FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project

Issue No. 6
November 2005

Message from Indonesia
Dear Colleagues,
In a country with high population like Indonesia, an increase in food crop production is the firstpriority for the government to fulfil the need for food. The use of plant genotypes with highproductivity and responsive to fertilizer input are the normal steps undertaken to achieve thispurpose. Referring to FAO Statistical database, it was reported that during the period of 21 years
fertilizer consumption in South East Asia increased 3.5 times, from 10 million ton in 1976 to 45
million ton in 1997.
Beside inefficient fertilizer use by crops, excessive application of N fertilizers imparts negative
impact to the environment. The remaining fertilizer N is stored in soil organic matter, some isconverted to atmospheric nitrogen, and some is leached into the groundwater as nitrate pollutant.
It is well accepted that the use of bio-fertilizer could reduce chemical fertilizer dependency andproduce equal grain yield as the recommended rate of chemical fertilizer.
Use of suitable bio-fertilizers to complement chemical fertilizer is to be encouraged as it will
improve the natural N balance in the environment.
Issue 6 of the FNCA Bio-fertilizer Newsletter presents results from the use of bio-fertilizer in the
agricultural sector in Indonesia. It is hoped that this sharing of information and the promoting of a“green biotechnology” will be beneficial to us and to those involved in the agriculture industry.
Regards,

Soertini Gandanegara
FNCA Biofertilizer Project Leaderof Indonesia

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FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project
DEVELOPMENT TECHNOLOGY AND COMMERCIALIZATION OF
EMAS (Enhancing Microbial Activity in the Soils) BIOFERTILIZER
D.H. Goenadi,1 Y.T. Adiwiganda,2 and L.P. Santi3
Indonesian Research Institute for Estate Crops (IRIEC), Department ofAgriculture, the Republic of Indonesia
2)
PT Bio Industri Nusantara
3)
Biotechnology Research Institute for Estate Crops, IRIEC
dgoenadi@indo.net.id1)

are basically microorganisms which can
improve the availability of nutrient to plants.
They have been believed to be an importantcomponent of sustainable agriculture, as they
can reduce significantly the use of chemicalfertilizers. In addition, microbial inoculant
is believed to be potentially effective ininducing microbial activities in therhizosphere. It is hypothesized that by
improving rhizospheric microbial activities
via
biofertilizer
application,
nutrientsolubilization can be enhanced andconsequently less conventional fertilizers will
be needed.

Introduction

Large areas of marginal
soils in Indonesia have beendeveloped
for
plantation.
These soils have been characterized by low
organic matter content, predominant low
activity clays, and strongly acid in reaction.
These in effect will depress the microbialactivities which in turn disturb the nutrient
cycle in the soils. On the other hand, underhumid-tropic conditions, fertilizer loss
through leaching, volatilization, and/or
fixation represents an economic loss as well
as a potential environmental contamination.
The inefficient use of conventional fertilizers
is still a part of the current agriculture
practices. However, crop productivity isclosely related to the ability of plant roots to
extract water and nutrients efficiently out of
the soils. The function of the roots isgoverned by an integrated set of biologicalprocesses.
A biotechnological approach is thenassumed to be able to enhance the emergenceof biotechnology in soil management
provides a new approach in tackling
efficiently many problems which remain
unsolved by current conventional technology.
Manipulation of soil microbes offers anefficient technique to stabilize soil aggregates,
increase nutrient uptake, control soil-borne
pathogen, and accelerate the decompositionof solid organic wastes, without adding newpollutants to the environment. Biofertilizers

Development of EMAS biofertilizer
technology

Research results in our laboratory(1994-1995) gave one prototype of
biofertilizer product. Our previous study
indicated that multi-isolate inoculums weremore effective in improving soil nutrient
availability and stable aggregate formation
than that of the single-isolate one. Based on
this evidence a biofertilizer formulation was
prepared in granular form consisting of nonsymbiotic N-fixing bacteria Azospirillum
lipoferum (BCC 2369) and Azotobacter
beijerinckii (BCC 2368), and phosphatesolubilizing Aeromonas punctata (BCC 2367)
and Aspergillus niger (BCC F194). The
formers have also the capability to solubilize
hardly soluble phosphate, whereas the latter
species can induce the formation of stable soil
aggregate that intended for increasing
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FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Projectapplicable to any types of crops. Finally,
multiplier effects of EMAS application could
be generated from cost savings on storage,
labour, transportation, etc. The process waspatented in 1998 in Indonesia with Patent No.
ID 0 000 206S.
Although the benefits offered are
remarkable, the implementation of EMAS
biofertilizer has some constraints. Being a
new product type it raised a doubt about its
effectiveness compared to conventional
products. There are a very large number ofdifferent microorganisms in biofertilizer
products. They tend to be heavily promoted
and this is a great need for a standard forsimple and accurate ways of measuring theireffectiveness.
Therefore,
successfulcommercialization of such product needs asmart strategy to be taken. For commercial
purposes, a consortium of PTP Nusantara III,V, VII, and VIII built a company called PT
Bio Industri Nusantara (Bio Nusa) on 10
November 1999. The company office locatesin Bandung, whereas manufactory of EMAS
is at Purwakarta, West Java. Production
technology of EMAS by PT Bio Industri
Nusantara is under license of Biotechnology
Research Institute for Estate Crops (BRIEC).
This product has been legally registered forcommercial production and marketing at
Department of Agriculture with registration
number of G 798/BSP/X/2001.

fertilizer use efficiency on marginally suitablesoils. Some of them have a capability to
improve other nutrients solubilization. All ofthese microbes were native to Indonesian
Ultisols soil.
Pilot scale development ofEMAS
biofertilizer
technology
wasconstructed based on the previous results incollaboration with Project Management of
the Ministry of Research and Technology,
Republic of Indonesia, and state –owned
plantation enterprises PTP Nusantara I, IV,
VII, VIII, and XIV (1996 -1999). The goal ofthis project is to provide the planters by anefficient biofertilizer product capable ofreducing conventional fertilizer requirements
commonly applied on marginally suitablesoils.
The pilot scale production technology
of EMAS biofertilizer is an intermediate step
before entering commercial scale within aseries of technology development that was
beginning from laboratory and green house
experiments. These activities were done in
1996-1999.
Development of pilot scale
EMAS production technology was carried out
on the basis of an efficient principle ofbioprocess technology that means a simple
technology, cheap and abundant of materials,
and an effective product output.
EMAS was prepared in granular form
having sizes of 2-4 mm in diameter by using
a mixture of minerals and in-land peat as
carrier, and packed in a 25 kg bag (Fig 1 and
2). Field experiments were conducted inplantation crops (tea, rubber, cocoa, oil palm,and sugarcane), food crops (paddy and corn),horticulture(potatoes),andspice.
Application of EMAS has shown that it can
reduce the use of conventional fertilizer up to50%.
In addition, introduction to soil
provides a more stable soil aggregate andimprove soil fertility status.
The otherbenefits of EMAS are : (i) economically offer
10-30% saving on fertilizer cost, (ii)environmentally friendly practice, and (iii)

Impact on productivity

Since 1997 the biofertilizer EMAS has being
produced semi commercially at 10 ton/day
capacity.
An up-scaled plant has been
constructed by PT BioIndustri Nusantara(Persero) with 10,000 ton/year capacity.
Unlike chemical fertilizer, EMAS biofertilizer
will only be produced after the users orderthe product, since microbes of EMAS
biofertilizer has shelf-life about 12 months.
EMAS biofertilizer has been proven to3

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project
highly potential for increasing fertilizer use
efficiency of hevea rubber through the
reduction of conventional fertilizer dosage on
highly weathered tropical soils. This result
indicated that the microbes had played an
important role in improving efficient of
fertilizer use.
Similar phenomena wereobserved at other crops species, i.e. tea, cocoa,
and sugarcane. In addition, at various oilpalm growing environments, combination of
250 g EMAS biofertilizer per tree persemester and complete conventional fertilizer
at a 75% reduced rate per tree can givecomparably similar fresh fruit bunch (FFB)
yield to full rate application of conventional
fertilizers.
Thejustificationofeconomic
feasibility for EMAS biofertilizer application
is based on cost reduction of fertilizer. EMAS
biofertilizer could save national fertilizer costat Rp. 1.5 T/year value. Results of financial
analyses indicated that the development of
EMAS manufacture with Rp. 9.625 billion
investment fund could reached NPV at Rp.
14.23 billion, 72.21% IRR, 1.30 B/C, andpositive cash flow cumulative in the fourthyears or twenty months after production.
These indicated that the manufacture of
EMAS biofertilizer could reached a positive
NPV, IRR value higher than bank loan(commercial) and B/C > 1 and therefore it isfeasible to be done.

Figure 2. EMAS biofertilizer in granular form with
one year stable quality guarantee.

Concluding Remarks

Lack of intensive microbial activities
in Indonesia marginal soils can be overcome
by introducing beneficial microbes. EMAS
biofertilizer that contained inoculants nativefrom Indonesian Ultisols are proven to becost effective, eco-friendly and renewable,
and generally capable of reducing chemicalfertilizers use in sustainable agriculture andplantation system. Development of EMAS
production technology was carried out on thebasis of an efficient principle of bioprocess
technology. This product has been legally
registered for commercial production andmarketing at Department of Agriculture.
Through appropriate management of EMAS
biofertilizer in agriculture and plantation soil
quality could be maintained and fertilizer use
efficiency of plantation crops, food crops,
and horticulture could be increased by
reduction of conventional fertilizer dosages
on highly weathered tropical soils.

Figure 1. EMAS biofertilizer packed in a 25 kg bag.

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FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project
Role of Ectomycorrhizal Fungi and Organic Fertilizer
To Improve the Productivity of Tailing Soil in Gold Mining Area
Supriyanto
Researcher at SEAMEO BIOTROP
and Lecturer at Faculty of Forestry, Bogor Agricultural University
Ujang Susep Irawan
Research Assistant, SEAMEO BIOTROP
Background

Gold Mining Area in Pongkor-Bogor, West
Java. The experiment was conducted for 8
months. Factorial experiment in Completely
Randomized Design 5 x 3, with 12 replicates,
was used at the greenhouse test. Two factorof this experiment consisted of type of media(five levels) and mycorrhizal fungi (threelevels). The type of media consisted of tailing
100% (T1), soil 100% (T2), mixed of tailing:
compost = 1:1 (v/v) (T3), mixed of tailing:
compost = 3: 1 (v/v) (T4), and mixed oftailing: soil: compost = 1: 1: 1 (v/v/v) (T5).
Species of ectomycorrhizae consisted of no
mycorrhizal inoculation (M1), inoculatedwith Scleroderma columnare (M2), andinoculated with Rhizopogon luteolus (M3).

Gold mining produces
two types of waste; those arerocky-dump soil and tailingsoil. One of serious problems in gold mining
activity is the over capacity of tailing dam tostore the tailing soil. In addition, restorationof tailing soil is the most difficult aspect onreclamation of mining area.
Chemicalcharacteristic such as acidity, alkalinity,
salinity, and high concentration of heavy
metals are common problem in gold mining.
The uptake of the metals, such as lead, by theplant is seldom a problem. Association oflead and cellular or sub-cellular membranesof the plant often results in the manifestationof toxic lead effects such as reductions in theactivity of phosphatase enzyme and enzymebound ATP-ase. To minimize negative effectof tailing, inoculation mychorrhizal fungi andaddition of organic fertilizer in the tailing
medium to accelerate biogeochemistry
processes was applied.

Seeds of Shorea selanica were
germinated in sterilized soil media at green
house conditions. Prior to the seedgermination, seeds were sterilized using
fungicide-containing Mankozeb 80% to kill
pathogenic fungi. Seedling transplantation
was done three weeks after seed germination
into culture media according to thetreatments. Mycorrhizal fungi inoculation
was done one week after seedlingtransplantation. Inoculum suspension of one
milliliter containing 10000 spores/ml was
injected into media closed to the root system.
The experiment was conducted at greenhouseconditions. Height and diameter growth ofseedlings was measured periodically, while
presence of Hartig’s net and mantle structure
and the seedling dry-weight were measured
at the end of the experiment. Histological

The aims of this research were to
study
the
interaction
betweenectomycorrhizal fungi and organic fertilizer
in the tailing medium to increase the plant
growth in greenhouse condition.
Material and Method

The research was conducted in
Silviculture
Laboratoryof
SEAMEOBIOTROP and tailing soil was taken from5

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project
analysis of suspected mycorrhizal roots wasdone at laboratory.

The Growth of Shorea selanica Seedlings

The highest growth of Shorea selanica
seedlings was obtained in the seedlings
inoculated with Rhizopogon luteolus andadded with 25 % organic fertilizer to thetailing media (T4M1). This height growth of
S. selanica increased 272% higher thancontrol (T1M1).
On the other hand,
inoculation of Scleroderma columnare andRhizopogon luteolus with addition 25%
organic fertilizer (T4M2 and T4M3)
stimulated the best diameter growth of
Shorea selanica compared to the othertreatments. In this case, the diameter growthof S. selanica increased until 93% and 100%
compared to control, respectively.
Application of mycorrizal fungi andorganic fertilizer increased the total dry
weight of S. selanica. Inoculation of
Rhizopogon luteolus increased the dryweight of Shorea selanica until 8.8% higher
than control or the seedlings inoculated with
Scleroderma columnare. Mixture of tailingand organic fertilizer (3: 1; v/v) increased
total dry weight of Shorea selanica until
196% higher than other composition of
tailing media and organic fertilizer. The
growth of Shorea selanica at different types oftailing media and mycorrhizal fungi ispresented at Figure 3, 4, 5, and 6.

Fig 1: Fruit-body of Scleroderma columnare (A) andRhizopgon luteolus (B)Results and Discussion

The result of this research showedthat the height growth, diameter growth, andmycorrhizal colonization of Shorea selanica
seedlings were affected by interaction
between mycorrhizal fungi and type oftailing media, but, total dry weight was only
affected by single factor of mycorrhiza ortype of media only.

Height Growth (cm)

10
8
M1
M2
M3

6
4
2
0
T1

T2

T3

T4

T5

Type of Media

Figure 3: Effect of media on height
growth of Shorea selanica seedlings

Figure 2: Effects of ectomycorrhizal fungi andtailing media on the growth of Shorea selanica
seedlings

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Diameter Growth(mm)

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project
auxin that will affect to root ramification andelongation (Brundret et al. 1996).

3
M1

2

M2

1

M3

0
T1

T2

T3

T4

T5

Type of Media

Total Dry Weight(gram)

Figure 4: Effects of media on diameter
growth of Shorea selanica

6
42

Figure 7: Non infected root (A), infected root by S.
columnare (B), infected root by R. luteolus (C) in tailing
media

0
T1

T2

T3

T4

T5

Type of Media

colonization of Scleroderma
columnare (M2) and Rhizopogon luteolus(M3) can be seen in Figure 8, while theiranatomical analysis can be seen in Figure 9.
The

4
3
2
1
0
M1

M2

Mycorrhizal
Colonization (%)

Total Dry Weight(gram)

Figure 5: Effects of media on total dry
weight of Shorea selanica seedlings

M3

Mycorrhizal Fungi

40
30

M2

20

M3

10
0
T1

T2

T3

T4

T5

Type of Media

Figure 6: Effects of mycorrhizal fungi on
total dry weight of Shorea selanica
seedlings

Figure 8: Colonization percentage of
Scleroderma columnare (M2) andRhizopogon luteolus (M3) in different media

Mycorrhizal Colonization

Mycorrhizal colonization is the key
success in the use of mycorrhizal technology.
Morphologically, the colonization success canbe observed through the presence of hyphae
enveloping the root system (Figure 7). Whitehyphae developed well in the seedling media.
Good colonization will strengthen thecompactness of culture media due to thedevelopment of hyphae and rooting system.
In this case, mycorrhizal fungi release the

Figure 9: Root of Shorea selanica infected by S.
columnare (A), non-infected root (B), and root
infected by Rhizopogon. luteolus (C)7

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project<References>
Brundret, M., N. Bougher, B. Dell, T. Grove
and N. Malajczuk, 1996, Working
with mycorrhizas in forestry andAgriculture. ACIAR. Australia.

Theaverage
percentage
ofcolonization by Scleroderma columnare andRhizopogon luteolus was 28.1% and 19.5%,
respectively. It can be classified as low
colonization percentage. Even though theircolonization percentage was low, but they
were able to increase the growth of Shorea
selanica seedlings in the culture media
containing tailing as a waste of mining
activity.
It means that mycorrhizal fungiplay important role to increase theproductivity of tailing media in its association
with root system of Shorea selanica. Good
colonization of Scleroderma columnare wasalso found in pure tailing media (28.1%) andin pure soil media (35.5%). On the other
hand, good colonization of Rhizopogon
luteolus was also found in mixed-media of
tailing and organic fertilizer = 3 : 1 (33.8%).
In cross-section of an ectomycorrhizal
roots of Shorea selanica showed the presenceof Mantle and Hartig’s Net (Figure 9). Mantle
sheets of both mycorrhizal fungi was formedin two layers, while in control was notformed. Colonization of mycorrhizal fungi in
Dipterocarpaceae is very specific (Pampolina
et al. 1994). The presence of auxin released
by mycorrhizal fungi was able to develop theelongation development of cortical cells. It is
not the case in Pinus merkusii or in Pinaceae
in general.

Pampolina, N.M., R.E. de la Cruz and
M.U.Garcia, 1994, Ectomycorrhizal
roots and fungi of Philippine
Dipterocarps. ACIAR Proc No. 62.
On Mycorrhizas for Plantation
Forestry in Asia.

Conclusions

Ectomycorrhizal
fungi
plays
important role to improve the productivity oftailing soils as a waste of gold mining activity.
Organic fertilizer amendment is still needed
to stimulate the biogeochemistry process.

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FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project

Azolla AS AN ALTERNATIVE NITROGEN SOURCEFOR RICE CROP PRODUCTION

Lowland rice is one of the main
sources of the staple diet in the east, south,
south-east Asia including Indonesia. The
main constrain of this lowland is the soil
fertility that is unsustainable. This is very
important due to the fact that currently andin the future the lowland rice production will
have to be accelerated, to meet the needs ofthe growing population in this region. It isobserved that at lowland soils, the soilorganic matter (SOM) and the soil nitrogencontent are limited if not in deficiency. One
effort needed to meet this limitation is to
apply chemical fertilizers especially Nfertilizer, at high rates. But the risk of using
N-fertilizers at high rates is its low efficiency
and the potential pollution of ground water
caused by nitrate.
To solve this problem is to decrease
the rates of the chemical fertilizers and to
replace a part of the chemical fertilizers by analternative N source. One of the alternatives
N source is biofertilizers. One of thebiofertilizers which have a great potential asa nitrogen source and to build SOM is green
manure. From years of study done by BATAN,
Azolla is a suitable green manure as analternative nitrogen source and a source oforganic matter.
Azolla is a water fern plant. What has
been studied by BATAN is the mini Azollawith a size of 3 – 4 cm. Azolla live insymbiosis with a Cyanobacteria which is able
to fix N2-air. The Cyanobacteria is Anabaena
Azollae. This symbiosis makes Azolla to havea quite high N content. Azolla is indigenous
in Asia, America, Europe and Africa. It has
been widely used for centuries in China andVietnam as nitrogen-source for lowland rice.

Azolla in lowland rice

Ability of Azolla a nitrogen-source
An international research sponsoredby the International Atomic Energy Agency(IAEA) where Indonesia (Batan) participatedhas used 15N to shown the ability of Azolla
Nutrients content of Azolla
Nutrients
content (%)
N
1.96 – 5.30
P
0.16 – 1.59
K
0.31 – 5.97
Ca
0.45 – 1.70
Mg
0.22 – 0.66
S
0.22 – 0.73
Si
0.16 – 3.53
Na
0.16 – 1.31
Cl
0.62 – 0.90
Al
0.04 – 0.59
Fe
0.04 – 0.59
ppm
Mn
66 – 2944
Co
0.264
Zn
26 - 989
to fix N2. It was found that Azolla, which
symbiosis with Anabaena Azollae, could fix
N2 at a rate of 70 – 90%. The N2-fix, whichaccumulated in the Azolla’s body, is used as
altternative N-source for lowland rice.
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FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project¾¾¾¾

Several studies showed that thegrowth rate of Azolla is 0.355 – 0.390 gramper day in the laboratorium and 0.144 – 0.860
gram per day in the field. Generally in thefield Azolla reach maximum biomass after 14–28 days after its inoculation. Studies done by
BATAN showed 3–4 weeks after inoculation
at rate of 200 g fresh Azolla per m2 the whole
field at the water surface would be covered.
When the whole field is covered by Azolla it
could be expected that it could contribute 30-45 kg N per ha or equal to 100 kg urea. It
was found that Azolla grow better in therainy season as long as water is available.
The benefit of Azolla¾ Source of N which could replace urea
up to 100 kg¾ Feed for fish and small animals,
especially chickens and ducks

Suppress growth of weeds
As an ornamental water plant
Control of mosquitos
After using of Azolla for four seasons
or more in lowland rice it could
replace P-fertilizer up to 50% fromthe usual rate applied.

Azolla in basin and Field trial

Research result by BATAN
A layer of Azolla at the water surface in lowland rice could replace 50 to 100 kg urea
depending on growth rate and season.
Production of lowland rice (ton ha-1)
1. Without Azolla layer + 50 kg urea
5.0.
2. Azolla layer + 50 kg urea
6.0.
3. Azolla layer + 150 kg urea
6.5
4. Without an Azolla layer + 150 kg urea
6.0.
1. Result from research in Special Province of Jogjakarta
The research in this location is still in progress. Preliminary data that can be shown fromthe experiment in this location are the dry weight data of grain and straw. For the time being,
the highest dry weight of grain and straw – as can be seen from table – are obtained from
Treatment B (100% urea).
Dry weight data of grain (kg ha-1)
Treatment
A
B
C
D
E
F

Replication
I
4292.5
8420
7450
8895
4676.255596.25

II
3871.256080
5673.757080
7527.57537.5

III
4805
5642.56238.75
5601.256138.755080
10IV
3543.75
6627.56671.254507.56558.756965

ρ4128.1256692.56508.438
6520.938
6225.313
6294.688

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project

2. Result from South Kalimantan
The research in this location is still being carried out until now. Preliminary data thatcan be shown from the experiment in this location are the dry weight data of grain.
Dry weight data of grain (kg ha-1)
Treatment
A
B
C
D

I
3520
3840
4000
4620

Replication
II
III
3440
3680
4240
4480
4400
4640
4520
4840

IV
3200
4320
4720
4680

ρ
3460
4220
4440
4665

For this time being, the highest dry weight of grain – as can be seen from table – is obtained
from Treatment D (50% urea + 50% Azolla).
Promising Technology
Azolla is one of green manure sources, because they are (1) easily mineralized (21 – 24days after inoculation), (2) capable of supplying enough N needed by plants (30 kg N/ha) and (3)capable of reducing the loss of N from urea and (4) could be grown by farmers going to use it.
Due to the high capability of Azolla to fix N2 in water, Azolla can be a promising source ofgreen manure for wetland rice.

Biofertilizer for soybean on acid soil
Due to continuing land conversion of fertile
soil to non agricultural purposes in Javaisland, the government move the areal
cultivation out of Java island which dominate
by low pH, low fertility and status of nutrientwith high of Mn and Al content.
Soybean is sensitive to acid acidity withcritical level of Al saturation around 20%.
Poor soybean plant growth and no nodule
formation due to ineffective indigenous
Bradyrhizobium occurred. Soil management
including liming to alleviate soil acidity is acommon practice.
were performed at upland soils in Sitiung,West Sumatra and Sembawa, South Sumatera,
and tidal swamp areas at Karang Agung Ulu,
South Sumatera during 1990-1996.

A program has been carried out through low
input (0.5 t/ha banded on plant rows at 5 –7
cm in depth and 2.t/ha broadcasted) withsoybean mutant lines and Bradyrhizobium
inoculant develop for acid soil. Field trials
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FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Projectability of 38 – 55% and increased productionof 47 – 59% over control. The production wasalmost double as a result of inoculation
reaching 1.0 – 1.0 t/ha.
Further field trials showed that those local
isolates and its mixed culture have show
equal effectiveness with strain TAL 102, on
nodule formation. Application of those
inoculant increased plant biomass and plant
N yield at R5, and grain and grain yield.
Nitrogen plant yield were 114 mg N/plantand 43 kg N/ha, respectively in control plants.
Nitrogen plant yield were 218 mg N/plant atstage R5 and 83 kg N/ha at harvest in
inoculated plants.

Inoculation with Bradyrhizobium improved
plant performance in all locations. Evaluationon the effectiveness of symbiotic N fixation
on pH ranged between 3.90 - 4.50 at low (26–39%) and high (59 – 68%) Al saturation at
Sitiung and Sembawa, Karang Agung Ulu
showedthat,interaction
betweenBradyrhizobium isolates – soybean genotypes
significantly expressed in soil with high Al
saturation. Inoculation significantly affected
the grain yield in both limed and unlimed
soil. It seemed that the Bradyrhizobium
introduced were not able to establish thesymbiosis in the rhizosphere in unlimed soil
as good as in limed soil. Limed plots
improved grain yield 136 – 161% and 135 –
246%, over control, in Sitiung and Sembawa,
respectively. The multistrain inoculant
showed superiority as compared with single
inoculant. The grain yield reached up to 2.6
t/ha by using this multistrain inoculant.

Soybean mutant lines responsive to
inoculation on ultisol soil were identified.
Further field trials showed that especiallymutant line no. 7 has some good characters.
Biological nitrogen symbiotic ability of thisline was not affected by high dose of N
fertilizer which indicated that this mutant
line is suitable for multiple cropping systems
with cereal. This large seeded soybean variety
has been released as Rajabasa variety in 2003.

Further screening of Bradyrhizobium isolates
effectiveness on soil with Al saturation (50 68%) was carried out at Sembawa withselected soybean mutant lines. No nodulation
formed in uninoculated plots has madepossible to determine the N fixation by N
difference method. Two local isolates (BtJ. No.
22 and BtJ. No. 37) were found to becomparable to strain TAL 102 with fixing
12

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project

Biofertilizer for cornusing local isolates. The effectiveness of
Azospirillum-corn association were evaluatedin green house and in field trials.

After the symbiotic N2 fixing bacteria
Rhizobium, another group of soil bacteria
i.e. plant growth promoting rhizobacteria(PGPR) draw attention as a biofertilizer
with huge potential. Amongst them, isAzospirillum a gram negative bacterium,that produces plant hormones, reduces
nitrate, and fixes atmospheric nitrogen(Burdman et al, 2000).
Early studies on Azospirillum-plant
associations indicated that plant growthpromotion on plant was believed to be fromits N2 fixing ability. Results from later
investigations showed that the positive effects
of inoculation are principally derived frommorphological and physiological changes in
the inoculated roots, leading to enhancementof water and mineral uptake (Fallik et al,1994). Worldwide data of field evaluation
showed that these bacteria are capable ofpromoting the yield of important crops indifferent soil and climatic regions. Around
70% occurrence of success with statisticallysignificant yield increase in the order of 530% (Okon and Labandera-Gonzales, 1994).
In a decade Azospirillum inoculant are
produced in several countries for use on
cereals such as rice, corn and wheat.

Some advantages of seed inoculation
including better plant growth at early growthstage and early flowering were commonly
observed.
Improvementofseedlings
emergence in inoculated plants wereobserved during drought. Three mixed
inoculants increase plant growth by 27%
above the control and 21% of the crop N
came from the atmosphere, as determined by
15N
dilution method. In the field thebiofertilizer could reduce fertilizer inputs,
either of N chemical fertilizers or farmyard
manure. Increase of production depends on
soil fertility status, which ranged between 17
and 22%, and save N fertilizer close to 45 kg
N/ha (100 kg urea).

In Indonesia corn is an importantfoodcrop after rice and soybean, covering
3.000.000 ha of cultivation area, and with aproduction of around 9.000.000 tonne in 2004.
The recommended nitrogen dose is 90 kg/ha
for variety corn and 135 kg/ha for hybrid
corn.

Currently, at a demonstration plot at AgroTechno Park, South Sumatra a trial isconducted to evaluate selected Azospirillum
mixed inoculant M5 on corn. Evaluation andpromotion on the use of M5 inoculant on
sweet corn will be carried out at farmer’s
field at Nanggung village, West Bogor withthe cooperation of Farmer’s Association.

One program to obtain Azospirillum
biofertilizer for corn was initiated in 2001

13

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project<References>
1. Burdman S, Okon, Y and Jurkevitch E
2000 Surface characteristics ofAzospirillum brasilense in relation to
Cell Aggregation and Attachment to
Plant Roots. Critical Reviews in
Microbiology, 26, 91-116.
2. Fallik E, Sarig S and Okon Y 1994
Morphology and Physiology of Plant
Roots associated with Azospirillum. In
Azospirillum/Plant Association. Ed . Y
Okon, Boca Raton, Fla, 77-85.
3. Okon Y and Labandera-Gonzales C A
1994 Review. Agronomic applicationof Azospirillum : an evaluation of 20
years world wide field evaluation. Soil
Sci. Biochem, 26, 1591-1601.

Sterilization of biofertilizer carrier by irradiation

Carrier material is an important factor inbiofertilizer production, since it is also usedasthegrowing
media
oftargetmicroorganisms during storage and coatedonto seeds (Date and Roughley, 1977).
Materials for carrier selected should be nottoxic to the organisms, abundant and cheap.
Better quality of sterile product, encouraging
better growth and survival at high storage
temperature, high optimum moisture content,
and ease in quality control are the advantagesof sterile carrier (Roughley, 1988).
Sterilization by gamma irradiation is one way
to get sterile carrier materials, it is thepreferred method for carrier sterilization overautoclaving where irradiation source isavailable (Somasegaran and Hoben, 1985).
Gamma irradiation process makes almost nochange in physical and chemical properties ofthe material. While in sterilization by

autoclaving, some material change theirproperties and produce toxic substance tobacterial strains (Somasegaran and Hoben,
1985, Senoo, in press).
In Indonesia, the use of irradiation to sterilize
biofertilizer carrier was started by a privatecompany, PT Rhizogen Indonesia, as early in1986 for sterilizing carrier of inoculants forlegumes. Irradiation was chosen due to its
practical and effectiveness. The product wasirradiated at the Center R&D of Isotopes andRadiation Technology, BATAN. Lower dosethan the recommended dose 50 kGy i.e. at 35
kGy was chosen to eliminate Bacillus, a spore
forming bacterium, found in dry peat. After
irradiation,
viabilityof
inoculated
Bradyrhizobium cells was found to be high,with low contaminant, after 6 month storage
time.
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FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project
An adhoc meeting on irradiation of carrierfor biofertilizer, sponsored by MEXT and
FNCA was held in Tokyo 19 - 22 June 2005,
to encourage the use of 60Co irradiation forsterilization of biofertilizer carrier in FNCA
member countries. Irradiation sterilization isbetter, more economical and more practicalthan heat autoclave especially in case oflarger production. Method of irradiation was
discussed to reduce the irradiation dose wasgiven by Takasaki Research Institute, JAERI
to reduce biofertilizer cost resulting fromsterilization by irradiation.

2.

Roughley R J 1988 Commercial
Application of biological nitrogen
fixation, In Biotechnology andWorkshop on Biotechnology of
Nitrogen Fixation in the Tropics(BIOnifT),
heldat
University
Pertanian, Malaysia, 25-29 August
1986, Eds Z R Shamsudin et al, pp
147-164 UPM Malaysia.
3. Somasegaran P and Hoben H J 1985
Method in Legume – Rhizobium
Technology, NiFTAL Project.
4. Senoo K Carrier for biofertilizer.
FNCA Biofertilizer Manual, FNCA
Biofertilizer Project. Japan Industrial
Forum, Tokyo, Japan (in press).

<References>
1. Date R A and Roughley R J 1977
Preparation of legume seed inoculants. In
A Treatise on Dinitrogen Fixation, Section
IV. Agronomy and Ecology, Eds. W. F.
Hardy and A. H. Gibson. John Willey,
New York, 243-275.

15

FNCA (Forum for Nuclear Cooperation in Asia) Biofertilizer Project

Project Leader of Indonesia

Ms. Soertini Gandanegara
Senior Research Scientist
Soil and Plant Nutrition Research Group
Division of Agriculture
Center for Research and Development of Isotope andRadiation Technology (P3TIR)
National Nuclear Atomic Energy (BATAN)
P. O. BOX 7020 JKSKL, JAKARTA 12070, INDONESIA
Tel: +62 –21- 7690709 Fax: +62-21 – 7513270/7691607
E-mail: tgandanegara@yahoo.com
Research Interest:
Studies on the Use of Nuclear Techniques (32P. 14C, and 15N) in plant nutrition of foodcrops (e.g. photosynthetic rates, nitrogen fixation in legume-Rhizobium symbiosis andcereal-Azospirillum association)
Participated in research on compatibility study between root and scion in some fruits plants,:
rambutan, durian and mangosteen
Screening plant tolerant to NOx application of biofertilizer on crop improrvement

Check FNCA Web Site!
http://www.fnca.jp/english/

FNCA Biofertilizer News Letter No. 6
Editor:Ms. Soertini Gandanegara
Leader of FNCA Biofertilizer Project of Indonesia

Secretariat:

Ms. Yuko Wada
Asia Cooperation Center (ACC)Japan Atomic Industrial Forum, inc. (JAIF)
Tel:+81-3-5777-0753 Fax:+81-3-5777-0757 E-mail: yuko@jaif.or.jp* This Biofertilizer Newsletter is issued by Japan Atomic Industrial Forum, Inc. (JAIF)
under the contract of the Ministry of Education, Culture, Sport, Science and Technology (MEXT)
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