel of thepleasant and peaceful atmosphere. These are my great
impression and they make the university worth being the
University of Agriculture. It is unbelievable that big city
like Tokyo has the university with a great number of big
trees. It can enounce that ones who love trees will love
this university like I do.
I do not only impress in the atmosphere of this university
but also in a lot of various persons, my Lab mate,professional who is very kind and warm, and my friend
both Japanese and other overseas. They all are lovely andhelpful. They also advice and teach me everything I do
not know, especially Japanese cultures and tradition. We
did many activities of the lab together including selling,
things in booth in the traditional fair of the university.
These help me not to feel lonely while living in foreigncountry. I love my professor and my friend very much.
Apart from what I said above. I would like to say thatliving in this university is so simple and convenient thatit becomes my very impressing thing that cannot
described. I promise that I am going to visit you again
TUAT, Fuchu, Tokyo.

Nuclea r Sa fety Resea rch Associa tion
FNCA Secreta ria t
5-18-7 Shimbashi, Minato-ku, Tokyo
105-0004, JAPAN
TEL:+81-3-5470-1983
FAX : +81-3-5470-1991
FNCA Website:
http://www.fnca.me xt.go.jp/english/inde x.html
Ms. Prongjunthuek with Members ofTokyo University of Agriculture and Technology

11