Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 × 104 g of soil-1 for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha-1 in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P ≤ 0.05). On average, inoculation increased yield by 62%. Soybean (G. max) responded to inoculation most frequently, while cowpea (V. unguiculata) failed to respond in all trials. Inoculation responses in the other legumes were site dependent. The response to inoculation and the competitive success of inoculant rhizobia were inversely related to numbers of indigenous rhizobia. As few as 50 rhizobia g of soil-1 eliminated inoculation response. When fewer than 10 indigenous rhizobia g of soil-1 were present, economic yield was significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil-1. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N2-fixing crops indicates a potential for improving inoculation technology, the N2 fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.

Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes.

Inoculant production and formulation

Symbiotic nitrogen fixation is carried out in specialized organs, the nodules, whose formation is induced on leguminous host plants by bacteria belonging to the family Rhizobiaceae. Nodule development is a complex multistep process, which requires continued interaction between the two partners and thus the exchange of different signals and metabolites. NH4+ is not only the primary product but also the main regulator of the symbiosis: either as ammonium and after conversion into organic compounds, it regulates most stages of the interaction, from the production of nodule inducers to the growth, function, and maintenance of nodules. This review examines the adaptation of bacterial NH4+ metabolism to the variable environment generated by the plant, which actively controls and restricts bacterial growth by affecting oxygen and nutrient availability, thereby allowing a proficient interaction and at the same time preventing parasitic invasion. We describe the regulatory circuitry responsible for the downregulation of bacterial genes involved in NH4+ assimilation occurring early during nodule invasion. This is a key and necessary step for the differentiation of N2-fixing bacteroids (the endocellular symbiotic form of rhizobia) and for the development of efficient nodules.

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Key role of bacterial NH(4)(+) metabolism in Rhizobium-plant symbiosis.

Inoculation of legumes under field conditions with superior nitrogen-fixing rhizobia does not always result in the desired yield increase. Often it is observed that the inoculum strain fails to occupy a significant proportion of the nodules. The introduced inoculant strains have to compete with the indigenous, often ineffective, nitrogen-fixing rhizobial population at different levels. The success of inoculation depends to a large extent on the ratio of the inoculant cells to indigenous rhizobia. However, intrinsic characteristics of the inoculant and indigenous rhizobia, and their responses to abiotic and biotic environmental variables, also influence the outcome of inoculation. In this review, the genetic basis for “efficient host-bacteria interaction” is reviewed. In addition, environmental factors that influence competition and saprophytic competence of rhizobia are discussed.

Factors Influencing Nodule Occupancy by Inoculant Rhizobia

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.

Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes

The competitiveness of a Rhizobium leguminosarum strain was investigated at two separate locations in field inoculation studies on commercially grown peas. The soil at each location (sites I and II) contained an indigenous R. leguminosarum population of ca. 3 × 104 rhizobia per g of soil. At site I it was necessary to use an inoculum concentration as large as 4 × 107 CFU ml−1 (2 × 106 bacteria seed−1) to establish the inoculum strain in the majority of nodules (73%). However, at site II the inoculum strain formed only 33% of nodules when applied at this (107 CFU ml−1) level. Establishment could not be further improved by increasing the inoculum concentration even as high as 109 CFU ml−1 (9.6 × 107 bacteria seed−1). The inoculum strain could be detected at both sites 19 months after inoculation. Analysis by intrinsic antibiotic resistance patterns and plasmid DNA profiles indicated that a dominant strain(s) and plasmid pool existed among the indigenous population at site II. Competition experiments were carried out under laboratory conditions between a dominant indigenous isolate and the inoculum strain. Both strains were shown to be equally competitive.

Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia.

A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm − ) was isolated following mutagenesis with N -methyl N -nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an ‘in vitro’ assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm − mutant class in Rhizobium spp.

Isolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation

Recovery of Rhizobium leguminosarum cells by centrifugation after growth in an industrial fermenter was 100-fold higher when cells were grown on yeast extract (5 g/1) as sole source of carbon and nitrogen when compared with the yields recovered when cells were grown in standard mannitol-yeast extract medium. Methods of storing concentrated suspensions of R. leguminosarum were investigated. Freeze-drying caused a marked decrease in viable cell numbers. Viable cell numbers of bacterial concentrates stored in peat decreased steadily from 1011-1012 cfu/g to 109 cfu/g or less during 26 weeks storage at room temperature or at 4°C. Cell concentrates stored in 40% glycerol at — 20°C maintained viable numbers higher than 1011 cfu/ml during a 76 week storage period.

Production and storage of Rhizobium leguminosarum cell concentrates for use as inoculants

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP4. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8 X 10(-6). Assimilatory GDH (NADP+) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm+ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by L-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum.

A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm−) was isolated following mutagenesis with N-methyl N-nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an ‘in vitro’ assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm− mutant class in Rhizobium spp.

James Meade

Papers

Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia.

Isolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation

Production and storage of Rhizobium leguminosarum cell concentrates for use as inoculants

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum.

Research letterIsolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation