Dự án nông nghiệp " Replacing fertiliser N with rhizobial inoculants for legumes in Vietnam for greater farm profitability and environmental benefits " MS7 doc

Executive Summary Objective assessment of capacity and competency of trained staff to produce high quality inoculants and to train farmers and extension workers in successful applicatio

Ministry of Agriculture & Rural Development

Collaboration for Agriculture & Rural

Development

013/06VIE Replacing fertiliser N with rhizobial inoculants for legumes in Vietnam for

greater farm profitability and

environmental benefits

MS7: Capacity Improvement Report

September 2009

5.2 Private sector distribution and extension of inoculants 25

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 Industry & Investment

University of New England University of Sydney

Australian Personnel Dr David Herridge; Dr Roz Deaker

Ms Elizabeth Hartley, Mr Greg Gemell

Completion date (original) March 2009

Completion date (revised) November 2009

2 Contact Officer(s)

In Australia: Team Leader

Name: Dr David Herridge Telephone: 02 67631143

Email: david.herridge@dpi.nsw.gov.au

In Australia: Administrative contact

Organisation Industry & Investment NSW Email: graham.denney@dpi

nsw.gov.au

In Vietnam

Name: Ms Tran Yen Thao Telephone: 08 9143024 – 8297336

Organisation Oil Plants Institute

(OPI)

Email: yenthao@opi.org.vn

yenthao@hcm.fpt.vnyenthao9@yahoo.com

3 Project Abstract

4 Executive Summary

Objective assessment of capacity and competency of trained staff to produce high quality inoculants and to train farmers and extension workers in successful application in the field

During 2007-08 the three collaborating institutes, OPI, IAS and SFI, produced inoculants and had them tested for quality by OPI Each institute used slightly different production technologies The number of samples tested was 261 from a total

of 465 samples received (60 from IAS, 180 from SFI and 225 from OPI) There were

36, 108 and 117 samples tested from IAS, SFI and OPI, respectively 117 samples were tested in 2007 and 144 samples in 2008

Results indicated that the number of rhizobia in the inoculants ranged between <106 to

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 annuallyin 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 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

The variation in rhizobial and contaminant counts for the different batches resulted from differences in procedures and expertise amongst the three laboratories The number of rhizobia/g inoculant was unstable between production batches in 2007 but improved during 2008 This mainly resulted from improvement in moisture content of inoculants at OPI and sterilization process at SFI At IAS, the problems with sterilization and moisture content were not properly resolved, resulting in less consistency and lower quality of inoculants However, IAS continues improving carrier formulations that will improve low quality peat

The training program for farmers and extension workers was based on their involvement in field demonstrations, attendance in workshops, exposure to extension literature and application of inoculants on their farms The field demonstrations were simple, multi-location inoculation experiments in the legume production areas of the country

During 2007–09, a total of 168 demonstration (extension) trials were conducted in 10 provinces The demonstration fields usually had two treatments: +inoculation and –inoculation (farmer’ practice with N fertilisers) 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 (3400+ person visits to the extension trials) Researchers at the project institutes trained farmers and extension workers on how inoculants work, how to apply inoculants to seed, how to determine if the inoculated crops are fixing nitrogen well and how to record results They observed development of soybean and groundnut and compared the health and growth of the plants in the inoculated and N-fertilised treatments They dug plants from the soil to observe nodules and learnt to recognise effective nodules with pink colour inside as distinct from the white ineffective nodules They learnt to evaluate inoculation benefits by sampling soybean and groundnut plants, weighing biomass and grains Farmers were very interested in learning about nitrogen fixation and asked many questions

As well, 20 training workshops for farmers were conducted in 10 provinces In each workshop, 30–50 farmers, extension workers/officers and other (agricultural persons, officers) participated, with around 800 persons in total at the workshops Farmers and extension workers who participated in the field experiments and demonstrations were usually involved At the workshop farmers were be supplied information on legume nitrogen fixation and its benefits, and on inoculants and how they are used Each of the three project institute prepared their own presentations Flyers (about 1500) were prepared and handed out to the farmers

Capacity of the private sector to provide high quality inoculant distribution systems, together with sound advice to farmers in their use

There is an increasing capacity of the private sector to distribute inoculants and support farmers in their use Three private-sector companies are now actively involved

in the project - Private Business Ngoc Trung at Son La Town, Son La province, Viet

A Nghia Dan Joint Stock Company at Nghia Dan, Nghe An province and Komix at Binh Duong province Cu Chi Bio-Chemical Fertiliser Joint Stock Company would like to distribute inoculants for farmers but are having problems arranging staff It is hopeful that IAS can collaborate with Cu Chi company in the future for distribution and marketing Ngoc Trung and Viet A Nghia Dan are young companies but they are interested in production of bio-products like rhizobial inoculants and have great potential for production and distribution With support from SFI about delivery of broth cultures and technology transfer, the two companies produced and supplied inoculants for 90 ha of groundnut and soybean, for Nghe An and Son La provinces

Komix now has a network for distributing their bio-fertilizers covering almost half of the country, from the center to the south Komix conducted field trials with inoculants produced by OPI During 2008, they conducted one soybean field experiment at DakNong province and completed 15 field demonstrations in DakNong and Tay Ninh provinces Komix supplied inoculants for several ha of soybean and groundnut where they conducted field trials

Assessment of the improved capacity of private sector companies in the production of high quality inoculant

The potential market for inoculants in Vietnam could be around 500 tonnes annually, assuming application rates of 1 kg inoculant/ha and about 50% of legume area inoculated This amount substantially exceeds the capacity of the project institutes for production (currently <20 tonnes annually) Therefore, it was considered necessary to involve the private sector in commercial production

Inoculant production at Son La and Nghe An companies – SFI produces the broth cultures and supplies them to the companies The broth cultures are injected into sterilized peat prepared by the companies SFI has transferred the technologies of peat sterilization, injection, packaging and storage The quality of inoculants has been monitored by SFI throughout the production process Final products were tested by an independent quality control laboratory Some pilot batch production was done and inoculants were supplied for 90 ha of groundnut and soybean in Son La and Nghe An However, the cost of production was found to be high due to high cost of

and extension documents, extension activities (field days at farms, farmer workshops) and together with Komix took care of field experiments and demonstrations The second step is to set up production at Komix It is proposed for medium scale production at Komix and then eventually increased production OPI manages quality control of inoculants produced by Komix, supplies mother culture annually, improves production technology and promotes the products OPI is arranging for a pilot production at Komix in December 2009 Inoculants will be supplied on order to Dong Thap province for soybean and groundnut (100 ha soybean and 20 ha groundnut)

Copies of training and extension materials including training reports and lessons learned from study tours

Copies of training and extension materials are attached as appendices In terms of training of institute personnel in inoculant technology, there were four major events:

• Quality assurance workshop, IAS, HCM City Feb-March 2007 There were 17 participants drawn from collaborating institutes and private sector companies The workshop was designed by ALIRU staff and presented by Elizabeth Hartley (ALIRU), Greg Gemell (ALIRU) and Rosalind Deaker (University of Sydney) Each participant was provided with a workbook and comprehensive notes on procedures:

Workbook: Quality Control of Legume Inoculants Workshop, 26th February –

9th March 2007, compiled by E Hartley, G Gemell, J Hartley (01/02/2007)

• Inoculant training workshop, Suranaree University of Technology, Thailand October 2008 Four project scientists participated Activities focussed on

practice rather than theory and consisted of recognition of Rhizobium strains,

small- and large-scale inoculant production and quality control Participants from each institute concentrated on particular aspects that were particularly relevant to them

• Study tour, Yen Thao to Australia November-December 2008 Activities focused on research on identification of rhizobial strains, survival of the Australian, Vietnamese strains as well as strains from other countries; participated in the review of quality control and QA system currently in

Australia and prepared documents for protocols of inoculant production and quality control of legume inoculants in order to apply in Vietnam

The research was conducted at the Department of Agriculture, Food and Natural resources, the University of Sydney, from 20 to 28 November and from 15 to 20 November, at the Australian Legume Inoculant Research Unit, Department of Primary Industries, NSW, Gosford

5 Technical Report

The technical report addresses the required reporting headings

1 Objective assessment of capacity and competency of trained staff to produce high quality inoculants and to train farmers and extension workers in successful application in the field

2 Capacity of the private sector to provide high quality inoculant distribution systems, together with sound advice to farmers in their use

3 Assessment of the improved capacity of private sector companies in the production of high quality inoculant

4 Copies of training and extension materials including training reports and lessons learned from study tours

5.1 Objective assessment of capacity and competency of trained staff to produce high quality inoculants and to train farmers and extension workers in successful application in the field

5.1.1 Testing inoculants produced by the three project institutes

All participating institutes produced inoculants using their own technologies and sent samples to OPI for quality testing We followed standardized methods of testing of ALIRU (Australia) with some change to suit laboratory conditions in Vietnam.During the two years 2007–2008, the number of samples tested was 261 from a total of 465 samples received (60 from IAS, 180 from SFI and 225 from OPI) There were 36, 108 and 117 samples tested from IAS, SFI and OPI, respectively 117 samples were tested

in 2007 and 144 samples in 2008

Moisture content is showed in the Table 2 Generally moisture content of inoculants

produced in Vietnam varied in the range 20–52%, because of different qualities of the

carriers and production technologies applied in the institutes However, moisture

content of most inoculants was 38–49% (80% samples) (Graph 1) For peat

inoculants, around 40–50% moisture content is optimum

At IAS moisture content of inoculants fluctuated from 20 to 52% (Graph 2) while it

was more stable at ISF and OPI, ranged 38–46% (79% samples)

M oistur e content of inoculants tested dur ing 2007-2008

Numbers of rhizobia in the inoculants were enumerated using direct plate counting

and plant-infection, most probable number (MPN) counting With direct plate

counting, rhizobial numbers ranged from <106 to >109 (Table 3) The plant-infection

MPN counts closely matched those of the plate counts and ranged from <105 to >108

(Table 4) Most inoculant samples tested (71%) had rhizobial counts between 108 to

109 cells/g, a very good result Twenty six percent of samples tested had counts in the

range 105 to 107 There were few inoculants in which rhizobial cells did not exist or

could not be detected because contaminants (mainly fungal) had overun the rhizobia

The necessity of MPN counting was highlighted with IAS Batch 3 and SFI Batch 1

when plate counting was impossible because of the large background populations of

contaminating organisms (Table 5)

Numbers of contaminants in the inoculants are shown in Table 5 Almost all samples

had number of contaminants from 105 to 106 cells/g peat (75%) Some samples had

very low contaminant counts, i.e <10 cells/g, while few other samples had high levels

of contaminants, around 107 cells/g

Table 1: General information of inoculants tested IAS-produced inoculants

Year 2007

Legume host Groundnut

Soybean

Groundnut Soybean

Groundnut Soybean Rhizobium strain

Lab code of samples IAS-P-B1-G-NC92 (1-5)

IAS-P-B1-S-BC1809 (1-5)

IAS-P-B2-G-NC92 (1-5) IAS-P-B2-S-BC1809 (1-5)

IAS-P-B3-G-NC92 (1-5) IAS-P-B3-S-BC1809 (1-5)

Year 2008

SFI-produced inoculants

Year 2007

Legume host Groundnut

Soybean

Groundnut Soybean

Groundnut Soybean Rhizobium strain

name

NC 92 GL1 GL2

CB 1809 SL1 SL2

NC 92 GL1 GL2

CB 1809 SL1 SL2

NC 92 GL1 GL2

CB 1809 SL1 SL2

Lab code of samples ISF-B1-NC92 (1-5)

ISF-B1-SL1 (1-5) ISF-B1-GL2 (1-5) ISF-B1-CB1809 (1-5) ISF-B1-SL1 (1-5) ISF-B1-SL2 (1-5)

ISF-B2 NC92 (1-5) ISF-B2 SL1 (1-5) ISF-B2 GL2 (1-5) ISF-B2-CB1809 (1-5) ISF-B2-SL1 (1-5) ISF-B2-SL2 (1-5)

ISF-B3—NC92 (1-5) ISF-B3 SL1 (1-5) ISF-B3-GL2 (1-5) ISF-B3-CB1809 (1-5) ISF-B3-SL1 (1-5) ISF-B3-SL2 (1-5)

Year 2008

Legume host Groundnut

Soybean

Groundnut Soybean

Groundnut Soybean Rhizobium strain

name

NC 92 GL1 GL2

CB 1809 SL1 SL2

NC 92 GL1 GL2

CB 1809 SL1 SL2

NC 92 GL1 GL2

CB 1809 SL1 SL2

Lab code of samples ISF-B1-G-NC92 (1-5)

ISF-B1-G-SL1 (1-5) ISF-B1-G-GL2 (1-5) ISF-B1-CB1809 (1-5) ISF-B1-SL1 (1-5) ISF-B1-SL2 (1-5)

ISF-B2-G-NC92 (1-5) ISF-B2-G-SL1 (1-5) ISF-B2-G-GL2 (1-5) ISF-B2-CB1809 (1-5) ISF-B2-SL1 (1-5) ISF-B2-SL2 (1-5)

ISF-B3-G-NC92 (1-5) ISF-B3-G-SL1 (1-5) ISF-B3-G-GL2 (1-5) ISF-B3-CB1809 (1-5) ISF-B3-SL1 (1-5) ISF-B3-SL2 (1-5)

IOOP-produced inoculants

Year 2007

Legume host Groundnut

Soybean

Groundnut Soybean

Soybean

Rhizobium strain

name

NC 92 GL1 GL2

CB 1809 SL1 SL2

NC 92 GL1 GL2

CB 1809 SL1 SL2

-

-

-

CB 1809 SL1 SL2

Lab code of samples OPI-G-B1-NC92 (1-5)

OPI-G-B1-SL1 (1-5) OPI-G-B1-GL2 (1-5) OPI-S-B1-CB1809 (1-5) OPI-S-B1-SL1 (1-5) OPI-S-B1-SL2 (1-5)

OPI-G-B2-NC92 (1-5) OPI-G-B2-SL1 (1-5) OPI-G-B2-GL2 (1-5) OPI-S-B2-CB1809 (1-5) OPI-S-B2-SL1 (1-5) OPI-S-B2-SL2 (1-5)

-

-

- OPI-L-S-B3-CB1809 (1-5) OPI-L-S-B3-SL1 (1-5) OPI-L-S-B3-SL2 (1-5)

Year 2008

Legume host Groundnut

Soybean

Groundnut Soybean

Groundnut Soybean Rhizobium strain

name

NC 92 GL1 GL2

CB 1809 SL1 SL2

NC 92 GL1 GL2

CB 1809 SL1 SL2

NC 92 GL1 GL2

CB 1809 SL1 SL2

Lab code of samples OPI-G-B1-NC92 (1-5)

OPI-G-B1-SL1 (1-5) OPI-G-B1-GL2 (1-5) OPI-S-B1-CB1809 (1-5) OPI-S-B1-SL1 (1-5) OPI-S-B1-SL2 (1-5)

OPI-G-B2-NC92 (1-5) OPI-G-B2-SL1 (1-5) OPI-G-B2-GL2 (1-5) OPI-S-B2-CB1809 (1-5) OPI-S-B2-SL1 (1-5) OPI-S-B2-SL2 (1-5)

OPI-G-B3-NC92 (1-5) OPI-G-B3-SL1 (1-5) OPI-G-B3-GL2 (1-5) OPI-L-S-B3-CB1809 (1-5) OPI-L-S-B3-SL1 (1-5) OPI-L-S-B3-SL2 (1-5)

Table 2: Moisture content of inoculants Moisture content (%)

43.3

54.6 49.5

39.4

19.8 20.4 20.6

42

34.9 38.6 34.7 37.0 31.8 34.5

35.2

45.6 43.2 44.8 46.1 43.7 42.5

36.0

41.3 31.9 30.2 41.6 38.2 45.3

38.9

41.3 31.9 30.2 41.6 38.2 45.3

38.1 IOOP

20.7

45.5 46.6 45.8 44.7 44.6 45.8

37.3 38.2 37.9 38.4

41.1 37.5 36.4 38.6

OPI-S-SL1

OPI-S SL2

Average

37.6 36.9

37.0

40.0 39.3

38.5

39.2 37.3

37.7

38.3 37.4 38.2 34.8 39.4 40.2

38.2

39.2 37.4 40.5 38.7 39.6 40.7

39.4

Table 3: Number of viable rhizobia by direct plate count

Number/g Samples

43 x 107

Fungi cont* Fungi cont*

20 x 105

6 x 105Fungi cont* Fungi cont*

22x10834x10812x10818x10812x10887x107

10x10840x10852x10890x10747x10764x107

12x10821x10843x10811x10825x10832x108

12x10823x10810x10818x10812x10876x107

23x10811x10848x10875x10721x10816x108

Table 4: Number of viable rhizobia by plant-infection MPN

Number/g Samples

30x10714x10822x108 30x10722x108 60x107

42x10734x108 23x107 60x10750x107 70x107

70x10717x10758x10726x10815x10880x107

11x1087x10824x10823x10810x10832x108

30x10790x10750x10710x10826x10750x107

17x10890x10740x10733x10750x10740x107

*¨

undetected: rhizobia could not be detected as samples were contaminated by a fungus that damaged groundnut root (turned to black color)

Table 5: Number of contaminants in inoculants

Number/g Samples

5.1.2 Institute capacity to produce high quality of inoculants

The results of QA on 261 inoculants produced by the three collaborating Vietnamese

institutes during 2007-2008 above showed that the number of rhizobia ranged between

<106 to >109 cfu/g inoculant and number of contamination was <105 to >106 cfu/g

However, the number of rhizobia/g was high in most inoculants (72% ), between 5 x

108 and 3 x 109 cfu/g, and low in contamination (≤106 cfu/g) Plant-injection MNP

counts were <105 to >108 rhizobia/g moist peat but most inoculants (75%) had ≥108

rhizobia/g Current standards for peat inculants in Australia are ≥ 1x109 rhizobial cfu/g

moist peat and contaminants are not detectable at a dilution level of 10-6; MPN

plant-infection counts are ≥108 Therefore, in relative terms, a high percentage of legume

inoculants now produced in Vietnam can meet the requirements for production of high

quality inoculants

The variation in rhizobial and contaminant counts for the different batches resulted

from differences in procedures and expertise amongst the three laboratories The

number of rhizobia/g inoculant was unstable between production batches in 2007

(Graph 3) but was stable during 2008 This resulted from improvement of production

technology In the second year of the project the quality of inoculants produced at SFI

and OPI was much improved, 108–109 rhizobia/g and contaminant <107 cells/g and no

batches were over-run with contaminants (Graphs 3 and 4, Tables 3–5) This mainly

resulted from improvement in moisture content of inoculants at OPI and sterilization

process at SFI At IAS, the problems with sterilization and moisture content were not

properly resolved, resulting in less consistency and lower quality of inoculants

However, IAS continues improving carrier formulations that will improve low quality

Graph 4 Number of Rhizobium/g inoculant by institute

The quality control process at OPI worked extremely well and, clearly, the foundation

has been laid for a formal and expanded QA program in Vietnam in line with the

proposed expansion of inoculant production

Graph 5 Number of contaminants in inoculants

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 played a large role in extension activities Data from field demonstrations has used to compare the economics of inoculant use versus the current practice of fertilizer N application In the future, after the project has officially ended, additional 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

During 2007–09, a total of 168 demonstration (extension) trials were conducted in 10 provinces The demonstration fields usually had two treatments: +inoculation and –inoculation (farmer’ practice with N fertilisers) 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 (3400+ person visits to the extension trials) Researchers at the project institutes trained farmers and extension workers on how inoculants work, how to apply inoculants to seed, how to determine if the inoculated crops are fixing nitrogen well and how to record results They observed development of soybean and groundnut and compared the health and growth of the plants in the inoculated and N-fertilised treatments They dug plants from the soil to observe nodules and learnt to recognise effective nodules with pink colour inside as distinct from the white ineffective nodules They learnt to evaluate inoculation benefits by sampling soybean and groundnut plants, weighing biomass and grains Farmers were very interested in learning about nitrogen fixation and asked questions, such as:

- How does the inoculant price?

- How much do inoculants used for 1000m2 or 1 ha?

- Where can we purchase inoculants?

- Do inoculants have other benefits besides urea (N) fertiliser?

- 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?

- Can we use inoculants together with urea (fertiliser N)?

And main requests:

- Supply inoculants to farmers to test inoculants in their fields

- Technical support for farmers to use inoculants

Farmers were given flyers introducing biological nitrogen fixation, benefits in terms

of chemical nitrogen fertiliser replacement, yield improvement and how to use inoculants for their fields

All participating institutes were successful with extension activities SFI and IAS staff organized field trials, invited farmers and extension workers to the fields and trained them OPI utilized extension officers more for the extension work with the extension workers playing a principal role in the field trials They prepared the trials from the beginning to the end and liaised with the farmers while OPI staffs gave them field trial procedures after discussions with them and extension documents The OPI staff and extension officers came together at the field demonstration sites at important times (sowing, nodulation sampling, biomass and harvest sampling, field days) and kept close contact during the course of the trials The main objective of the collaboration was that extension workers would become familiar in field trials and demonstrations of legume inoculation and will be the key persons to train others when inoculant use is spreading As they are local extension persons they know very well the current agricultural packages for peanut or/and soybean in an area, i.e best varieties, the package for plant protection, irrigation, fertiliser applications etc This is the case in Binh Dinh province (central coast), Dong Thap, An Giang and Tra Vinh provinces (Mekong Delta)

In regarding to extension activities, 20 training workshops for farmers were conducted

in 10 provinces In each workshop, 30–50 farmers, extension workers/officers and other (agricultural persons, officers) participated, with around 800 persons in total at the workshops Farmers and extension workers who participated in the field experiments and demonstrations were usually involved At the workshop farmers were

be supplied information on legume nitrogen fixation and its benefits, and on inoculants and how they are used Each of the three project institute prepared their own presentations Flyers (about 1500) were prepared and handed out to the farmers

5.2 Capacity of the private sector to provide high quality inoculant distribution systems, together with sound advice to farmers in their use

At this stage three companies are actively involved in the project - Private Business Ngoc Trung at Son La Town, Son La province, Viet A Nghia Dan Joint Stock Company at Nghia Dan, Nghe An province and Komix at Binh Duong province Cu Chi Bio-Chemical Fertiliser Joint Stock Company would like to distribute inoculants for farmers but are having problems arranging staff It is hopeful that IAS can collaborate with Cu Chi company in the future for distribution and marketing Ngoc

fixation in terms of nodulation, biomass and seed yields and they completed 15 field demonstrations in DakNong and Tay Ninh provinces The data from the trials were yield, income from inoculation and willingless to use inoculants by farmers Komix supplied inoculants for several ha of soybean and groundnut where they conducted field trials

In Vietnam there is no history of private companies producing substantial amounts of quality legume inoculants Initially Fitohoocmon was the proposed major private company to be involved in the project for inoculant production as the profile supplied

by the company showed that it was producing soybean and groundnut inoculants However, the company soon withdrew from the project after proposing to produce inoculants the same way as bio-fertilizers in Vietnam, i.e based on unsterilised peat and mixed with a huge amount of bio-fertilisers (up to 5 tonnes/ha) This does not fit with the objectives of the project to produce and market high quality of inoculants, applied at rates of around 1 kg/ha

In the North, Private Business Ngoc Trung at Son La Town, Son La province (Son La) and Viet A Nghia Dan Joint Stock Company at Nghia Dan, Nghe An (Nghe An) and

in the South the Komix company are interested in the objectives of the project and they have become involved

Inoculant production at Son La and Nghe An companies – SFI produces the broth cultures and supplies them to the companies The broth cultures are injected into sterilized peat prepared by the companies SFI transferred the technologies of peat sterilization, injection, packaging and storage The quality of inoculants has been monitored by SFI throughout the production process Final products were tested by an independent quality control laboratory Some pilot batch production was done and inoculants were supplied for 90 ha of groundnut and soybean in Son La and Nghe An However, the cost of production was found to be high due to high cost of transportation Nghe An and Son La are around 300 km far from Hanoi where the broths were produced by SFI In addition, there is always the risk that the broths might

be damaged during transport from Hanoi to Son La

The Komix company works in the project with OPI There are 2 steps of the process

In the first step they field-test inoculants produced by the institute, and market and distribute them to farmers Komix gets feedback of the potential of the inoculant based

on evaluation of biological and economic benefits of inoculation in the target

provinces OPI transferred technologies to Komix including field experiments/trials and extension documents, extension activities (field days at farms, farmer workshops) and together with Komix took care of field experiments and demonstrations Komix were invited to workshops OPI organised for farmers and extension workers There were 16 field experiments and demonstrations organized by Komix in DakNong (soybean), Tay Ninh (groundnut) provinces Komix persons participated in the workshop in Tra Vinh province The second step is to set up production at Komix It is proposed for medium scale production at Komix and then eventually increased production OPI manages quality control of inoculants produced by Komix, supplies mother culture annually, improves production technology and promotes the products OPI is arranging for a pilot production at Komix in December 2009 Inoculants will be supplied on order to Dong Thap province for soybean and groundnut (100 ha soybean and 20 ha groundnut)

5.4 Copies of training and extension materials including training reports and lessons learned from study tours

Inoculant Production Training Workshops, Suranaree University, Thailand

The aim of this training workshop (4–22 June 2007) was to improve the skills of Vietnamese scientists in order to improve inoculant production techniques in Vietnam

It was expected that the practical and theoretical nature of the training would mean rapid application of the technologies in Vietnam Activities focussed on lectures, laboratory training and visits to production facilities in Thailand The training covered background information on rhizobia, fermentation technologies including large systems and the micro production unit (MPU), carbon source for rhizobia in fermentation, other plant growth promoting (PGP) organisms and counting techniques (see Appendix 2 for details) Three Vietnamese scientists participated in the training There was a second training program for 4 Vietnamese researchers from collaborating institutes on inoculant production technology and QA at the Suranaree University of Technology (Thailand), from 5–19 October 2008 The training report is attached as Appendix 3 The aim of this training was to improve skills of Vietnamese scientists in order to improve inoculant production techniques and inoculant quality control in Vietnam The training was focussed on practice rather than theory and consisted of

recognition of Rhizobium strains, small- and large-scale inoculant production and

QA Workshop, IAS, HCM City

The workshop (26 Feb – 9 March, 2007) aimed were to provide the Vietnamese scientists and technicians with an overview of the requirements and protocols for quality control of legume inoculants, and to improve their skills in order that legume inoculants in Vietnam could be routinely assessed for quality It was expected that understanding the parameters of quality would improve inoculant production techniques and increase availability and adoption of high quality legume inoculants in Vietnam The protocols presented were based on the quality control program used by the NSW Department of Primary Industries - Australian Legume Inoculant Research Unit (ALIRU) in Australia The workshop was divided into two components Firstly, participants gained practical experience in quality control protocols and secondly, quality control procedures were discussed and adapted to suit local conditions in Vietnam

Lists of workshop personnel and the 17 participants are provided in Appendix 1 The workshop was designed by ALIRU staff and presented by Elizabeth Hartley (ALIRU), Greg Gemell (ALIRU) and Rosalind Deaker (University of Sydney) Each participant was provided with a workbook and comprehensive notes on procedures:

Workbook: Quality Control of Legume Inoculants Workshop, 26th February – 9thMarch 2007, compiled by E Hartley, G Gemell, J Hartley (01/02/2007) 37 pages

Procedures Section: Quality Control of Legume Inoculants Workshop, compiled by E Hartley, G Gemell, J Hartley (01/02/2007) 32 pages

Organisation of the workshop and selection of the participants was facilitated by the Vietnamese project leader Tran Yen Thao Workshop participants were selected to represent all institutes and private-sector inoculant manufacturers/distributors involved

in project 013/06VIE All participants were skilled in general microbiological procedures

Study tour Australia by Tran Yen Thao Activities focused on research on

identification of rhizobial strains, survival of the Australian, Vietnamese strains as well as strains from other countries; participated in the review of quality control and

QA system currently in Australia and prepared documents for protocols of inoculant production and quality control of legume inoculants in order to apply in Vietnam

The research was conducted at the Department of Agriculture, Food and Natural resources, the University of Sydney, from 20 to 28 November and from 15 to 20 November, at the Australian Legume Inoculant Research Unit, Department of Primary Industries, NSW, Gosford

A collection of rhizobia for soybean and groundnut originated from Australia, U.S.A, Achentina, Korea, Thailand and Vietnam (34 strains) were used in the research The strains first were streaked on CRYMA medium and incubated in 7 days for purity check The strains after that were cultured on YEM for preservation and research process The strains were also freeze-dried for long-term preservation

In research and application of rhizobial inoculants, identification of strains plays an important role The morphological characteristics are related to colony and cellular appearances Colonies and gram stains of the strains on YEMA medium were determined Cells were observed under microscope PCR of all strains were done The process consisted of DNA extraction, PCR 16S rDNA multification, DNA check on gel agarose The experiment is continued at the Sydney University for genome analysis Peat is most frequently used as carrier materials for inoculant production, in sterilized or un-sterilized forms Inoculants based on sterilized peat are mainly products in many countries due to higher number of viable cells per gram peat, more than 100 times compared to un-sterilized peat inoculants Number of rhizobia is up to 109 cells/g of moist peat In Australia almost inoculants are produced from sterilized peat even though the demand of market has been changed (inoculated seed or granular inoculants) Peat is sterilized by gamma irradiation However, the sterilize method is quite expensive and might be a problem for small and medium producers in Vietnam It should have a survey

to know if the method can be applied for inoculant production in Vietnam or not, especially in term of the sterilize cost

The quality of inoculants depends much on quality of peat If the numbers of rhizobium

at production are low, the number will decrease quickly during preservation due to contaminant development Good properties of a peat are summarized (see Appendix 4 for details)

Quality of inoculants can be defined as a complex of some factors The factors affected

on quality of inoculants were described by Roughley and Pulsford (1982) and this was a background of quality control They are number of rhizobia, nitrogen fixation, moisture content and number of contaminants The quality control is doing by the Australian Legume Inoculant Research Unit (ALIRU)

Standards were based on the number of viable rhizobia on seeds In practice, the number

is expressed by the number of rhizobium/g moist inoculant The number of 109 cells/g moist peat was calculated by 108cells/g at selling which was based on at least 300 cells/seed (Roughley 1964; Date 1970) Roughley (1964) proposed an increase in standard from 100 to 300 cells/seed After that the standard changed to 1000 cells/seed These changes were due to technology improvement of inoculant production See the current standard of Australia for legume inoculants the table 1 of Appendix 4

maintain high quality of inoculants Some of inoculant samples were out of expiry dates and these samples did not meet the national standards

The current national standard for inoculated seeds is minimum 106 rhizobium/g at the end

of expiry dates However, many samples did not meet the requirements The quality of inoculants depends much on moisture content therefore environmental factors are very important One of the most important advantages of lyophilized inoculants is long preservation Freeze-dried inoculants produced in Australia can keep up to 1.8 years to fafa bean

One of the most advantages of the project is to release the two superior strains for soybean and groundnut inoculant production This is one main key for a successful production of inoculants in Vietnam However, at this time, technology should be further improved Inoculant production in Vietnam depends on institutes so far, quality is from

104-109 cfu/g Quality of inoculants has been changed depending much on peat quality, peat moisture and purity of sterilized peat (contamination) The quality also depends on facilities and expertise between institutes In the near future we need to select good and sustainable peat sources for production, determine sterilization and preservation when delivery Determination of standards for production is very important This assures high quality of inoculants supplied to farmers The national standards and QA system proposal should be submitted to government A dependent laboratory probably is a suitable model for QA program in Vietnam

The lessons learnt from the tour of inoculant production and QA together with actual results of research of the project are the background of the protocol for inoculant production, establishment of a national standard and procedures of quality control of inoculants for legumes in Vietnam

APPENDIX 1 Report on Workshop on Quality Control for Legume Inoculants

Institute of Agricultural Science, Ho Chi Minh City

February 26 th – March 9 th

1 Introduction and workshop objectives

A workshop was held on quality control of legume inoculants as part of AusAID CARD project number 013/06VIE ‘Replacing fertiliser N with rhizobial inoculants for legumes in Vietnam for greater farm profitability and environmental benefits’ The workshop was designed to provide participants with an overview of the requirements and protocols for quality control of legume inoculants The aim of the workshop was to improve skills of Vietnamese scientists so that legume inoculants in Vietnam can be routinely measured for quality It is expected that understanding the parameters of quality may improve inoculant production techniques and increase availability and adoption of high quality legume inoculants in Vietnam The protocols presented were based on the quality control program used by the Australian Legume Inoculant Research Unit (ALIRU) in Australia The workshop was divided into two components Firstly, participants gained practical experience in quality control protocols and secondly, quality control procedures were discussed and adapted to suit local conditions in Vietnam

2 Workshop personnel and participants

A list of workshop personnel and participants is provided in Appendix 1 The workshop was designed by ALIRU staff and presented by Elizabeth Hartley, Greg Gemell (ALIRU) and Rosalind Deaker (University of Sydney) Organisation of the workshop and selection of the participants was facilitated by the Vietnamese project leader Tran Yen Thao

Workshop participants were selected to represent all institutes and inoculant manufacturers involved in project 013/06VIE All participants were skilled in general microbiological

3 Workshop activities

The workbook (with schedule of workshop activities included) and procedures manual are included in hardcopy/CD Rom and not electronically due to the large size of the files The workshop commenced on Monday 26 th February with an introductory lecture and discussion session covering the definition of quality control and quality assurance in relation to legume inoculants Quality control of legume inoculants in Australia was reviewed with particular reference to the setting of standards, the importance of high numbers of viable effective rhizobia, low levels of contaminant organisms and peat quality and conditions (ie moisture potential) The participants were involved in an active discussion session on the inoculant quality parameters and their responses indicated a good understanding of the issues

For the practical component of the workshop the participants were divided into three groups each lead by one of the workshop coordinators The workbook outlined the activities for each day and included a check list of activities to be reviewed at the end of each day At various points throughout the workbook participants were asked to refer to the procedures manual for details of

a particular procedure Extra material was also demonstrated by the group leader and time was allowed for discussion of various points Most of the materials for the workshop were shipped from Australia to give the participants as close to the ‘Australian experience’ of quality control as possible

The activities were designed to expose participants to commonly used QC protocols for legume inoculants and to become familiar with the morphological and growth characteristics of bradyrhizobial strains commonly used to inoculate soybean and groundnut In particular the participants were given the opportunity to observe differences in morphological and growth characteristics of these strains when isolated from different environments such as peat and broth cultures Experience with visual observation of cells and colonies of rhizobia from many different environments will improve identification skills Participants also gained experience in serological agglutination tests for verification of strains Antibody production in Vietnam through the inoculation of rabbits is readily available and affordable

The workshop covered counting techniques from both sterile and non-sterile peat carriers As pre-sterilisation of the peat carriers in Vietnam will not be possible, participants were given experience in the plant infection most probable number technique For the purposes of the

workshop plants were grown in plant tubes with Jensen’s agar slopes Participants were able to observe the suitability of this growing method for small seeded legumes but not for large seeded legumes Alternative growing systems were demonstrated and there was much discussion about modification of the system using materials readily available in Vietnam

4 Implementation of QC in Vietnam

During the second week a two day workshop was organised to adapt the QC protocols to Vietnamese conditions The workshop covered processes and protocols as well as facilities required for QC in Vietnam

4.1 QC protocols

The participants were guided through a discussion to outline the major components of the QC process This was done in order to establish an overall structure by which QC could be implemented in Vietnam Participants had already experienced protocols in the workshop and had discussed the context and relevance of various QC protocols The guided discussion was a means of assessing their understanding of the objectives and rationale of the QC process The results of the session are recorded in Appendix 2

Three main areas of responsibility for QC laboratories were identified; maintaining a culture collection, verifying and testing strains for issue to manufacturers and measuring the quality of manufactured legume inoculants It was acknowledged that identification and testing protocols were important before preserving cultures and before release to manufacturers It was clear that participants were also aware of the importance of a good recording system to track samples throughout the QC process and to produce reports for manufacturers Also identified was the need to set standards by which results of QC tests are compared Results of QC tests performed on manufactured inoculants should be interpreted in terms of quality standards There is a notional standard of 109 cells/g moist peat in Vietnam but there is

substitution of disposable items (eg plastic petri-dishes and Eppendorf tubes) with disposable items (eg glass petri dishes and glass test tubes) will be made The QC laboratory will use plant tubes for MPN counts rather than Gemell tubes or similar growth systems for large seeded legumes due to space restrictions In order to do this they will acquire siratro seeds for testing of groundnut rhizobia and small seeded soybean seeds from ALIRU

non-4.2 QC Laboratory design and equipment

The participants were asked to design a preparation laboratory, QC laboratory and plant growth room including all relevant equipment for QC in Vietnam The designs and lists

of equipment required are included in Appendix 3 The designs indicated high degree of understanding of conditions and facilities required for QC of legume inoculants The lists

of equipment and laboratory design can be used as a guide for the organisation of the QC laboratory in Vietnam and the purchase of equipment

The preparation area was designed to allow a logical flow of activities involved in preparing media and reagents, receiving and processing used materials and equipment from the QC laboratory and maintaining a clean area for handling sterile media The QC laboratory was designed with areas dedicated to the common QC protocols The laminar flow cabinet was orientated so that it was not adjacent to a main thoroughfare Gas was included to allow the use of Bunsen burners which are more efficient than spirit burners commonly used in Vietnam The plant growth room included plant growth benches as well as adequate means of controlling and monitoring temperature and light Exhaust fans would help reduce the build up of heat from growth lights and a balance was included to allow gravimetric application of water during growth of plants All workspaces had air-conditioning oriented in such a way that fans would not create air currents or lift dust compromising purity of cultures

5 Assessment and survey of workshop participants

Towards the end of the workshop participants were asked to complete an assignment to apply the knowledge and skills they had learnt in the workshop and a questionnaire

designed to assess the overall success of the workshop and aspects that could be improved

5.1 Problem solving assignment

A problem solving assignment was prepared to assess the ability of workshop participants

to apply QC protocols demonstrated in the workshop to hypothetical situations The assignment problems and suggested solutions are included in Appendix 4 The completed assignments were not given a mark and participants will receive the suggested solutions and individual feedback via email due to time constraints during the workshop This will facilitate on-going communication between scientists in Vietnam and Australia The majority of questions were answered well and indicated a high level of understanding

of the principles of the QC protocols

5.2 Survey of participants

Participants were asked to rank various aspects to represent their experience of the workshop The ranking system was designed based on the 5 point Likert scale where 5 was the highest ranking (strongly agree) and 1 the lowest (strongly disagree) The survey

is included in Appendix 5 and the results in Appendix 6 All aspects of the workshop were rated highly and were never ranked below 4 reflecting the opinion that the workshop was highly successful and relevant for all participants The highest score was achieved for the usefulness of the workbook and procedures manual after the workshop All participants gave a ranking of 5 for this question The weakest score was achieved for application of the information and techniques to each laboratory This was expected

as only one laboratory in Vietnam will be responsible for QC of legumes inoculants However, most other laboratories will use some of the protocols Suggestions made by

List of workshop personnel and participants

Workshop Personnel Affiliation

Elizabeth Hartley Australian Legume Inoculant Research Unit

(ALIRU), NSW Department of Primary Industries Greg Gemell Australian Legume Inoculant Research Unit

(ALIRU), NSW Department of Primary Industries Rosalind Deaker Faculty of Agriculture, Food and Natural Resources,

University of Sydney

Workshop Participants Affiliation

1 Tran Yen Thao Oil Plants Institute (OPI)

2 Ngo Thi Kieu Duong Oil Plants Institute (OPI)

3 Tran Ngoc Thao Oil Plants Institute (OPI)

4 Nguyen Thị Phuong Tam Can Tho University (CTU)

5 Pham Thi Khanh Van Can Tho University (CTU)

6 Le Thi Thanh Thuy National Institute of Soil Fertility (NISF)

7 Tran Minh Hien Institute of Agricultural Science (IAS)

8 Tran Dang Dung Institute of Agricultural Science (IAS)

9 Tran Thi Kim Cuc Institute of Agricultural Science (IAS)

10 Do Thi Thanh Truc* Institute of Agricultural Science (IAS)

11 Mai Thanh Truc Institute of Agricultural Science (IAS)

12 Nguyen Duc Hoang Institute of Agricultural Science (IAS)

13 Pham Thi Thu Hien Fitohoocmon

14 Nguyen Thi Bich Lien Fitohoocmon

15 Nguyen Thi Tuyet Trinh* Humix Company

16 Nguyen Truong Tho* Humix Company

17 Cao Manh Hung* Cu Chi Company

Discussion on Inoculant Quality Control (Wed 7 th March)

QC laboratory culture collection

• Strain tests – growth rate, purity, plant nodulation, stability in culture, match with host legume, field tests? (QC lab or agronomists)

• Store preserved cultures (temperature, duplicate copies etc.)

QC laboratory strains for issue

• Information should come with strain (host legume, photo of colonies on agar, date received, strain name, growth speed)

• Record all information in laboratory log/database

• Subculture strains many times for working cultures

• Need to describe colony morphology (does it match description from source?)

• Gram stain

• Serology (if antisera available)

• Check growth in fresh broth (high cfu/mL, purity/contaminants, speed of growth, colony morphology/consistency)

• Ability to nodulate host legume

• How long strain survives in culture

• Survival in peat carrier (will it grow/survive in peat? How long?)

• Monitor stability of strain for growth, morphology, plant effectiveness, nodulation

• Preserve many copies of original culture (Freeze dry, store under paraffin, liquid nitrogen, subculture regularly)

• Supply fresh subculture of stored cultures to manufacturers (after testing)

• Tell manufacturers to check purity of culture

QC laboratory incoming legume inoculants

• Record in log book/database (source, strain, date of manufacture, host legume, arrival date, date of test, batch identification number, weight or size, check packet for damage)

• Test some of packets received and save some packets for re-testing if packets fail

• Test inoculant quality (contamination, number/g - MPN/plate count)

• Authenticate strains (reaction with specific antisera, gram stain, nodulation)

• Test peat environment (Moisture content)

• Determine if inoculant product meets standards (pass/fail)

• Complete report (for manufacturers and own records)

• How will farmers know that the inoculant has passed?

Fig A2.1 Design of preparation room

Equipment included in preparation laboratory

1 Water purification unit

2 Drying oven for glassware and for determining moisture content

3 Two balances (2 and 3 decimal places)

4 Chemical storage including desiccators for hygroscopic chemicals

5 Magnetic stirrer and pH meter for media preparation

6 Fume cupboard

7 A fridge for chemical storage

8 Water bath for maintaining temperature of agar media

9 A fridge/freezer for storage of prepared media

Dirty glassware

Washing area

Distilled water

Clean glassware storage

Chemical storage with desiccators (list)

Balance 1 (0.00)

Balance 2 (0.000)

Magnetic stirrer

pH meter

Fume cupboard

Chemical fridge

Window and fire exit Oven

Racks for drying

Bench for media

Gas/flame First aid kit

Dirty glassware

Washing area

Distilled water

Clean glassware storage

Chemical storage with desiccators (list)

Balance 1 (0.00)

Balance 2 (0.000)

Magnetic stirrer

pH meter

Fume cupboard

Chemical fridge

Window and fire exit Oven

Racks for drying

Bench for media

Gas/flame First aid kit

Fire extinguisher