Progress 07/01/07 to 06/30/09
Outputs
OUTPUTS: Prior to his death, on May 9th, 2009, Dr. Peter Graham was conducting research to determine the factors involved in successful nodulation and enhanced nitrogen fixation by Rhizobia species on a variety of legumes, including edible bean and native prairie legumes, particularly Dalea purpurea, purple prairie clover. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Prairie legumes are an important component of sustainable native prairie restoration because they provide long-term nitrogen inputs to prairie grass ecosystems. Successful nodulation and enhanced nitrogen fixation capacities greatly enhance the early success of prairie restoration projects.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project considers plant and rhizobial variation affecting nodulation and nitrogen fixation in crop, prairie and pasture species in Minnesota. In 2008, this emphasized the importance of inoculation and inoculation method in prairie establishment and function, evaluated in a three year old prairie at the Becker Sandplain experiment station. Uninoculated plots or plots with relatively low numbers of rhizobia applied via seed inoculation, showed very little change in soil microbial biomass N over the three years from prairie establishment. However, where peat- or clay- based granular inoculants were used and Rhizobium application rate increased,, microbial biomass N increased from 6.96 to 9.57 ppm over the same period. Similarly, the number of legumes per plot and biomass of legumes per quadrat in the third year after establishment varied from 68 and 1.21g in the seed inoculated treatment to 133 and 5.15 g in the granular clay-based treatment. Over the course of the study there was marked decrease in weed species and corresponding development in prairie forbs and grasses. 315 Rhizobium strains trapped from soil in these prairie areas using Dalea purpurea as host, and fingerprinted using BoxA1R-PCR could be divided into eight clusters, with four of these showing similarities to the inoculant strains used. While only 2% of strains recovered from the seed inoculated treatment showed homology to the inoculant rhizobia, 92-100% of strains in the granular peat and wheat-cover crop treatments showed homology to these strains. Further, when strains taken at random from those trapped from soil were tested for ability to enhance the growth of Dalea purpurea, plants inoculated with strains subsequently identified as similar to the inoculant cultures achieved an average weight of 130.6 mg plant-1 whereas strains that did not identify with the inoculant strains yielded only 64.9 mg plant-1. Results with slow-growing rhizobia from Desmodium canadense were less satisfactory, with only 13.7% of the strains identifying with the inoculant strains used. To better understand the influence of host trapping on the diversity of rhizobia recovered from soil, we have developed a culture medium permitting direct recovery of large numbers of rhizobia from soil, and so the comparison of diversity in host-plant trapped vs directly isolated rhizobia. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our laboratory continues to be one of the few in the USA that produce and distribute rhizobial inoculants for the legumes used in prairie restoration and for lesser pasture legumes such as Kura clover, In 2008 we supplied such cultures to research workers in university projects, USDA, restoration projects, MNDOT, the Mdewakaton Sioux community, and to a range of prairie practitioners, seed producers and farmers. Ability to supply and use such inoculants has also resulted in numerous requests for information, and in speaking engagements in Minnesota and beyond. A consequence of the increased request for inoculants is that we have needed to identify and prove a peat source in Northern Minnesota that could be used for additional inoculant supply, as needed
Publications
- Graham, P.H. (2008). Ecology of the root-nodule bacteria of legumes. IN: Nitrogen fixation: Origin, applications and research progress. Volume 3. Leguminous nitrogen-fixing symbioses. M.J.Dilworth et al.(eds.) Springer, Dordrecht, 23-58.
- Mangan, M., Sheaffer, C., Wyse, D., Graham, P.,Tschirner, U. and Weisberg, S.(2008) Native perennial grassland species for bioenergy: Monocultures vs Mixtures. Forage Focus pp 19-20.
- Graham, P.H., Beyhaut, E. and Tlusty, B. (2008) Improved methodologies for the inoculation of prairie legumes in roadside/revegetation settings. MNDOT Final Report,79 pp.
- Graham, P.H. (2008) Soil Biology with an emphasis on symbiotic nitrogen fixation. In: D. Emerich and H.B. Krishnan (eds.) Nitrogen fixation in crop production. ASA/CSSA/SSSA publishers, Madison. In press.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This project emphasizes plant genotype and rhizobial variation affecting nodulation and nitrogen fixation in crop, prairie and pasture species. Inoculation of bean and soybean is of increasing interest to farmers in the Central Lakes region of Minnesota following demonstration of dramatic yield increases with inoculation, and with increases in the cost of fertilizer N probable. Farmers want liquid inoculants, to inoculate in advance of sowing, and to combine inoculation with fungicide seed treatment. This is feasible with soybean, but does not work with beans. Studies in this area continued in 2007 with striking survival responses to inclusion of trehalose in inoculant broths, and with methyl cellulose as sticker. Good survival on seed has been obtained up to 30 days after inoculation, but not when inoculant and fungicide are in contact. Because bean cultivars are commonly weak in nodulation and N2 fixation we have collaborated with Ken Grafton at NDSU to breed locally
acceptable dark red kidney bean lines with enhanced ability to fix nitrogen. BC-F4 populations screened in 2007 identified four lines outyielding the agronomically acceptable parent Montcalm by more than 50% when plants were totally dependent on nitrogen fixation for growth. Hydroponic systems developed earlier have been used to contrast bean varieties in tolerance to acid and low P stress. RNA extracted from lines differing in low P tolerance is being used for genomic studies in beans. Plants are being grown to identify common responses in lines tolerant to low P and pH stresses. Studies on the rhizobia of prairie legumes have identified strains with chromosomal characteristics of R.etli but with much wider host range. Fast growing rhizobia for Robinia and Psoralea have been identified and overlap in host range with isolates from Dalea and Desmanthus. Studies are underway to characterize these strains using nod and nif probes, as well as probes designed from genes important both for
the growth of rhizobia and for nodulation of prairie legumes. We are also actively exploring evidence for genetic interchange in prairie soils. Our program continues to be essentially the only one involved with the nodulation and nitrogen fixation of prairie legumes in the central USA, and supplies inoculant rhizobia to a range of seed producers and restoration activities.
PARTICIPANTS: Elena Beyhaut and Maina Martir, Graduate Students Pedro Balatti, Visiting Post Doctoral Fellow, Universidad Mar de Plata Collaboration in RNA extraction from lines differing in low P tolerance Michelle Graham, USDA-ARS Ames IA Collaboration in primer design and genomics Gina Hernandez, Nacional University, Cuernavaca, Mexico Carrol Vance, USDA-ARS St Paul Collaboration on Gene expression in beans exposed to low P supply Maggie Mangan, Agronomy.
TARGET AUDIENCES: Inoculant manufacturers and farmers interested in inoculant application Restoration practitioners and people interested in restoration of natural landscapes. Prairie areas maintaintained at the Becker Sandplain Experiment Station serve to show restoration and inoculation principles to target audience
Impacts
Rhizobia identified by this project continue to be important as inoculant cultures for beans and prairie legumes, with UMR1899 used for bean inoculation in Brazil and Canada. Inoculants for prairie legumes, identified through a project with MNDOT are being supplied to seed producers and restoration experts. Similarly we have been a source of high N-fixing germplasm for breeding programs in the USA and Argentina. Our experience with inoculation of legumes and especially in prairie legume inoculation led to invitations to speak at national and international meetings in 2007. These included a)Becker Underwood Collaborators Meeting, St Joseph, Mo March 5-7th 2007. Invited presentation " Native legumes and their rhizobia" b) Reunion Latino Americana de Rhizobiologia, Los Cocos, Argentina. March 25-29, 2007. Invited presentation. " Leguminosas indigenas y sus rhizobias: Cuestiones en restoracion y la ecologia de restoracion". c) North American Conference on Symbiotic
Nitrogen fixation, Milwaukee, WI, July 10-15, 2007. Invited presentation "Native legumes and their rhizobia: Issues in biogeography, restoration and restoration ecology. The Rhizobium Research Laboratory website (http://www.Rhizobium.umn.edu) continues to draw attention
Publications
- Graham, P.H. (2007).Ecology of the root-nodule bacteria of legumes. In: Nitrogen fixation: Origin, applications and research progress. Volume 3. Leguminous nitrogen-fixing symbioses. W.E.Newton et al., (eds.) Kluwer Academic Press, Dordrecht, In press(December, 2007)
- Martir, M.C., Tlusty, B., van Berkum, P. and Graham, P.H. (2007) The genetic diversity of rhizobia associated with Dalea purpurea Vent. in fragmented grasslands of West-Central Minnesota. Can. J. Microbiol. 53, 351-363
- Grange, L., Hungria, M., Graham, P.H. , et al. (2007) New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil. Soil Biol. Biochem 39, 867-876.
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Progress 01/01/06 to 12/31/06
Outputs
This project considers plant and rhizobial variation affecting nodulation and nitrogen fixation in crop, pasture and prairie plants of significance in Minnesota. Liquid inoculant formulations used for soybean fail to protect bean and other fast-growing rhizobia after their application to the seed. Improvement in rhizobial survival on seed has been achieved by inclusion of malt extract in the fermenter medium; by addition of modified dilution fluid to the inoculant, and by use of methyl cellulose as sticker. We have had only limited success in overcoming effects of seed-applied pesticides on inoculant rhizobia. Studies with prairie rhizobia added to wheat seeds show good early-growth benefits with specific wheat cultivars and the potential for both crop benefit from inoculation , and easier prairie-legume inoculant establishment using inoculated wheat as a cover crop.The range of prairie legumes for which inoculant quality rhizobia is available is being extended to
include Robinia, Baptisia and Psoralea. Backcross progeny from the cross between the high nitrogen fixing bean line EL34 and the locally adapted Montcalm show substantial differences in growth and yield when dependent on inoculation with rhizobium. Studies on the rhizobia associated with a range of prairie legumes show overlap in their host range, and the dominance of a limited number of genotypes in each environment. Unexpectedly R.etli - like rhizobia have been recovered from both Dalea and Desmanthus. This could be the result of genetic interchange between rhizobia in the soil, and warrants study on the evolution of these organisms. Fragmentation in the prairie environment has been shown to influence genetic richness of Dalea rhizobia in these areas, but not Shannon-Weaver diversity measures. A hydroponic culture system has been developed for evaluatiuon of differences in bean host and Rhizobium tolerance of acid pH, low soil P and other environmental stresses, and good nodulation
shown by some cultivars to a pH of 4.5.
Impacts
Our laboratory continues to be one of few in the US that produce and distribute rhizobial inoculants needed in prairie restoration. Inoculated wheat used as a cover crop should facilitate establishment of these legumes, while the possibility that wheat crops might benefit from inoculation is also highly significant. Improvement of bean yield potential through selection for enhanced nitrogen fixation and for stress tolerance would be of significance not only in the USA, but in many developing regions of the world.
Publications
- Beyhaut, E., Tlusty, B., van Berkum, P. and Graham, P.H. (2006) Rhizobium giardinii is the microsymbiont of Illinois bundleflower (Desmanthus illinoensis (Michx.) MacMillan in midwestern prairies. Can. J. Microbiol. 52, 903-907.
- Grossman JM, Sheaffer C, Wyse D, et al. (2006) An assessment of nodulation and nitrogen fixation in inoculated Inga oerstediana, a nitrogen-fixing tree shading organically grown coffee in Chiapas, Mexico. Soil Biol. Biochem. 38, 769-784.
- Hungria, M., Campo, R.J., Mendes, I.C. and Graham, P.H. (2006). Contribution of biological nitrogen fixation to the N nutrition of grain crops in the tropics: The success of soybean (Glycine max L. Merr.) in South America. In: Focus on Plant Agriculture 1. Nitrogen nutrition in plant productivity. R.P.Singh et al. (Eds.) Studium Press, Houston TX, pp 43-93.
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Progress 01/01/05 to 12/31/05
Outputs
This project emphasizes plant genotype and rhizobial variation affecting nodulation and nitrogen fixation in crop, prairie and pasture species. Inoculation of bean and soybean is of increasing interest to farmers in the Central Lakes region of Minnesota following demonstration of dramatic yield increases with inoculation, and with increases in the cost of fertilizer N probable. Farmers want liquid inoculants for ease of application, to inoculate well in advance of sowing, and to comine inoculation with fungicide seed treatment. This is feasible with soybean, but current soybean formulations do not work on beans. In formulation studies with bean rhizobia a modified dilution fluid/polyvinylpyrrolidine-based inoculant has permitted survival of bean rhizobia up to 8 days after inoculation;, with R.tropici more tolerant of dessication stress than is R.etli. Because bean cultivars are commonly weak in nodulation and N2 fixation we have collaborated with Ken Grafton at NDSU
to breed locally acceptable dark red kidney bean lines with enhanced ability to fix nitrogen. Advanced glasshouse of seed lines is under way; further field testing will be done in 2006. Inoculation response with the dual forage/grain legume Desmanthus illinoensis has been striking, but with several unexpected results. Thus a) regrowth under Minnesotan conditions was only obtained with inoculated plants, and b) The organism responsible for fixation with Desmanthus has been identified as R..giardinii, previously only recovered from ineffectively nodulated bean plants in France.
Impacts
Study on the inoculation of prairie legumes continues. A new planting compares different inoculant formats, including the use of winter wheat as a cover crop to deliver prairie rhizobia. Fragmentation effects have been shown in the diversity of rhizobia recovered from Dalea spp at 10 sites around Hoffman/Kensington, MN. The effect of fragmentation on the diversity of Dalea plants in these remnant prairies is also being studied using AFLP. Hydroponic systems have been developed to study response of inoculated bean cultivars to acid , P, Mn and Al stress, and to follow changes in stress response.
Publications
- Graham, P.H. 2005. Practices and isssues in the inoculation of prairie legumes used in revegetation and restoration. Ecol. Restor. 23, 186-194.
- Grossman, J.M., Sheaffer, C.C., Wyse, D, and Graham, P.H. 2005. Characterization of slow growing root nodule bacteria from Inga oestediana in organic coffee ecosystems in Chiapas, Mexico. Appl. Soil Ecol 29, 236-251
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Progress 01/01/04 to 12/31/04
Outputs
Inoculation of bean and soybean is of increasing interest to farmers in the Central Lakes region of Minnesota following the demonstration of dramatic yield increases with inoculation. However farmers would prefer preinoculated and fungicide-treated seed. This appears feasible with soybean, less likely with beans where current inoculant formulations do not appear to adequately protect the rhizobia. Many bean cultivars also appear weak in nodulation and N2 fixation. Because of this we have collaborated with Ken Grafton at NDSU in the development of locally acceptable dark red kidney bean lines with enhanced ability to fix nitrogen. F2 backross seed from four distinct crosses are currently being multiplied and will be ready for field evaluation in 2005. Very marked inoculation response was obtained with the dual forage/grain legume Desmanthus illinoensis at the Becker Sandplain experiment station in 2003. In summer 2004 regrowth from these plants was only obtained in the
inoculated treatments. Surprisingly, the organism responsible for fixation with Desmanthus has been identified using rRNA gene sequence analysis as R..giardinii, previously only recovered from ineffectively nodulated bean plants in France. A major study on the inoculation of prairie legumes has been completed, with nitrogen fixation by the prairie legumes involved a major factor in prairie establishment. A new study comparing inoculant formats, and with winter wheat used as a cover crop to deliver prairie rhizobia, is being planted. The study of fragmentation effects on the diversity of prairie legumes and their rhizobia continues at 10 sites in the area of Hoffman/Kensington, MN. So to does a study of the diversity of bean rhizobia in Hidatsa and Double Ditch Indian gardens.
Impacts
Diversity experiments with the rhizobvia of beans and Dalea are close to completion, while the RRL website has been updated to service groups interested in the field of inoculation.
Publications
- Hungria, M., J.C. Francini, R.J. Campo, and P.H. Graham. 2004. The importance of nitrogen fixation to soybean cropping in South America. In: D. Werner (Ed.) Nitrogen fixation research: Origins and Progress. Vol 7. In Press.
- Tlusty, B., J.M. Grossman, and P.H. Graham. 2004. Selection of rhizobia for prairie legumes used in restoration and reconstruction programs in Minnesota. Can. J. Microbiol. In press.
- Bernal, G.R., B. Tlusty, C. Estevez de Jensen, P. van Berkum, and P.H. Graham. 2004. Characteristics of rhizobia nodulating beans in the Central region of Minnesota. Can. J. Microbiol. In press.
- Tlusty, B., P. van Berkum and P.H. Graham. 2004. Characteristics of the rhizobia associated with Dalea spp. in the Ordway, Kellogg-Weaver-Dunes and Hayden prairies. Can. J. Microbiol. In press.
- Graham, P. H. 2004. Nitrogen fixation: symbiotic. In: Sylvia, D.M. et al (eds.) Principles and applications of soil microbiology. Pearson/Prentice hall Second edition, pp. 405-432.
- Graham, P.H., M. Hungria, and B. Tlusty. 2004. Breeding for better nitrogen fixation in grain legumes: where do the rhizobia fit in? Crop Management Electronic Manuscript ISSN 1543-7833. March 2004.
- Hungria, M., I.C. Mendes, M.F. Loureiro, J.C. Rubens, and P.H. Graham. 2004. Inoculant preparation, production and application. In: D. Werner (Ed.) Nitrogen fixation research: Origins and Progress. Vol 7. In press.
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Progress 01/01/03 to 12/31/03
Outputs
This project evaluates plant genotype and rhizobial variation affecting nodulation and nitrogen fixation in crop, prairie and pasture species. Inoculation of bean and soybean is of increasing interest to farmers in Central Minnesota. Farmers want inoculants that can be applied some time before planting, to use liquid rather than peat based inoculants, and to apply inoculants to fungicide treated seed. In experiments with soybean, inoculation 10-15 days before sowing, or fungicide seed treatment lowered numbers of rhizobia alive at sowing, but not sufficiently to affect a yield increase of as much as 60% under favorable sowing conditions. Marked strain responses were also shown. Very marked inoculation response was also obtained with the dual forage/grain legume Desmanthus illinoense at the Becker Sandplain experiment station. Monitoring of small grass prairies established at the Becker site continued in 2003. Prairie areas established in 1999 and 2000 are increasing
grass/legume dominant, with notable increase in microbial biomass N, soil respiration, and soil pH. For Dalea spp. essentially all rhizobia recovered were from the inoculant supplied. For the second year running the prairie legumes studied differed in %NDFF (estimated using δ15 N from 40 to 100%. Soil solution experiments have been used to identify bean cultivars tolerant of low pH, low P levels and Mn and Al toxicity effects when dependent on N2 fixation for growth. Diversity experiments with the rhizobia of beans and Dalea are close to completion, while the RRL website has been updated to service groups interested in the field of inoculation.
Impacts
Diversity experiments with the rhizobvia of beans and Dalea are close to completion, while the RRL website has been updated to service groups interested in the field of inoculation.
Publications
- Beauregard, M.S., P. Seguin, C.C. Sheaffer, and P.H. Graham. 2003. Characterization and evaluation of North American Trifolium ambiguum-nodulating rhizobia. Biol. Fertil. Soils. 38, 311-318.
- Graham, P.H. 2003. Nodule formation in legumes. In: The desk encyclopedia of microbiology. ISBN 0-12-621361-5, pp 713-722.
- Graham, P.H., A.E. Hall, and D.P. Coyne.(Eds). 2003. Contributions and impacts of the Bean/Cowpea CRSP project 1982-2002. Field Crops Res. 82, 79-242.
- Graham, P.H. et al. 2003. Addressing edaphic constraints to bean production: the Bean/Cowpea CRSP project in perspective. Field Crops Res. 82, 179-192.
- Graham, P.H. and C.P. Vance. 2003. Legumes: Importance and constraints to greater utilization. Plant Physiol. 131, 872-877.
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Progress 01/01/02 to 12/31/02
Outputs
This project evaluates plant genotype and rhizobial variation affecting nodulation and nitrogen fixation in crop, prairie and pasture species. Indigenous rhizobia recovered from bean grown in soils from Central Minnesota were evaluated for tolerance to the common seed treatments captan and streptomycin. Surprisingly no indigenous strain was resistant to these antibiotics, though the R.tropici UMR1899 was highly tolerant. This is perhaps a reason for its success as an inoculant strain in a region where chemical seed treatment is common. When other R.tropici Type IIA and IIB strains were evaluated only the type IIB strains proved tolerant to these seed treatment chemicals. Experiments are being undertaken to determine differences in the nodulation patterns of chemically treated and untreated bean seeds. 71% of the rhizobia recovered from soils in this region belong to R.leguminosarum. This is a much higher figure than found in other soils where beans are traditionally
grown, the source of the inoculum has to be further studied. We are now compiling strain data from 8 other historical, organic and commercial midwestern sites. In studies using delta 15N to estimate N sources for prairie legumes, the %NDFF varied with the legume species used from 36-100%. In 2 year old prairies, legume establishment was greater where plants were inoculated with rhizobia than where they were not inoculated. Rhizobia from the species Desmanthus illinoiense that are effective in symbiosis have been identified, and will be tested in the field next spring.
Impacts
Nitrogen supply is a limiting factor in crop production in more than 50% of world agriculture, with subsistence farmers particularly dependent on nitrogen fixation for reasonable crop yield. Varieties identified in this project as superior in nitrogen fixation are now widely used in Ecuador, others are available as breeding lines in the USA. Rhizobium tropici UMR1899, a strain identified by the PI, is a widely used inoculant strain for beans, especially under acid soil conditions in Brazil and Africa. IT has been extensively studies for its pH and salt tolerance and broad host range. Micronutrient fertilizer recommendations to farmers in Ecuador have been adopted by over 70% of farmers; about 50% use inoculants. The RRL website has been updated and continues to be widely used.
Publications
- Seguin, P., Sheaffer, C.C., Ehlke, N.J., Russelle, M.P. and Graham, P.H. (2001) Nitrogen fertilization and rhizobial inoculation effects on Kura clover growth. Agron.J. 93, 1262-1268.
- Ballen, K.G. and Graham, P.H. (2002) The role of acid pH in symbiosis between plants and soil organisms. In: Z.Rengel (Ed.) pH as a master variable in plant growth. Marcel Dekker, New York, page numbers not known .
- Christiansen, I. and Graham, P.H., (2002) Variation in nitrogen (N2) fixation among Andean bean (Phaseolus vulgaris L.) genotypes grown at two levels of phosphorus supply. Field Crops Res. 73, 133-142.
- Estevez de Jensen, C., Percich, J.A. and Graham, P.H. (2002). Dry bean root rot control with Bacillus subtilis in Minnesota. Field Crops Res. 74,107-115.
- Graham, P.H. and Vance, C.P. (2002) Symbiotic nitrogen fixation in soil. In: The Encyclopedia of Environmental Microbiology, G. Bitton (Ed.), John Wiley and sons. Pp 2201-2210.
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Progress 01/01/01 to 12/31/01
Outputs
This project evaluates plant genotype and rhizobial variation affecting nodulation and nitrogen fixation in crop, prairie and pasture species. The bean lines ANT22 and E295, previously identified as effective in nitrogen fixation at low soil P were shown to maintain similar nodule Rhizobium at low and high P; less P tolerant cultivars showed variation in nodule rhizobia according to soil P level. Rhizobia associated with antibiotic and captan treated bean plants grown in the central region of Minnesota were predominantly strains of R.leguminosarum. They were effective in N2 fixation with bean but quite variable in speed of nodulation with that host. The potential during historical times for the movement of bean rhizobia from Central America to the northern USA as contaminants on bean seed was examined. Rhizobia were recovered from four of seven freshly harvested seed samples from Central and South America, but numbers per seed were very low. Rhizobia from the species
Desmanthus illinoiense are being collected and will be studied for use as inoculants
Impacts
Nitrogen supply is a limiting factor in crop production in more than 50 per cent of world agriculture, with subsistence farmers particularly dependent on nitrogen fixation for reasonable crop yield. Varieties identified in this project as superior in nitrogen fixation are now widely used in Ecuador, others are available as breeding lines in the USA. Rhizobium tropici UMR1899, a strain identified by the PI, is a widely used inoculant strain for beans, especially under acid soil conditions in Brazil and Africa. IT has been extensively studied for its pH and salt tolerance and broad host range. Micronutrient fertilizer recommenations to farmers in Ecuador have been adopted by over 70 per cent of farmers; about 50 per cent use inoculants. The RRL website has been updated and continues to be widely used.
Publications
- Bernal, G, and Graham, P.H. 2001. Diversity in the rhizobia associated with Phaseolus vulgaris L. in Ecuador, and comparisons with Mexican bean rhizobia. Can. J.Microbiol. 47, 526-534.
- Graham, P.H. 2001. Nitrogen fixation. In: Plant Science for Students, R.Robinson et al. (Eds.) Macmillan Reference, Vol 3, pp 91-95.
- Graham, P.H. and Swenson, J. 1998/2001. Rhizobium Research Laboratory: http://www.rhizobium.umn.edu.
- Sadowsky, M.J. and Graham, P.H.,2001. Root- and stem-nodule bacteria. In: The prokaryotes. http://link.springer-ny.com/link/service/books/10125/index.html
- Seguin, P., Graham, P.H., Sheaffer, C.C., Ehlke, N.J. and Russelle, M.P. 2001. Genetic diversity of rhizobia nodulating Trifolium ambiguum in North America. Can. J. Microbiol. 41, 81-85.
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Progress 01/01/00 to 12/31/00
Outputs
This project evaluates plant genotype and rhizobial variation affecting nodulation and nitrogen fixation in crop, prairie and pasture species. The bean lines ANT22 and E295 were identified as effective in nitrogen fixation at low soil P. Contributing factors included rhizosphere populations active in phosphatase production and rhizosphere acidification. Rhizobia from Staples, Verndale and Park Rapids differ from those normally associated with beans. We are concerned that streptomycin seed treatment may have been a contributing factor, and are studying this possibility.
Impacts
Nitrogen supply is a limiting factor in crop production in more than 50% of world agriculture, with subsistence farmers particularly dependent on nitrogen fixation for reasonable crop yield. Varieties identified in this project as superior in nitrogen fixation are now in use in Ecuador, others are available as breeding lines in the USA. UMR1899 is a widely used inoculant strain for beans; isolates for prairie rhizobia are increasingly sought. The RRL website has now had more than 200,000 hits, with visitors from 80 different countries.
Publications
- Zhang, X., G. Nick, S. Kaijalanen, Z. Terefework, L. Paulin, S.W. Tighe, P.H. Graham, and K. Lindstrom. 1999. Phylogeny and diversity of Bradyrhizobium strains isolated from the root nodules of peanut (Arachis hypogaea) in Sichuan, China. System Appl. Microbiol. 22:378-386.
- Espinosa-Victoria, D., C.P. Vance and P.H. Graham. 2000. Host variation in traits associated with crown nodule senescence in soybean (Glycine max L. Merrill). Crop Sci. 40:103-109.
- Graham, P.H. 2000. Nodule formation in legumes. In: J. Lederberg et al. (Eds.) Encyclopedia of Microbiology. Prentice Hall Publishers, 3:407-417.
- Graham, P.H. and C.P. Vance. 2000. Nitrogen fixation in perspective: An overview of research and extension needs. Field Crops Res. 65:93-106.
- Graham, P.H. and C.P. Vance. (Eds.). 2000. Applied aspects of symbiotic nitrogen fixation. Field Crops Res. 65, 91-272.
- Vance, C.P., P.H. Graham, and D.L. Allan. 2000. Biological nitrogen fixation: Phosphorus a critical future need. In: Pederosa, F. et al (Eds.) Nitrogen fixation from molecules to crop productivity. Kluwer Publishers, Dordrecht, The Netherlands. Pp. 509-514.
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Progress 01/01/99 to 12/31/99
Outputs
This project examines cultivar variation in the nodulation and N2 fixation of soybean, bean and prairie legumes, and looks to improve inoculant strains and inoculation methods. Four lines from a recurrent selection program to enhance N2 fixation in beans were again superior in biomass and yield to all varieties evaluated in field tests at Becker, MN outyielding other varieties by as much as 46%. In another study at this low N site, 100 recombinant inbred lines from the cross between BAT477 x DOR365 were also evaluated for differences in N2 fixation; marker studies on these RILS are underway at CIAT. Three lines with Andean seed type (ANT22, E295, and MA7-8) that differ in growth and N2 fixation at low P have been identified in glasshouse and field studies. Mechanisms contributing to these differences are under study. Rhizobia associated with beans from mesoamerica and the Andean region show significant differences in diversity. Rhizobia from Dalea have been recovered
from effectively nodulated bean plants in Mexico and the USA, and occur on bean seed after harvest. Identification of superior inoculant strains for prairie legumes is important to plant establishment in revegetation situations.
Impacts
Nitrogen supply is a limiting factor to crop production over large areas of the world. Varieties identified in this project as superior in N2 fixation are now in use in Ecuador; others are available as breeding lines in the USA. Bean inoculant strains identified in the Rhizobium Research laboratory are now used in Brazil, Canada, Ecuador, and the UK; prairie strains are increasingly sought for revegetation activities. The RRL website initiated in August, 1998 has been visited by more than 30,000 viewers from more than 60 countries.
Publications
- Elisondo Barron, Pasini, R.J., Davis, D.W., Stuthmann, D.D. and Graham, P.H. 1999. Recurrent selection for improved nitrogen fixation in beans. Field Crops Research 62, 119-128.
- Graham, P.H. 1999. Nitrogen fixation. In: Handbook of Soil Science. (M.J. Sumner et al., Eds.), CRC press. C139-148.
- Graham, P.H., et al. 1999. Characterization of rhizobia associated with Dalea sp in natural prairies and revegetation areas in Minnesota. In: Highlights in Nitrogen Fixation Research (E. Martinez and G. Hernandez [Eds.}. Plenum Publishing, New York, 69-75.
- Zhang, X., Nick, G., Kaijalainen, S., Terefework, Z., Paulin, L., Tighe, S.W., Graham, P.H. and Lindstrom, K. 1999. Phylogeny and diversity of Bradyrhizobium strains isolated from the root nodules of peanut (Arachis hypogaea) in China. Syst. Appl. Bacteriol. 22, 378-386.
- Zhu, Y., Sheaffer, C.C., Vance, C.P., Graham, P.H., Russelle, M.P. and Montealegre, C. (1999. Inoculation and nitrogen affect herbage and symbiotic properties of annual Medicago species. Agron. J. 90, 781-786.
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Progress 01/01/98 to 12/31/98
Outputs
This project examines cultivar variation in nodulation and nitrogen (N2) fixation by soybean, bean and prairie legumes, and looks to improve inoculant cultures and inoculation methods. It also considers the biodiversity of rhizobia associated with prairie legumes in relation to the contribution of these legumes to prairie remnants and revegetation projects. A project to improve BNF in beans by recurrent selection for enhanced N2 fixation was completed in 1998. Significant gains in BNF in Mesoamerican bean types was demonstrated with four lines identified as breeding lines for enhanced N2 fixation. Studies to develop marker- assisted selection for enhanced N2 fixation in common bean have been initiated, as have studies to identify Andean lines superior in their ability for nodulation and nitrogen fixation. Studies with prairie legumes used in revegetation activities progressed significantly in 1998. We now hold more than 1000 strains of rhizobia for prairie legumes,
and have identified inoculant-quality strains for Dalea, Chamaecrista, Desmodium, and Astragalus. New inoculant methodologies for these prairie legumes are under study and appear highly promising. A surprising result from the studies with rhizobial isolates from Dalea is that these organisms are similar to the newly-reported Rhizobium gallicum, first identified on beans in France, and are highly promiscuous nodulating Phaseolus, Coronilla, Onobrychis and Leucaena. A possibility is that these organisms were transferred to France as seed contaminants on bean seed.
Impacts
(N/A)
Publications
- Sadowsky, M.J. and Graham, P.H. (1998) Biology of the Rhizobiaceae. In: The Rhizobiaceae. (H.Spaink et al., Eds.) Biological nitrogen fixation. Kluwer Academic, Dordrecht, pp 155-172.
- Ballen, K.G., Graham, P.H., Jones, R.K. and Bowers, J.H. (1998) Acidity and calcium interaction affecting cell envelope stability in Rhizobium. Can. J. Microbiol. 44:582-587.
- Graham, P.H. (1998) Symbiotic nitrogen fixation. In: Soil microbiology: Environmental and agricultural perspectives. (D.M. Sylvia et al., Eds.) Prentice Hall pp 322-345.
- Graham,P.H. (1998) Internationalization of the course work in Soil Science and Agronomy, with an emphasis toward Latin America. In: J.Mestenhauser (Ed.) American council on education/Onyx Press. pp 125-134.
- Graham, P.H. (1998) Nitrogen fixation. In: Handbook of Soil Science. (M.J. Sumner et al., Eds.), CRC Press. December 1998, pp III.1-III.10.
- Graham, P.H. and Swenson, J. (1998) Rhizobium Research Laboratory Website. (http://rhizobium.umn.edu)
- Graham, P.H., et al.(1998) Characterization of rhizobia associated with Dalea spp in natural prairies and revegetation areas in Minnesota. In: Highlights in Nitrogen Fixation Research (E.Martinez and G. Hernandez [Eds.]. Plenum publishing, New York, December 1998, In press.
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Progress 01/01/97 to 12/31/97
Outputs
This project examines variation in nodulation and N2 fixation in beans and soybeans, and seeks to improve their ability to fix N2. In 1997 it also began studies on the rhizobia for native prairie legumes, including strain and host diversity. Lines from a recurrent selection program for enhanced BNF were evaluated on a low N site at Becker, MN (where plants are fully dependent on N2 fixation), with yields for selected lines superior to that of lines not previously evaluated for ability in BNF. Crosses between soybean lines differing in traits that affect overall N2 fixation have been advanced to the F2, and will be further screened in 1998. Acid tolerance in R.tropici UMR1899 was shown to include both enhanced proton dependent ATP-ase activity and measurable changes in membrane composition. Collection and characterization of rhizobia associated with prairie legumes, such as Dalea purpurea was initiated. 500+ strains were collected, with differences in rhizobial
biodiversity between prairie and revegetation sites shown for both Dalea and Chamaecrista.
Impacts
(N/A)
Publications
- Graham, P.H. 1997. Symbiotic nitrogen fixation. In: Soil microbiology: Environmental and agricultural perspectives. D.M.Sylvia et al (eds.) Prentice Hall pp 322-345.
- Graham, P.H., and Ranalli, P. 1997. Common bean (phaseolus vulgaris). In: P. Ranalli and P.H. Graham (eds.) Grain Legumes. Field Crops Res. 53:131-146.
- Pazdernik, D.L., Vance, C.P., Sadowsky, M.J., Graham, P.H. and Orf, J.H. 1997. A host-controlled serogroup specific ineffective nodulation system in the Bradyrhizobium-soybean (Glycine max) symbiosis. MPMI (November) 10:994-1001.
- Pazdernik, D.L., Graham, P.H. and Orf, J.H. 1997. Variation in the pattern of dinitrogen fixation and nitrogen distribution of soybean. Crop Sci. 37:1482-1486.
- Pazdernik, D.L., Graham, P.H. and Orf, J.H. 1997. Heritability in the early nodulation of F3 and F4 soybean lines. Can.J.Plant. Sci. 77, 201-205.
- Rannali, P. and Graham, P.H. (eds.) 1997. Grain legumes. Field Crops Research Special Volume 53, 218 pp.
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Progress 01/01/96 to 12/30/96
Outputs
This project examines cultivar variation in nodulation and nitrogen fixation of beans and soybeans, and seeks to improve the ability of these crops to fix N2. The second cycle of a recurrent selection program in beans has now been completed, with materials identified which are superior to Puebla 152 in N2 fixing ability. A number of these have been crossed to grain types more acceptable in Minnesota. In soybean, studies on early differences in nodulation and N2 fixation have been completed, with two groups of plants giving high yield under N-deficient field conditions. One of these was active in N2 fixation, but limited in N translocation to the grain, the other less effective in N2-fixing ability but with very high harvest index for N. Crosses between lines from each group have been made and will be used in studies to identify the genes responsible for these two traits. Studies on the acid-pH tolerance of Rhizobium tropici UMR1899 have been facilitated by the
identification of a buffer highly effective in pH regulation over the range pH 4.3 to 4.8.
Impacts
(N/A)
Publications
- Montealegre, C. and Graham, P.H. 1996. Preference in the nodulation of Phaseolus vulgaris L. II. Can. J.Microbial. 42:844-850.
- Pazdernik, D.L., Graham, P.H., Vance, C.P. and Orf, J.H. 1996. Host genetic variation in the early nodulation and dinitrogen fixation of soybean. Crop Sci. 36, 1102-1107.
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Progress 01/01/95 to 12/30/95
Outputs
This project examines cultivar variation in nodulation and N2 fixation in beans and soybeans, and to pyramid desirable traits affecting N2 fixation in these crops. In beans a recurrent selection program is now in its second cycle. Studies continue on the preference in nodulation exhibited by cultivar RAB 39 for Rhizobium tropici UMR1899. Strain preference in nodulation is not affected by pH or temperature, and UMR1899 forms the majority of nodules even when applied up to 8 hours after a non-preferred strain. The presence of UMR1899 reduces nodulation by UMR1632. Studies on the acid pH tolerance also emphasize UMR1899. Differences in FAME composition have been shown between UMR1899 and a Tn5 pH mutant derived from it, and we are currently examining outer membrane composition and serology as a function of pH and calcium availability. In soybean, recent studies have emphasized cultivar variation in early nodulation and N2 fixation, and initially examined differences in
nodulation using the RTM procedure, as well as difference in onset of N2 fixation, and of nodule enzyme activity 15-20 days after planting. Nodule content of soluble protein and nodule fresh weight were early indicators of active N2 fixation. Correlations were obtained between early nodulation and N2 fixation with N accumulation by the plant to the R6 growth stage, and between glasshouse and field results.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
This project examines cultivar variation in nodulation and N2 fixation in beans and soybeans, and aims to pyramid desirable traits affecting fixation in agronomically acceptable lines. Transgressive segregants for N2 fixation from a cross between Puebla152 x BAT271 have been used as a starting point for a recurrent selection program to enhance N2 fixation in Phaseolus beans. Studies in soybean are perhaps one year behind those with beans; cultivar variation in traits affecting early nodulation and N2 fixation have been demonstrated, and hybrid materials developed using this germplasm. The varieties Mendota (soybean) and Durango222 (Phaseolus) have been confirmed as lines nodulating reasonably in the presence of nitrate. Studies of host preference in nodulation with the bean cultivar RAB39 continue. It was shown that preference is evidence even when the preferred strain is applied several hours after initial inoculation, and that preference is not due to differences in
attachment. Studies on acid pH tolerance in Rhizobium tropici CIAT899 also continue. Tn5 GUS mutants which express glucosidase activity at low pH have been identified, and a clone bank of CIAT899 in pLA2917 screened for clones conferring acid pH tolerance.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
Studies on bean (Phaseolus vulgaris) cultivar variation in traits related to N(superscript 2) fixation continued. F(subscript 3) progeny of the cross between Puebla 152 x BAT 271 showed transgressive segregation for N accumulation in N(subscript 2)-fixation dependent plants, but attempts to select for N(subscript 2) fixation in the F(subscript 2) were not successful. RAB 39 a line identified at CIAT as showing preference for modulation with R. tropici UMR 1899, was evaluated for this preference under a range of temperature and pH regimes. A strong preference for UMR 1899 over the more common R. etli, was evident under all environmental conditions. Sixty-eight bean cultivars were also evaluated for tolerance of modulation to high levels of soil nitrate Mantequilla tropical, Imbabello, Durango 222 and Bola 60 were among the varieties which were well modulated at high N0(subscript 3)-. Studies on soybean cultivar variation in traits related to N(subscript 2) fixation
parallel those with beans. Cultivar variation has been demonstrated in both earliness of nodulation and in duration of N(subscript 2) fixation in the first formed nodules. The basis for such differences are being explored and crosses have been made to examine the genetic basis for such differences. Studies on pH tolerance in R. tropici continued. Tolerance in this organism is not an adaptive response, nor is it plasmid encoded or due to the production of EPS R. tropici UMR 1899 is able to maintain a cytoplasmic pH differential of up to 1.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Studies on bean cultivar variation in traits affecting nodulation and nitrogen fixation by this crop continued in 1991-1992. Cultivars identified as varied in the traits contributing to active nodulation and nitrogen fixation were intercrossed and their F(subscript 2) progeny evaluated for earliness in nodulation and for ability to fix nitrogen under N limited conditions. Selected lines are being advanced and will be field tested in 1993. More than 300 accessions were also evaluated for their ability to restrict nodulation by Rhizobium etli, the more common bean microsymbiont. Several cultivars have been identified which show preference for Rhizobium tropici and studies are underway to determine the nature of this restriction and the factors which influence it. Studies on the early nodulation of soybean parallel those being undertaken with bean but were started somewhat later. Cultivars have been identified which differ in early nodulation with selected Bradyrhizobium
strains and these have been crossed to selected soybean genotypes. Studies on the pH tolerance of host and Rhizobium continue, with nuclear magnetic resonance spectroscopy being used to estimate the cytoplasmic pH of the acid tolerant strain UMR 1899 and Tn5 GUS mutants being used to evaluate regulation of pH tolerance in this organism.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
Studies on bean cultivar variation in traits affecting early nodulation were continued in 1990-91. The root-tip marking (RTM) procedure was used with variation between cultivars demonstrated in all parameters including uppermost nodule position, percentage of nodule initials developing producing nodules, and plant growth and N-accumulation in the period 26 DAP. Lines were identified which differed in early nodulation, date to flowering and maturity and in fixation post-flowering, and crossing between these accessions initiated. Four hundred bean lines were also evaluated for restriction with the Type 1 bean strain UMR 1632, and approximately 30 identified which appear restricted in nodulation with this strain. For soybean restriction has been shown to vary with inoculant dosage, while apparently restricted strains able to produce nodules on the lateral but not crown region of seed-inoculated plants. We view this as another indication that mobility in soil rather
than competitive ability per se is the factor limiting inoculant strain nodule occupancy. In studies on pH tolerance, clone banks of R. tropici UMR 1899 in pSUP 205 and pVK 102 have been used to identify regions of DNA coding for pH tolerance, while cells exposed to medium of pH 5.5 have been shown to accumulate both K+ and glutamate. Effects of pH on cell surface characteristics have also been shown.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
Competition for nodulation sites is a major factor limiting nitrogen fixation ingrain legumes. We have evaluated the ability of B. japonicum seed inoculant to stay abreast of the developing root system, and shown that strains compete effectively for nodulation sites in the region where they are placed. However, they are essentially immobile in the soil and poorly competitive for lateral root sites. We have used the root tip marking technique to determine the efficiency in nodule formation of Bradyrhizobium japonicum and correlated competitiveness of strains with the position of the uppermost nodule and with the percentage of nodules formed above the root tip mark. When indigenous populations of Bradyrhizobium japonicum serocluster 123 were evaluated for competitiveness using the RTM procedure in growth pouches, nodule occupancies were similar to those normally encountered in field studies. The competitiveness of B. japonicum USDA 38 against USDA 110 was evaluated at
20(degree)C than at 30(degree)C using immunofluorescence. USDA 38 was not competitive at either temperature but co-occupied more nodules at 20(degree)C than at 30(degree)C. Pre-treatment of this strain with soybean root exudate arrested cell growth and induced significantly poorer nodulation. Studies have also contrasted the restriction patterns of selected soybean cultivars and Bradyrhizobium strains.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Competition for nodulation sites is a major factor limitinggains in N(2) fixation in a number of grain legumes. We evaluated the ability of B. japonicum supplied as seed-inoculant to stay abreast of the developing root system. Rhizoplane populations declined rapidly with distance from the seed,and was paralleled by reduced inoculant-strain nodule occupancy. Inoculant rhizobia were competitive for crown nodulation sites but produced very few lateral root nodules. Lateral root nodules were shown to contribute more than 50%, of nodule number and mass and were a significant factor in acetylene reduction activity post-flowering. For soil applied rhizobia competitive ability is closely related to speed of nodulation in both bean and soybean. Temperature affects, but does not invalidate this relationship. For soybean UMR 161 has been shown to be highly competitive and effective, while for bean the superior strains include UMR 1102, 1165, and 1084.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Studies in 1988 have emphasized competition for nodulation sites between inoculant and indigenous rhizobia. In soybean, nodule occupancy by the inoculant strain UMR161 decreased with distance from the root crown. In both field and growth chamber studies, the inoculant strain contributed little to lateral root nodulation, which at pod fill accounted for more than 50% of nodule mass and number, and was a major contributor to overall acetylene reduction activity. When the movement of UMR161 in the rhizosphere was monitored, the strain failed to keep abreast of the expanding root system. In Phaseolus beans the root-tip marking procedure has been used to estimate speed in nodulation, and a strong correlation obtained between this parameter and strain competitiveness. Temperature and inoculant level can affect this relationship, explaining the highly variable results obtained in early studies.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
Strains of Bradyrhizobium japonicum applied as inoculants competed well for nodulation sites in the crown region of the plant, but formed only a low percentage of lateral and lower root nodules. Nodule distribution and function were studied at sampling times throughout the growing season. N(2) fixation at 50-76 DAP was highly dependent on lateral root nodules, more than 80% of N(2) fixation achieved by soybean at this stage of growth being due to secondary and lower root nodulation.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
Twenty group 00 and 27 group 0 soybean cultivars were evaluated for differences in N(2) fixation at two locations in each of two years. Total N(2) fixed (estimated by the difference method using a Clay maturity non-nodulating line) was positively correlated with shoot dry weight at R(2) and R(6), and with seed yield, but was not correlated with N concentration of tissues. There were highly significant differences within maturity groups for all traits, with some Group 00 lines fixing as much N(2) as high-fixing Group 0 lines. F(2) derived F(4) families from five crosses of lines differing in N(2) fixation were evaluated at 3 locations in Minnesota, using shoot dry weight at R6 as an indirect estimate for N(2) fixation. Heritabilities for shoot dry weight ranged from 5-54% and for yield from 30-82%. There were numerous transgressive segregants in the cross of two high N(2) fixing lines. Evans x PI438265, with lines identified having higher shoot dry weight and
yield than either parent N determinations for these lines are underway to determine improvements in N(2) fixation. In parallel studies, bacteroid viability in soybean nodules was shown to vary significantly both with strain used and cultivar. USDA 38, in particular showed low bacteroid viability. Despite differences in bacteroid viability, numbers of rhizobia surviving in soil after 3 months incubation, were not significantly different.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 12/30/85
Outputs
Forty-six soybean varieties from maturity groups 0 and 00 were tested at Becker and St. Paul, MN for differences in N(2) fixation. Significant differences between cultivars were found for all parameters used to estimate fixation. N(2) fixation (by the difference method using a Clay no-nod line) ranged from 206 to 1595 mg/plant at Becker, and from 70 to 1065 mg/plant at St. Paul. The %N varied from 31:1 to 80.0% and from 8.6 to 57.8% at the respective sites. Similar differences were detected among 46 Phaseolus vulgaris lines also tested at Becker. Among 12 strains of B. japonicum tested, the % viability of bacteroids, measured 30 and 67 days after planting, varied from 0.7-42.2 and from 1.9-139.1% respectively. Values in excess of 100% arose because of the difficulty of adequately separating cells prior to effecting total counts. EPAGRO 587 and USDA 123 had the highest, and USDA 38 and USDA 122 the lowest % viability. Percent bacteroid viability was also
influenced by cultivar used. Manenitol concentration in the planting medium had a variable effect on bacteroid viability. The number of bacteroid cells/envelope tended to be highest in strains with high percent bacteroid viability.
Impacts
(N/A)
Publications
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Progress 01/01/84 to 12/30/84
Outputs
Five hundred soybean accessions from maturity groups 00, 0, and I were evaluatedfor N(2) fixation by growth under low nitrogen conditions at the Becker sand plain station. Accessions were uninoculated, inoculated with USDA 110 or supplied fertilizer N. 60 accessions showing high yield under all condition; or strong response to N and/or inoculation have been selected for more detailed study. 10 accessions selected from similar studies in 198o were reevaluated in replicated trials with repeated assay of N(2) (C(2)H(2)) fixation, nodule score, biological development and finally yield. Results were generally consistent with the previous years data and justified the use of these lines in a crossing program to enhance N(2) fixation in soybean of the earlier maturity groups. Hybrid material from these crosses is currently being grown in winter nurseries in Chile. Fifty-six accessions of P. vulgaris have also been evaluated for N(2) fixation in replicated field plots,
with a further 250 materials evaluated in observation plots. In these studies, Wisconsin BNF lines 21-58, 21-43 and 21-19 all yielded well.
Impacts
(N/A)
Publications
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Progress 01/01/83 to 12/30/83
Outputs
Ninety soybean plant introduction lines and 400 lines of dry bean from the CIAT program were evaluated for growth, yield and N(2) fixation at the Becker location (<10 ppm N). The soybean lines were grown in replicated plots, the beans in single row observation and seed increase nurseries. The soybean lines differed in both yield and N(2) (C(2)H(2)) fixation. The four top ranked and three bottom ranked lines, plus the varieties--Evans, Simpson and Ossie--were included as parents in a 10 x 10 diallel. Strain differences in number of rhizobia surviving within nodules were evaluated with 12 strains of R. japonicum. When strains showed considerable variation in this trait, a more detailed study was undertaken with 4 strains, UMR 30, 62, 123, and 150. Strain UMR 62 was significantly worse than the others in survival within the nodule, perhaps explaining its poor persistence under field conditions. 40 strains of Rhizobium have been isolated from soybean nodules
collected in China. These are currently under investigation for efficiency in N(2) fixation and host inoculation pattern.
Impacts
(N/A)
Publications
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Progress 01/01/82 to 12/30/82
Outputs
This program was initiated June 30, 1982. Accomplishments to date are: An automatic irrigation system and improved lighting system has been installed in the glasshouse. Approximately 200 strains of R. japonicum and R. phaseoli have been freeze dried, reducing their maintenance cost and opportunity for mutation. Sixty strains of R. japonicum have been evaluated in leonard jar trials with soybean. Effective strains will be further evaluated. Seventy sites in Minnesota were assayed for nodule development on soybean. Rhizobia from nodule collections at these sites are currently being isolated and evaluated. Seventy-five accessions of P. vulgaris from the CIAT bean program have been evaluated for symbiotic N(2) fixation with Rhizobium. Selections active in N(2) fixation have been identified for further field testing.
Impacts
(N/A)
Publications
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Progress 01/01/81 to 12/30/81
Outputs
Persistence of a superior nitrogen fixing strain of Rhizobium japonicum (USDA 110) was studied in a soil containing a naturalized population of R. japonicum. Field plots were established utilizing three liquid inoculum treatments (2x10 6, 2x10 5, and 2x10 4 cells/g) and 2 granular inoculum treatments (2x10 4 and 4x10 2 cells/g). The level of the inoculum strain (USDA 110) was followed using a fluorescent antibody counting technique. The liquid inoculation at 2x10 6 cells/g showed an initial population rise to 1x10 7 cells/g after 1 week followed by a decline to 6x10 5 cells/g after 19 weeks. Similar but less dramatic trends were noted in the other inoculum treatments. The data indicated that this soil was able to support a population of USDA 110 of 2x10 5 cells/g since population levels approached this value by the end of the first growing season. A continuation of these studies in 1981 showed the population of USDA 110 observed for the inoculation treatments was
not significantly different from that observed for the uninoculated control. In neither year did inoculation increase the yield of soybeans. Studies were continued in 1981 at Waseca to determine the effect of tillage and weed control on soybean growth. Tillage had no effect on soybean root growth despite large differences in soil strength. No-tillage plots exhibited higher weed populations than did other tillage systems. When weed populations were adequately controlled the seed yield was not affected by the tillage system.
Impacts
(N/A)
Publications
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Progress 01/01/80 to 12/30/80
Outputs
Persistence of a superior nitrogen fixing strain of Rhizobium japonicum (USDA 110) was studied in a soil containing a naturalized population of R. japonicum. Field plots were established utilizing three liquid inoculum treatments (2x10 6, 2x10 5 and 2x10 4 cells/g) and 2 granular inoculum treatments (2x10 4 and 4x10 2 cells/g). The level of the inoculum strain (USDA 110) was followed using a fluorescent antibody counting technique. The liquid inoculation at 2x10 6 cells/g showed an initial population rise to 1x10 7 cells/g after 1 week followed by a decline to 6x10 5 cells/g after 19 weeks. Similar but less dramatic trends were noted in the other inoculum treatments. The data indicated that this soil was able to support a population of USDA 110 of 2x10 5 since population levels approached this value as a limit. Symbiotic dinitrogen fixation by the soybean cultivar Hodgson-78 was measured at Rosemount and Becker by the use of the stable isotope N-15 at two
fertilizer rates (10 and 100 kg N/ha) and by total Kjeldahl nitrogen. At Becker N(2) fixed at the 10 kg N/ha rate ranged from 110 to 261 kg/ha as measured by N-15 dilution techniques. At the 100 kg N/ha rate value of N(2) fixed ranged from 141 to 274 kg/ha as determined by Kjeldahl analysis and 124 to 245 kg/ha as measured by N-15 dilution. The percentage of the total plant nitrogen derived from fixation ranged from 53 to 90. Isotope data from the Rosemount Station is unavailable at this time.
Impacts
(N/A)
Publications
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Progress 01/01/79 to 12/30/79
Outputs
Rhizobium japonicum strains USDA 110 and USDA 138 were introduced (inoculated) into a soil containing an indigenous R. japonicum population in growth chamber studies. Recovery (percent f plant nodules) of the introduced strains was dependent on soil temperature with their highest recoveries at 15 degrees C (110=19%; 138=15%) and 30 degrees C (110=17%; 138=20%). The remainder of the soybean nodules were derived from indigenous R. japonicum present in the soil from previous inoculation. The indigenous population was most difficult to displace at 20 degrees C (110=4%; 138=10%) which approximates the soil temperatuare at normal planting dates. The recovery of R. japonicum strain 140 was low (3% or less) at all temperatures. These results indicate that soil temperature is a factor to be considered in soybean inoculation studies. R. japonicum strains should be selected for tolerance to the average soil temperature at planting time. Soybean inoculation studies at two
locations where the soils contain an indigenous rhizobia population indicated a wide range of recovery (based on percent plant nodules) of Rhizobium japonicum (rhizobia) strain USDA 110 depending on soybean genotype. These soybean genotypes were selected for inferior nodulation with the indigenous soil rhizobia. Average recovery of strain 110 was 41% at Waseca (ranged from 28% to 58%) and 25% at Rosemount (ranged from 3 to 43).
Impacts
(N/A)
Publications
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Progress 01/01/78 to 12/30/78
Outputs
Foliar fertilization studies were conducted at 5 field locations during 1978 using 2 adapted cultivars at each location. Folian and the Iowa State-TVA formulation were applied during flowering and during the podfilling period (1 to 4 applications were made containing 14 plus 5 plus 5 plus 0.6 kg/ha of N plus P plus K plus S with 0.1% Tween 80). In addition, other non-traditional fertilizers (Seaborn plus F and Eco-Gro) were applied during flowering according to the label. No significant yield increases were obtained from any of the materials at any location. In addition, 9 soybean cultivars, 11 experimental soybean lines and 6 semideterminate-indeterminate isoline pairs received Folian at the rate of 14 plus 5 plus 5 plus 0.6 kg/ha of N plus P plus K plus S during early podfilling at 3 field locations (not all soybeans were grown at every location because of maturity differences). The seed yield of one experimental line was increased 420 kg/ha; otherwise, no
significant yield increases were obtained. Field studies were conducted with P 3 3 and S 3 5 to determine the movement of these nutrients within the soybean plant. Nutrient elements applied to leave went maintly to pods in the axils of treated leaves with minimal amounts of nutrients transferred to adjacent nodes. These results indicate that further research must be conducted before the process of foliar fertilization can produce consistent and economical yield increases.
Impacts
(N/A)
Publications
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Progress 01/01/77 to 12/30/77
Outputs
Foliar application (4 times during pod-filling) of N, P, K and S with surfactantat the rate of 20, 2, 6 and 1.5 kg/ha (elemental basis) increased soybean seed yields from 3760 to 4370 kg/ha (with irrigation). The recovery of PG1N labelled urea applied to the foliage was 56% (45 of the 80 kg/ha applied). The recovery of foliar-applied calcium nitrate, ammonium sulfate and ammonium chloride added with P, K and S was 36%, 23% and 24%, respectively, based on PG1N analyses. Studies are underway to increase the recovery of foliar-applied N by the soybean plant. In other studies, the following observations were reflected in soybean seed yields: 1) adding increasing biuret levels up to 4% of urea had no adverse effects; 2) the lowest seed yields were from foliar-applied N, P, K and S at noon and 3 PM compared to other times every 3 hours for 24 hours; 3) none of the 20 soybean genotypes examined for response to foliar fertilization showed a yield increase at any of 3
locations. The "A(N)" value concept provided an estimate of N(2) fixation by soybeans which was somewhat higher than acetylene reduction measurements made weekly and Kjeldahl measurements. The acetylene reduction technique estimated N(2) fixation at the given point in time when sampled whereas the "A(N)" value concept gives an integrated value over the entire growing season.
Impacts
(N/A)
Publications
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Progress 01/01/76 to 12/30/76
Outputs
A comparison of soil and foliar PG1N labelled urea applied during early flowering, late flowering and pod-fill showed that plant recovery of soil applied nitrogen was inferior only during the pod-filling period (47% recovery for foliar-applied N and 6% for soil-applied). During the earlier stages of plant growth, the recovery of soil and foliar applied N was similar (averaged 45%). These results indicate the decrease in nutrient uptake during pod-fill and suggest a potential for adding nutrients by foliar fertilization during pod-fill. Foliar application (4 times during pod-filling) of N (urea), P, K andS with surfactant at the rate of 20, 1.9, 5.8 and 1.3 kg/ha (elemental basis) increased soybean seed yields from 3760 to 4370 kg/ha (with irrigation). AddingN alone (soil or foliar-applied) did not increase seed yield. Foliar-applied calcium nitrate, ammonium sulfate and ammonium chloride added with P, K and S decreased soybean seed yield by as much as 1475 kg/ha.
Spraying with surfactantonly did not alter yield. Inoculation with Rhizobium japonicum (5 commercial humus inoculants containing 4.9 x 101 - 1.3 x 1014 rhizobia/gm applied to the seed and 5 commercial granular inoculants containing 2.3 x 10, - 1.1 x 1014 rhizobia/gm applied to the soil with the seed) did not increase soybean seed yields significantly. At planting the soil contained 1.2 x 10, rhizobia/gm fromprevious soybean crops.
Impacts
(N/A)
Publications
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Progress 01/01/75 to 12/30/75
Outputs
Fifteen high yielding soybean genotypes were analyzed for Ny fixation with the acetylene assay and nutrient uptake at various stages of growth. Considerable soybean genetic variability was found for Ny fixation and total P, K and Ca uptake. Maximum Ny fixation/plant/day varied by as much as 270%. Ny fixation patterns (rate/day and duration) during the season varied among soybean genotypes even though the total kg Ny fixed/ha was the same in some cases. The correlation of total Ny fixation with seed yield was highly significant (0.88**). Depending on soybean genotype, total P uptake ranged from 20 to 28 kg/ha, total K uptake ranged from 89 to 113 kg/ha and total Ca uptake ranged from 60 to 84 kg/ha. Kg seed/total kg nutrient uptake ranged from 102-133 for P, from 24 to 31 for K and from 36 to 45 for Ca. The kg seed/ha was highly correlated with total nutrient uptake (P=0.59**, K=0.82** and Ca=0.58**). Some exceptions were noted which produced relatively more seed
yield per amount of nutrient and these soybean genotypes will be used in future nutrient uptake and fertilizer utilization studies. Fertilizer N (100 kg N/ha as urea) increased soybean seed yield from 4840 to 5480 kg/ha in 38 cm spaced rows (13% increase) and from 3670 to 4150 in 75 cm spaced rows (13% increase). However, decreasing row spacing from 75 to 38 cm increased seed yield by 32% and increased Ny fixation by about 80 kg N/ha.
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Progress 01/01/74 to 12/30/74
Outputs
Fertilizers labelled with P-33 (ordinary superphosphate) and N-15 (urea) were used to study the efficiency of fertilizer utilization by field-grown soybeans. Seed yield was increased significantly with all fertilizer placements (mixed in top 10 cm soil, banded near or between rows, and surface broadcast at the rate of 35 kg P/ha). No differences were measured among placements either in seed yield or P derived from fertilizer. Recovery of N from 30 kg N/ha ranged from 43% to 67% and recovery from 100 kg N/ha ranged from 25% to 54% depending on time of application and placements. When applied at planting, mixing 100 kg N/ha in the top 10 cm soil reduced nodulation and N(2) fixation the least followed by surface broadcast, banded midway between the rows (22 cm to side) and banded near the row (5 cm to side) in order of increasing detrimental effects. Seed yield and total plant N were increased with 100 kt N/ha applied at planting only when mixed in the top 10 cm soil
or surface broadcast. Recovery of fertilizer N ranged from 49% to 54% for the different placements. Nrecovery was lower when applied later in the season. As little as 3% (7 kg N/ha) of the total plant N was supplied by the nodules when 100 kg fertilizer N/ha was banded near the row while 26% (55 kg N/ha) of the plant N came from thenodules with no N applied. Compared to Kjeldahl and N-15 estimates of N(2) fixation, the acetylene reduction technique overestimated N(2) fixation in some cases and underestimated in other cases.
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Progress 01/01/73 to 12/30/73
Outputs
Application of fertilizer nitrogen increased soybean seed yields 49%, 26% and 8%at Crookston, Morris and Waseca, respectively (check yields were 1640, 2298 and 3676 kg/ha, respectively). In a comparison of determinate and indeterminate isolines of Corsoy and Hark background, seed yields were increased up to 18% with 224 kg N/ha (the larger yield increases were obtained with the determinate isolines). N(2) fixation, nodule number and nodule weight were reduced by all Ntreatments in all experiments (although the reductions were less severe when theN was plowed-down compared to broadcast on the surface). These results suggest that soybean yields may be limited by inadequate nitrogen from nodulation under some conditions. Yield increases from N fertilizer have been obtained in more than half of the trials conducted during the past seven years. In P and K placement studies, equal quantities of P or K were more efficient in terms of yield increase when plowed down than
when banded 5 cm beside and 5 cm below the seed.
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Progress 01/01/72 to 12/30/72
Outputs
Plowing down 224 kg N/ha in the fall generally increased the yield of low-yielding soybean genotypes (from 2204-2735 kg/ha) & decreased the yield of high yielding genotypes (from 2903-2480 kg/ha). In another study adding 56 & 112 kg N/ha increased the yield of Corsoy soybeans 250 & 500 kg/ha when planted on May 8. Other varieties planted on May 8 & all varieties planted on June 7 did not respond positively on N fertilizer. Nodule weight and N(2) fixation perplant were increased by 56 kg N/ha while 112 kg N/ha was about the same as the control. In a comparison of N sources applied to the surface in the spring (ammonium nitrate, urea and soybean meal) at the rate of 112 kg N/ha, only soybean meal increased yields significantly (195 kg/ha; check yield was 2450 kg/ha). All N sources increased yields significantly at the rate of 224 kg N/ha(276, 188 & 464 kg/ha for ammonium nitrate, urea and soybean meal, respectively). N(2) fixation, nodule weight and nodule number
per plant were reduced by all N treatments. Leaf Mn increased from 56-82 ppm with increasing Nrates while the concentration of other elements was not changed. Check plants in all studies were well-nodulated. In comparison of nonnodulating and nodulating soybean isolines, 50 & 51% of the seed yield of nodulating isoline was attributed to nodulation at Morris and Lamberton, respectively.
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Progress 01/01/71 to 12/30/71
Outputs
Soybean yield responses to P and K fertilizer with no nitrogen were all less than 270 kg/ha at Lamberton, Morris and Waseca in 1971. With 60 kg N per ha, P and K increased yields 470 kg/ha at Morris and Waseca and 340 kg/ha at Lamberton. Highest yields were obtained with 60 or 120 kg N per ha plus P and Krather than with P and K alone. Small amounts of N placed with the seed (5.6, 11.2 and 16.8 kg/ha) resulted in small increases (up to 200 kg/ha) or decreases (up to 134 kg/ha) in seed yield. Soybean inoculation studies with Rhizobium japonicum were equivocal. Seed yields were not increased significantly with about as many small yield decreases as increases. Inoculating with 400 times normal placed with the seed increased the percentage of nodules on the plant by strains 110 and 138 by 38 and 34 percent, respectively. In comparisons of nonnodulating and nodulating soybean isolines, about 16, 38 and 33 percent of the seed yield was attributed to nodulation at
Lamberton, Morris and Waseca, respectively. Soil nitrogen contributed 164, 105 and 119 kg N per ha, respectively. Nodule nitrogen contributed the remainder of the nitrogen available to the plant (86, 139 and 133 kg N per ha at Lamberton, Morris and Waseca, respectivly, resulting in 220 plus or minus 3 kg N per ha in the soybeanat the various locations). These results indicate that N fixation in nodules isvery dependent on the inversely related to soil N level.
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Progress 01/01/70 to 12/30/70
Outputs
Soybean inoculation studies utilizing coating agents applied to Corsoy seed withthe inoculum at planting time increased seed yields at the two locations studied. The treatment which increased seed yields 400 kg/ha at St. Paul increased seed yields 200 kg/ha at Lamberton. A treatment which increased seed yield 315 kg/ha at Lamberton yielded no better than the uninoculated control treatment at St. Paul. Soybean host genotype - Rhizobium japonicum combinationswhich allow up to 80% of the nodules to be formed on the root of the host plant by the added strain when the soil already contains a naturalized population of R. japonicum at St. Paul did not follow the same pattern at Lamberton. Only 5-10% of the nodules were formed by the added strain at Lamberton. Acetylene reduction studies of field-grown soybeans indicated differences in N fixation among different varieties. Considerable variation from plant to plant in the same row was noted. Nonnudulating and nodulating
soybean seeds were blended together in eight combinations. Seed yield increased gradually from 2000 to 3000 kg/ha as the percentage nodulating seed in the mixture increased from 0 to 100%.
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Progress 01/01/69 to 12/30/69
Outputs
Soybean inoculation tests were conducted at Morris, Waseca and St. Paul on soilscontaining a naturalized population of Rhizobium japonicum. A total of 40 strains of R. japonicum were applied to two soybean varieties and the nodules are being serotyped to determine how competitive the added strains were. In addition 13 coating agents were used to apply 5 strains of R. japonicum to the soybean seed in an attempt to selectively introduce effective strains of Rhizobia. Results to date indicate that some strains are more competitive then others. Some strains are present in 25-33% of the nodules while many strains are present in 0-10% of the nodules serotyped. Growth chamber studies indicate that the added strains are more effective nitrogen fixers than the R. japonicum in the soil.
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Progress 01/01/68 to 12/30/68
Outputs
Soybean inoculation tests conducted at Big Lake, Rosemount, Waseca, Lamberton, and Morris showed no yield increase due to inoculation when the soil contained anaturalized population of soybean rhizobia. Each of the legume inoculant companies preinoculated two samples of soybean seed and provided two samples of humus inoculants to be applied to the seed before planting. The average yields of the uninoculated and inoculated plots were 29.5 and 29.4 bushels per acre, respectively. Inoculation increased yields in 10 cases, decreased yields in 12 cases and had no effect in one case (based on the average yield for each treatment at all five locations). The uninoculated soybeans were adequately nodulated by soybean rhizobia already in the soil. Soybean plants grown in the laboratory with sterile root systems in sterile soil, were compared with plants similarly grown but inoculated with R. japonicum, with respect to use of aspartic acid supplied to roots. The free amino
acid pool of the stems was essentially similar in inoculated and uninoculated plants except for serine. Serine concentrations were consistently higher in inoculated plants.
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Progress 01/01/67 to 12/30/67
Outputs
Soybean inoculation tests at Grand Rapids provided data on the importance of inoculation when soybeans are to be grown in soil essentially free of rhizobia. Seed yield was increased by as much as 7-8 bushels per acre due to inoculation. Yield increases due to inoculation could not be matched by nitrogen fertilization of uninoculated plants. Such field data are of substantial significance in view of the expansion of soybean culture into rhizobia-free soils. Comparisons of nodulating and non-nodulating soybean isolines of Chippewa maturity in soils of southern and south-central Minnesota with rhizobiaalready present, showed that the contribution of nodulation to yield amounted toabout 15 bushels per acre of a 40-bushel crop. These data point up the practical importance of nodulation to soybean production and provide ready justification for research on rhizobium populations in soils. A survey indicated that many of the serological groups of R. japonicum found in Iowa
occurred also in Minnesota, together with some additional strains not yet characterized as to serology or importance. The fluorescent antibody technique was applied successfully to study of R. japonicum in soil. It seems likely thatthis technique will provide investigators, for the first time, with means to observe Rhizobium in soil apart from the host plant.
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