Báo cáo khoa học nông nghiệp " Replacing fertiliser N with rhizobial inoculants for legumes in Vietnam for greater farm profitability and environmental benefits " MS6 potx

The best strains were NC92 Australian commercial strain, GL1 and GL2 local strains for groundnut and CB1809 Australian commercial strain, SL2, SL1, CJ2 and U110 old US commercial strain

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

MS6: High Quality Inoculants Technical Report

September 2009

5.1 Introduction of two Australian strains into Vietnam 6

5.2 Protocols for inoculant production, QA and use 16

5.3 Results and evaluation of on-farm demonstration trials 32

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

Dr Roz Deaker

Ms Elizabeth Hartley, Mr Greg Gemell

Completion date (original) March 2009

2 Contact Officer(s)

In Australia: Team Leader

Position: Professor, Soil Productivity Fax: 02 67631222

Organisation University of New England -

PIIC

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

In Australia: Administrative contact

Position: Manager External Funding Fax: 02 63913327

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

In Vietnam

8297336

Organisation Oil Plants Institute (OPI) Email: yenthao@opi.org.vn

yenthao@hcm.fpt.vnyenthao9@yahoo.com

In the potted field soil trial, there were 13 treatments for groundnut (11 groundnut strains, a +N control without inoculation and –N uninoculated control) and 18 treatments for soybean (17 soybean strains, a +N control without inoculation and –N uninoculated control) All strains increased groundnut and soybean nodulation and yield compared to the control treatments There were close correlations between nodule number, nodule weight and plant biomass while correlations between nodulation and plant height were poor The best strains were NC92 (Australian commercial strain), GL1 and GL2 (local strains) for groundnut and CB1809 (Australian commercial strain), SL2, SL1, CJ2 and U110 (old US commercial strain) for soybean

The total number of field experiments during 2007–09 was 36 in the 10 provinces The experiments were conducted in the main legume-growing areas in Vietnam, from the highlands in the North, to the Central Coast area to the highlands in the South and Mekong Delta The provinces involved were Son La, Nghe An, Binh Dinh, Binh Thuan, DakLak,

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

DakNong, Tay Ninh, Dong Thap, An Giang and Tra Vinh There were at least 5 treatments in each experiment:

1 Farmer’s practice without N fertiliser

2 Farmer’s practice with N fertiliser

3 Inoculation with Australian strains CB1809 (soybean) or NC92 (groundnut), -N fertiliser

4 Inoculation with local strain: SL1 (for soybean) or GL1 (groundnut), -N fertiliser

5 Inoculation with local strain: SL2 (for soybean) or GL2 (groundnut), -N fertiliser The Australian strains were the most effective in terms of nodulation, biomass yield and grain yield Compared with the uninoculated control, CB1809 and NC92 increased nodulation of soybean and groundnut, respectively, by an average of 58%, biomass yield by 30% and grain yield by 29% Compared to the local Vietnamese strains, CB1809 and NC92, increased soybean and groundnut nodulation by an overall average of 22% Biomass yields were increased by an average of 10% and grains yields increased by an average of 13%

Protocols for production of high quality inoculants including QA, packaging, storage, distribution and on-farm application of inoculants

During the two years of the project, technology for inoculant production at the three institutes (SFI, OPI and IAS) was developed The principal aim was production of high quality of inoculants containing >5 x 108 rhizobia/g and a maximum 1 x 108 contaminants/g Some details of the technologies are different between the collaborating institutes depending on facilities and expertise To some extent, the inoculant technologies have been adapted from those used in countries with existing successful inoculant industries, e.g Australia, US

The project team has decided that CB1809 and NC92 will be used for inoculant production in Vietnam as multi-field trials throughout the country showed these strains are the best for soybean and groundnut They increased nodule weight, crop biomass and grain yield compared to local strains tested In the future, more strain evaluation will likely be done to try to develop even more effective inoculant strains It is also proposed that cultures of these strains will be supplied annually from the independent QA laboratory to private and public sector laboratories producing inoculants together with protocols for strain maintenance and production of broth cultures Details are provided in Section 5.2

It is likely that peat will be the major inoculant carrier for Vietnam Details are provided (Section 5.2) on different peats in Vietnam, their efficacy as an inoculant carrier and the usefulness of various additives in improving efficacy Guidelines are also provided on optimum pH and water content

An experiment was done to compare different methods of sterilization of peat After peat samples were sterilized, some samples were used for direct determinations of contamination Other peat samples were cultured in Glucose Peptone broth and then the broths were assessed for contamination after 3 hrs, 24 hrs, 36 hrs and 72 hrs Samples were also assessed for rhizobial numbers The best treatments in terms of highest numbers of rhizobia and lowest numbers of contaminants were the autoclaved treatments with autoclaving for 60 min and the irradiated treatments with 30 kGy the best overall

There are currently no specific standards for rhizobial inoculants in Vietnam, rather there are standards for nitrogen fixing microbial fertilizers However, it is very important to have effective QA of legume (rhizobial) inoculants A number of modifications to the Vietnam National Standard for Nitrogen-Fixing Microbial Fertilizers (TCVN 6166-1996) were justified to make it more relevant to rhizobial inoculants, based on production technology and efficacy requirements The new standards largely utilize the well-constructed and

comprehensive framework of the current standard The proposed name of the standard is the

Vietnam National Standard for Legume Inoculants and contains details on the technical requirements of the inoculants including labelling as well as methods of testing and

reporting

Results of demonstration trials and effectiveness of demonstration trials in

improving farmer’s awareness of benefits

A total of 168 demonstration trials have now been conducted in 10 provinces The demonstration fields had two treatments: +inoculation with nil or very low amounts of fertiliser N and –inoculation with farmer’s rate of fertiliser N Results are summarised in Appendix 3

Generally, inoculation of soybean and groundnut increased the profit for farmers, on average

by 4.500.000VNĐ/ha The size of the benefit varied across the different sites The increase was around 500.000VNĐ/ha at the demonstration field of groundnut at Bau Don, Tay Ninh province, and as high as 14.200.000VNĐ/ha at Chau Thanh, Tra Vinh province Similarly for soybean, the profit from inoculation was as much as 11.640.000VNĐ at Duong Minh Chau, Tay Ninh province In Dong Thap province the benefit from inoculation was on average 4.900.000VNĐ/ha

In Dong Thap province, at the Phu Huu vllage, Chau Thanh district where demonstrations were conducted on a large area of land (61.5 ha) with the participation of 120 local farmers, yields of soybean increased on average 12.5%, equal to 300 kg seed/ha Farmers produced higher incomes around 4.900.000 VNĐ/ha compared to their normal cultivation with N fertilisers

The demonstration trials and associated extension/training activities were very effective in increasing farmer’s awareness of the benefits of inoculants for legume production Farmers were invited to the demonstration sites at least once and, in many fields they came for nodule and biomass samplings as well as at grain harvest time Overall, there were a total of 3400+ person visits 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 legume nitrogen fixation

5 Technical Report

The technical report includes comprehensive details of experiments on rhizobial strains, protocols for production, QA, distribution and application of inoculants and results and evaluation of demonstration trails, according to the required report headings:

• Introduction of two Australian strains into Vietnam, their impact on legume production and productivity and comparative analysis between local and introduced strains

• Protocols for production of high quality inoculants, packaging, storage, distribution and on-farm application of inoculants and for quality assurance of production

• Results of field demonstration trials, including assessement of physical and financial performance, indication of on-going benefits in cropping rotations and the effectiveness of demonstration trials in improving farmer’s awareness of benefits

5.1 Introduction of two Australian strains into Vietnam, their impact on legume production and productivity and comparative analysis between local and introduced strains

Introduction

Research on legume inoculants in Vietnam has been done since the 1980’s at the Hanoi University and SFI (VASI) in the North and, in the South, at Can Tho University (CTU), IAS and OPI (now named IOOP) Generally, the objectives of the research were selection of strains, small-scale production of inoculants and field trials evaluating efficacy of the inoculants Each institute focussed on target regions and particular legume crops, such as CTU in the Mekong Delta with soybean, IAS in the Southern East Region with groundnut and OPI in the Central Coast and Highlands with groundnut and soybean Strains proposed for inoculant production were not tested throughout the country and outcomes of associated research on production technologies were not shared between the institutions Thus, even with a history of legume inoculant research and production in Vietnam, inoculants are currently not available in the market and farmers are to a large extent unaware of their potential benefits Instead, farmers use expensive N fertilisers on their legume crops Part of this project was to evaluate elite international strains across the country and to compare them with national strains Included were local and imported strains from Vietnamese institutes, from NifTAL (USA), ALIRU (Australia), DOA (Thailand), Korea and Argentina Several of these strains are currently used in commercial inoculants in Australia such as CB1809 (soybean) and NC92 (groundnut) We conducted two experimental sets; the first was in a potted field soil and the second in field trials

Methodology

Screening rhizobial strains in pots

The experimental design was a randomized complete block design with three blocks There were 13 treatments for groundnut (11 groundnut strains, a +N control without inoculation and –N uninoculated control) and 18 treatments for soybean (17 soybean strains, a +N control without inoculation and –N uninoculated control) Information of strains is given in the Table

1 Each strain was grown up in yeast mannitol broth (YMB) for 5–7 days to reach maximum turbidity (approx 1 x 109 cells/ml) The broths were then injected into sterilized peat and allowed to stabilise for 1 week Seeds were inoculated with the peats at the rate of 105–106

cells/seed just before sowing

The sandy, infertile soil used in the pots was from Trang Bang district, Tay Ninh province The soil was obtained from a depth of 10–15 cm and transported to Binh Thanh experimental station of OPI The soil was mixed thoroughly, then sieved using a 5-mm mesh screen The soil was then mixed with coir dust (1:1) and lime and allowed to equilibrate for 7 days Each pot contained 1.7 kg of the soil mixture

The soil moisture content at field capacity was determined, then each pot adjusted to field capacity by adding water Application of fertiliser was as follows: KH2PO4 - 195 mg/pot; KCl - 168.4 mg/pot; MgSO4.7H2O - 22.21 mg/pot; ZnSO4.7H2O - 20.63 mg/pot; (NH4)6Mo7O24.7H2O - 0.81 mg/pot

We planted 5 seeds/pot and removed 2 young plants after 7 days The plants were harvested

at 30 days for soybean and at 45 days for groundnut Numbers of nodules, dry weight of nodules and dry weight of biomass was determined at harvest

Table 1 Rhizobial strain information

Field experiments

The experiments were conducted in 10 main legume-growing areas in Vietnam, from the highlands in the North, to the Central Coast area to the highlands in the South and Mekong Delta The provinces involved were Son La, Nghe An, Binh Dinh, Binh Thuan, DakLak, DakNong, Tay Ninh, Dong Thap, An Giang and Tra Vinh There were at least 5 treatments:

6 Farmer’s practice without N fertiliser

7 Farmer’s practice with N fertiliser

8 Inoculation with CB1809 (for soybean) or NC92 (groundnut), -N fertiliser

9 Inoculation with local strain: SL1 (for soybean) or GL1 (groundnut), -N fertiliser

10 Inoculation with local strain: SL2 (for soybean) or GL2 (groundnut), -N fertiliser Source of strains:

SL1: local strain (soybean) from Can Tho University

SL2: local strain (soybean) from SFI (VASI - from the national microbial strain program) GL1: local strain (groundnut) from OPI

GL2: local strain (groundnut) from SFI (VASI - from the national microbial strain program)

CB1809: Australian commercial inoculant strain (soybean) from ALIRU

NC92: Australian commercial inoculant strain (groundnut) from ALIRU

Measurements were: dry weight of nodules, biomass and grain yield Plot size was at least 20

m2 with 4 replications A randomized complete block design was used Depending on growing areas, sowing date, land preparation, fertiliser inputs, date of sampling were different Details can be sent if required

Peat inoculants were made by the three Vietnamese institutes (OPI, IAS and SFI) and in some experiments Australian commercial inoculants were use as a positive control treatment Inoculant rates were 1–2 kg/ha for Vietnamese inoculants and 0.25 kg/ha for Australian commercial inoculants The inoculants were tested for quality by OPI before conducting experiments The seed inoculation method was used throughout Methods of sampling and processing of nodules, biomass and grain yield can also be sent if required

Results and Discussion

Screening rhizobial strains in pots

All strains increased groundnut nodulation and yield compared to control treatments (Table 2) Based on assessments of plant nodulation, there were 3 groups:

- Highest nodulation: NC92, GL1, GL2

- Average nodulation: P12, GL14, P03818

- Lower nodulation: P08183, CTP, P31, LAC1, Tal179

There were close correlations between nodule number, nodule weight and biomass (r2=0.82) while correlation between nodulation and plant height was not significant (r2=0.27) The results showed that NC92, GL1 and GL2 were the best strains They produced more nodules and more biomass than other strains

Table 2 Nodulation and growth of inoculated groundnut with different rhizobial strains

Rhizobial strains nodules/plant Number of nodules/plant (mg) Dry weight of Plant height (cm) Dry biomass (g/plant)

Control 1: uninoculated, without N fertilizer

Control 2: uninoculated, plus N fertilizer (100ppm)

Source: OPI

Table 3 Nodulation and growth of inoculated soybean with different rhizobial strains

Rhizobial strains nodules/plant Number of nodules/plant (mg) Dry weight of Plant height (cm) Dry biomass (g/plant)

Control 1: uninoculation, without N fertilizer

Control 2: uninoculation, plus N fertilizer (100ppm) - Source: OPI

Similarly, inoculated soybean produced more nodules and more biomass compared to controls (Table 3) with substantial differences amongst the strains Grouping strains based on effectiveness of nodulation and yield as follows:

- Highest effectiveness: SL2, CB1809, SL1, CJ2, U110

- Average effectiveness: DT2, CJ1, S37, S01015, SEMÍA 5019, S1059

- Lower effectiveness: YCK, ACH, DL1, DL2, S6

In the first group, biomass amounts were 3 times higher than control treatments The close correlation between nodulation and biomass showed that nodules were very effective and contributed to higher biomass yield (r2=0.86) and will likely play an important role in seed yield increase The 5 strains CB1809, U110, SL1, SL2 and CJ2 were the best strains for soybean

In another experiment conducted in a green house, CB1809 showed high effectiveness in term of nodulation (Table 4) The strain produced more nodules compared to 3 other local strains

Nodule number

on main roots

Nodule number on lateral roots

Graphs 1, 2 and 3 summarise responses to inoculation with strains CB1809 (soybean) or NC92 (groundnut) for plant nodulation, biomass and grain yield, respectively Responses ranged from small to large, depending on field sites

There were large effects of inoculation on nodulation at 72% of the field sites (Graph 1) At those sites, nodule weight increased by 43–166% Nodulation responded moderately at 11% and, at the rest (17%) of the sites, response were small with an average increase of 11% The overall average increase in nodulation using the superior Australian strains was 58%

Graph 2 Range of crop biomass responses to inoculation

remaining 22% 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 (41–70%) at 20% of sites Moderate responses (20–40%) were recorded at 62% of the sites and small responses (4–18%) at the remaining 18% of sites The overall average increases in biomass yield and grain yield using the superior Australian strains were 30% and 29%, 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 91%, 94 and 97% 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

The two Australian strains, CB1809 and NC92, increased soybean and groundnut nodulation

by an overall average of 22%, relative to the local Vietnamese strains (Graph 4) Biomass yields were increased by an average of 10% (Graph 5) and grains yields increased by an average of 13% (Graph 6), relative to the local strains

Graph 3 Range of grain yield responses to inoculation

Graph 4 Nodulation increases with CB1809 and NC92 compared to

Graph 5 Crop biomass increases with CB1809 and NC92 compared

Graph 6 Yield increases with CB1809 and NC92 compared to

5.2 Protocols for production of high quality inoculants, packaging, storage, distribution and on-farm application of inoculants and for quality assurance of production

During the two years of the project, technology for inoculant production at the three institutes (SFI, OPI and IAS) was developed The principal aim was production of high quality of inoculants containing >5 x 108 rhizobia/g and a maximum 1 x 108 contaminants/g In the following we present the current technologies for inoculant production in Vietnam Some details of the technologies are different between the collaborating institutes depending on facilities and expertise To some extent, the inoculant technologies have been adapted from those used in countries with existing successful inoculant industries, eg Australia, US

Strains for production

The project team has decided that CB1809 and NC92 will be used for inoculant production in Vietnam as multi-field trials throughout the country showed these strains are the best for soybean and groundnut They increased nodule weight, crop biomass and grain yield compared to local strains tested In the future, more strain evaluation will likely be done to try to develop even more effective inoculant strains

Maintenance of strains and preparation of mother cultures

It is proposed for production of inoculants in Vietnam that mother cultures will be provided annually by an independent quality control laboratory where the strains for production are maintained in terms of purity, viability and effectiveness (nodulation and nitrogen fixing ability) After receiving the mother cultures, producers (manufacturers) have to maintain them for that year of production

The first step after receiving the mother cultures is for the cultures to be transferred into culture tubes containing YMA (yeast mannitol agar) medium (called sub-mother cultures), at the same time streaking the cultures onto YMA and CRYMA (congo red yeast mannitol agar) Petri dishes for purity checking If the result show that the mother culture is contaminated then a request to the quality control lab for a new mother culture will be made The mother culture should be pure before sending to manufactures but there are risks associated with transport or handling Pure sub-mother cultures will then be kept in a refrigerator by the manufacturers at low temperature (1–4oC) until use They need to be sub-cultured after 6 months The number of sub-cultures prepared will depend on the demand of production Culturing temperature for the rhizobia is 30oC in an incubator Another method can be applied when there is a higher demand for production Transfer the mass culture from the mother culture to a flask with YM broth, then inoculate at 30oC for 5–7 days Two ml of the broth culture then are dispensed into 2 ml of 50% glycerol, previously sterilized in bottles These sub-mother cultures are stored in a domestic deep-freeze for use before checking purity by streaking on CRYMA and YMA plates

Growth of broth cultures

Starter culture: Streak out the culture of the strain to be used onto CRYMA and YMA Petri plates to check for purity Place the plates in an incubator at 30°C for 6–8 days If the plate streak is pure, aseptically pour about 10 mL of sterile water onto the slope sub-culture Close the lid and gently wash the slope with the water Aseptically, pour the liquid containing the rhizobia into an appropriately-sized flask containing YM broth Reseal the flask with the cotton wool bung and paper bag Gently mix the broth, and place in an incubator shaker at 30°C until the broth becomes milky in colour (5-7 days), and the rhizobial population reaches at least 109/ml broth The broth in the flask can then be aseptically poured into a fermentor

Broth growth media: Medium for rhizobial growth consists of a carbon source, nitrogen source and minerals Most rhizobia can utilize pentoses, hexoses, disacharides, polysaccharides and sugar alcohols even though the carbon utilization properties of rhizobia vary Generally, improvements in medium for inoculant production is based on the basic medium, YMB (yeast mannitol broth) Large scale inoculant production requires cheap and available ingredients, such as corn steep liquor and proteolyzed pea husks Table 5 below

shows effects of different growth media on growth of 6 strains of rhizobia The alternatives

tested by SFI were green bean extract, saccharose and glucose instead of expensive yeast extract and mannitol The number of rhizobial cells of the 6 strains yielded higher than

109/ml in the proposed media SX1 and SX2, similar to YMB The SX1 medium is the best one in terms of efficacy and economic benefit

The data are promising and need to be repeated If rhizobial growth rates on media SX1 and

SX2 are again high, then they can be considered for inoculant production in Vietnam

Duration of growth: The results showed that the numbers of rhizobial cells increased over time and reached a maximum at the 7th day for all strains (Table 6) Counts were as high as 7

x 109 cells/ml

Table 5 Number of cells/ml culture in improved media compared to basic YMB medium

pH effects:

Table 7 Effects of pH on the growth of rhizobial strains

Growth of rhizobial strains pH

-: no development

+: weak development (10 4 –10 5 CFU/ml)

++: nomarl development (106–107 CFU/ml)

+++: good development (108–109 CFU/ml

+: weak development (10 4 –10 5 CFU/ml)

++: nomarl development (106–107 CFU/ml)

+++: good development (108–109 CFU/ml

Source: SFI

Results from Tables 7 and 8 showed pH of medium and temperature effects on the

growth of rhizobia All 6 strains tested developed well at pH 6.5–7 and at 300C

Fermentation: Depending on capital investment or available equipment and expertise,

manufacturers will need to consider how big the fermentors should be Large-scale batch

fermentation has efficiency in terms of time but may be more prone to contamination

Fermentors can vary in size from 1–2 L (glass flasks on a rotary shaker or with an aerator) to

>1600 L (purpose-built steel fermentors)

Broth cultures can be produced in simple glass fermentors, such as a slightly modified 4-L

Erlenmeyer flask with a sampling port fitted close to its base A maximum of 2–3 L of

culture medium is added to the flask To prevent the entry of contaminants, the cotton

wool-packed filters are connected via the air lines All rubber stoppers and outlet tubes are

autoclaved The large rubber stopper which holds the air inlet and outlet tubes with their

filters is inserted firmly into the neck of the flask An aquarium pump is used to supply

aeration It is connected with the air outlet tube Air will flow freely through both filters

while bubbling through the broth The cotton wool in the filters needs to be packed uniformly

but loosely Over packing the air inlet filter can cause resistance to incoming air and lead to

poor aeration Over packing the outlet filter can lead to poor air escape and pressure build up

in the fermentor

The pump is disengaged from the fermentor for sterilisation Sterilise for 40 minutes at 1 atm

if the broth is 2 L and increase time by 10 minutes for each additional 1 L After the

fermentor has cooled, remove the clamp from the air inlet tubing and connect the air supply

then check for proper aeration and for leaks in the system The system is ready for

inoculation with the starter culture Inoculation is conducted through the latex air inlet with a

sterilized syringe fitted with a needle The air inlet tubing is surface sterilised with 70%

alcohol or 3% hydrogen peroxide about 2–3 cm above its connection to the glass tube The

needle is inserted downwards into the tubing and the starter culture is injected The culture

then is incubated at 30oC Use of small fermentors would be appropriate for Vietnam,

particularly when combined with the broth dilution – solid state fermentation With this, the

broth can be diluted 100 fold Thus, a 2 L broth can be used to inoculate 10,000 packets (at

rate of 20 mL/packet)

Small steel fermentors are common in industry and are usually sterilized by autoclave A

fermentor system for culturing rhizobia was designed by NifTAL and uses direct heating by

gas and cooling tubes for time-saving after sterilisation The body of the NifTAL fermentor

is a pressure vessel with a 141 L total capacity Working (broth) capacity is 20–100 L

Details of this fermentor can be accessed through Prof Nantakorn Boonkerd’s laboratory at

Suranaree University of Technology, Thailand

Selection of carriers

Carrier plays an important role in solid-based inoculant production Most inoculants are

produced based on the mixture of the broth culture and a finely milled, neutralized carrier

material The properties of the good carrier are:

- Supportive to rhizobial growth and survival

- Good moisture absorption capacity

- Easy to process

- Easy to sterilize by autoclave or gramma-irradiation

- Available in adequate amounts

- Inexpensive

- Good adhesion to seeds

- Good pH buffering capacity

Table 9 Characteristics of Sedge Peat used for commercial inoculant production

in the United States

Peat is the best researched and most frequently used as carrier materials for inoculant

production A large number of studies have showed that rhizobia are protected and survive

well in peat Tables 9 and 10 show physical and chemical analysis of well-researched peats

These peat were used for commercial inoculant production in U.S.A and Australia However,

physical and chemical analyses of a peat are only a partial assessement of its suitability as a

carrier Only a test related to growth and survival of rhizobia can confirm its acceptability

Apart from characteristics at the source such as salinity, clay, organic matter and 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 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.

Table 10 Characteristics of Bendenoch Peat used for commercial inoculant production in

Source: Peat in Vietnam and use in agriculture (Agriculture Publish House, 1997)

Results in Table 12 show effects of peat sources on inoculant quality Except Komix 2 the three other Vietnamese peats produced good growth of rhizobia The number of rhizobia in these peat reached ≥ 109 cfu/g moist peat, equal to commercial Australian peat The number

of rhizobia in Komix 2 was only 3.4 x 107 cfu/g moist peat at 6 months

Similarly, Son La and Thai Nguyen peat of the North supported good growth and survival of rhizobial strains CB1809, NC92, GL2, SL2 (Table 13) The number of rhizobia was ≥ 109cfu/g moist peat

Table 12 Number of rhizobia in different peat sources of the South

Table 13 Number of rhizobia in two peat sources of the North

Number of rhizobia/g moist peat inocualnts

Thái Nguyên 2,4 x 109 1,3 x 109 3,6 x 109 7,6 x 109 4,0 x 109 6,8 x 109 Sơn La 1,6 x 10 9 1,2 x 10 9 1,8 x 10 9 2,1 x 10 9 1,4 x 10 9 1,3 x 10 9 GL2

Thái Nguyên 1,2 x 10 9 2,4 x 10 9 1,2 x 10 9 1,1x 10 9 3,6 x 10 9 1,2 x 10 9 Sơn La 6,5 x 109 1,8 x 109 7,5 x 109 3,4x 109 3,2 x 109 3,6 x 109 CB1809

Thái Nguyên 5,3 x 10 9 2,8 x 10 9 5,3 x 10 9 2,2 x 10 9 2,4 x 10 9 1,4 x 10 9 Sơn La 6,6 x 10 9 1,4 x 10 9 1,6 x 10 9 1,5 x 10 9 5,2 x 10 9 1,1 x 10 9 SL2

Thái Nguyên 4,0 x 109 1,2 x 109 4,8 x 109 1,0 x 109 1,0 x 109 1,2 x 109 Source: ISF

Table 14 Number of rhizobial cells in different carriers after one month

Number of rhizobial cells (CFU/g)

5 NC 92 Peat + worm casts + coconut coir dust 2.5 x 109

6 CB 1809 Peat + worm casts + coconut coir dust 6.8 x 108

Source: IAS

3 NC92 Peat + worm casts + coconut coir dust 3,8 x 109

Source: ISF

Carrier processing

Particle size of carriers: Peat is currently the preferred carrier for inoculant production in Vietnam The project research has shown some additives can be used as mixtures with peat They are worm cast and coconut coir dust Molasses and “rare soil” can be also added as nutrient additives The peat is mined, drained if wet peat, screened to remove stones and roots and then shredded and dried The peat then is ground in high-speed harmer and passed through a sifting machine which has a set of sieves: 1mm, 355µm, 150 µm and 75 µm The milled carrier will go through the sieves Particles of 75 µm or thinner make carriers suitable

for seed coating

pH of carriers: The pH was shown to be critical and acid peats could be amended with calcium or magnesium carbonate The suitable pH of inoculant carrier is around 6.5–7.0

Fine agricultural lime is used for adjusting carrier pH

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

The amount of lime is calculated as following:

- Suspend 10 g of carrier into 90 ml of water in a 400 ml-glass beaker

- Stir the mixture on a magnetic stirrers while monitoring the pH with the electrode of a

pH meter and gradually add lime until a pH of 6.5 has been reached

- Record the amount of lime needed to neutralize 10 g of the carrier

- Add the corresponding amount of lime to amount of the carrier Mix well