Improvement of biological nitrogen fixation in Egyptian winter legumes through better management of Rhizobium

The diversity of rhizobia nodulating common bean ( Phaseolus vulgaris), berseem clover (Trifolium alexanderinum) and lentil (Lens culinaris) was assessed using several characterization techniques, including nitrogen fixation efficiency, intrinsic antibiotic-resistance patterns (IAR), plasmid profiles, serological markers and rep-PCR fingerprinting. Wide diversity among indigenous rhizobial populations of the isolates from lentil, bean and clover was found. Strikingly, a large percentage of the indigenous rhizobial population was extremely poor at fixing nitrogen. This emphasizes the need to increase the balance of highly efficient strains within the rhizobial population. Use of high-quality inocula strains that survive and compete with other less-desired and less-efficient N2-fixing rhizobia represents the best approach to increase biological nitrogen fixation of the target legume. In field-grown lentils, the inoculant strains were not able to outcompete the indigenous rhizobia and the native lentil rhizobia occupied 76–88% of the total nodules formed on inoculated plants. Nitrogen fixation by lentils, estimated using the ¹⁵N isotope dilution technique, ranged between 127 to 139 kg ha-1 in both inoculated and un-inoculated plants. With berseem clover, the inoculant strains were highly competitive against indigenous rhizobia and occupied 52–79% of all nodules. Inoculation with selected inocula improved N2 fixation by clover from 162 to 205 kg ha-1 in the three cuts as compared with 118 kg ha-1 in the un-inoculated treatment. The results also indicated the potential for improvement of N2 fixation by beans through the application of efficient N2-fixing rhizobia.     

Use of the gusA gene marker in a competition study of the Rhizobium strains nodulating the common bean (Phaseolus vulgaris) in Senegal soils

Indigenous rhizobial population is among the factors which influence increased crop yield through inoculation with elite strains. In this study, we compared in greenhouse conditions the competitiveness of Rhizobium strain ISRA 355 for nodulation of the common bean (Phaseolus vulgaris) cultivated in different unsterile Senegal soils in terms of pH, N and C contents. The strain ISRA 355 produced a stable GUS+ transconjugant which was used for competition with indigenous soil rhizobia in six localities. At Bayakh, the transconjugant ISRA 355gusA was less competitive than the indigenous rhizobial strains, whereas in the other localities, it was more competitive since it occupied more than 90% of the nodules. Thus the Rhizobium strain ISRA 355 should be used for successfully inoculating the common bean in Senegal soils.     

Symbiotic effectiveness and plant growh promoting traits in some Rhizobium strains isolated from Phaseolus vulgaris L.

The ability of Rhizobia to colonize roots of certain legumes and promote their growth has been proven previously. In this study the symbiotic efficiency of 47 Rhizobium strains with 6 common bean cultivars was evaluated under greenhouse condition. Fourteen strains showed the best symbiotic efficiency, whereas some isolates could not induce nodules on host plants. The ability of fourteen superior strains to solubilize phosphorus and zinc and to produce auxin, HCN and siderohores was evaluated in the laboratory assays. Rhizobium strain Rb102 produced the highest amount of auxin (14.2?mg?l^?1) in the medium containing l-tryptophan. None of the isolates were able to solubilize ZnO and ZnCO₃ on solid medium but in liquid medium some of them had negligible solubilization. The highest P solubility in liquid and solid medium was observed in strains Rb113 and Rb130, respectively. Strain Rb102 produced the highest amount of siderophores. None of the isolates were able to produce HCN. This study showed that there was a great diversity between the strains of Rhizobium in terms of their plant growth promoting traits symbiotic efficiency which supports the importance of screening rhizobia for selecting the most efficient strains. The genetic diversity of the isolates was analyzed by PCR–RFLP of the 16S rDNA. Our rhizobia were clustered into 10 groups showing high levels of diversity.     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

Influence of the Size of Indigenous Rhizobial Populations on Establishment and Symbiotic Performance of Introduced Rhizobia on Field-Grown Legumes

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.     

Genomic insight into the taxonomy of Rhizobium genospecies that nodulate Phaseolus vulgaris

Due to the wide cultivation of bean (Phaseolus vulgaris L.), rhizobia associated with this plant have been isolated from many different geographical regions. In order to investigate the species diversity of bean rhizobia, comparative genome sequence analysis was performed in the present study for 69 Rhizobium strains mainly isolated from root nodules of bean and clover (Trifolium spp.). Based on genome average nucleotide identity, digital DNA:DNA hybridization, and phylogenetic analysis of 1,458 single-copy core genes, these strains were classified into 28 clusters, consistent with their species definition based on multilocus sequence analysis (MLSA) of atpD, glnII, and recA. The bean rhizobia were found in 16 defined species and nine putative novel species; in addition, 35 strains previously described as Rhizobium etli, Rhizobium phaseoli, Rhizobium vallis, Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium spp. should be renamed. The phylogenetic patterns of symbiotic genes nodC and nifH were highly host-specific and inconsistent with the genomic phylogeny. Multiple symbiovars (sv.) within the Rhizobium species were found as a common feature: sv. phaseoli, sv. trifolii and sv. viciae in Rhizobium anhuiense; sv. phaseoli and sv. mimosae in Rhizobium sophoriradicis/R. etli/Rhizobium sp. III; sv. phaseoli and sv. trifolii in Rhizobium hidalgonense/Rhizobium acidisoli; sv. phaseoli and sv. viciae in R. leguminosarum/Rhizobium sp. IX; sv. trifolii and sv. viciae in Rhizobium laguerreae. Thus, genomic comparison revealed great species diversity in bean rhizobia, corrected the species definition of some previously misnamed strains, and demonstrated the MLSA a valuable and simple method for defining Rhizobium species.     

Growth and survival of cowpea rhizobia in bauxitic silt loam and sandy clay loam soils

Survival of 4 cowpea Rhizobium strains, IRC291, MI-50A, JRW3 and JRC29, in two soil types (bauxitic silt loam and sandy clay loam) undergoing drying at 30°C and 37°C was examined. While all strains except JRW3 showed a general pattern of increase in their numbers during the first 3 weeks in sterile soils, none of the strains showed any increase in their population in non-sterile soils. Cowpea rhizobia showed better survival in non-sterile bauxitic silt loam than in clay loam soils at 30°C. However, the long-term survival (examined up to 6 months) of rhizobia in both soils was poor at 37°C as compared to 30°C. We also found that cowpea rhizobia survived better in soils undergoing drying than in moist soils at 30°C. Our results suggest that (a) cowpea rhizobia survived better in bauxitic silt loam than in clay loam soil and (b) the low indigenous cowpea rhizobial population in Jamaican soils may be due to their poor long-term survival and weak saprophytic competence.     

Diversity and Symbiotic Effectiveness of Indigenous Rhizobia-Nodulating Adesmia bicolor in Soils of Central Argentina

Native perennial legume Adesmia bicolor reveals characteristics that are key to securing persistence under grazing. Literature on the diversity and symbiotic effectiveness of indigenous rhizobia-nodulating A. bicolor in central Argentina is limited. The purpose of this study was therefore to determine phenotypic and genotypic variability as well as biological N-fixation effectiveness in rhizobia isolated from A. bicolor nodules. To this end, repetitive genomic regions were analyzed using ERIC primers. In the greenhouse, plants were grown under a (i) N-fertilized treatment, (ii) N-free control treatment, and (iii) rhizobia inoculation treatment. Dry weight and N-content were analyzed. All isolates belonged to Rhizobium genus and showed high symbiotic effectiveness. The N-content/subterranean N-content ratio in aerial and subterranean parts of inoculated plants was higher than that observed in N-fertilized plants during the vegetative stage. Results from this study demonstrate that symbiosis between native rhizobial strains and A. bicolor is very effective.     

Phylogenetic diversity of Rhizobium strains nodulating diverse legume species growing in Ethiopia

The taxonomic diversity of thirty-seven Rhizobium strains, isolated from nodules of leguminous trees and herbs growing in Ethiopia, was studied using multilocus sequence analyses (MLSA) of six core and two symbiosis-related genes. Phylogenetic analysis based on the 16S rRNA gene grouped them into five clusters related to nine Rhizobium reference species (99–100% sequence similarity). In addition, two test strains occupied their own independent branches on the phylogenetic tree (AC86a2 along with R. tibeticum; 99.1% similarity and AC100b along with R. multihospitium; 99.5% similarity). One strain from Milletia ferruginea was closely related (>99%) to the genus Shinella, further corroborating earlier findings that nitrogen-fixing bacteria are distributed among phylogenetically unrelated taxa. Sequence analyses of five housekeeping genes also separated the strains into five well-supported clusters, three of which grouped with previously studied Ethiopian common bean rhizobia. Three of the five clusters could potentially be described into new species. Based on the nifH genes, most of the test strains from crop legumes were closely related to several strains of Ethiopian common bean rhizobia and other symbionts of bean plants (R. etli and R. gallicum sv. phaseoli). The grouping of the test strains based on the symbiosis-related genes was not in agreement with the housekeeping genes, signifying differences in their evolutionary history. Our earlier studies revealing a large diversity of Mesorhizobium and Ensifer microsymbionts isolated from Ethiopian legumes, together with the results from the present analysis of Rhizobium strains, suggest that this region might be a potential hotspot for rhizobial biodiversity.     

An assessment of a method based on intrinsic antibiotic resistance for identifying Rhizobium strains

A method based on intrinsic antibiotic resistance (IAR) for identifying large numbers of Rhizobium strains was assessed and found to be unsatisfactory for R. phaseoli and isolates from Cicer arietinum (Rhizobium spp.). Our data showed that the number of different IAR patterns always exceeded the number of strains tested. With 90 nodule isolates from plants inoculated with a mixture of three strains of R. Phaseoli, the technique gave 18 different resistance patterns. When 24 strains of Rhizobium spp., each replicated three times, were examined 68 different resistance patterns were obtained. Single colony isolates from one strain also gave several different IAR patterns. All strains tested with fluorescent“ antibody were readily identified. Attempts to obtain correct strain identification with IAR by simplifying the scoring systems or allowing up to two differences in the resistance patterns were unsuccessful. We were unable to define the source of this variation although incubation time and inoculum concentration were shown to affect the IAR patterns     

Genetic diversity of rhizobia associated with root nodules of white lupin (Lupinus albus L.) in Tunisian calcareous soils

With a view to introducing white lupin (Lupinus albus L.) for cultivation in Tunisian calcareous soils, compatible indigenous rhizobia for nitrogen-fixing symbiosis were investigated and characterized. Two L. albus varieties, Mekna and Lumen, were used to trap rhizobia in soil samples collected from 56 sites with high active lime contents (0–49%). Nodulation occurred in only 15 soils. The local variety, Mekna, developed significantly more root nodules and had a trapping capacity in more soils than the imported variety Lumen. A phylogenetic analysis based on the partial 16S-23S ribosomal RNA internal transcribed spacer region (ITS) and multi-locus sequence analysis (MLSA) of three chromosomal housekeeping genes, recA, atpD and dnaK, showed that strains were affiliated to Agrobacterium, Rhizobium, and Neorhizobium, with large internal diversity, including separate lineages. Infectivity tests highlighted some nodulation specificity at the plant variety level, since the strains originating from Mekna could only nodulate this variety, while strains trapped in Lumen could nodulate both varieties. When inoculated, almost all strains resulted in a significant increase in plant shoot dry weight on L. albus. Although Agrobacterium sp. strains isolated from L. albus could nodulate and had a plant growth promoting effect, no nodA and nodC genes could be amplified. This is discussed together with the absence of bradyrhizobia and the general infrequency of L. albus–nodulating rhizobia in Tunisian soils. The adapted and efficient rhizobial strains reported here were promising candidates for inoculant development and represent a contribution towards successful cultivation of L. albus in Tunisia, especially the most promising Mekna variety.     

Genetic diversity of indigenous Bradyrhizobium nodulating promiscuous soybean [Glycine max (L) Merr.] varieties in Kenya: Impact of phosphorus and lime fertilization in two contrasting sites

While soybean is an exotic crop introduced in Kenya early last century, promiscuous (TGx) varieties which nodulate with indigenous rhizobia have only recently been introduced. Since farmers in Kenya generally cannot afford or access fertilizer or inoculants, the identification of effective indigenous Bradyrhizobium strains which nodulate promiscuous soybean could be useful in the development of inoculant strains. Genetic diversity and phylogeny of indigenous Bradyrhizobium strains nodulating seven introduced promiscuous soybean varieties grown in two different sites in Kenya was assayed using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 16S-23S rDNA intergenic spacer region and 16S rRNA gene sequencing. PCR-RFLP analysis directly applied on 289 nodules using Msp I distinguished 18 intergenic spacer groups (IGS) I–XVIII. Predominant IGS groups were I, III, II, IV and VI which constituted 43.9%, 24.6%, 8.3% 7.6% and 6.9% respectively of all the analyzed nodules from the two sites while IGS group VII, IX, X, XI, XII, XIV, XVI, XVII, XVIII each constituted 1% or less. The IGS groups were specific to sites and treatments but not varieties. Phylogenetic analysis of the 16S rRNA gene sequences showed that all indigenous strains belong to the genus Bradyrhizobium. Bradyrhizobium elkanii, Bradyrhizobium spp and Bradyrhizobium japonicum related strains were the most predominant and accounted for 37.9%, 34.5%, and 20.7% respectively while B. yuanmigense related accounted for 6.9% of all strains identified in the two combined sites. The diversity identified in Bradyrhizobium populations in the two sites represent a valuable genetic resource that has potential utility for the selection of more competitive and effective strains to improve biological nitrogen fixation and thus increase soybean yields at low cost.     

Mass Spectrometric Quantification of Indole-3-Acetic Acid in Rhizobium Culture Supernatants: Relation to Root Hair Curling and Nodule Initiation

Indole-3-acetic acid (IAA) has been unequivocally identified in culture supernatants of Rhizobium strains by gas chromatography-mass spectrometry. A method for accurately quantitating IAA in bacterial culture supernatants, employing deuterium-labeled IAA as an internal standard, has been developed. Similar IAA concentrations were found in culture supernatants of chosen Rhizobium mutants (defective in nodule formation) and their corresponding parent strains. Since some of the mutants are known to adhere to root hairs, it can be concluded that root hair curling is not simply a consequence of IAA production by rhizobia.     

Effectiveness of Rhizobium Strains Used in Inoculants after Their Introduction into Soil

Rhizobium strains used in inoculants for Trifolium spp., Medicago spp., Glycine max, and Lotus pedunculatus were isolated from nodules of these legumes grown in soils into which the rhizobia had been introduced 4 to 8 years before. Isolations were made from a total of 420 nodules. Nodule occupancy by the inoculant strains varied from 17.7% for a soybean strain to 100% in the case of L. pedunculatus whose specific rhizobia did not occur in the soils studied. In general, inoculant strains isolated from nodules did not differ in effectiveness from cultures of the same strains concurrently maintained in lyophilized form. The average effectiveness of all of the isolates (identified and unidentified) from a legume was 7.1 to 73.3% higher than that of the unidentified isolates alone, demonstrating the prolonged effect that a single-seed inoculation has on the rhizobial population in a soil which had not been planted with legumes before. Relatively weak recovery of a Rhizobium japonicum strain introduced into soil 4 years after soybean seed inoculated with a different strain had been planted in the same soil confirmed the advantage of a resident population over an introduced inoculant strain.     

Rhizobium qilianshanense sp. nov., a novel species isolated from root nodule of Oxytropis ochrocephala Bunge in China

During a study of the diversity and phylogeny of rhizobia isolated from root nodules of Oxytropis ochrocephala grown in the northwest of China, four strains were classified in the genus Rhizobium on the basis of their 16S rRNA gene sequences. These strains have identical 16S rRNA gene sequences, which showed a mean similarity of 94.4 % with the most closely related species, Rhizobium oryzae. Analysis of recA and glnA sequences showed that these strains have less than 88.1 and 88.7 % similarity with the defined species of Rhizobium, respectively. The genetic diversity revealed by ERIC-PCR fingerprinting indicated that the isolates correspond to different strains. Strain CCNWQLS01^T contains Q-10 as the predominant ubiquinone. The major fatty acids were identified as feature 8 (C18: 1ω7c and/or C18: 1ω6c; 67.2 %). Therefore, a novel species Rhizobium qilianshanense sp. nov. is proposed, and CCNWQLS01^T (= ACCC 05747^T = JCM 18337^T) is designated as the type strain.     

Use of Two-Dimensional Polyacrylamide Gel Electrophoresis to Identify and Classify Rhizobium Strains

Fifty-seven strains of various Rhizobium species were analyzed by two-dimensional gel electrophoresis. Since the protein pattern on such gels is a reflection of the genetic background of the tested strains, similarities in pattern allowed us to estimate the relatedness between these strains. All group II rhizobia (slow growing) were closely related and were very distinct from group I rhizobia (fast growing). Rhizobium meliloti strains formed a distinct group. The collection of R. leguminosarum and R. trifolii strains together formed another distinct group. Although there were some similarities within the R. phaseoli, sesbania rhizobia, and lotus rhizobia, the members within these seemed much more diverse than the members of the above groups. The technique also is useful to determine whether two unknown strains are identical.     

Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agricultural-forestry ecosystem

To investigate the diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in the same habitat, a total of 97 rhizobial strains isolated from nine legume species grown in an agricultural-forestry ecosystem were identified into seven genomic species and 12 symbiotic genotypes within the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium based upon analyses of genomic DNA regions and symbiotic genes. The results evidenced that the symbiotic genotypes of rhizobia were consistent with their hosts of origin; revealed that vertical transfer was the main mechanism in rhizobia to maintain the symbiotic genes but lateral transfer of symbiotic genes might have happened between the closely related rhizobial species; suggested the existence of co-distribution and co-evolution among the legume hosts and compatible rhizobia. All of these data demonstrated that the biogeography of rhizobia was a result of interactions among the host legumes, bacterial genomic backgrounds and environments.     

Growth of Indigenous Rhizobium leguminosarum and Rhizobium meliloti in Soils Amended with Organic Nutrients

The ability of indigenous Rhizobium leguminosarum and Rhizobium meliloti to use organic nutrients as growth substrates in soil was assessed by indirect bacteriophage analysis. A total of 17 organic compounds, including 9 carbohydrates, 3 organic acids, and 5 amino acids, were tested (1,000 μg g⁻¹) in three soils with different cropping histories. Four additional soils were screened with a glucose amendment. Nutrient amendments stimulated growth of indigenous rhizobia, allowing subsequent replication of indigenous bacteriophages. Phage populations were enumerated by plating soil extracts on 19 R. leguminosarum and 9 R. meliloti indicator strains, including root nodule isolates from the soils assayed. On the basis of indirect phage analysis, all soils contained native rhizobia similar to one or more of the indicator strains, although not all indicator strains were detected in soil. All organic compounds stimulated growth of indigenous rhizobia, but the growth response varied for each rhizobial strain depending on the nutrient, the nutrient concentration, and the soil. Indigenous rhizobia readily utilized most organic compounds except phenylalanine, glycine, and aspartic acid. The ability of indigenous rhizobia to utilize a wide range of organic compounds as growth substrates in situ indicates their ability to successfully compete with other soil bacteria for nutrients in these soils.     

Diversity of a Soybean Bradyrhizobial Population Adapted to an Indian Soil

Soybean (Glycine max) is an introduced crop in India. Over the years it has been regularly inoculated with indigenous rhizobia. In this study genetic diversity has been studied at a site where soybean has been regularly grown with inoculation. Rhizobia were plant trapped using soybean varieties as host, and fingerprinted using BOX-PCR. BOX-PCR genomic fingerprints of 69 isolates from the nodules of 4 soybean varieties Pusa22, Bragg, PK1041 and PK1142 showed a high level of genetic diversity. The population profiles of the 69 isolates clustered them into 10 groups. Root nodule isolates from the four varieties were Bradyrhizobium japonicum types, growing in 4–7 days with typical colonies which were found to be genetically distinct from the USDA and SEMIA strains of B. japonicum and B. elkanii. Also the genotype of the host plant seemed to be one of the factors determining the diversity. The high diversity could be attributed both to lateral transfer of genetic material between inoculant and indigenous strains and to genomic rearrangements during the adaptation to the Indian soils.