Progress 10/01/93 to 09/30/05
Outputs
In 1975 at the peak of the oil crisis, national economies worldwide contended with high costs of fossil fuels. The nitrogen-fixing symbiosis between legumes and rhizobia was recognized as an attractive, low-cost alternative to petroleum-based nitrogen fertilizers for increased crop production in the tropics. The United States Agency for International Development (USAID) drew on expertise in the University of Hawaii's College of Tropical Agriculture and Human Resources. NifTAL was dedicated to the application of technologies based on BNF to international development goals. NifTAL's ultimate purpose was to help farmers maximize BNF inputs to their cropping systems, and thereby increase the production and quality of high protein foods while reducing their dependence on expensive nitrogen fertilizers. NifTAL was designated a UN Microbiological Resource Center (MIRCEN); rhizobial germplasm resource was collected and tested for symbionts for 50 target legumes. A network of
more than 200 collaborators participated in NifTAL's International Network of Legume Inoculation Trials (INLIT) to conduct standardized experiments in countries around the globe. This effort verified the benefits of legume inoculation in many parts of the tropics. From 1982 to 1986, NifTAL developed appropriate BNF technologies for tropical agriculture. Technology transfer and training activities increased, particularly those related to production and use of inoculants. In 1983, the BNF Resource Center (BNFRC) for South and Southeast Asia was established to extend NifTAL's efforts through a regional center. NifTAL also initiated WREN, the Worldwide Rhizobial Ecology Network of select researchers to conduct standardized field trials to link the response to inoculation to measurable environmental parameters. One of NifTAL's major accomplishments was the development of models to predict when, where, and to what extent legumes will respond to inoculation. NifTAL also strengthened its
capacity to transfer BNF technology to extension personnel, and thereby forged another conduit through which NifTAL expertise can serve developing country farmers. NifTAL was instrumental in the establishment of inoculant production facilities throughout the tropics, and developed tools for more efficient and economic inoculant production and quality control. The Future. 1) A continued commitment to meeting the needs of the US and developing country inoculant production industry, with increased emphasis on serving the private sector through improved production, quality control, distribution, and promotion of inoculant products. 2) The integration of NifTAL's legume management research into information synthesis through simulation models and decision support systems. 3)Increased collaboration with CGIARs to provide regional BNF expertise and augment resources. 4) New training and communications efforts to meet a broader spectrum of clients in nutrient management, technology extension
through private voluntary organizations and nongovernment organizations, and market and enterprise development. 5) Emphasis on assessments of the economic impacts of BNF technologies on national scales.
Impacts
NifTAL programs had an impact in many places around the world. Below is an economic impact assessment from an integrated program of research, training and facilities development on the production of soybean in Zambia. This data is typical of NifTAL impact in other locations. NifTAL's Zambia program was a joint effort with the University of Illinois ZAMARE Project (USAID/Zambia) consisting of four years of technical assistance, research, facilities design and inoculant market development. Results represent increases in farm income due to inoculation of soybean. Results calculated by average response to inoculation and yearly price data. Year Area Production Increased Value (ha) (90 kg bags) $ U.S. 1984: 6,550 131,031; 1985: 7,069 141,386 532,005; 1986: 5,983 119,666 509,048; 1987: 9,542 190,848 993,937; 1988: 9,145 182,916 1,229,196; 1989: 11,906 238,134 2,639,054; 1990: 20,000 488,889 5,648,136; 1991: 22,786 455,716 5,756,412. TOTAL 92,981 1,948,558 $37,248,652.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Support for this project ceased several years ago. We continued to provide technical assistance to several firms and parastatals to aid their quest to adopt products originating from this project. This year firms manufacturing legume inoculants, other agricultural inputs and biotechnology products have sought information about formulating and manufacturing liquid legume inoculant products we developed. Entities in India, the U.S. and Peru received our technical assistance. We continue to upgrade our web based information system on applied and plan to have it posted in 2005. During the year we added approximately 15 Mb of information and rearranged critical presentation in the research information and support section. The web now totals approximately 800 Mb of information ranging from our publications and MSc and PhD theses to practical training guides for the laboratory and extension service. The project made its final move from its headquarters in 2005 but much of
the disposal of equipment, supplies, hazardous materials and environmental assessment, securing and maintaining the 20 acre property and managing tenants took place in the 2003-2004 period. We disposed of approximately 100 cubic yards of materials to the landfill, removed 14 vehicles, wind generators, large towers and other heavy equipment items to metal recycling facilties. We catalogued and removed approximately 200 lbs of chemicals both hazardous and non-hazardous to either disposal venues or to proper CTAHR storage until their final disposal. We inventoried and distributed over 3000 equipment and supply items to other CTAHR units, public and private schools, non-profits, government agencies, UH Manoa and other entities. We continue to work with several graduate students from CTAHR and overseas. Most are involved in molecular work with Rhizobium in relation to ecological adaptation and interaction with host legumes.
Impacts
Most agricultural technologies go through various phases of adoption including facets of building awareness among users of the technology's possibilites, developing manufacturing techniques and marketing. We beleive our liquid inoculant product has the potential to be adapted widely in areas where legumes are inoculated. Not only do our extensive field performance data show its efficacy but we know from our interaction with end users and the consistant interest shown by manufacturers that the product has the potential to be commercialized. Inoculant producers and growers alike can benefit from switching to liquid formulations from solid based carrier materials. Production costs are significantly lower albeit the liquid formulations require more stringent handling during manufacture. Growers benefit from easier handling and inoculation procedures while increasing yield compared to traditional product formulations. Companies in India and elsewhere have already added
these formulations to their product mix.
Publications
- Leary, J.K., Singleton, P.W. and Borthakur, D. 2004. Canopy nodulation of the endemic tree legume Acacia koa in the mesic forests of Hawaii. Ecology. 85(11):3151-3157.
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Progress 10/01/02 to 09/30/03
Outputs
The project's long-range goals are to improve the capability of developing country farmers and legume inoculant producers to capture atmospheric nitrogen through Biological Nitrogen Fixation. Specifically, we aim to 1) develop liquid inoculants for commercial use with field performance that rivals inoculants manufactured with finely ground (300 mesh), neutralized, sterile sedge peat carriers, 2) field test these formulations with a global network of collaborators and, 3) digitize most of the project's technical and training publications that have been developed in the last 25 years. In 2002 reports we described the development and preliminary field-testing of two liquid inoculant formulations. One formulation is based on standard yeast mannitol media and another uses glycerol as a C source. Both formulations are impregnated with polyvinylpyrrolidone (K=30) that promotes cell survival during storage in the formulation and after application to seed. Earlier field tests
showed these formulations outperformed locally produced inoculants in terms of number and total weight of root nodules, seed yield and total seed N. Field trials in 2001 - 2002 continued in this vein but evaluated the liquid formulations against inoculants produced with sterilized Canadian peat carrier. In 2003 we completed these extensive trials and summarized the data. Out network reached 22 collaborators from 18 countries who carried out the trials under a diverse set of environmental conditions. In 42 field observations the average seed yield of our two liquid formulations was 579 kg/ha greater than un-inoculated controls and 398 kg/ha greater than yield of soybean inoculated with a sterile peat based inoculant impregnated with the same strain. There was no appreciable difference between yield of the two liquid formulations: G5 formulation mean yield was 2130 kg/ha and G6 formulation was 2073 kg/ha. The G6 formulation is based on glycerol as a carbon source with 20 g/L pvp K30
added; the G5 uses a combination of mannitol, arabinose and gluconate with pvp. Nodulation measurements followed a similar trend as yields except the G6 formulation produced 200,000 more nodules/ha and slightly more nodule weight than the peat inoculant. The G5 formulation produced slightly less nodule weight and numbers of nodules than the peat. We have scanned, performed OCR and edited another 600 Mb of documents and translated them into Adobe PDF, WORD and HTML files. These include primarily scientific papers but also some parts of manuals and technical bulletins. Nearly all have been inserted into a web and this web should be posted in 2004.
Impacts
From the data generated by more than 5 years of field experimentation, we believe inoculant producers and growers alike can benefit from switching to liquid formulations from solid based carrier materials. Production costs are significantly lower albeit the liquid formulations require more stringent handling during manufacture. Growers benefit from easier handling and inoculation procedures while increasing yield compared to traditional product formulations. Companies in India and elsewhere have already added these formulations to their product mix.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
The project's long-range goals are to improve the capability of developing country farmers and legume inoculant producers to capture atmospheric nitrogen through Biological Nitrogen Fixation. Our more immediate goals are to 1) develop liquid inoculants for commercial use with field performance that rivals inoculants manufactured with finely ground (300 mesh), neutralized, sterile sedge peat carriers, 2) field test these formulations with a global network of collaborators and, 3) digitize and post on the web most of the project's technical and training publications that have been developed in the last 25 years. In 1997-2001 reports, we described the development and preliminary field-testing of two liquid inoculant formulations. One formulation is based on standard yeast mannitol media and another uses glycerol as a C source. Both formulations are impregnated with polyvinylpyrrolidone (K=30) that promotes cell survival during storage in the formulation and after
application to seed. Earlier field tests showed these formulations outperformed locally produced inoculants in terms of number and total weight of root nodules, seed yield and total seed N. Field trials in 2001-2002 that compared these formulations with inoculants produced with sterilized Canadian peat carrier are mostly complete. We employed a network of 22 collaborators from 18 countries to carry out the trials under a diverse set of environmental conditions. We are still waiting for results from several sites. The average yield increase from the two liquid formulations over uninoculated controls was 746 kg soybean seed/ha. The average yield produced by our two liquid formulations was 131 kg/ha greater than the crops inoculated with the Canadian peat based inoculant. There was no appreciable difference between the two liquid formulations (one uses glycerol as a C source the other a combination of mannitol, arabinose and gluconate). Nodulation measurements followed a similar trend.
We have completed scanning, editing and translating into Adobe PDF, MS WORD and HTML files approximately 4 Gb of scientific papers, theses, technical bulletins, and laboratory, extension and field training materials. About 80% of the materials have been inserted into our Web format and design. Several pictorial tutorials on laboratory and inoculation techniques have also been developed. Most materials are in word processing formats that meet the ADA requirements applied to information derived from public institutions. We are still waiting for copyright permission from about 80% of the externally published work derived from the project. We expect to post the bulk of the materials we are processing by the end of 2003.
Impacts
We expect two long run impacts from this project. First, there is a rapid by inoculant producers to switch production methods from solid carrier materials to liquid formulations. Interest is evident by the fact that several firms have contacted us about our results and are evaluating the potential to use liquid inoculant products in their markets. Another long-term impact will be the ready availability of a massive amount of practical information about legume BNF, legume management and inoculant production, quality control and use.
Publications
- Abaidoo, R.C., Keyser, H.H., Singleton, P.W. and Borthakur, D. 2002. Comparison of molecular and antibiotic resistance profile methods for the population analysis of Bradyrhizobium spp. (TGx) isolates that nodulate the new TGx soybean cultivars in Africa. J. App Micro. 92:109-117.
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Progress 10/01/00 to 09/30/01
Outputs
This project's current goals are to 1) develop liquid inoculants for commercial use with field performance that rivals inoculants manufactured with finely ground (300 mesh), neutralized, sterile sedge peat carriers and, 2) digitize and post on the web the vast array of technical and training publications developed in the last 25 years. In earlier reports we described the development and preliminary field-testing of two liquid inoculant formulations. One formulation is based on standard yeast mannitol media and another uses glycerol as a C source. Both formulations are impregnated with polyvinylpyrrolidone (K=30) that promotes cell survival during storage in the formulation and after application to seed. Earlier field tests showed these formulations outperformed locally produced inoculants in terms of number and total weight of root nodules, seed yield and total seed N. Our preliminary results from field trials at multiple sites around the world show that these
formulations perform as well as the best sterile peat based carrier formulations when the same strain (USDA 110) is used to manufacture the liquids and the peat based inoculants. A web design has been developed for the resource materials developed by this project. To date more than 2000 pages of scientific papers, theses, technical bulletins, and laboratory, extension and field training materials have been digitized and edited. All materials are in a downloadable format including word processing formats that meet the ADA requirements applied to information derived from public institutions. We have received copyright permission from about 80% of the externally published work derived from the project. We expect to post the bulk of the materials we are processing by the end of 2002.
Impacts
Given the 6% yield response recorded in our field trials and assuming up to half the world's producers may be interested in adopting liquid inoculant formulations, this technology could result in aggregate yield increases of 1.2 million mt/year worth $360 million U.S.
Publications
- No publications reported this period
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Progress 10/01/99 to 09/30/00
Outputs
The project is developing liquid inoculants for commercial use. The project's rationale is derived from the desire of inoculant producers for a simpler, less costly carrier that supports rhizobial survival during storage and after application to the seed. We developed six generations of liquid formulations by evaluating additives, strains and cell physiological conditioning that enhances cell survival. Only the first two approaches appear to be commercially viable in the short run. Performance of a fifth generation formulation was evaluated in a series of 47 field experiments conducted by 27 collaborators in 16 countries. The fifth generation formulation (G5) increased crop seed yield above local products 68% of the time producing an average seed yield increase of 6%. Nodule number was increased by 20% in 77% of the trials when G5 inoculant was compared to local inoculants. Increased nodule number indicates the G5 inoculant probably supported more viable cells between
seed inoculation and the time roots emerged and colonization and infection by the inoculant began. Shelf life of the formulations with protecting additives was also superior to standard formulations. After 180 days of storage at 25 C, the number of viable cells remained nearly constant for the G6 medium with 20 g PVP/l, yeast mannitol medium (YM) and the G5 medium (Figure 1). When glycerol, FeEDTA or glycerol + FeEDTA + PVP was added to the G6 medium survival was poor and cell numbers declined to levels approaching the washed cells suspended in de-ionized water. Research has shown that carbon or nitrogen starvation at the beginning of the stationary growth phase creates stress resistance in rhizobia cells.
Impacts
Given the 6% yield response recorded in our field trials and assuming up to half the world's producers may be interested in adopting liquid inoculant formulations this technology could result in aggregate yield increases of 1.2 million mt/year worth $360 million U.S.
Publications
- No publications reported this period
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Progress 10/01/98 to 09/30/99
Outputs
The broad objective of this project is to improve agricultural productivity by enhancing legume biological nitrogen fixation. Several approaches address this goal including improving legume management, genetic improvement of Rhizobium/Bradyrhizobium and developing improved inoculant products. Present funding limitations restrict activities only to the specific objective to develop new liquid inoculant product with improved field performance. Summary of Research Output: 1) two prototype liquid media support cell numbers in excess of 1 X 109 mL-1 when stored for six months at 25 C. 2) reduced the rate of cell death after application to seed by 50% compared to earlier media. 3) material cost of the liquid formulations 60% lower than peat carriers. 4) in 42 experimental comparisons obtained from field trials conducted in 16 countries the liquid formulation nodulated legumes more prolifically (freq. = 77%) and increased seed yield 68% of the time compared to local
inoculant products. We developed the liquid inoculant formulation by finding additives that improved cell survival, selecting superior strains and preconditioning cells for survival with oxidative stress. Components of the formulation that appear to promote cell survival after the inoculant is applied to seed are polyvinylpyrrolidone. FeEDTA, and glycerol. Strain genotype contributes about 50% of the variance in formulation performance. Oxidative stress while shown to improve cell survival is not a practical approach for commercial inoculant producers.
Impacts
A survey of producers indicates many are interested in liquid formulations. With the 6 percent yield response in field trials and 50 percent of producers adopting liquid formulations this technology could result in soybean yield increases of 1.2 million metric tons/year worth $360 million U.S. Multiplier effects could raise the net contribution of this research to annual global GNP to more than one billion dollars.
Publications
- Abaidoo, R., Singleton, P., Keyser, H., Borthakur, D. and Dashiell, K. 1999. Distribution and characteristics of Bradyrhizobium spp. nodulating African soybeans. In Highlights of Nitrogen Fixation Research, E. Martinez and G. Hernandez (eds.). Kluwer Academic/Plenum Publishers, New York. pp. 77-84.
- Woomer, P.L., Savala, C.N., Ndakidemi, P.A., Kizza, C. and Nyika, M. 1999. An evaluation of liquid formulation legume inoculants in East and Southern Africa. African Crop Science Conference Proceedings. 4:1-10.
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Progress 10/01/97 to 09/30/98
Outputs
A first generation liquid inoculant formulation was for soybean. Preliminary tests show the formulation performs as well as the best commercial liquid product available today in the world market. A series of experiments evaluated additives to inoculant that provided physical protection to B.japonicum (rhizobia for soybean) cells upon application to seed. Protectants were identified that ameliorated against rapid dessication and the effects of toxic seed exudates on cell survival. Strains of B. japonicum were also identified that exhibited superior survival when inoculated onto soybean seed. This will allow us, in the next phase of research, to exploit the potential synergy between genetic mediated survival and physical protectants and produce an inoculant product with superior field performance. To field test this and future generations of inoculant products, we have developed a network of 22 experimental sites in 17 countries. Results of field trials of this first
generation product should be available in late 1999. Field tests will continue with the next generation inoculant formulation in 1999.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/96 to 09/30/97
Outputs
USAID funds were terminated between April 1997 and January 1998. Outputs in this progress report reflect this funding reduction. Program 1, (Improved Methods for Quality Control of Legume Inoculant) developed a 10-min immunological assay to detect Bradyrhizobium japonicum strains in fermentor broth. The method is being introduced to inoculant producers in the U.S. and abroad to reduce the incidence of contamination in fermetors. Program 2 (Improved Inoculant Formulations) seeks to improve the performance of liquid rhizobial inoculants. We assessed the survival characteristics of rhizobia on soybean seed over time at constant relative humidity (63%) and temperature (25 C). Results indicate that the 2-h period after inoculation is a period of rapid cell death and the rate of decline is a predictor of both longer-term survival and performance of rhizobia inoculated to seed planted in soil. The rapid reduction in inoculant viability may be caused by two factors; rapid and
large reductions in water potential and exposure of the cells to mobile toxic elements released from the seed coat at inoculation. Substances such as Fe EDTA and PVP seem to deactivate toxic substances mobilized from seed coats at inoculation and PVP also acts as an osmoprotectant. An initial formulation containing Fe EDAT, PVP, trehalose, gylcerol in addition to standard media salts and yeast extract improved survival of rhizobia on seed compared to several formulations and water. Data collected from more than 100 farms in Laos, Vietnam and the Philippines demonstrated a high frequency of soil P deficiency. New upland rice varieties from IRRI produced significantly higher yields than unimproved varieties on these when no P-inputs were added to the soil. Results indicate that a new soil management strategy for the upland rice production in the tropics that relies on higher inputs and long term cultivation of these fragile lands may be possible.
Impacts
(N/A)
Publications
- LINQUIST, B.A., SINGLETON, P.W., and CASSMAN, K.G. 1997. Inorganic and Organic Phosphorus Dynamics During a Build-up and Decline of Available Phosphorus in an Ultisol. 1967. Soil Sci. 162:254-264.
- PARVEEN, N. WEBB, D.T., and BORTHAKUR, D. 1997. The Symbiotic Phenotypes of Exopolysaccharide-Defective Mutants of Rhizobium sp. Strain TAL 1145 Do Not Differ on Determinant- and Indeterminant- Nodulating Tree Legumes. Microbiol. 143:1959-1967.
- LINQUIST, B.A., SINGLETON, P.W., YOST, R.S., and CASSMAN, K.G.. 1997. Aggregate Size Effects on the Sorption and Release of Phosphorus in an Ultisol. Soil Sci. Soc. Am. J. 61:160-166.
- HACIN, J.I., BOHLOOL, B.B. and SINGLETON, P.W.. 1997. Partitioning of 14C-Labeled Photosynthate to Developing Nodules and Roots of Soybean. New Phytol. 137:257-265.
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Progress 10/01/95 to 09/30/96
Outputs
Objectives: 1) distribute rhizobial germplasm; 2) improve performance of legume inoculant; 3) increase legume BNF through improved legume management. Distributed more than 800 cultures and research inoculants to facilities in 35 countries. Evaluated inoculant quality in global markets and found more than 50% in developing countries had less than 10 million rhizobia/g. Quality control kits based on ELISA technology were evaluated by producers in seven countries. New packaging technology maintained inoculant quality for periods exceeding 2 y. Continued long term field trials on N and P management of upland legume/rice systems in Asia. Developed liquid inoculant that increases rhizobia survival after application to legume seed above most commercial liquid formulations. Found the greatest rate of decline in rhizobia survival occurs within 2 h after inoculating seed. This rapid population decline is related to: 1) the physiological state of rhizobia at the time of
inoculant manufacture, and; 2) the rapid release of toxic substances from the seed at imbibition. Large molecular weight PVP compounds alleviate some effects of seed released antimicrobial compounds.
Impacts
(N/A)
Publications
- GEORGE, T., LADHA, J. K., GARRITY, D.P. and TORRES, O. 1995. Nitrogen dynamics ofgrain legume-weedy fallow-flooded rice sequences in the tropics. Agron. J. 87:1-6.
- DOBERMAN, A., GOOVAERTS, P. and GEORGE, T. 1995. Sources of soil variation in anacid ultisol of the Philippines. Geoderma. 68:173-191.
- BECKER, M. and GEORGE, T. 1995. Nitrogen fixation response of stem- and root- nodulating Sesbania species to flooding and mineral nitrogen. Plant and Soil. 175:189-196.
- LINQUIST, B. 1995. The role of phosphorus cycling and soil physical properties in governing the bioavailability of phosphorus in a tropical ultisol. Ph.D. Dissertation. Univ. California, Davis. 99p.
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Progress 10/01/94 to 09/30/95
Outputs
Improve and distribute rhizobial germplasm: 1) Sent 500 cultures, 220 inoculant packs requested by inoculant producers, researchers, and farm- groups in more than 40 countries; 2) Identified genes controlling polysaccharide production that affect rhizobial competition for nodule formation. Improve performance of inoculant: 1) New packaging technology improves long-term shelf-life of inoculant products; 2) Efficacy of electron- beam sterilization of inoculant carriers investigated as low-cost alternative to gamma irradiation; 3) Developments in polycolonal antibody kit allow quantification of rhizobia in inoculants and soil. Improve performance of legumes: 1) Research on interaction between soil-aggregate size and residual P-availability will affect soil testing procedures and interpretations for tropical soils; 2) Genetic characterization of rhizobial populations in African soils will affect strategies for legume breeding in Africa; 3) Field tests initiated in Asian
upland soils network in five countries to develop strategies for managing N, P, and organic matter. Technical assistance outreach: Technical assistance, training provided to firms and institutes from 8 African, 7 Asian, 2 Central American countries. Technical information sent to more than 1500 persons/institutes in more than 90 countries.
Impacts
(N/A)
Publications
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Progress 10/01/93 to 09/30/94
Outputs
Objective-Improve and distribute rhizobia germplasm: 1) Sent more than 1500 strains to 50 countries; 2) Described genes in R. spp. controlling mimosine metabolism, polysaccharide production, and heat tolerance. Objective-Improve performance of inoculant: 1) New packaging system developed. System used by local producer; 2) Two new quality control methods (selective media and polyclonal antibody kit for B. japonicum) developed and tested in global network of inoculant producers. Objective-Improve performance of legumes: 1) Quantified effect of N on BNF and C & N partitioning by three field grown legumes to develop better N subroutines for crop growth simulation models; 2) Measured influence of aggregate size and their distribution on immediate and residual P availability (as determined by soil extraction and buffering tests) for tropical soils; 3) Identified root growth characteristics and internal P use efficiency as mechanisms conferring P tolerance to tropical tree
species; 4) Assessed dynamics of B. spp. and B. japonicum populations in W. Africa for nodulation efficiency on freely nodulating soy varieties; 5) Competed site characterization related to soil heterogeneity in an Asian upland soils network. Objective-technical assistance to inoculant producers: 1) Technical assistance and training provided to firms and institutes from more than 15 countries. U.S. Patent serial # 193,394.
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
The University of Hawaii NifTAL (Nitrogen Fixation by Tropical Agricultural Legumes) Center is an interdisciplinary resource institute for biological nitrogen fixation (BNF) technologies. NifTAL receives funding from USAID to promote BNF for sustainable agricultural development through five program areas: 1) genetic technologies to improve the rhizobia/legume symbiosis; 2) methods to monitor microorganisms; 3) environmental database to maximize performance of BNF; 4) regional resource center; 5) assistance to inoculant producers. Activities in each program integrate research, training, outreach, and communications. During this reporting period, NifTAL developed new inoculant production and quality control methods; conducted an impact assessment of BNF technologies in Zambia that revealed BNF has had huge economic benefits for that country; developed BNF management strategies for low-fertility soils; established international collaboration with private and public sector
inoculant producers; provided training for graduate students and developing country scientists and technicians; and provided long-term technical support to international agricultural centers.
Impacts
(N/A)
Publications
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Progress 10/01/91 to 09/30/92
Outputs
The University of Hawaii NifTAL (Nitrogen Fixation by Tropical Agricultural Legumes) Center is an interdisciplinary resource institute for biological nitrogen fixation (BNF) technologies. Under a new agreement with USAID, NifTAL operates under five defined programs: 1) Genetic technologies for improvement of the Rhizobium/legume symbiosis; 2) Development of methods to monitor microorganisms; 3) Environmental database to maximize performance of BNF; 4) Regional resource centers; 5) Assistance to inoculant producers. NifTAL activities in each program include research, training, outreach, and communications. There is a new program focus on meeting the needs of the private sector to develop and market quality legume inoculant products to farmers and natural resource managers. New inoculant production and quality control methods, market assessment, and financial planning services are at the core of this new approach. The Nitrogen Fixing Tree Association has relocated to the
NifTAL headquarters and the two organizations have started to merge some program activities. The recent merge of NifTAL into the Soil Management Collaborative Research Support Program (TROPSOILS) increases the integration of NifTAL technologies and programs into whole systems approaches of responsible soil management.
Impacts
(N/A)
Publications
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Progress 10/01/90 to 09/30/91
Outputs
Improvement of biological nitrogen fixation in legumes, and successful promotionof its use in developing countries, depend on approaches which involve research, education, training and outreach. The research thrust of this project was directed towards defining the environmental factors controlling legume response to inoculation and the fate of inoculum introduced into the soil, and development of a financial analysis program for inoculant production enterprises. Specificity for nodulation and nitrogenfixation was found in several tree legumes, and response to inoculation in soil was highly influenced by the level of indigenous rhizobia and available nitrogen. Education and trainingactivities included graduate and post-doctoral student support in t he U.S. and abroad, and training courses in Thailand, Egypt and Hawaii for developing country scientists, technicians and extension workers. Outreach activities included: 1) distribution of rhizobial germplasm, antisera and
inoculants, 2) information dissemination and communications assistance, 3) assistance with establishment of private inoculant production enterprises, 4) needs assessment for inoculation and technology development, and 5) coordination of efforts with other public and private voluntary organizations.
Impacts
(N/A)
Publications
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Progress 10/01/89 to 09/30/90
Outputs
Improvement of biological nitrogen fixation, and successful promotion of its usein developing countries, depend on approaches which involve research, education, training and outreach. The research thrust of this project was directed towards defining the environmental factors that control legume response to rhizobial inoculation and the fate of the inoculum rhizobia in the soil. Mathematical models were constructed that incorporate the most critical of the factors for predicting symbiotic success and saprophytic competence. Education and training activities included graduate student support and technical training courses in Thailand and Morocco for developing country scientists, technicians and extension workers. The outreach effort of this project included: 1) distribution of rhizobial cultures, antisera and inoculants; 2) information dissemination and communications assistance; 3) assistance with establishment of private inoculant production enterprises; 4) need
assessment for inoculation and technology development; and 5) formation of a consortium with several private voluntary organizations and the Peace Corps.
Impacts
(N/A)
Publications
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Progress 10/01/88 to 09/30/89
Outputs
Ecological models that define response of 9 different legumes to rhizobial inoculation were developed. Background populations of native rhizobia, soil N status and the legume demand for nitrogen were found to be the most important factors contributing to inoculation success/failure. Other mathematical models were developed for persistence and dispersal of 18 strains belonging to 6 species of Rhizobium and Bradyrhizobium. Among environnmental factors that affect persistence of these species, temperature, rainfall and soil pH had the most significant influence. The rate of dispersal was related most to site slope, rainfall and the size of the source population. In another effort, the influence of temperature and applied N on rhizosphere colonization, nodulation and rhizobial interstrain competition was examined along an elevational transect on Maui. Site differences and applied N had asignificant effect on nodule number and mass and above ground biomass, but no
significant effect on rhizosphere numbers or nodule occupancy by different strains.
Impacts
(N/A)
Publications
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Progress 10/01/87 to 09/30/88
Outputs
An IBM Compatible Computer Program, MPNS, has been developed for analyzing rhizobial enumeration data. Rhizobial populations in the rhizosphere of various tropical grasses varied significantly. Persistence of eighteen rhizobial strains introduced into diverse tropical soils was significantly different for strains and different environments, with organic carbon, water holding capacity and temperature having the greatest influence on survival of introduced Bradyrhizobium japonicum and R. leguminosarum, bvs. trifolii and viceae. Available soil N was found to have differential effects of nitrogen fixation by soybean, bushbean and chickpea. In solution culture studies, bushbean assimilated NO3 more readily than soybean. N2 fixation was undetectable in bushbean at NO3 concentrations that allowed the process to function in soybean.
Impacts
(N/A)
Publications
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Progress 10/01/86 to 09/30/87
Outputs
We evaluated Rhizobium germplasm for 10 species of nitrogen-fixing tree legumes;screened 10 species of tree legumes for Ureide transport; conducted inoculation response trials on four grain legumes at 2 sites; established persistence trials of 18 strains belonging to 6 Rhizobium spp. at 14 sites and evaluated the nitrogen fixing capabilities of mutants with copies of their nif genes inactivated by Tn-5 mutagenesis; over 200 most probably number (MPN), data points were analyzed for a test of internal cohesion. Results indicated that the soil type had no significant effect on precision of MPN counts, but plant species did: a quantitative model to describe the ecological determinants of native populations of Rhizobium spp. has been developed; a quantitative model of plant response to inoculation with Rhizobium spp. has been developed; and soybean varieties from 4 maturity groups, and their non-nondulating isolines were tested for nitrogen fixation and phenological
development at 3 sites along an elevational transect.
Impacts
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Progress 10/01/85 to 09/30/86
Outputs
Major conclusions of scientific research are: A low cost steel fermentor (141) designed at NifTAL proved appropriate for producing Rhizobium and other industrial microorganisms; Bacillus thuringensis and Lipomyces starkii are compatible with rhizobia co-cultured in peat; Peat had a greater capacity for adsorbing and inactivating streptomycin than did Indian coal; Ureides were found in xylem sap, leaf and stem extracts of 35 species of leguminous trees, but no direct relationship with nitrogen fixation could be discerned; the use of value for atom % N for measuring N(2) fixation in leucaena by the natural abundance method was unsuitable; The INLIT trials revealed that of 8 legume crops used in international network of field trials; 68% showed increase in yield by 59 kg/ha due to inoculation; R. fredii were found to be the dominant nodule occupants on soybean grown in soils from a soybean growing region of China. New isolates proved highly effective on New-World
cultivars; In split-root systems, suppression of nodulation by a secondary inoculum was related to the effectiveness of the primary inoculum; The pea symbiotic plasmid functioned in R. trifolii and R. leguminosarum backgrounds, but not in R. fredii; All R. fredii isolates tested were able to use ethanol as a sole source of carbon and energy.
Impacts
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Progress 01/01/85 to 09/30/85
Outputs
Conclusions of the major scientific experiments are: 1) At least 10,000 rhizobia per seed were needed to ensure adequate nodulation of chick pea in soils free of chick pea rhizobia; 2) Nitrogen was transferred from a legume (soybean) to a non-legume (corn) via VA mycorrhizal fungi; 3) Inoculation responses of soybean were likely when there were four native rhizobia/g soil or less and rare if the native population rose about 100 cells/g soil; 4) 15N depleted material can be used in both field and greenhouse studies to measure nitrogen fixation by herbaceous and woody legumes; 5) Greenhouse and field studies with soybeans revealed that low phosphorus availability reduced nodulation and nitrogen fixation by reducing plant growth and photosynthesis; 6) Field experiments designed to assess the effect of intercropping on nitrogen fixation by cowpeas showed that although intercropped maize took up more soil nitrogen than intercropped cowpea, the relative amount of nitrogen
derived by cowpeas from fixation was similar in mono and intercropped systems. The following activities were also carried out by NifTAL during this reporting period: the NifTAL Regional Resource Center in Bangkok co-sponsored training courses for agricultural extension workers in the Philippines (with North Carolina State University), Bangladesh (with Bangladesh Agricultural Research Council) and Thailand (with the Thailand Department of Agricultural Extension); 29 INLIT experiments were dispatched, and 25 completed tests were returned.
Impacts
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Progress 01/01/84 to 12/30/84
Outputs
The NifTAL Germplasm Resource Center was increased slightly, bringing the total number of strains in the collection to 1774. A computerized data base management system, designed to store and retrieve strain data, was used to generate a strain catalogue. This catalogue is now available on request. Antisera and inoculants continued to be produced for 14 targeted legumes and sent out on request. Additional INLIT trials have been distributed. A regional meeting for Latin American INLIT co-operators was held in Brazil. Composite data analysis for completed trials was begun. Five training courses were held in 4 countries, training a total of 105 persons representing 25 countries. The NifTAL Rhizobium Manual is in publication. Three issues of the BNF bulletin were published and 5700 copies distributed. Eight intern trainees from eight countries studied at NifTAL for a total of 10 months of training. A Regional Resource Center, staffed by NifTAL professionals, was
established in Bangkok, Thailand to provide services and training in BNF technology for south and southeast Asia.
Impacts
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Progress 01/01/83 to 12/30/83
Outputs
1) The NifTAL Germplasm Resource Center has increased its collection to 1800 strains of Rhizobium including isolates from legumes, and most recently wing bean. Computerized strain profiles and a comprehensive catalog are 90% completed. 2) A serum bank now stocks antisera and gamma globulins for the identification of agglutination of 42 Rhizobium strains selected for the International Network of Legume Inoculation Trials (INLIT). 3) Inoculants are produced for a total of 14 targeted legumes. 4) Several INLIT cooperators were visited by NifTAL staff and data is being processed for those "A" experiments that have been completed. 5) Four training courses were held in 4 countries, training a total of over 50 persons representing 18 countries. 6) Revision of the NifTAL Rhizobium Technology Manual has been completed based on reviews and field testing. Potential publishers have been contacted. 7) Three issues of the BNf Bulletin were produced and over 4500 issues were
distributed 8) Ten intern trainees from 7 countries studied for a total of 22 months at the NifTAL site.
Impacts
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Progress 01/01/82 to 12/30/82
Outputs
The NifTAL Germplasm Research Center continues to house over 1,500 strains of Rhizobium including isolates from various economically important grain legumes, pulses, forages, and tree legumes. A serum bank now stocks antisera and gamma globulins for the identification by agglutination of all 36 Rhizobium strains initially selected for INLIT. Inoculants are produced for all 12 original legumes. Several INLIT cooperators were visited by NifTAL staff and many "A" experiments have been completed and INLIT "A" data has been analyzed. "B" and "C" experiments are in process. One 6-week training course in Rhizobium technology was undertaken; the Thailand course trained 17 people from 8 countries. The NifTAL Handbook of Rhizobium Technology was revised and is being reviewed by outside experts prior to publication. Three issues of the BNF Bulletin were produced. Audio visual modules on Nitrogen-Fixing Trees of Maui; What is NifTAL; and Pasture Legumes and Nitrogen
Fixation were produced. Ten intern trainees from 10 countries studied for a total of 204 months at the NifTAL site.
Impacts
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Progress 01/01/81 to 12/30/81
Outputs
The NifTAL Germplasm Research Center has over 1500 strains of Rhizobium. The collection contains isolates from various economically important grain legumes, pulses, forages, and tree legumes. Identification of Rhizobium in oven dried nodules by immunofluorescence and agglutination was undertaken. A serum bank was started and stocks antisera and gamma globulins for the identification by agglutination of all 36 Rhizobium strains initially selected for International Legume Inoculation Trials. There were requests for 21 antisera. Inoculants are produced for all 12 original legumes. Several INLIT cooperators were visited by the NifTAL staff and many "A" experiments have been completed. Procedures were developed for handling INLIT A data; materials were sent to all cooperators explaining the specific procedures. "B" experiments, strain testing have been produced. Two, 6-week training courses in Rhizobium technology were undertaken; the West Malaysia course trained 14
people from 9 countries; the Mexican course trained 13 people from 9 countries. Other activities included: translation of the NifTAL training manual and INLIT trials booklet into Spanish; expansion of the training manual into a Handbook of Rhizobium technology. Three issues of the BNF Bulletin were produced. Three A-V units were produced on Rhizobium technology and translated into Spanish and Mandarin; three more units are in development.
Impacts
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Progress 01/01/80 to 12/30/80
Outputs
A new legume inoculant production system has been devised in which high density rhizobial broths are cultured in shaken flasks and diluted (1,000-fold) prior to incorporation in a peat carrier. Water is as satisfactory as three other diluents tested. Both fast- and slow-growing rhizobia, and peats of different origins (three tested) can be used. The (International Network of Legume Inoculation Trials (INLIT) was extended to 95 institutions in 34 countries that are implementing 202 trials with the 14 agriculturally important tropical legumes offered. Significant inoculation responses occurred with some legume species at some locations. Fourteen three-strain combinations of antigenically distinct rhizobia of diverse origines that are fully effective with the INLIT legumes have been developed. Acid tolerance was a stable, heritable characteristic in the strains tested. Rhizobium for soybeans and peanuts have generally greater tolerance of low pH than their
respective plant hosts. Many strains of rhizobia tested are tolerant of such low P levels that it is unlikely that they would ever be P-limited under field conditions. Demonstration that inhibition of nodulation in the presence of nitrates can be overcome using exceedingly high numbers of rhizobia, as well as the demonstration of strain variation in the ability to nodulate in the presence of nitrates, highlights the complexity of the control of nodulation in the presence of free nitrate.
Impacts
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Progress 01/01/79 to 12/30/79
Outputs
Rhizobium Germplasm Resource at NifTAL now exceeds 2,000 strains. Reported lossof viability in cultures preserved by the porcelain bead method is being investigated and lyophilization adopted for selected strains. A serum bank for 36 widely used strains for tropical legumes has been established. Effectiveness of strains of Rhizobium for Vigna aconitifolia, Vigna umbellata, Pachyrrhizus erosus, Dolichos lablab, Voandzeia subterranea, Phaseolus lunatus, Desmodium canum and Glycine wightii was assessed. Strain testing indicated that cultivar-specific recommendations are necessary for Phaseolus vulgaris, Vigna unguiculata, Cajanus cajan and Arachis hypogaea. Among strains of R. leguminosarum selected for nitrogen fixing effectiveness with Lens culinaris, Hawaii 5-0 was highly competitive against native soil rhizobia in an Ustic Humitropept. An enzyme-linked immunosorbent assay was developed to identify strains of Rhizobium in culture and in nodules of Lens culinaris.
The method offers advantages over other used techniques. High temperature damaged inoculants shipped to tropical locations. Maximum temperature experienced by an inoculant was 44 degrees C. Temperature sensitivity was greatest in R. leguminosarum and R. phaseoli, and least in R. japonicum and cowpea-type rhizobia. Scientists and administrators convened in Hawaii to plan an international Network of Legume Inoculation Trials.
Impacts
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Progress 01/01/78 to 12/30/78
Outputs
More than 250 strains were added to the Rhizobium (RH) collection. Screening ofthe effectiveness of RH strains on target legumes identified several cowpea-type strains tolerant to acid soils and two highly competitive lentil strains. Field trials involving 11 soybean RH strains were completed on Hawaii with two strains (TAL 377 and 379) being superior. Field trials with cowpeas revealed that large levels of introduced cowpea inoculum were required to replace in part the less effective native RH strains. Symbiotic nitrogenase activity of field grown Pachyrhizus erosus, yam bean, was found to be diurnal. Testing of several lines of the winged bean, Psophocarpus tetragonolobus, revealed large differences in yield parameters. Effectiveness of six cowpea RH strains on winged bean did not differ significantly. Soil fertility trials with soybeans (SB) and cowpeas (CP) revealed that external P requirements of SB increased as soil N decreased; CP was more efficient at
fixing N at low soil P than SB; and at optimum soil fertility levels, SB fixed more N than CP. Trials with several target legumes revealed positive responses to pH 5.0-5.3 were due to reduced A1 and, above this pH, to mineralization of soil N.
Impacts
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Progress 01/01/77 to 12/30/77
Outputs
Rhizobium Strain Collection now totals over 650 strains. Effective strains for peanut, cowpea, mungbean (Vigna radiata), adzuki bean (V. angularis), tepary bean (V. acutifolius), chick pea (Cicer arietinum), Bambarra groundnut (Voandzeia subterranea), winged bean (Psophocarpus tetragonolobus), Mexican yam bean (Pachyrhizus erosus), and Leucaena leucocephala have been selected. Grain legumes intercropped with maize did not affect maize yields, but forage legumes reduced maize yields. Legume yields were low resulting in little or no increase in total production per unit area. Yields of wheat following maize-legume mixtures were higher than wheat following maize alone. More residual N was present where maize was intercropped with Leucaena or fertilized with Leucaena leaf instead of urea. Legumes grown across a lime gradient on a high-aluminum Humoxic Tropohumult showed a broad range of tolerance to soil acidity. Stylo grew well in acid soil: soybean, winged bean,
lima bean responded primarily to lime rates which reduced available AlRGDT to moderate levels; other legumes required high lime rates to provide increased levels of available calcium. Liming increased the rate of mineralization of nitrogen and other nutrients from organic matter. Outstanding winged beans tested varieties were a long podded selection from the Philippines and "Mardi" from Malaysia.
Impacts
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Progress 01/01/76 to 12/30/76
Outputs
The germplasm collection of Rhizobium strains now contains over 700 cultures, ofwhich approximately 150 strains have been tested to some degree. Growth room, greenhouse, and field trials are in progress to evaluate the effectiveness of selected Rhizobium strains for tropical legumes with emphasis on soybean, cowpeaand mungbean. Rhizobium japonicum mutants from the University of Wisconsin showed high levels of nitrogen-fixing activity in growth room and preliminary field trials, but did not result in substantially higher levels of N-fixation activity in a larger field experiment. Three forage-type legumes were composed with two grain-type legumes as intercrops in corn. Stylosanthes guayanesis severely inhibited; Desmodium intorum, Leucaena leucocephala, and soybean slightly inhibited; and cowpea had no effect on corn growth. Legume growth was better in association with brachytic than medium height corn, apparently due to the greater shading by the latter.
Impacts
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Progress 01/01/75 to 12/30/75
Outputs
Nodules were collected from approximately 45 native and introduced Nigerian legumes, and seeds were obtained of 15 varieties of 10 grain legume species fromNigeria for testing in Hawaii. Rhizobium strains were isolated from approximately 25 Nigerian legumes and 50 Hawaii-grown legumes. Testing facilities have been set up to screen Rhizobium strains for host range and tolerance to stress factors.
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