Ecology and genetics of tropical rhizobia species

Biological nitrogen fixation (BNF) technology with special reference to Rhizobium-legume symbiosis is growing very rapidly with the hope of combatting world hunger by producing cheaper protein for animal and human consumption in the Third World. One can see rapid progress made in the biochemistry and molecular biology of symbiotic nitrogen fixation in general; however, less progress has been made on the ecological aspects despite the fact that an enormous amount of literature is available on inoculation problems and on agronomic aspects of symbiotic nitrogen fixation. So far most information on Rhizobium concerns fast-growing rhizobia and their host legume. Although it is essential that food production using BNF technology should be maximized in the Third World, the least work has been done on slow-growing rhizobia, which are generally found in tropical and sub-tropical soils. The majority of the developing countries are in tropical and sub-tropical regions. Except for R. japonicum, a microsymbiont partner of soybean (Glycine max), the majority of the slow-growing rhizobia belong to the cowpea group, and we refer to cowpea rhizobia as tropical rhizobia species. In this review we have tried to consolidate the recent progress made on ecology and genetics of tropical rhizobia. By using recombinant DNA technology techniques it is expected that super strains of rhizobia with desirable characteristics can be produced. One must evaluate the efficiency and effectiveness of these genetically manipulated laboratory strains under field conditions. In conclusion, if one aims at combatting hunger in the Third World using BNF technology, an intensive research programme on fundamental and applied aspects of tropical rhizobia species is suggested. This involves close cooperation between molecular biologists and microbial ecologists.     

Relationships Among Rhizobia from Native Australian Legumes

Isolates from 12 legumes at three sites in Victoria showed a wide range of morphological, cultural, symbiotic, and serological properties. Isolates from Acacia longifolia var. sophorae and Kennedia prostrata were fast growing but nodulated ineffectively Macroptilium atropurpureum and all native legumes except Swainsonia lessertiifolia. Isolates from S. lessertiifolia showed anomalous properties intermediate between fast- and slow-growing rhizobia. All isolates from the other two sites were slow-growing “cowpea” rhizobia. Symbiotic effectiveness was usually poor, and there was no relationship between effectiveness and host taxonomy or serological affinities of the isolates. This is the first report of fast-growing rhizobia from temperate Australian woody legumes and the first report of the symbiotic effectiveness of native Australian legumes with indigenous rhizobia.     

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48^T and B. liaoningense USDA 3622^T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.     

Effect of soil salinity level and zinc application on growth,yield, and nutrient composition of rice

Summary Physiological and symbiotic characteristics were identified in fast-growing (FG)Rhizobium japonicum. Carbon nutritional patterns linked these rhizobia to other FG rhizobia. They were able to use hexoses, pentoses, disaccharides, trioses, and organic acids for growth, but they were unable to use dulcitol or citrate. These rhizobia produced acid with all carbon sources except intermediates of the Krebs cycle. FGR. japonicum showed no vitamin requirements and were tolerant to 1% NaCl but not to 2%. They nodulated cowpea, pigeon pea, and mung bean but not peanut. Effective, nitrogen-fixing symbioses were observed only with cowpea and pigeon pea. In addition, FGR. japonicum formed effective symbioses with Asian-type soybeans. We concluded that although the physiological characteristics of FGR. japonicum were similar to other FG rhizobia, their symbiotic properties were similar to slow-growing rhizobia of the cowpea miscellany.     

Invalidity of the Concept of Slow Growth and Alkali Production in Cowpea Rhizobia

A total of 103 rhizobial strains representing the cowpea miscellany and Rhizobium japonicum were studied with regard to growth rate, glucose metabolic pathways, and pH change in culture medium. Doubling times ranged from 1.4 ± 0.04 to 44.1 ± 5.2 h; although two populations of “fast-growing” and “slow-growing” rhizobia were noted, they overlapped and were not distinctly separated. Twenty-four strains which had doubling times of less than 8 h all showed NADP-linked 6-phosphogluconate dehydrogenase (6-PGD) activity, whereas only one slow-growing strain (doubling time, 10.8 ± 0.9 h) of all those tested showed 6-PGD activity. Doubling times among fast growers could not be explained solely by the presence or absence of 6-PGD activity (r² = 0.14) because the tricarboxylic acid cycle and the Emden-Meyerhoff-Parnas pathway were operative in both 6-PGD-positive and 6-PGD-negative strains. Growth rate and pH change were unrelated to each other. Fast- or slow-growing strains were not associated with any particular legume species or group of species from which they were originally isolated, with the exception of Stylosanthes spp., all nine isolates of which were slow growers. We conclude that 6-PGD activity is a more distinctive characteristic among physiologically different groups of rhizobia than doubling times and that characterization of the cowpea rhizobia as slow-growing alkali producers is an invalid concept.     

Root nodules of some tropical legumes in Singapore

Summary A survey of 35 legume species comprising 25 of Papilionoideae, 7 of Mimosoideae and 3 of Caesalpinioideae was made. Nodulation was found in all the species except for 2 (Caesalpinnia pulcherrima and Cassia siamea) of Caesalpinioideae, both of which possessed dark coloured roots. Nodulation is reported for the first time for Adenanthera pavonina and Delonix regia. Nodule shapes were described and classified into different types. The isolates of rhizobia obtained belonged largely to the slow growing group (17 isolates) isolated mainly from members of Papilionoideae; some belonged to the fast growing group (14 isolates), and only 3 isolates belonged to the very slow growing group. The slow growing group isolates were confirmed to be cowpea type rhizobia on the basis of positive nodulation with cowpea plants. re]19750829     

Comparison of the host range of fast-growingR. japonicum strains with a fast-growing isolate from Lablab

Summary Fast-growingRhizobium japnicum strains derived from the People's Republic of China were compared with a fast-growingRhizobium isolate from Lablab for their ability to nodulate tropical legumes grown in Leonard-jars and test tube culture. Fast-growingR. japonicum strains were all effective to varying degrees in their symbiosis withVigna unguiculata. Two strains USDA 192 and USDA 201, effectively nodulatedGlycine whightii and one strain, USDA 193, effectively nodulatedMacroptilium atropurpureum. Other nodulation responses in tropical legumes were ineffective. The fast-growing isolate from Lablab was more promiscuous, effectively nodulating with a larger host range. The fast-growing Lablab strain was considered more akin, on a symbiotic basis, to the slow-growing cowpea type rhizobia than the fast-growing China strains ofR. japonicum whilst maintaining physiological characteristics of other fast-growing rhizobia.     

Phenotypic characteristics and composition of rhizobia associated with woody legumes growing in diverse Kenyan conditions

Over 480 rhizobia were isolated from root nodules of woody legume and herbaceous trap host species grown in soils collected from 12 different Kenyan sites. The isolates were differentiated by growth and morphological characteristics, intrinsic antibiotic resistance (IAR) and salt (NaCl) tolerance levels (STL) when grown on yeast mannitol mineral salts agar and broth media.The bulk of the isolates (91%) were watery, milky-translucent and curdled milk types with moderate to copious extracellular polysaccharide (EPS). The rest were creamy or white opaque with little to moderate EPS production. Overall, they showed a wide range of growth rates: very fast-growing (mean generation time 1.6–2.5 h), fast-growing (2.8–4.8 h), intermediate between fast- and slow-growing (5.6–5.7 h) and slow- and very slow-growing (6.4–8.8 h). The isolates were tentatively grouped into Rhizobium spp., to include very fast, fast and intermediate (acid-producing) types; and Bradyrhizobium spp., to include very slow, slow and intermediate (alkali-producing) types.Bradyrhizobium spp. were more sensitive to antibiotics (40 g mL^-1) than Rhizobium spp., contrary to the general opinion which indicates that they are normally resistant. Cluster analysis based on sensitivity responses of IAR and STL could not distinguish Rhizobium spp. from Bradyrhizobium spp., neither was there any association by site nor host of isolation except for those isolates trapped with Phaseolus vulgaris at Kibwezi.Our data demonstrated a high diversity of tropical rhizobia associated with trees.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.     

Morphology and host range of virulent phages of lotus rhizobia.

Nineteen virulent bacteriophages for fast- and slow-growing rhizobia were isolated. Most of the phage isolates were of two morphological types, and these showed specificity for either the fast- or the slow-growing rhizobia. The ecological distribution, morphology, and host range of the phages is presented. Classification of lotus rhizobia is discussed on the basis of phage typing.     

Patterns of Sole‐Carbon‐Source Utilization by Fast‐Growing Coastal Sand Dune Rhizobia of the Southwest Coast of India

Coastal sand dunes harbor a variety of free living and symbiotic microorganisms, which are adapted to stress conditions such as temperature, salinity and pH. The current paper explores the ability of sole‐source‐carbon utilization by symbiotic rhizobia isolated from tropical coastal sand dune wild legumes. Fast‐growing rhizobia isolated from five legume plant species (Canavalia cathartica, Canavalia maritima, Crotalaria retusa, Crotalaria verrucosa, and Derris triflorum) grown on the two coastal sand dunes of the southwest coast of India (Someshwara, S1–S5; Padubidri, P1–P5) were assessed for sole‐carbon‐source utilization patterns based on BIOLOG GN2 microplate technique. All the isolates showed high extents of utilization of the different carbon sources within 24 h of incubation at 30 °C. Cluster analysis based on quantitative and qualitative utilization of a sole carbon source revealed two distinct clusters. Cluster I consists of four isolates (S1, P2, P4, and P5), of which P2 and P5 showed greater similarities. The cluster II encompasses six isolates (S2–S5, P1 and P3), among them S3 and S5 showed high similarities. Based on the utilization of six guilds by the isolates S1, S2, P1, and P2 (polymers, carbohydrates, carboxylic acids, amides and amines, amino acids and miscellaneous), S2 and P1 exhibited high functional diversity. Principal component analysis revealed a close catabolic potential between the isolates S5 and P1; S1 and P3; S3, P2, and P5. The symbiotic rhizobia of the southwest coastal sand dune wild legumes of India studied might serve as novel inoculants to achieve legume production through nitrogen fixation under the varied conditions of tropical soils. These rhizobia were able to utilize a wide range of carbon sources, possessed functional diversity, withstood extreme conditions (temperature, salinity, pH), exhibited non‐host specificity and induced high biomass in edible legumes.     

Indigenous plasmids and cultural characteristics of rhizobia nodulating chickpeas (Cicer arietinum L.)

We examined 27 strains of chickpea rhizobia from different geographic origins for indigenous plasmids, location and organization of nitrogen fixation (nif) genes, and cultural properties currently used to separate fast- and slow-growing groups of rhizobia. By using an in-well lysis and electrophoresis procedure one to three plasmids of molecular weights ranging from 35 to higher than 380 Mdal were demonstrated in each of 19 strains, whereas no plasmids were detected in the eight remaining strains. Nitrogenase structural genes homologous to Rhizobium meliloti nifHD, were not detected in plasmids of 26 out of the 27 strains tested. Hybridization of EcoRI digested total DNA from these 26 strains to the nif probe from R. meliloti indicated that the organization of nifHD genes was highly conserved in chickpea rhizobia. The only exception was strain IC-72 M which harboured a plasmid of 140 Mdal with homology to the R. meliloti nif DNA and exhibited also a unique organization of nifHD genes. The chickpea rhizobia strains showed a wide variation of growth rates (generation times ranged from 4.0 to 14.5 h) in yeast extract-mannitol medium but appear to be relatively homogeneous in terms of acid production in this medium and acid reaction in litmus milk. Although strains with fast and slow growth rates were identified, DNA/DNA hybridization experiments using a nifHD-specific probe, and the cultural properties examined so far do not support the separation of chickpea rhizobia into two distinct groups of the classical fast- and slow-growing types of rhizobia.     

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I–V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.     

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.     

Survey of Chickpea Rhizobia Diversity in Portugal Reveals the Predominance of Species Distinct from Mesorhizobium ciceri and Mesorhizobium mediterraneum

Several Mesorhizobium species are able to induce effective nodules in chickpea, one of the most important legumes worldwide. Our aims were to examine the biogeography of chickpea rhizobia, to search for a predominant species, and to identify the most efficient microsymbiont, considering Portugal as a case study. One hundred and ten isolates were obtained from continental Portugal and Madeira Island. The 16S ribosomal RNA gene phylogeny revealed that isolates are highly diverse, grouping with most Mesorhizobium type strains, in four main clusters (A–D). Interestingly, only 33% of the isolates grouped with Mesorhizobium ciceri (cluster B) or Mesorhizobium mediterraneum (cluster D), the formerly described specific chickpea microsymbionts. Most isolates belong to cluster A, showing higher sequence similarity with Mesorhizobium huakuii and Mesorhizobium amorphae. The association found between the province of origin and species cluster of the isolates suggests biogeography patterns: most isolates from the north, center, and south belong to clusters B, A, and D, respectively. Most of the highly efficient isolates (symbiotic effectiveness >75%) belong to cluster B. A correlation was found between species cluster and origin soil pH of the isolates, suggesting that pH is a key environmental factor, which influences the species geographic distribution. To our knowledge, this is one of the few surveys on chickpea rhizobia and the first systematic assessment of indigenous rhizobia in Portugal.     

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.     

Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.     

Relationships Amongst the Fast-growing Rhizobia of Lablab purpureus, Leucaena leucocephala, Mimosa spp., Acacia farnesiana and Sesbania grandiflora and their Affinities with Other Rhizobial Groups

Colony characteristics, growth in litmus milk, precipitation in calcium glycerophosphate medium and utilization of carbon sources of the root-nodule bacteria isolated from the tropical legumes Leucaena, Mimosa, Acacia, Sesbania and Lablab were similar to fast-growing rhizobia of temperate legumes, particularly Rhizobium meliloti. In agglutination tests, isolates from each host shared antigens with one or more of five Rhizobium strains from Leucaena. Infective characteristics of the fast-growing rhizobia were studied in modified Leonard jars and in agar culture. Cross-infections by rhizobia between these plants were common and the association often effective. Lablab was effectively nodulated by its own fast-growing isolate but only formed root swellings, possibly ineffective pseudonodules, with the other isolates. Slow-growing rhizobia which were able to nodulate Macroptilium atropurpureus were unable to form nodules on these legumes except Lablab which was considered more akin to the cowpea group. All fast-growing isolates nodulated, often effectively, Vigna unguiculata and V. unguiculata ssp. sesquipedalis. The isolate from Lablab also effectively nodulated a number of other tropical legumes which have previously only been reported to nodulate with slow-growing nodule bacteria and it also produced ineffective nodulation on Medicago sativa. This is the first record of an effective fast-growing isolate from Lablab.     

Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the Western Amazon by using cowpea as the trap plant

Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burkholderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.     

Nodule formation and haemoglobin content in some species of Papilionaceae

Summary Characteristics of nodule formation in eleven genera of the Leguminosae, belonging to the tribes Galegeae, Genisteae, Hedysareae and Phaseoleae of the sub-family Papilionaceae are described. Variation existed in the type, size and weight of nodules formed on the legumes when inoculated with effective cowpea rhizobia, in field conditions of plant growth. Among the legumes, the haemoglobin content of nodules indicated their possible effectiveness. Dolichos lablab L., had a higher haemoglobin content per unit nodule volume than other legumes. This host may have a greater potential than the other species in symbiotic activity with legume bacteria.