Harold H. Keyser

Potential for increasing biological nitrogen fixation in soybean

The importance of soybean as a source of oil and protein, and its ability to grow symbiotically on low-N soils, point to its continued status as the most valuable grain legume in the world. With limited new land on which to expand, and emphasis on sustainable systems, increases in soybean production will come mostly from increased yield per unit area. Improvements in biological nitrogen fixation can help achieve increased soybean production, and this chapter discusses research and production strategies for such improvement.The soybean-Bradyrhizobium symbiosis can fix about 300 kg N ha-1 under good conditions. The factors which control the amount of N fixed include available soil N, genetic determinants of compatibility in both symbiotic partners and lack of other yield-limiting factors. Response to inoculation is controlled by the level of indigenous, competing bradyrhizobia, the N demand and yield potential of the host, and N availability in the soil.Research efforts to improve BNF are being applied to both microbe and soybean. While selection continues for effective, naturally occurring bradyrhizobia for inoculants and the use of improved inoculation techniques, genetic research on bradyrhizobia to improve effectiveness and competitiveness is advancing. Selection, mutagenesis and breeding of the host have focused on supernodulation, restricted nodulation of indigenous B. japonicum, and promiscuous nodulation with strains of bradyrhizobia from the ‘cowpea’ cross-inoculation group. The research from the host side appears closer to being ready for practical use in the field.Existing knowledge and technology still has much to offer in improving biological nitrogen fixation in soybean. The use of high-quality inoculants, and education about their benefits and use can still make a significant contribution in many countries. The importance of using the best adapted soybean genotype with a fully compatible inoculant cannot be overlooked, and we need to address other crop management factors which influence yield potential and N demand, indirectly influencing nitrogen fixation. The implementation of proven approaches for improving nitrogen fixation in existing soybean production demands equal attention as received by research endeavours to make future improvements.

Biochemical Characterization of Fast- and Slow-Growing Rhizobia That Nodulate Soybeans

Fast-growing, acid-producing soybean rhizobia were examined to determine their biochemical relatedness to each other, to typical slow-growing Rhizobium japonicum strains, and to other fast-growing species of Rhizobium. Although both the fast- and slow-growing soybean rhizobia were positive for catalase, urease, oxidase, nitrate reductase, and penicillinase, the fast-growing strains grouped with other fast-growing species of Rhizobium in that they tolerated 2% NaCl, were capable of growth at pH 9.5, utilized a large variety of carbohydrates (notably disaccharides), and produced serum zones in litmus milk. In addition, these fast-growing strains were similar to other fast-growing species of Rhizobium in that they produced appreciable levels of β-galactosidase and nicotinamide adenine dinucleotide phosphate-linked 6-phosphogluconate dehydrogenase but had no detectable hydrogenase activity. The fast-growing soybean rhizobia share symbiotic host specificity with Bradyrhizobium japonicum, but appear to be related biochemically to the other fast-growing species of Rhizobium.

Bradyrhizobium spp. (TGx) isolates nodulating the new soybean cultivars in Africa are diverse and distinct from bradyrhizobia that nodulate North American soybeans

The newly developed cultivars of soybean in Africa, known as Tropical Glycine cross (TGx), are nodulated by bradyrhizobia indigenous to African soils, here designated Bradyrhizobium spp. (TGx). Isolates of Bradyrhizobium spp. (TGx) obtained from nodules of TGx soybeans that were inoculated with soils from 65 locations in six African countries were characterized and grouped into 11 phylogenetic clusters on the basis of RFLP of the 16S rRNA gene. Five restriction enzymes (RsaI, HinfI, MspI, CfoI and HaeIII) established RFLP groups within these Bradyrhizobium spp. (TGx) isolates, which were used to construct a phylogenetic tree showing their genetic relationship with other Bradyrhizobium species. RFLP analysis indicated that Bradyrhizobium spp. (TGx) is a heterogeneous group with some isolates related to Bradyrhizobium japonicum and Bradyrhizobium elkanii strains and some to Bradyrhizobium spp. (misc.) reference strains isolated from a variety of tropical legumes. The heterogeneity within the large phylogenetic clusters was further examined through analysis of randomly amplified polymorphic DNA (RAPD) using GC-rich PCR primers. The RAPD analysis showed additional heterogeneity in the Bradyrhizobium spp. (TGx) phylogenetic clusters, which was not revealed by separations based on RFLP analysis. The Bradyrhizobium spp. (TGx) isolates were classified into effective and ineffective types based on their symbiotic performance on TGx soybean. The isolates were randomly distributed throughout the phylogenetic clusters regardless of their symbiotic effectiveness on TGx soybean.

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

Aims: Comparison of molecular and antibiotic resistance profile methods to identify an easy method that can differentiate between strains of introduced Bradyrhizobium japonicum and the indigenous Bradyrhizobium spp. (TGx) isolates which nodulate the newly developed TGx soybean cultivars in Africa.

Time-course of dinitrogen fixation of promiscuous soybean cultivars measured by the isotope dilution method

Abstract Soybean cultivars capable of nodulating with indigenous Bradyrhizobium spp. have been developed by the International Institute of Tropical Agriculture (IITA) and national programs in Africa in order to avoid artificial inoculation by resource-poor farmers in Africa. The current selection procedure for enhanced N2 fixation is based on an assessment of nodule formation which does not directly quantify the proportions of crop N derived from the atmosphere. We have monitored N accumulation patterns and N2 fixation in nine promiscuous soybean cultivars with different maturity periods, using the 15N dilution technique. Nodule development generally peaked at the early podfill stage for all cultivars except Tgx 1519-1D and Tgx 1447-2D in which it continued to increase. The proportion of crop N derived from fixation (%NDFA) ranged between 51% and 67%, 77% and 84%, and 66% and 73% at full bloom, early podfill, and physiological maturity stages, respectively. Total N accumulation increased in all soybean genotypes with increasing plant age. Significant correlations (P<0.001) were established between nodule weight and %NDFA, even though this did not explain the relationship between nodule development and N2 fixation in cultivars such as Tgx 1519-1D. Promiscuous soybean cultivars retained between 10% and 19% of total N accumulated at the final harvest, in belowground biomass. Our results indicated that these soybean cultivars can derive substantial proportions of plant N from N2 fixation in soils where compatible indigenous bradyrhizobia populations are adequate and effective. Also, we have substantiated the claims that qualitative nodulation parameters currently used to select varieties with a high N2 fixation capacity need to be validated with other measurements of N2 fixation.

Accuracy of most-probable-number estimates of rhizobia for tree legumes

Abstract Rhizobia that nodulate tree legumes have rarely been enumerated by the most-probablenumber (MPN) plant infection assay. We compared MPN estimates of pure cultures of rhizobia with plate counts, using as hosts seedlings of 14 tree legume species grown separately in growth pouches or glass tubes. Reasonable agreement was obtained with 11 of the 14 tree species in one or both growth systems, with closer agreement in glass tubes than growth pouches for small-seeded species. Weekly assessment of MPN assays indicated that glass tubes and growth pouches should be scored for nodulation at least 7 and 5 weeks after inoculation, respectively.

Distribution and Characteristics of Bradyrhizobium Spp. Nodulating African Soybeans

The size and effectiveness of indigenous rhizobia populations influence the legume yield response to inoculation. Some African soybean varieties (e.g. TGx lines) were developed to nodulate with indigenous Bradyrhizobium spp. as well as B. japonicum ostensibly to eliminate the need for inoculation with B. japonicum. We characterized the bradyrhizobia populations from 70 soils in 9 African countries according to nodulation phenotypes: B. spp. effectively nodulate cowpea, B. spp. (TGx) effectively nodulate TGx soybean and cowpea, B. japonicum nodulates N. American and TGx soybean and cowpea. Populations of B spp.(TGx) and B. japonicum were ≤102 g-1 in 85% and 91% of the soils indicating inoculation should increase soybean yields. B spp.(TGx) and B. japonicum were not detected in 26% and 67% of the soil samples. Population size was not related to soil physiochemical properties but was related to prior management. B. spp.(TGx) and B. japonicum populations≥103 g -1 soil were more frequent at research stations than farmers’ fields and where soybean had previously been grown. Of 258 isolates we made using TGx soybean as a trap host, only 27% were highly effective on TGx soybean. Most of the effective isolates were also effective on N. American soybean. RFLP analysis of 16S rDNA from B. spp.(TGx), B. japonicum, and B. spp. strains showed that most B. spp.(TGx) strains were phylogenetically related to B. spp. Like B. elkanii strains, B. spp.(TGx) strains do not have the nodY gene. Additional genetic analyses, evaluation of cross inoculation with tropical legumes and IAR analysis indicated the B. spp.

Genomic Arrangement of Nod Gene Sequences of Bradyrhizobium Isolates from TGx Soybean Genotypes in Relation to Bradyrhizobium USDA110

An article published by Current Plant Science and Biotechnology in Agriculture, vol. 38 pp 297-297, 2000

Gene-for-gene interaction in the legume-Rhizobium symbiosis

The gene-for-gene hypothesis of plant-pathogen interaction as proposed by H.H. Flor states that for each resistance gene in the host there is a specific, complementary gene conditioning pathogenicity in the parasite. Numerous examples of this type of plant-pathogen interaction have been elucidated in the past 40 years. This model of plant-microbe interaction also provides a simple framework in which to view symbiotic plant-microbe interactions. We have identified 10 instances in the literature in which interaction between plants in the family Leguminosae and bacterial symbionts in the genera Rhizobium or Bradyrhizobium appear to function in a gene-for-gene fashion. In addition, we describe two further instances of apparent gene-for-gene interaction in the soybean-B. japonicum symbiosis. These last examples were identified in our laboratory as a result of an intensive search for soybean germplasm that would restrict or resist nodulation by highly competitive, but relatively poor nitrogen fixing strains of B. japonicum that are indigenous to soybean production areas of the U.S.A. The practical goal of this work is firstly, the development of a soybean genotype with genetic factors conferring ‘resistance’ to a broad spectrum of ineffective indigenous bradyrhizobia and secondly, the construction of a highly effective inoculant strain possessing the complementary genes necessary to optimally nodulate such a soybean genotype. This dual approach to develop both partners in the legume-rhizobial symbiosis represents a new application of the gene-for-gene system.