1 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 1 Table of Contents 1. Institute Information 2 2. Contact Officers 2 3. Project Abstract 2 4. Executive Summary 3 5. Technical Report 5 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 2 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 Date commenced March 2007 Completion date (original) March 2009 Completion date (revised) November 2009 Reporting period December 2008 – September 2009 2. Contact Officer(s) In Australia: Team Leader Name: Dr David Herridge Telephone: 02 67631143 Position: Professor, Soil Productivity Fax: 02 67631222 Organisation University of New England - PIIC Email: david.herridge@dpi.nsw.gov.au In Australia: Administrative contact Name: Mr Graham Denney Telephone: 02 63913219 Position: Manager External Funding Fax: 02 63913327 Organisation Industry & Investment NSW Email: graham.denney@dpi.nsw.gov.au In Vietnam Name: Ms Tran Yen Thao Telephone: 08 9143024 – 8297336 Position: Researcher Fax: 08 8243528 Organisation Oil Plants Institute (OPI) Email: yenthao@opi.org.vn yenthao@hcm.fpt.vn yenthao9@yahoo.com 3 3. Project Abstract 4. Executive Summary Impact of two Australian strains in Vietnam on legume production and productivity and comparative analysis between local and introduced strains 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. 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 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 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. 4 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 10 8 rhizobia/g and a maximum 1 x 10 8 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. 5 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: 6 • 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 10 9 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 10 5 –10 6 cells/seed just before sowing. 7 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: KH 2 PO 4 - 195 mg/pot; KCl - 168.4 mg/pot; MgSO 4 .7H 2 O - 22.21 mg/pot; ZnSO 4 .7H 2 O - 20.63 mg/pot; (NH 4 ) 6 Mo 7 O 24 .7H 2 O - 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 No. Strain name Target crop Source 1 NC92 Groundnut Australia 2 Tal 179 Groundnut NifTAL 3 P088183 Groundnut Thailand 4 P03818 Groundnut Thailand 5 GL1 Groundnut OPI – local strain 6 GL2 Groundnut SFI – local strain 7 GL14 Groundnut OPI – local strain 8 LAC1 Groundnut SFI – local strain 9 P3 Groundnut OPI – local strain 10 P1 Groundnut OPI – local strain 11 CTP Groundnut CTU – local strain 12 CB1809 Soybean Australia 13 U110 Soybean NifTAL 14 SEMIA 5019 Soybean NifTAL 15 S01015 Soybean Thailand 16 S1059 Soybean Thailand 17 Ach Soybean Argentina 18 YCK Soybean Korea 19 SL1 Soybean SFI – local strain 20 DT2 Soybean SFI – local strain 21 SL2 Soybean SFI – local strain 22 DL1 Soybean OPI – local strain 23 DL2 Soybean OPI – local strain 24 CJ1 Soybean OPI – local strain 25 CJ2 Soybean OPI – local strain 26 S6 Soybean OPI – local strain 27 S37 Soybean OPI – local strain 8 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 m 2 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 (r 2 =0.82) while correlation between nodulation and plant height was not significant (r 2 =0.27). The results showed that NC92, GL1 and GL2 were the best strains. They produced more nodules and more biomass than other strains. 9 Table 2. Nodulation and growth of inoculated groundnut with different rhizobial strains Rhizobial strains Number of nodules/plant Dry weight of nodules/plant (mg) Plant height (cm) Dry biomass (g/plant) 1. NC92 79 b 93 b 33 abcd 2.7 b 2. P08183 29 gh 35 fg 29 cd 1.7 ghi 3. CTP 44 e 52 d 36 ab 2.2 def 4. GL1 113 a 136 a 35 abc 3.8 a 5. P31 35 fg 42 ef 31 bcd 1.7 hij 6. GL2 77 b 91 b 35 ab 2.5 bc 7. P12 54 cd 66 c 35 ab 1.9 fgh 8. GL14 60 c 72 c 32 abcd 1.9 efg 9. LAC1 39 ef 47 de 38 a 1.6 ij 10. Tal 179 27 h 33 g 35 ab 1.5 jk 11. P03818 53 d 63 c 32 abcd 2.4 cd Control 1 11 i 12 h 27 d 1.2 k Control 2 5 i 6 h 32 abcd 2.2 de CV% 10.6 11.4 12.7 8.6 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 Number of nodules/plant Dry weight of nodules/plant (mg) Plant height (cm) Dry biomass (g/plant) 12. U110 47 cd 129 d 38 defg 2.8 d 13. DT2 45 de 121 e 35 defg 2.7 e 14. CB1809 52 bc 149 b 43 abcd 3.3 bc 15. CJ1 35 fg 121 e 41 bcde 2.7 e 16. DL2 30 g 72 h 34 efg 1.6 hi 17. S37 16 h 103 f 50 a 2.3 f 18. SL2 66 a 170 a 42 abcd 3.7 ab 19. CJ2 49 cd 136 cd 42 abcde 2.9 c 20. DL2 35 fg 114 e 37 defg 2.5 e 21. SL1 57 b 141 c 37 defg 3.1 cd 22. YCK 33 fg 77 gh 48 ab 1.7 gh 23. S01015 35 fg 121 e 46 abc 2.6 e 24. SEM 5019 34 fg 118 e 39 cdef 2.6 e 25. DL1 39 ef 83 g 39 cdef 1.8 g 26. S6 21 h 80 g 39 cde 1.7 gh 27. ACH 22 h 69 h 30 g 1.5 i 28. S1059 32 fg 97 f 31 fg 2.2 f Control 1 9 i 35 i 43 abcd 1.3 j Control 2 8 i 29 i 37 defg 1.4 i CV% 13.2 5.1 14.6 4.1 Control 1: uninoculation, without N fertilizer Control 2: uninoculation, plus N fertilizer (100ppm) - Source: OPI [...]... 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... financial performance, indication of on-going benefits in cropping rotations and the effectiveness of demonstration trials in improving farmer’s awareness of benefits Field demonstration trials, including assessement of physical and financial performance A total of 168 demonstration trials have now been conducted in 10 provinces The demonstration fields had two treatments: +inoculation with nil or very... Indication of on-going benefits in cropping rotations Major economic benefits in the cropping systems of replacement of fertiliser N inputs with inoculants is covered in the previous section (Graphs 9, 10, 11 and Appendix 3) Other potential benefits were not (and could not be) assessed because the foci of the project was inoculant production, quality, distribution and marketing and farmer education... 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... provided in the next report on Project Validation 32 Appendix 1 Summary of project field experiments during 2007–09 Inoculation effects of CB1809 and NC92 on nodule, biomass and grain yield Region Province Village Crop Sowing date Inoculation (NC92 or CB1809) effects on - Northern Highland Son La Muong Chum Co Noi Phu Cat Soybean Soybean Soybean Soybean Soybean Sobean Groundnut Groundnut Groundnut Groundnut... efficacy requirements The new standard largely utilized the wellconstructed and comprehensive framework of the current standard The proposed name of the standard : Vietnam National Standard for Legume Inoculants 1 Definition - Legume inoculants are bio-products: • Contain one or more viable rhizobial strains, which were selected for fixing nitrogen and for being able to grow in culture media • Produced... provided extension materials (see Appendices 4 and 5) Also, researchers and extension officers explained how rhizobia work and the conditions for successful inoculation Farmers were very interested in learning about nitrogen fixation A quantitative assessment of the effectiveness of the demonstration trials in improving farmer’s awareness of the benefits of inoculants was gained in the March-August... 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 In Vietnam currently, sterilisation by autoclaving is used The moisture content of the peat before autoclaving should be adjusted to approximately 20% Depending on contamination in terms... 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 14 The first step after receiving... 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 . 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. 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. 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