Báo cáo khoa học nông nghiệp " Replacing fertiliser N with rhizobial inoculants for legumes in Vietnam for greater farm profitability and environmental benefits " MS4 docx

1

Ministry of Agriculture & Rural Development

Collaboration for Agriculture & Rural Development



Project Progress Report

MS4: Second Six-monthly Report




013/06VIE
Replacing fertiliser N with rhizobial
inoculants for legumes in Vietnam for
greater farm profitability and
environmental benefits




July 2008
Table of Contents

1. Institute Information ___________________________________________________ 1
2. Project Abstract _______________________________________________________ 2
3. Executive Summary ____________________________________________________ 2

4. Introduction & Background _____________________________________________ 3
5. Progress to Date _______________________________________________________ 6
5.1 Implementation Highlights ________________________________________________ 6
5.2 Smallholder Benefits_____________________________________________________ 22
5.3 Capacity Building _______________________________________________________ 23
5.4 Publicity_______________________________________________________________ 23
5.5 Project Management ____________________________________________________ 23
6. Report on Cross-Cutting Issues__________________________________________ 24
6.1 Environment ___________________________________________________________ 24
6.2 Gender and Social Issues _________________________________________________ 24
7. Implementation & Sustainability Issues ___________________________________ 23
7.1 Issues and Constraints ___________________________________________________ 23
7.2 Options________________________________________________________________ 23
7.3 Sustainability___________________________________________________________ 24
8. Next Critical Steps ____________________________________________________ 24
9. Conclusion __________________________________________________________ 25
10. Statuatory Declaration__________________________Error! Bookmark not defined.
1. Institute Information
Project Name
Replacing fertiliser N with rhizobial inoculants for
legumes in Vietnam for greater farm profitability
and environmental benefits
Vietnamese Institution
Oil Plants Institute (OPI)
Vietnamese Project Team Leader
Ms Tran Yen Thao
Australian Organisation
NSW Department of Primary Industries
University of Sydney
Australian Personnel

Dr David Herridge
Dr Roz Deaker
Ms Elizabeth Hartley
Mr Greg Gemell
Date commenced
March 2007
Completion date (original)
March 2009
Completion date (revised)
As above
Reporting period
October 2007 – March 2008

Contact Officer(s)
In Australia: Team Leader
Name:
Dr David Herridge
Telephone:
02 67631143
Position:
Principal Research Scientist
Fax:
02 67631222
Organisation
NSW Department of
Primary Industries
Email:


In Australia: Administrative contact

Name:
Mr Graham Denney
Telephone:
02 63913219
Position:
Manager External Funding
Fax:
02 63913327
Organisation
NSW Department of
Primary Industries
Email:


In Vietnam
Name:
Ms Tran Yen Thao
Telephone:
08 9143024 –
8297336
Position:
Researcher
Fax:
08 8243528
Organisation
Oil Plants Institute (OPI)
Email:

2
2. Project Abstract






















3. Executive Summary

Increased production of high-quality inoculants and QA
Much of the R&D effort during the reporting period was focussed on evaluating rhizobial
strains for soybean and groundnut in both growth room and field. The Australian commercial
strains CB1809 (soybean) and NC92 (groundnut) were found to be more effective than local
Vietnamese strains at almost all of the 20 field sites for which data are available. Thus, when
the crops were inoculated with CB1809 or NC92, nodule weight, biomass yield and grain
yield increased relative to the local strains at 85%, 85 and 90% field sites respectively.
However, the extent of the increase was different depending on sites and local strains. The
two Australian strains increased soybean and groundnut nodulation, biomass yield and grain
yield by an average of 62%, 34% and 27%, relative to uninoculated plots and 26%, 11% and
10% relative to inoculation with local Vietnamese strains.

R&D on inoculant production technology focussed on additives to both broth and peat, on
survival of rhizobia in peat, and on temperature and pH effects on rhizobial growth and
survival. The IAS examined effects of broth (fermentor) culture dilution prior to inoculating
the peat carrier as a means of extending the broth.

Issues with rhizobial strain purity and maintenance, discussed at the Project Review
Workshop in February 2008, prompted new recommendations for strain maintenance. Other
issues raised and discussed were procedures for optimising the peat carrier through
adjustment of pH and moisture and for sterilisation of the peat. There was no training
Farmers in Vietnam currently fertilise legumes such as soybean and groundnut with N,
rather than inoculate with rhizobia. Replacing fertiliser N with rhizobial inoculants
would save Vietnamese farmers A$50-60 million annually in input costs and, at the same
time, help facilitate the desired expansion in legume production. There would also be
positive environmental outcomes. This project aims to increase production of high-
quality legume inoculants in Vietnam through enhanced production capacity,
implementation of a national quality assurance (QA) program at and increased inoculant
R&D. Participating in the project in Vietnam are the Oil Plants Institute (OPI), the
Institute of Agricultural Science (IAS) and the National Institute for Soils and Fertilisers

(NISF; now known as the Soils & Fertilisers Institute (SFI)). Institutions in Australia are
NSW Department of Primary Industries and the University of Sydney. Legume inoculant
use by farmers in Vietnam will be increased through the development and
implementation of an effective extension and training program for researchers, MARD
extension officers and farmers. The benefits of inoculants and legume nitrogen fixation
will be demonstrated in the field and communicated through workshops, meetings and
publications. To ensure sustainability of inoculant production and use, the project will
engage the private sector in marketing and ‘pilot production’ of legume inoculants, with
the aim that they may scale-up production and progressively take over supply as the
technology and markets are developed.

3
conducted during the reporting period, although training scheduled for August-September
2008 in Thailand and for later in 2008 in Australia was discussed at the February 2008
Project Review Workshop.
Extension and training of farmers and advisors
Extension and training of farmers and advisors is a major focus of the project as a means of
facilitating adoption of legume inoculation in Vietnam. The extension-training program is
built around simple, multi-location inoculation experiments in the legume production areas of
the country. The experiments involve participation of farmers and extension officers in all
aspects, from the design of experiments to sowing, sampling, harvesting and interpretation of
results. The MARD extension service plays a large role in extension activities. Data from
field demonstrations will be used to produce an economic model for production and use of
legume inoculants in Vietnam. In addition, training courses will be organised for farmers,
extension workers and researchers in methods of inoculant use, and economic as well as
environmental benefits of inoculation.

A total of 28 demonstration (extension) trials have now been conducted in 9 provinces. At
the time of writing this report, data were available for 15 demonstration trials. The
demonstration fields usually had two treatments: +inoculation and -inoculation. Overall,

inoculation of soybean and groundnut increased potential income to farmers by an average of
3.5000.000VNĐ. The size of the profit varied with different sites.

Farmers were invited to the demonstration fields at least once. At many fields such as in
DakNong and DakLak, they also came to the fields 2–3 times at nodule and biomass harvest
as well as grain harvest time. At each trial site, at least 20 farmers, extension officers,
agriculture advisors came and made evaluation of the trial (600–800 person visits to the
extension trials). They observed development of soybean and groundnut and compared the
health and growth of the plants in the two treatments. They took nodule samples to observe
nodules and learnt to recognise effective nodules with pink colour inside and the white
ineffective nodules. They evaluated the inoculation benefits by themselves by sampling
soybean and groundnut plants, weighing biomass and grains. They were provided extension
materials. Also, researchers and extension officers explained how rhizobia work and the
conditions for successful inoculation. Farmers were very interested in learning about nitrogen
fixation.

Questions were often asked by farmers were:
- How do the inoculants cost?
- How much do inoculants used for 1000m
2
or 1 ha?
- Where can we purchase inoculants?
- Do inoculants have other benefits besides urea (N) fertilizer?
- Can we use inoculants together with plant protection products?
- Can we use legume inoculants for other crops?
- Are inoculants effected by bad weather such as heavy rain, hot weather when inoculation
and during plant growth?
- Can we use inoculants together with urea (fertiliser N)?

And main requests:

- Supply inoculants for farmers to test inoculants in their fields
- Technical support for farmers to use inoculants

4
Involvement of the private sector in inoculant production, distribution and
marketing
Careful selection of private sector partners for commercial production of legume inoculants
is critical for ensuring the sustainable supply of high quality legume inoculants to farmers in
Vietnam. After visiting potential inoculant producers in February 2008 and the withdrawal of
Fitohoochmon from the project it was initially concluded that the prospect of the private
sector producing high quality inoculants in Vietnam in the near future was low and that large
scale production should be carried out by the institutes. However, following this, contact was
made with the Thien Sinh (Komix) company which is much better equipped to adopt legume
inoculant production and a strategy has been developed for the transfer of technology from
the institutes to this company. A site visit in February 2008 revealed very good potential for
legume inoculant production. The facilities were well equipped for careful industrial
microbiological application. Technology transfer from the research institutes to the company
was discussed and a clear strategy was determined. The company also agreed to conduct
extension activities through their country-wide network.
4. Introduction & Background
Project Objectives and Expected Outputs
The Vietnamese government (MOI, MARD) is committed to increase the area sown to
legumes from the current 780,000 ha to >1,000,000 ha by 2010, with particular focus on
soybean and groundnut in the Mekong Delta, the Central Coastal region and upland
(highland) areas of the North, Central and North. The legumes are used for production of
food, oil and protein meal, and are grown as rotation crops with rice (Mekong Delta), as
intercrops in the upland areas with cassava, sugar cane, rubber, fruit and maize and as cover
crops in the sandy coastal soils. ACIAR Small Project LWR2/98/27 (Increasing yield and
nitrogen fixation of soybeans, groundnuts and mungbean in Vietnam through Rhizobium
inoculation) identified that legume production in Vietnam currently relies on expensive

imported fertiliser N, rather than cost-effective inoculants containing rhizobia. Replacing
fertiliser N with rhizobial inoculants would save Vietnamese farmers A$50–60 million
annually in input costs and, at the same time, help facilitate the desired expansion in legume
production. There would also be positive environmental outcomes.
Details of the economic benefits of replacing fertiliser N with rhizobial inoculation were
outlined in the proceedings of the technical workshop to terminate LWR2/98/27. However,
for this to happen, high-quality inoculants need to be readily available in the market. The
current capacity of inoculant production in Vietnam is about 40,000 packets annually, and
would need to be increased to about 500,000 packets annually to meet potential demand.
Inoculant quality is also poor (LWR2/98/27 project) and would need to be improved. Shelf
life and distribution and marketing are issues that would also need to be addressed.
Moreover, there is limited awareness of the benefits of inoculants and methods of application
among Vietnamese farmers and extension workers.
Capacity gaps are evident at the national and institutional level. The major gap at the national
level is the lack of a coordinated, focussed national legume inoculant program. At the
institutional level, the gaps are capacity for medium-scale inoculant production and
associated quality assurance (QA) as well as R&D and training capacity. The proposed
project would address these issues of production, quality, distribution and marketing and
farmer education. Involvement of the private sector in both production and marketing will
ensure the long-term viability of the concept. The project objectives are to:

5
1. Increase production of high-quality inoculants for soybean, groundnut and other
legumes in Vietnam through enhancement of production capacity (personnel and
equipment) at participating institutions, implementation of QA, and increased
inoculant R&D;
2. Increase farmer interest and use of inoculants in Vietnam through development and
implementation of an effective extension and training program on inoculants and
legume nitrogen fixation for researchers, MARD extension officers and farmers
through demonstration trials, workshops and meetings, and publications;

3. Ensure the long-term viability of the project through involvement of the private sector
in this ‘pilot production’ of legume inoculants, with the aim that the private sector
would progressively take over production as the technology and markets are
developed.
The Project is aligned with the CARD Program Strategic Objective 2 ‘Improved productivity
and links to markets for the rural poor in the Mekong Delta and Central Coast regions’,
through Objective 2.1 ‘Increase rural productivity’, using principally Strategy 1 ‘Increase
productivity and competitiveness of the agricultural system’.
Project Approach and Methodology
The project strategy is to enhance inoculant production, quality, distribution and marketing
and farmer education through the collaborating institutions. It will involve both Government
institutions – Oil Plants Institute (OPI), the Institute of Agricultural Science (IAS) and the
Institute for Soils and Fertilisers (ISF) – as well as private sector companies (Fitohoocmon
Fertiliser JSC, Cu Chi Bio-Chemical Fertiliser JSC and Humix). The latter would be
involved initially in marketing and distribution of inoculants and would be provided with
advice and technical expertise to improve and expand their inoculant production capabilities.
In time, it is envisaged that the private sector would take over inoculant production, leaving
QA to the public institutions. Involvement of the private sector in both production and
marketing will ensure the long-term viability of the concept.
Increased production of high-quality inoculants and QA
The focus will be on rhizobial strains and their maintenance, inoculant production
(fermentation) technologies, quality assurance of the production process and products and
training in the production technologies and QA.
Rhizobial strains - Existing strains from the collections in Vietnam and/or other institutions,
eg. ALIRU (Australia), NifTAL (University of Hawaii), Suranaree University (Thailand) will
be utilised where appropriate. Decisions will be made about the most appropriate strains for
inoculant production through a program of research and development.
Strain maintenance - Protocols and operation manuals for maintaining strain effectiveness
and recognition to ensure stability of inoculant quality during long-term storage will be
developed and implemented. During the course of the project a decision will be made about

which institute/s will take responsibility for the maintenance of a culture collection and
verification of strains for inoculant production. The institute/s will be equipped with the
necessary materials to continue processing germplasm for inoculant production.
Production technology – Draw on experiences from Thailand and Australia to develop
production technology of inoculants at medium-scale in Vietnamese institutes through:
• Modifications to broth formulations and experiments in procedures for maintaining sterility
and dispensing broths into the inoculant carrier.

6
• Testing appropriate forms of inoculant (peat, granular, liquid) that allow compliance with
quality control standards and are easy to use, supply and transport. Economic benefits will
be determined by assessing their effectiveness in laboratory and field trials.
• Strain selection: Different strains will be tested for survival in inoculant products and
during delivery of products to the field.
Quality assurance - Australian QA protocols will be used initially as a model. From that,
QA protocols, training and working manuals specific to inoculant production in Vietnam will
be developed jointly between Australian and Vietnamese project scientists. Decisions will be
made about which institute will be equipped to carry out on-going routine quality control of
inoculants in Vietnam.
Training on inoculant production and QA - Vietnamese researchers from institutes will be
trained in Vietnam by Australian collaborators and at Suranaree University of Technology
(Thailand) in inoculant production, QA and laboratory management, as well as R&D in
rhizobiology.
Extension and training of farmers and advisers
The extension-training program for farmers and extension officers will be built around
simple, multi-location inoculation experiments in the legume production areas (Mekong
Delta, the Central Coastal region and upland (highland) areas of the North and Central and
the South East). They will involve participation of farmers and extension officers in all
aspects, from the design of experiments to sowing, sampling, harvesting and interpretation of
results. It is expected that the MARD extension service will play a large role in extension

activities. Data from field demonstrations will be used to produce an economic model for
production and use of legume inoculants in Vietnam. In addition, training courses will be
organised for farmers, extension workers and researchers in methods of inoculant use, and
economic as well as environmental benefits of inoculation. This extension-training program
will be conducted by Vietnamese researchers in collaboration with Australian counterparts,
who will assist in collating and preparing extension materials for translation and transfer to
Vietnam.
Involvement of the private sector in production, distribution and marketing
Two (and possibly three) Vietnamese companies will be involved in the project,
Fitohoocmon Fertiliser JSC and Cu Chi Bio-Chemical Fertiliser JSC (and Humix). It is
envisaged that the market for inoculants will grow during the course of the project from the
current low level and that the private sector will progressively become involved as the
production technology is developed and the market for the inoculants expands. The
companies will initially be involved in marketing and inoculant distribution. Training
workshops will also be open to researchers from the collaborating private companies. Project
scientists will provide technical expertise to the companies throughout the project to
troubleshoot production problems and increase production capacity and product quality.
5. Progress to Date
5.1 Implementation Highlights
5.1.1 Increased production of high-quality inoculants and QA
The focus is on rhizobial strains and their maintenance, inoculant production (fermentation)
technologies, QA of the production process and products and training in the production
technologies and QA.

7
5.1.1.1 Rhizobial strains
The efficacies of local strains for soybean and groundnut were compared with that of
Australian commercial inoculant strains CB1809 (soybean) and NC92 (groundnut). Pot trials
at ISF indicated that inoculation with CB1809 resulted in more nodules on soybean plants
grown in sterile sand when compared with three local strains (Table 1). The ability for the

different strains to grow in different fermentation conditions was also tested at ISF (see
Section 5.1.1.3).

Table 1: Nodulation of CB1809 and local strains on soybean

Nodule number/plant No Strains
Total nodule
number
Main roots Secondary roots
1 Uninoculated 0 0 0
2 CB1809 50 25 25
3 Local strain SL1 32 13 19
4 Local strain SL2 39 13 26
5 Local strain SL3 32 11 22
Field experiments were also conducted in 2007–08 to evaluate strain efficacy and effects of
inoculation on nodule, biomass and grain yield. The total number of field experiments was 29
in 10 provinces; 23 experiments have already been harvested, with 6 experiments to be
harvested in July 2008 (two experiments in Binh Thuan, one in Tra Vinh and 3 other ones in
Son La). In each province, experiments involving soybean and groundnut and at least 3
rhizobial strains (CB1809 or NC92 and two local strains) were evaluated. Treatments are
control without inoculation and without N fertilization, control with chemical N fertilizer,
inoculation with CB1809 or NC92 and inoculation with local strains. A summary of project
field experiments during 2007–08 and inoculation effects of CB1809 or NC92 on nodule,
biomass and grain yield is shown in Appendix 1. A small response to inoculation was
determined as less than 20%, from 20 to 40% was a moderate response and large response
was more than 40%.
Graphs 1, 2 and 3 summarise responses to inoculation with strains CB1809 (soybean) or
NC92 (groundnut) of plant nodulation, biomass and grain yield, respectively. Responses
ranged from small to large to inoculation depending on field sites.

Graph 1. Range of nodulation responses
to inoculation
-20
0
20
40
60
80
100
120
140
160
180
0 5 10 15 20
Field site
% response

8
There were large effects of inoculation on nodulation at 70% of the field sites (Graph 1). At
those sites, nodule weight increased by 43–166%. Nodulation responded moderately at one
site (34%) and, at the rest (25%) of the sites, response were small with an average increase of
10%. The overall average increase in nodulation using the superior Australian strains was
62%.














With crop biomass, there were large responses (44–60%) to inoculation at 50% of the field
sites, moderate responses (22–37%) at 30% of the sites and small responses (5–19%) at the
remaining 20% of sites (Graph 2). Increases in grain yield from inoculation were smaller
than the increases in nodulation and biomass yield (Graph 3). There were large responses
(40–68%) at 20% of sites. Moderate responses (20–37%) were recorded at 55% of the sites
and small responses (4–19%) at the remaining 25% of sites. The overall average increases in
biomass yield and grain yield using the superior Australian strains were 34% and 27%,
respectively.

There were large differences in nodulation, biomass yield and grain yield responses amongst
the rhizobial strains. Australian commercial strains CB1809 (soybean) and NC92
(groundnut) were more effective than local Vietnamese strains at almost field sites
(Appendix 2). Data analysis shows that when the crops were inoculated with CB1809 or
NC92, nodule weight, biomass yield and grain yield increased relative to the local strains at
85%, 85 and 90% field sites respectively. However, the extent of the increase was different
depending on sites and local strains. Graphs 4, 5 and 6 show the increase in nodulation,
biomass and grain yield of soybean and groundnut when inoculated with CB1809 and NC92,
respectively, compared with inoculation using local strains.
Graph 2. Range of crop biomass
responses to inoculation
0
10
20
30
40
50
60

70
0 5 10 15 20
Field site
% response
Graph 3. Range of grain yield responses
to inoculation
0
10
20
30
40
50
60
70
80
0 5 10 15 20
Field site
% response

9

The two Australian strains, CB1809 and NC92, increased soybean and groundnut nodulation
by an overall average of 26%, relative to the local Vietnamese strains (Graph 4). Biomass
yields were increased by an average of 11% (Graph 5) and grains yields increased by an
average of 10% (Graph 6), relative to the local strains.

For each of the measures, there were large variations according to the particular site. For
nodulation, the range was 0–70%. For biomass yield, the range was 0–30% and for grain
yield, the range was 0–32%.

Graph 4. Nodulation increases with CB1809
and NC92 compared to local strains
0
10
20
30
40
50
60
70
80
0 5 10 15 20
Field site
% Increase
Local Str 1
Local Str 2
Graph 5. Biomass increases with CB1809 and
NC92 compared to local strains
0
5
10
15

20
25
30
35
0 5 10 15 20
Field site
% Increase
Local Str 1
Local Str 2
Graph 6. Grain yield increases with CB1809 or
NC92 compared to local strains
0
5
10
15
20
25
30
35
0 5 10 15 20
Field site
% Increase
Local Str 1
Local Str 2

10
5.1.1.2 Rhizobial strain maintenance
There is a need to ensure that the rhizobial cultures used in inoculant production are
maintained as authentic, pure strains that originate from the same source and are effective in
fixing nitrogen.

On reviewing the QA of inoculant strains within each institute (i.e. OPI, IAS, SFI) during the
project visit to Vietnam in February 2008, it was determined that there was a need for more
training in the identification and maintenance of pure rhizobial cultures. The lack of in-depth
knowledge of the morphological identification of the rhizobial strains may have resulted in
the production of poor quality of legume inoculants for the field trials.

It was therefore recommended that for future research (laboratory, growth room and
fieldwork), only commercial strains from Australia would be used. These were the strains
that were most effective in field trials conducted in 2007–08.

As the purity of the cultures of NC92 and CB1809 used previously in research was doubtful,
it was decided that all existing cultures of these strains be discarded. OPI was selected as the
curator of the strains to be used in the commercial production of legume inoculants in
Vietnam. It was recommended that ALIRU re-supply OPI with fresh agar cultures and
freeze-dried cultures of NC92 and CB1809.

It was decided that:
• on receipt of the cultures issued from ALIRU, scientists of OPI would check the
purity of cultures by streaking onto YMA and CRYMA plates. Once the purity of
the strains had been established, sub cultures of the strains would be made, and
copies issued to the other institutes.
• when OPI issued cultures to the other institutes, they would be accompanied by
photographs of cultural growth showing colony morphology on agar in Petri plates.

In conjunction with this strain identification, each institute would streak out the culture that
they were using in the starter broths, photograph colony morphology and send it to the other
institutes including ALIRU. This cross checking between laboratories would ensure that
cultures of the rhizobial strains used in the production of inoculants were identical.
5.1.1.3 Inoculant production technology - experimental

Experiments on inoculant production were conducted at SFI, IAS and OPI. Each institute
prepared inoculants using different sources of peat and measured survival over time. The IAS
measured survival of rhizobia in peat amended with worm casts and coconut coir dust thus
changing the water holding capacity of peat. Both OPI and IAS investigated the effect of
different additives on rhizobia in inoculants formulated as liquids, a technology that would
reduce the high cost of sterilisation of peat carrier, while the ability to culture rhizobia using
cost effective ingredients was investigated at SFI.

The SFI produced inoculants using sterilised peat with three local strains of soybean rhizobia
and two local strains of groundnut rhizobia. The Australian commercial inoculant strains
CB1809 (for soybean) and NC92 (for groundnut) were also included as reference strains.
The aim of the experiment was to compare the survival of local strains and Australian strains
in peat culture.

11

Broth cultures were grown in erlynmeyer flasks and transferred to a fermentor after growth.
The broth was checked for contamination after growth in both the erlynmeyer flask and
fermentor. Peat was collected, milled, mixed with lime and sterilised by autoclave in the
packet. The pH of the peat was 3.0 to 4.0 after collection and increased to 6.8 to 7.0 after the
addition of lime. After sterilisation, peat was suspended in sterile water, diluted to 10
-6
and
plated on glucose-peptone media to check for contamination. The packet was then sealed
and injected with broth. Broth was added to peat at a ratio of one part broth to four parts peat.
The initial moisture content was less than 10% and increased to 40% after injection. The
inoculant was incubated for one week and then stored at room temperature.

The number of rhizobia in peat was measured at time intervals up to 6 months, immediately
after production, after 2 weeks, 1, 2, 3 and 6 months (Table 2). The initial counts for soybean

rhizobia, CB1809 and local strains SL1, SL2, SL3 were 5 x 10
9
, 2.3 x 10
9
, 1.8 x 10
9
and 2.1 x
10
9
respectively and decreased to 2.7 x 10
8
, 2.5 x 10
8
, 1.2 x 10
8
and 1.3 x 10
8
after 6 months.
At 3 months the numbers of viable cells were still over 10
9
and ranged from 1.1 to 2.2 x 10
9
.
However, all numbers were determined from viable plate counting and no plant infection
tests were used to confirm presence of rhizobia. Numbers can be overestimated if only
determined using plate culturing methods because of poor distinction between rhizobial and
contaminant colonies. Although contamination of peat was low after sterilising, contaminants
may grow quickly upon addition of moisture and out-compete rhizobia.

Table 2. Number of rhizobia in peat during storage time

Number of rhizobia (CFU/g) Rhizobial strains
Innitial 2 weeks 1 month 2 month 3 month 6 month
CB1809
Local strain SL1
Local strain SL2
Local strain SL3
5.0 x 10
9
2.3 x 10
9

1.8 x 10
9

2.1 x 10
9

6.2 x 10
9
4.8 x 10
9

3.4 x 10
9

3.2 x 10
9
5.6 x 10
9

2.9 x 10
9
2.2 x 10
9

3.0 x 10
9
3.5 x 10
9

3.0 x 10
9

2.0 x 10
9

1.8 x 10
9
2.2 x 10
9

1.3 x 10
9

1.3 x 10
9

1.1 x 10
9

2.7 x 10
8
2.5 x 10
8

1.2 x 10
8

1.3 x 10
8

Ingredients for growth of rhizobia in large scale fermentors can be expensive. The SFI
investigated the replacement of laboratory grade yeast extract with industrial yeast extract
and soybean extract. All rhizobial strains grew to 10
9
cfu/mL indicating that fermentation
media can be prepared using more cost effective and accessible ingredients (Table 3).

Table 3. Number of rhizobia grown on different growth media

Media Strains
YEM Soybean extract Crude yeast extract
CB1809
SL1
SL2
SL3
6.1 x 10
9

3.1 x 10

9

6.6 x 10
9

3.8 x 10
9
4.2 x 10
9

1.6 x 10
9

2.7 x 10
8

2.6 x 10
8
2.8 x 10
9

3.4 x 10
9

5.6 x 10
9

5.2 x 10
9

A comparison of strain tolerance to temperature and pH revealed that there was little
difference in growth of strains in media at different temperatures and growth of all strains
was best at 30
o
C. Numbers were low at 25
o
C and lowest at 37
o
C. No cells grew at >45
o
C
(Table 4). Differences were observed in growth of strains at pH 5.5 with Vietnamese strains
achieving 10 fold higher numbers than Australian strains indicating acid tolerance that may
be an advantage in acid soil conditions (Table 5).

12

Table 4. Effect of temperature on growth of strains

Growth of strains Temperature (
0
C)
CB1809 SL1 SL2 SL3
25
30
37
>45
++
+++
++

-
+++
+++
++
-
+
+++
++
-
+
+++
++
-
- no growth; + weak growth, around 10
4
– 10
5
CFU/ml; ++ moderate growth, 10
6
– 10
7
CFU/ml
+++ good growth, 10
8
– 10
9
CFU/ml

Table 5. Effect of pH on growth of rhizobial strains

Growth of rhizobial strains pH
CB1809 SL1 SL2 SL3
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
-
-
-
+
++
+++
+++
++
-
-
-
-
-
++
++
+++
+++
++

-
-
-
-
-
++
++
+++
+++
++
-
-
-
-
-
++
++
+++
+++
++
-
-
- no growth; + weak growth, around 10
4
– 10
5
CFU/ml; ++ moderate growth, 10
6
– 10
7

CFU/ml
+++ good growth, 10
8
– 10
9
CFU/ml

The aims of laboratory experiments at the IAS were to determine the factors influencing the
number of viable rhizobia in inoculants. Higher numbers of rhizobia in the inoculant have
greater potential to outcompete resident soil microflora after inoculation of legumes and
increase N
2
fixation. The cost of inoculation would also decrease because of the need for
smaller volumes.

The IAS prepared inoculants using the Australian inoculant strains NC92 (groundnut) and
CB1809 (soybean). Experiments were carried out to determine the effect of dilution of the
broth culture on final numbers of rhizobia in peat mixed with worm casts and coconut coir
dust after incubation (Table 6). Growth in the peat mixture of broth cultures diluted using
yeast mannitol broth (YMB, to give final broth concentrations of 0.1%, 1%, 10% and 30%)
was compared with growth of undiluted cells containing 10
9
cfu/mL. The peat mixture (70 g)
was injected with 38 mL of broth. After four weeks of growth, the results indicated that
injection with undiluted broth cultures was necessary to achieve maximum numbers of 6.18 x
10
7
for NC92 and 5.85 x 10
8
for CB1809 in the peat mixture. The ratio of viable cells of

CB1809 injected at 0.1%, 1 % broth dilutions with undiluted broth indicated that no growth
had occurred in the peat mixture possibly due to the presence of contaminant organisms (in
the order of 10
6
/g peat mixture). Contaminants may have been encouraged by the addition of
YMB to dilute broths before injection. All counting was done using viable plate counts on
yeast mannitol agar with and without Congo red (YMA and CRYMA respectively) and cell
morphology of colonies was observed after gram staining under the light microscope.




13
Table 6. Rhizobial cell number at different culture dilutions

No Rhizoial strains Culture dilution (%)
Cell number
(CFU/g)

1 NC 92 0,1 5.23 x 10
5

2 NC 92 1,0 8.78 x 10
5

3 NC 92 10 4.25 x 10
6

4 NC 92 30 6.90 x 10
6

5 NC 92 100 6.30 x 10
7

6 CB 1809 0,1 4.68 x 10
5

7 CB 1809 1,0 9.95 x 10
5

8 CB 1809 10 4.73 x 10
6

9 CB 1809 30 6.18 x 10
7

10 CB 1809 100 5.85 x 10
8


Many inoculant producers dilute broth cultures before injection into peat as a means of
extending the broth culture and reducing the requirement for very large fermentor volumes.
Large volumes of broth are more expensive to produce and are easily contaminated during
production. Thus, smaller volumes are highly desirable particularly in the less developed
countries. In Thailand, inoculants are produced using broth cultures that have been diluted to
1/1000. It is recommended that experiments at the IAS be repeated with special attention to
sterilisation of peat and dilution of broths in water. At the SFI peat was less than 10%
moisture before sterilisation and at the IAS it was only 1% moisture. This was identified as a
potential problem resulting in inefficient sterilisation. It was recommended that the moisture
content of peat be adjusted to 20% before sterilisation. However, not all peat and peat

mixtures were capable of holding 20% moisture highlighting the need for selection of peat
with higher water holding capacity and/or the use of emendments (e.g. coir etc).

In another experiments at the IAS, survival of rhizobia was measured after injection of broth
into mixtures of peat with worm casts and coconut coir dust (Table 7). The viable numbers of
rhizobia in peat, peat (70%) mixed with worm casts (30%) and peat (40%) mixed with worm
casts (30%) and coconut coir dust (30%) were counted after growth in carriers for one month.
It was perceived that both worm casts and coconut coir dust increased the water holding
capacity of peat. Only 15 mL broth could be added to 70 g peat while 20 mL could be added
to peat mixed with worm casts and 35 mL to peat, worm cast and coconut coir dust mixture
(by feel only moisture content not measured). It was also observed that coconut coir dust
absorbs and loses moisture very easily. Survival of rhizobia improved when peat had been
amended with worm casts and both casts and coconut coir dust. Although the carriers were
injected with different quantities of broth, the numbers after one month did not reflect the
different inoculum ratios at injection. There was, however, higher numbers of contaminants
in peat mixtures than in peat alone and as counting was done by viable plate counts it is not
clear if rhizobial numbers were overestimated due to incorrect identification of colonies. It
was recommended that experiments on carrier mixtures be carried out after adjusting carriers
to different moisture contents using protocols outlined later in this report. It was also

14
recommended that all experiments include confirmation of the presence of rhizobia using
plant infection tests also outlined later in this report.

Table 7. Number of rhizobial cells in different carriers after one month
No Rhizobial strain Carriers
Number of rhizobial cells
(CFU/g)
1 NC 92 Peat 2.7 x 10
7

2 CB 1809 Peat 5.4 x 10
6

3 NC 92 Peat + worm casts 8.3 x 10
8

4 CB 1809 Peat + worm casts 5.4 x 10
8

5 NC 92 Peat + worm casts + coconut coir dust 2.5 x 10
9

6 CB 1809 Peat + worm casts + coconut coir dust 6.8 x 10
8


Liquid inoculants at the IAS were prepared by adding PVA (0.5%), gum Arabic (0.17%) and
sodium alginate (0.5%) to broth as adhesives for better survival of rhizobial cells on seed.
After one month rhizobial numbers were highest in PVA (4.15 x 10
8
/mL for NC92 and 6.88
x 10
7
/mL for CB1809) and Na alginate (1.04 x 10
8
/mL for NC92 and 5.38 x 10
7
/mL for
CB1809) and lower in gum Arabic (4.42 x 10

6
/mL for NC92 and 8.73 x 10
7
/mL for
CB1809) (Table 8).

Table 8. Effect of different additives on number of rhizobial cells

No
Rhizobial
strains Additives Amount of additives Cell number (CFU/ml)
1 NC 92 PVA 0,5 4.15 x 10
8

2 CB 1809 PVA 0,5 6.88 x 10
7

3 NC 92 Gum Arabic 0,17 4.42 x 10
6

4 CB 1809 Gum Arabic 0,17 8.73 x 10
7

5 NC 92 Sodium Aginate 0,5 1.04 x 10
8

6 CB 1809 Sodium Aginate 0,5 5.38 x 10
7

5.1.1.4 Inoculant production technology - issues arising in February 2008
Project Review Workshop, OPI, HCM City
Present at the Project Review Workshop at OPI were personnel from all collaborating
institutes – OPI, IAS and SFI (Vietnam) and NSW Department of Primary Industries and the
University of Sydney (Australia). In the workshop, personnel from each Vietnamese institute
presented 2007-08 research and extension reports. The major outcome of the Workshop and
subsequent discussions was a clear R&D plan for inoculant production, and
recommendations for revised methods for conducting experiments.
The following section outlines those recommendations related to experimental design and
protocols for testing of peat-based legume inoculants. Some suggestions are made to improve
the designs of experiments presented by institutes at the project meeting at OPI on 18
th
and

15
19
th
February 2008. New strains of rhizobia CB1809 and NC92 will be supplied from
ALIRU to OPI. These will then be distributed for use in all research in 2008. Sterile
Australian peat will also be supplied to institutes as a reference to compare the quality of
Vietnamese peat. All demonstration trials will be set up using Australian peat cultures of
CB1809 and NC92.

General information about peat quality
The quality of inoculants in Australia was improved, following widespread nodulation
failures, by the amelioration of five main factors affecting survival in peat (Roughley and
Vincent, 1967). Firstly, the origin of the peat was shown to be important. Survival of clover,
lucerne and cowpea rhizobia varied according to the location and depth of the peat source.
The peats tested varied according to their colour and texture but no explanation was given by
the authors as to the cause of variation in survival. Secondly, pH was shown to be critical and

acid peats could be amended with calcium or magnesium carbonate. Thirdly, peat
sterilisation, preferably by gamma irradiation, was considered essential particularly for the
growth and survival of slow-growing rhizobia presumably allowing them to out-compete
faster-growing contaminants. Fourthly, when rhizobia were added to peat previously dried at
100
o
C, they survived poorly due to both the heat of wetting generated upon inoculation and
the production of inhibitory substances originating from the heat treatment. Finally, moisture
contents of 40 to 50% proved optimal for growth and survival of a range of rhizobial strains
prepared as peat cultures. Later, accumulation of salt in the peat deposit, caused by several
dry seasons, was found to adversely affect rhizobial survival (Steinborn and Roughley,
1975). There may be many other factors affecting the quality of peat used for legume
inoculants and the ultimate measure is the growth and survival of rhizobia.

Adjusting the pH of peat
Adjusting pH of peat should be done carefully allowing time for equilibration. The reaction
between limestone and H
+
in peat will depend on particle size of both limestone and peat.
The more finely milled the ingredients the faster the reaction. Moisture content of peat is
also important to allow the reaction to occur. The amount of limestone required to change the
pH will depend on organic matter and clay content as well as buffering capacity of the peat.
After mixing peat and limestone should ideally be allowed to react for several weeks before
pH is tested. It may also be necessary to measure pH over a longer period of time. Finely
milled agricultural lime (Aglime, calcium carbonate with some impurities passing through a
150 µm mesh) is the best limestone to use to adjust pH. Builders lime is too caustic and
other lime may be too weak.

Water holding capacity of peat and optimizing moisture content
Particle size, organic matter and clay content of peat will play a role in water holding

capacity and water potential. It is desirable to increase the water holding capacity of the peat
so that larger amounts of broth culture (and hence more cells) can be introduced to peat
before incubation. Experimental design to test survival at different moisture contents is
detailed as follows:

Optimum moisture content of peat or mixtures of peat with other ingredients should be
determined at both IAS and SFI using only one strain of rhizobia to minimise the number of
samples and the treatments listed in Table 9. Before injecting peat with broth, peat must be
sterilised with a moisture content of 20%. The efficacy of sterilisation should be measured by
injecting broth without bacteria and measuring growth of contaminants over time for one

16
month. Suspend peat and dilute as is done when counting rhizobia and spread onto the
surface of glucose peptone media. Record the dilutions at which growth occurs.

Table 9 Treatments for measuring optimum moisture content for legume inoculants
Moisture content (%) Liquid added (mL) Volume of broth (mL)
Volume of sterile water
(mL)
40 29.5 29.5 0
50 52.5 29.5 23
60 87.5 29.5 58

Calculations based on 70 g dry peat after adjusting to 20% moisture content for sterilisation

Equation [1] is used to calculate moisture content of 70 g dry peat. The same equation can
be used for any quantity of peat but if the peat is moist the mass of dry peat must first be
calculated.

70 100

x
y
x
=
+
[1]

Where x is the amount of liquid added and y is the final percent moisture (eg. 50).

To adjust 70 g of dry peat to 20% for sterilisation 17.5 mL of water should be added.

An example calculation: How much liquid (eg. broth) is added to 150 g peat with 20%
moisture to get a final moisture content of 35%?

a) Mass of dry peat

20
100
x 150 g = 30 g

150 g – 30 g = 120 g

b)
Moisture to add to dry peat

35
120 100
x
x
=

+


0.35(120 )
x
x
=
+

42 0.35
x
x
=
+

0.35 42xx
−
=

0.65 42x
=


64.6x
=



17
64.6 g moisture should be added to 120 g dry peat to achieve 35%. If peat already has 30 g

moisture then 64.6 g – 30 g = 34.6 g should be added to 150 g peat.

Experiments testing the efficacy of mixtures of peat, worm cast and coconut coir dust should
have the design outlined in Table 10. All carriers should be adjusted to 20% moisture before
autoclaving and effectiveness of sterilisation measured as described in section 2.1. The
number of viable cells per g of carrier should be measured at 1 week and 1 month to indicate
the relative growth and survival. Counts of viable rhizobia should be done using both plate
counts and the presence of rhizobia confirmed using plant infection tests by inoculating 2
plants from the 10
-5
and 10
-6
dilutions from each carrier suspension as outlined in Figure 7.
There will be 36 counts at each time (ie. 4 carriers x 3 moisture contents x 3 replicates).

Table 10. Experimental design to measure effectiveness of different carriers
Moisture content (%)
Carrier
40 50 60
Peat
Peat + worm cast
Peat + coir dust
Peat + worm cast + coir dust
There should be 3 replicates of each treatment. Counts to
be done at 1 week and 1 month. Counts should be on plates
as well as plant infection tests. Two plants should be
inoculated from each of the 10
-5
and 10
-6

dilutions.























Figure 7. Counting and confirmation of viable rhizobia from contaminated peats


Sources of peat
Apart from characteristics at the source such as salinity, clay, organic matter or
contamination with chemical residues, some unknown factors will affect suitability of peat

for use as an inoculant carrier. Peats from different sources should be tested after adjusting to
10
-2
10
-3
10
-4
10
-5
10
-6
Carrier suspended in sterile water (10 g in 90 mL, 10
-1
dilution)
Spread 0.1 mL on the surface of duplicate
CRYMA plates and count colonies after growth
taking note of diltuions with contamination
Prepare dilution series to 10
-6
Inoculate 2 plants
from each of the
10
-5
and 10
-6
dilutions and
check for nodules
to confirm colonies
are rhizobia on the
corresponding

plates
10
-2
10
-3
10
-4
10
-5
10
-6
Carrier suspended in sterile water (10 g in 90 mL, 10
-1
dilution)
Spread 0.1 mL on the surface of duplicate
CRYMA plates and count colonies after growth
taking note of diltuions with contamination
Prepare dilution series to 10
-6
Inoculate 2 plants
from each of the
10
-5
and 10
-6
dilutions and
check for nodules
to confirm colonies
are rhizobia on the
corresponding

plates

18
the same particle size distribution and moisture content (if possible). It is not possible to
judge the suitability of peat from the colour or texture.

Sterilisation of peat
In Australia peat is packaged and then sterilized using gamma irradiation. The efficacy of
sterilization is dependent on the arrangement of the packets when exposed to the cobalt 60
rods, the moisture content of the peat during sterilization and the contaminant load. If peat is
wet for prolonged periods after mining, growth of contaminant organisms may be
encouraged. Some of these organisms are resistant to heat and irradiation and can become a
problem for legume inoculant manufacturers by growing quickly after nutrients are added at
injection. Gamma sterilization increases the cost of inoculants considerably. Sterilisation by
autoclaving may be a good alternative but toxic substances may be mobilized during
autoclaving that are detrimental to the survival of rhizobia. If this is the case autoclaving may
be carried out several times at a lower temperature. The moisture content of the peat before
autoclaving should be adjusted to approximately 20%.

Solid state fermentation
To determine the minimum number of cells required for maximum cell growth in peat broth
cultures of a single strain of rhizobia should be diluted with sterile water to achieve 1:10,
1:100 and 1:1000 dilutions. Triplicate bags of sterilised peat should be injected with these
dilutions as well as undiluted broth to the optimum moisture content determined previously
taking into account the 20% moisture of peat for sterilisation. Viable cells should be counted
at 1 week, 1 month, 3 months and 6 months after injection of peat using plate counts and
confirming the presence of rhizobia with plant infection as described in section 2.1.2 and
Figure 7.
5.1.1.5 Inoculant QA (Quality Assurance) Program
This was dealt with in detail in the First Six-Monthly Report, submitted September 2007, and

in the Baseline Information Report, submitted May 2008.
5.1.1.6 Training in inoculant production and QA
There were no specific training programs during the reporting period. It is intended that
training of 3–4 Vietnamese researchers from collaborating institutes and commercial
companies will be conducted at the Suranaree University of Technology (Thailand) in
inoculant production, QA and laboratory management, as well as R&D in rhizobiology
during July–August 2008. Additionally, there will be training for one Vietnamese scientist in
Australia during the latter part of 2008. Training during 2007 was reported in the First Six-
Monthly Report, submitted September 2007.

5.1.2 Extension and training of farmers and advisers
The extension-training program for farmers and extension officers is built around simple,
multi-location inoculation experiments in the legume production areas (Mekong Delta, the
Central Coastal region and upland (highland) areas of the North and Central and the South
East). They involve participation of farmers and extension officers in all aspects, from the
design of experiments to sowing, sampling, harvesting and interpretation of results. The
MARD extension service plays a large role in extension activities. Data from field
demonstrations will be used to produce an economic model for production and use of legume

19
inoculants in Vietnam. In addition, training courses will be organised for farmers, extension
workers and researchers in methods of inoculant use, and economic as well as environmental
benefits of inoculation. This extension-training program will be conducted by Vietnamese
researchers in collaboration with Australian counterparts, who will assist in collating and
preparing extension materials for translation and transfer to Vietnam.
5.1.2.1 Field Surveys of farmer knowledge and attitudes to inoculation
The Surveys were conducted during April – August and again during December – February
to determine the level of knowledge about inoculants, inoculant use by farmers and advisers
and their attitudes to inoculant use in the future, and, finally, current production and QA of
inoculants in Vietnam. Results were reported in full in the Baseline Information Report,

submitted May 2008.
5.1.2.2 Field Extension Program – biological and economic effects of inoculation
A total of 28 demonstration trials have now been conducted in 9 provinces. In Binh Dinh,
based on discussion with researchers at the Agricultural Science Institute for Southern
Coastal Centre of Vietnam (ASISCV) and provincial extension officers, demonstration trials
are to be conducted after field experiments. The reason was that inoculation field trials had
not been done there before and the local research/extension agents wanted to get experience
in doing such experiments before doing demonstration fields. The remaining demonstration
trials will be done next season during Summer/Spring 2008–09. In this report we present
results of 15 demonstration fields only as some fields are yet to be harvested and data from
other fields are not yet analysed. The demonstration fields usually had two treatments:
+inoculation and -inoculation. With the +inoculation plots, N chemical fertiliser was not used
or, if used, at not more than 10 kg N/ha. The –inoculation plots, on the other hand, had
fertiliser N (as urea) applied at either recommended rates or at farmer rates. Results to date
are summarised in Appendix 3.

Generally, inoculation of soybean and groundnut increased income to farmers, on average
3.5000.000VNĐ. The profit depended on different sites (Graphs 8 and 9). The increase was
around 500.000VNĐ/ha at the demonstration field of groundnut at Bau Don, Tay Ninh but as
much as 7.180.000VNĐ/ha at Cau Ngang, Tra Vinh. Similarly for soybean, the profit from
inoculation was up to about 6.000.000VNĐ at Quang Hiep, DakLak.

Graph 8. Profit from soybean inoculation
0
1
2
3
4
5
6
7
0246810
Field site
million VNĐ
Graph 9. Profit from groundnut inoculation
0
1
2
3
4
5
6
7
8
02468

Field sites
million VNĐ

20
Farmers were invited to the demonstration fields at least once. At many fields such as in
DakNong and DakLak, they also came to the fields 2–3 times at nodule and biomass harvest
as well as grain harvest time. At each trial site, at least 20 farmers, extension officers,
agriculture advisors came and made evaluation of the trial (600–800 person visits to the
extension trials). They observed development of soybean and groundnut and compared the
health and growth of the plants in the two treatments. They took nodule samples to observe
nodules and learnt to recognise effective nodules with pink colour inside and the white
ineffective nodules. They evaluated the inoculation benefits by themselves by sampling
soybean and groundnut plants, weighing biomass and grains. They were provided extension
materials (see Appendix 4). Also, researchers and extension officers explained how rhizobia
work and the conditions for successful inoculation. Farmers were very interested in learning
about nitrogen fixation.

Questions were often asked by farmers were:
-
How do the inoculants cost?
-
How much do inoculants used for 1000m
2
or 1 ha?
-
Where can we purchase inoculants?
-
Do inoculants have other benefits besides urea (N) fertilizer?
-
Can we use inoculants together with plant protection products?

-
Can we use legume inoculants for other crops?
-
Are inoculants effected by bad weather such as heavy rain, hot weather when inoculation
and during plant growth?
-
Can we use inoculants together with urea (fertiliser N)?

And main requests:
-
Supply inoculants for farmers to test inoculants in their fields
-
Technical support for farmers to use inoculants

5.1.3 Involvement of the private sector in inoculant production, distribution
and marketing
When the project was initiated, it was envisaged that 2–3 Vietnamese companies would be
involved in the project. It was also envisaged that the market for inoculants would grow
during the course of the project from a low level and that the private sector would
progressively become involved as the production technology was developed and the market
for the inoculants expanded. The companies would initially be involved in marketing and
inoculant distribution, rather than production. Training workshops would also be open to
researchers from the collaborating private companies. Project scientists from the Government
institutes would provide technical expertise to the companies throughout the project.
5.1.3.1 Commercialisation of inoculant production and marketing in Vietnam
The following analysis was done during and following the February 2008 Project Review
and assumed that the retail cost of a product is made up of one third production, one third
distribution and one third profit. It is only meant as a rough guide to understand the possible
motivation of inoculant manufacturers in Vietnam. There are many companies in Vietnam
that produce biofertilisers (products that contain one or several microorganisms that are

claimed to have beneficial effects on plant growth). There are several major differences
between inoculant biofertilisers and legume inoculants. Inoculant biofertiisers are usually
produced using non-sterile peat, quality control is difficult and almost never done, very large
quantities are applied to soil and their effectiveness is questionable. The quality of legume

21
inoculants benefits from pre-sterilising peat and rhizobial numbers should be sufficiently
high that only small quantities are required for effective nitrogen fixation. The association
between rhizobia and the host plant is specific and the positive effects of inoculation are
easily demonstrated and dependent on several well characterised conditions.

In order that legume inoculants remain economically competitive the cost of production
should be kept to a minimum. Currently, N fertiliser costs the farmer approximately 600,000
dong per hectare. Legume inoculants should cost less than this but the exact market tolerance
is not clear.

Minimising cost of production by reducing inputs will be dependent on improved quality
(Table 10). This can be achieved by increasing the number of viable rhizobia per g of peat.
To increase numbers peat quality (particle size, organic matter, pH, water holding capacity),
sterilisation process, final moisture content (and perhaps packaging) of products in Vietnam
should be improved. Minimising the cost of production will allow for more flexibility in the
profit margin and be of much greater economic benefit to farmers. Manufacturers of high
quality inoculants may also be motivated by an opportunity to access overseas markets such
other countries in the region and even Australia.

Careful selection of private sector partners for commercial production of legume inoculants
is critical for ensuring the sustainable supply of high quality legume inoculants to farmers in
Vietnam. After visiting potential inoculant producers in February 2008 and the withdrawal of
Fitohoochmon from the project it was initially concluded that the prospect of the private
sector producing high quality inoculants in Vietnam in the near future was low and that large

scale production should be carried out by the institutes. However, following this, contact was
made with the Thien Sinh (Komix) company which is much better equipped to adopt legume
inoculant production and a strategy has been developed for the transfer of technology from
the institutes to this company.

It is important to ensure through the extension program that legume inoculants maintain a
perception of high quality and that they are different from other inoculant biofertilisers.
Vietnamese companies currently producing inoculant biofertilisers applied at rates of 0.2–5
tonnes per hectare may be attracted by large profits from poor quality inoculant. Production
of high quality inoculant by private companies would require a substantial investment in
capital and training. While their capacity to produce large volumes of inoculants exceeds that
currently required for production of rhizobial inoculants for soybean and groundnut in
Vietnam, the inoculant quality would be very poor and difficult to control. The attitude
towards production of microbial inoculants would need to be changed completely.
Biofertiliser production techniques and equipment indicate a very poor understanding of
microbial requirements for growth and survival (see attached reports on individual company
site visits as Appendix 5).

While there are national standards, the quality of biofertiliser products is not effectively
controlled and it is almost certain that they are of poor quality. To our knowledge, the only
biofertiliser produced with any attention to bacterial numbers and product quality is BioGro,
developed by Professor Nguyen Thahn Hien at the Hanoi University of Science with
contributions from AusAID and ACIAR over 8 years. Separate starter peat cultures for each
biofertiliser organism are produced by Professor Hien and distributed to biofertiliser
companies who mix the organisms into a multistrain inoculant extended with non-sterile peat
to achieve large volumes. Current application rates of BioGro are 0.2 tonnes/ha and above.

22
Rhizobial inoculants do not suit this kind of production as peat should ideally be sterilised
and therefore the quantity should be kept to a minimum to reduce sterilisation costs.

Application rates of legume inoculants are not as high as that of biofertilisers as the
symbiotic interaction between rhizobia and their legume host is highly selective compared
with that of plant-associated microorganisms. However, a minimum standard of 2.5 x 10
11

cells per ha should be achieved to maximise the potential for nitrogen fixation. This relates to
the application rates of inoculant, shown in Table 11.

Contrary to the original information from Fitohoochmon, legume inoculants are not currently
produced in any capacity in Vietnam. Initially, it was recommended that institutes build their
capacity to produce high quality inoculants and that new private companies be formed
possibly as joint-ventures in the future. The current capacity of institutes to produce legume
inoculants is listed in Table 12. However, it should be noted that current quality of inoculants
produced by institutes is variable and needs improvement (details provided in Part 3,
Baseline Information Report, submitted May 2008). Details of recommendations for
improvements in inoculant production procedures can be found in Section 5.1.1.4 of this
report.

In the longer term, production (as well as marketing and distribution) of legume inoculants is
better placed with the private sector. This should happen as soon as possible. A very positive
step in that direction was the Thien Sinh (Komix) company expressing an interest in being
involved in production. A site visit in February 2008 revealed very good potential for legume
inoculant production. The facilities were well equipped for careful industrial microbiological
application (see Appendix 5). Technology transfer from the research institutes to the
company was discussed and a clear strategy was determined. The company also agreed to
conduct extension activities through their country-wide network.

Table 11. Minimising retail cost by improving inoculant quality (note: this has only been
calculated proportional to the quantity of peat which is not an accurate measurement of the
real values. A more accurate analysis defining labour costs, packaging etc is necessary)

Inoculant application rate (to achieve 2.5 x 10
11
cells/ha)
3 kg/ha

1 kg/ha 250 g/ha
Cells/g moist peat
1
0.8 x 10
8
2.5 x 10
8
1 x 10
9
Cost of peat
2
840 dong 280 dong 70 dong
Total moist peat
3
3000 tonnes 1000 tonnes 250 tonnes
No. packets
4
30 million 10 million 2.5 million
Amount of broth
5
900 L 300 L 75 L
Retail cost/ha
6
540,000 dong 180,000 dong 45,000 dong

Company profit
7
180 billion dong 60 billion dong 15 billion dong

1
Farmers currently apply 3 kg legume inoculant per hectare. To reduce application rate number of cells per g
peat should be increased (i.e. quality increased).
2
Peat costs 400,000 dong per tonne (moisture content of peat undefined). Cost of peat calculated based on 20%
moisture.
3
Total area sown to groundnut and soybean in Vietnam >1,000,000 ha by 2010
4
Number of packets for entire Vietnamese market based on packet weight of 100 g.
5
Broth for entire market calculated on 50% final moisture content in peat. Peat sterilised with 20% moisture and
30 mL broth added to each bag at a 1:100 dilution.
6
Retail cost calculated by multiplying cost of production by 3. Cost of production of 1 kg peat inoculant estimated
at 60,000 dong. The cost of producing 1 kg may vary with different quantities of product as overheads and
capital investment costs will change (i.e. size of equipment, storage and transport, packaging costs etc.). It may

23
be more expensive to produce 1 kg of inoculant at an application rate of 3 kg/ha because of the larger scale of
the operation.
7
Company profit calculated by multiplying retail cost by 1,000,000 ha and dividing by 3. Decreasing cost of
production allows more flexibility for profit margin. Peat inoculants with 1 x 10
9
cells/g would be of export quality

and open up more markets.

Table 12. Current and future capacity of institutes to produce high quality legume inoculant in
Vietnam.

Current capacity (tonnes/year) Institute
Current
production
t/year
1 10 100 250 1000 3000
IAS
*
1.1
9 8 8 8 8 8
SFI 12
9 9 8 8 8 8

Full production capacity for highest quality inoculants is 250 tonnes per year followed by 1000. Quantity of 3000
tonnes per year is required at current quality.
*
IAS is interested in increasing capacity to meet demand.

5.2 Smallholder Benefits
Potential benefits for smallholders are valued at A$50–60 million p.a., principally through
reduced use of fertiliser N. Benefits for the smallholders should start to flow through after the
second year of the project.


5.3 Capacity Building
Capacity building has commenced with participation of 17 Vietnamese scientists/technicians

at the QA Workshop in HCM City in February–March, the Inoculant Production
Technologies Workshop in Thailand in June 2007, and the purchase of materials and
equipment for R&D and inoculant production
. This will continue during 2008 with additional
training in inoculant production technologies in Thailand and in rhizobial R&D training in
Australia. Further equipment and operating purchases are planned for 2008.

5.4 Publicity
We propose to submit articles to the CARD Newsletter and the Newspaper of Agriculture
(see Appendix 6 for the article preparation).


5.5 Project Management
The project is progressing smoothly, although Can Tho University, originally identified in
the project as a collaborator, pulled out immediately following the 2007 Inception Workshop
and QA training in HCM City. Its place in the project has been taken by
the Institute of
Agriculture and Forestry for Tay Nguyen (IAF) and the Agricultural Science Institute for
Southern Coastal Centre of Vietnam (ASISCV). As well, Fitohoocmon, one of the
commercial companies originally identified in the Project, have ceased to be a participant as
the aim of this company is to produce biofertilizers but not high quality inoculants. Komix
company has joined the Project and has a detailed plan to work with Project personnel to
promote legume inoculant production and use in Vietnam (see Appendix 5).