Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N₂ fixation.     

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

Evaluation of the intrinsic competitiveness and saprophytic competence of Rhizobium tropici IIB strains

Inoculation of beans (Phaseolus vulgaris L.) with strains of R. tropici IIB and R. etli resulted in the disappearance of the R. tropici IIB stains from the nodule population and their replacement by other (non R. tropici IIB) bean symbionts (Vlassak et al. 1996). Coinoculation studies in monoxenic conditions and in soil core microcosms with plants harvested at two different growth stages indicated that the inoculated R. tropici IIB strains CIAT899 and F98.5 possess a good intrinsic competitiveness which declines, however, at a later plant growth stage and in soil conditions. The poor saprophytic competence of R. tropici IIB strain CIAT899 was further demonstrated by its poor survival in soil core microcosms after bean harvest. Strains were isolated from the field plots with a 3-year bean-planting history, characterized and evaluated for their competitiveness against R. tropici IIB strain CIAT899. Isolates from field plots, which had been repeatedly inoculated with R. tropici IIB strain CIAT899, showed a higher nodule occupancy compared to R. tropici IIB strain CIAT899, and this higher competitiveness exhibited by the field isolates might be an additional reason for the poor performance of R. tropici IIB strain CIAT899 in the field study. Plots with and without a history of bean production revealed after 3-year bean cultivation an almost totally different population that also significantly differed in competitiveness. Received: 12 February 1996     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

Genotypic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris L. grown in Tunisian soils

 One hundred and sixty isolates of rhizobia were sampled from the root nodules of common bean (Phaseolus vulgaris L.) cultivated in Tunisian soil samples originating from three geographically distinct fields. Plasmid profiling was used as a primary method to rapidly screen the isolates, and then 38 plasmid types were recorded among the 160 isolates. A sample representing the majority of plasmid types was chosen for further characterization by restriction fragment length polymorphism (RFLP) analysis of genomic DNA using chromosomal and symbiotic gene probes, and by their ability to nodulate a potential alternative host, Leucaena leucocephala. One third of the isolates showed a high similarity to Rhizobium gallicum isolated from common bean in France, another third showed the same characteristics as the R. etli-R. leguminosarum group, while the remaining isolates could not be related to any of the five species nodulating bean. When reexamined for nodulation, some of these isolates, showing similarities to R. tropici and Agrobacterium with respect to colony morphology and growth in different media, failed to nodulate their original host. The R. gallicum-like isolates, R. etli-like isolates, and R. leguminosarum-like isolates were recovered from regions where bean is frequently grown, while in fields which had not been cultivated with beans for at least the 10 previous years, solely unrecognized taxa of ineffective isolates were recovered. We detected variations in the symbiotic regions, but certain pSym RFLP patterns for nifH were conserved between Tunisian, French, and Austrian populations of bean rhizobia. Evaluation of symbiotic effectiveness showed that R. gallicum-like isolates and R. etli-like isolates were effective, whereas some R. leguminosarum-like isolates were ineffective. Furthermore, effective isolates were also found among the unrecognized taxa. Received: 10 March 1998     

Differences in common bean rhizobial populations associated with soil tillage management in southern Brazil

Progressive adoption of no-tillage (NT) agriculture in the tropics is finally reversing physical, chemical, and biological erosion of soil and in Brazil, an estimated 19 Mha are now devoted to NT. Common bean (Phaseolus vulgaris L.) is a main component of Brazilian agriculture, and enhancement of yields has been achieved under NT as a result of mitigation of environmental stresses, resulting in higher N₂ fixation. However, the effects of NT on rhizobial diversity are poorly understood. This study evaluated rhizobial diversity in soils planted to common bean under NT or conventional tillage (CT) systems that were compared with natural grassland used for grazing, in the State of Santa Catarina, southern Brazil. Genetic diversity was assessed by the amplification of the DNA by PCR with specific primers (BOX-PCR) and by RFLP-PCR analyses of the 16S rDNA region. A high level of diversity was observed among strains from all three systems, such that the similarity in the clustering analysis of BOX-PCR products ranged from 36% under natural grassland to only 23% for CT strains. High polymorphism was confirmed in the RFLP-PCR analysis; forty-seven different profiles were obtained, none sharing high similarity with the profiles of reference species of common bean rhizobia. These results indicate that other tropical rhizobial species remain to be described. Genetic diversity was higher among the NT than the CT rhizobial strains, especially when the RFLP-PCR profiles were considered. Genetic diversity in the natural grassland was lower than in the cropped systems, possibly due to absence of the host plant and stubble burning in winter. Average yields in the area under NT (e.g. common bean, approximately 1500 kg ha⁻¹) have been about 30% higher than under CT, therefore high rhizobial diversity may be a parameter indicative of superior soil quality.     

Novel strains of nodulating Burkholderia have a role in nitrogen fixation with papilionoid herbaceous legumes adapted to acid, infertile soils

We investigated the taxonomic position and symbiotic capabilities of two root-nodule bacterial strains isolated from the South African herbaceous, papilionoid legume Rhynchosia ferulifolia. The 16S rRNA gene sequence of the two strains was determined along with intragenic sequences of nodA and nifH, together with their symbiotic capabilities when inoculated onto the papilionoid legumes R. ferulifolia, Rhynchosia caribaea, Rhynchosia minima and Macroptilium atropurpureum (Siratro). Burkholderia phymatum STM815^T, Cupriavidus taiwanensis LMG 19424^T and root-nodule bacteria isolated from R. minima and Rhynchosia totta were included in the study. Root-nodule bacteria isolated from R. ferulifolia, WSM3937 and WSM3930, belong to the genus Burkholderia and are most closely related to Burkholderia terricola (98.8% similarity). The phylogenetic analysis of nodA and nifH revealed substantial similarity of the novel strains with Burkholderia tuberum STM678^T, a β-rhizobium also originated from South Africa, and only a distant relationship with South American Mimosa-nodulating β-rhizobia. R. ferulifolia was effectively nodulated only by Burkholderia sp. WSM3937 and WSM3930 and not by bradyrhizobia isolated from Rhynchosia minima and Rhynchosia totta or STM815 and LMG 19924. Nodules induced by the novel strains were determinate and hosted well organized symbiosomes within infected cells. In this study we describe a new symbiotic N-fixing relationship between Burkholderia sp. and the South African legume R. ferulifolia. This is the first report of N-fixation between β-rhizobia and an herbaceous, papilionoid legume from which the strains were originally isolated. The level of N-fixation in this symbiosis approached that achieved by effectively nodulated Medicago sativa and suggests that the β-rhizobia may have a role in N-fixation in agricultural systems.     

Nodulation competitiveness of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion

Summary The nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici strains was analysed in growth pouches, at pH 5.2 and 6.4. All 20 strains were coinoculated with a gus ⁺ strain of R. leguminosarum bv. phaseoli strain KIM5s. The gus⁺ phenotype, carrying the glucuronidase gene, was used to type nodules directly in the growth pouches. Nodule occupancy ranged from 4% for the least competitive to 96% for the most competitive R. leguminosarum bv. phaseoli strain. The R. tropici strains showed low rates of nodule occupancy at pH 6.4 but their competitiveness improved significantly under acid conditions. CIAT 895 was the only R. leguminosarum bv. phaseoli strain that was less competitive (P<0.05) at the lower pH. The competitiveness of all the other R. leguminosarum bv. phaseoli strains was unaffected by pH. Various physiological and genetic properties of the strains were analysed in search of correlations with nodulation competitiveness. Hybridisation patterns with three different DNA probes (nif KDH, common nod genes, and hup genes) and the metabolism of 53 different C sources were compared. No general correlations were found between hybridisation or growth pattern and competitiveness. The less competitive R. tropici strains had a unique DNA hybridisation pattern and were not able to use shikimate, ferulate, coumarate, or asparagine as C sources. Most of the less competitive R. leguminosarum bv. phaseoli strains could not metabolize either ferulate or coumarate. This might indicate a relationship between nodulation competitiveness and the ability to degrade aromatic compounds.     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.     

Effect of sewage sludge on suppressiveness to soil-borne plant pathogens

The objective of this study was to evaluate the effect of sewage sludge on soil suppressiveness to the pathogens Fusarium oxysporum f. sp. lycopersici on tomato, Sclerotium rolfsii on bean, Sclerotinia sclerotiorum on tomato, Rhizoctonia solani on radish, Pythium spp. on cucumber, and Ralstonia solanacearum on tomato. Soil samples were collected from an experimental corn field in which sewage sludge had been incorporated once a year, since 1999. Sludge from two sewage treatment stations in Brazil (Franca and Barueri, SP) were applied at the rates of one (1N), two (2N), four (4N) and eight (8N) times the N recommended doses for the corn crop. Soil suppressiveness was evaluated by methods using indicator host plants, baits and mycelial growth. There was no effect of sewage sludge on soil suppressiveness to Fusarium oxysporum f. sp. lycopersici in tomato plants. For S. rolfsii, reduction of the disease in bean was inversely proportional to the dose of Franca sludge. The incidence of dead plants, caused by S. sclerotiorum, was directly proportional to sludge doses applied. For R. solani and R. solanacearum, there was a linear trend with reduction in plant death in soils treated with increasing amounts of sludge from Franca. There was an increase in the pathogen community of Pythium spp., proportional to the amounts of sewage applied. The effects of sewage sludge varied depending on the pathogen, methodology applied and on the time interval between the sewage sludge incorporation and soil sampling.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.     

Co-inoculation of selected nodule endophytic rhizobacterial strains with Rhizobium tropici promotes plant growth and controls damping off in common bean

Production of common bean(Phaseolus vulgaris)is limited by the occurrence of damping off(rhizoctoniosis),which is caused by the fungus Rhizoctonia solani.However,the co-inoculation of plant growth-promoting rhizobacteria(PGPR)involved in biological control along with diatomic nitrogen(N2)-fixing rhizobia can enhance N nutrition and increase production.In this context,finding microorganisms with synergistic effects that perform these two roles is of fundamental importance to ensure adequate yield levels.The aim of this study was to evaluate the effects of co-inoculation of nodule endophytic strains of the genera Bacillus,Paenibacillus,Burkholderia,and Pseudomonas with Rhizobium tropici CIAT 899,an N2-fixing rhizobial strain,on the biocontrol of damping off and growth promotion in common bean plants.Greenhouse experiments were conducted under axenic conditions using the common bean cultivar Pérola.The first experiment evaluated the potential of the 14 rhizobacterial strains,which were inoculated alone or in combination with CIAT 899,for the control of R.solani.The second experiment evaluated the ability of these 14 rhizobacterial strains to promote plant growth with three manners of N supply:co-inoculation with CIAT 899 at low mineral N supply(5.25 mg N mL^-1),low mineral N supply(5.25 mg N mL^-1),and high mineral N supply(52.5 mg N mL^-1).The use of rhizobacteria combined with rhizobia contributed in a synergistic manner to the promotion of growth and the control of damping off in the common bean.Co-inoculation of the strains UFLA 02-281/03-18(Pseudomonas sp.),UFLA 02-286(Bacillus sp.),and UFLA 04-227(Burkholderia fungorum)together with CIAT 899 effectively controlled damping off.For the common bean,mineral N supply can be replaced by the co-inoculation of CIAT 899 with plant growth-promoting strains UFLA 02-281/02-286/02-290/02-293.Nodule endophytes UFLA02-281/02-286 are promising for co-inoculation with CIAT 899 in the common bean,promoting synergy with rhizobial inoculation and protection against disease.     

Influence of soil type and indigenous pathogenic fungi on bean hypocotyl rot caused by Rhizoctonia solani AG4 HGI in Cuba

Calcisol, ferralsol and vertisol soils, representative of different bean production areas of Villa Clara province in Cuba, were selected to determine the impact of soil type on bean hypocotyl rot severity caused by Rhizoctonia solani AG4 HGI (isolate CuVC-Rs7). In inoculated autoclaved soil, hypocotyl rot was most severe in calcisol soil, followed by ferralsol soils and then vertisol soils. In inoculated natural soils, disease severity was lower in vertisol and calcisol soils and higher in ferralsol soil, indicating that biological factors are suppressing or stimulating the pathogenic efficiency of R. solani. Native binucleate Rhizoctonia AGF, Sclerotium rolfsii and R. solani AG 4 HGI were isolated from bean plants grown in natural calcisol, vertisol and ferralsol soils, respectively. Subsequent studies about the interaction between these fungi and R. solani indicated that they were involved in the variability of disease severity caused by R. solani. The addition of R. solani AG4 HGI (isolate CuVC-Rs7) into each autoclaved soil inoculated with binucleate Rhizoctonia or S. rolfsii resulted in a reduction of disease severity caused by this pathogen while in soils inoculated with native R. solani AG4 HGI, disease severity increased. Irrespective of fungal interactions, calcisol was always the most disease conducive soil and vertisol the most disease repressive soil. The mechanisms by which native pathogenic fungi could influence disease severity caused by R. solani are discussed.     

The abundance of nitrogen cycle genes amoA and nifH depends on land-uses and soil types in South-Eastern Australia

Nitrogen is a critical nutrient in plant-based primary production systems, therefore measurements of N cycling by microorganisms may add value to agricultural soil monitoring programs. Bacterial-mediated nitrogen cycling was investigated in soils from two broad land-uses (managed and remnant vegetation) across different Soil Orders from three geomorphic zones in Victoria, Australia, by examining the abundance of the genes amoA and nifH using quantitative polymerase chain reaction (qPCR). The aim of the study was to identify parameters influencing bacterial populations possessing the genes nifH and amoA, and examine their distribution at a regional scale across different management treatments. The gene amoA was most abundant in the neutral to slightly alkaline surface soils from Calcarosols in North-West Victoria. There was a highly significant (P < 0.001) interaction between land-use and geomorphic zones in terms of the abundance of amoA. Detection of the gene nifH was site specific with low copy number (less than 100 copies per nanogram of DNA) observed for some strongly acidic surface soil sites in North-East Victoria (Dermosols) and South-West Victoria (Sodosols/Chromosols), while nifH was more abundant in selected Calcarosols of North-West Victoria. The gene amoA was detected across more sites than nifH and was strongly influenced by land-use, with almost consistently greater abundance in managed compared to remnant sites, particularly for North-West and South-West Victoria. The abundance of nifH was not related to land-use, with similar copy numbers observed for both managed and remnant sites at some locations. For the gene nifH, there was no significant interaction between land-use and geomorphic zones, between managed and remnant sites or between the three geomorphic zones. Regression tree analysis revealed a number of likely soil chemical and microbial variables which may act as drivers of gene abundance of amoA and nifH. Variables identified as drivers for amoA included pH, Olsen P, microbial biomass carbon, nitrate and total nitrogen while for nifH the variables were microbial biomass carbon, electrical conductivity, microbial biomass nitrogen, total nitrogen and total potassium. Measures of N cycling genes could be used as an additional indicator of soil health to assess potential ecosystem functions. The spatial scale of the current study demonstrates that a landscape approach may assist soil health monitoring programs by evaluating N cycle gene abundance in the context of the different microbial and chemical conditions related to Soil Order and land-use management.     

Nodulation and growth of common bean (Phaseolus vulgaris) under water deficiency

Three experiments were conducted in order to investigate the effect of water deficiency on nodulation, rhizobial diversity and growth of common bean. In the first experiment, the effect of water deficiency was studied on two soil samples under glasshouse conditions. A significant decrease in nodulation and shoot dry weight production was observed. The molecular characterization of the root nodule isolates by PCR-RFLP of 16S rRNA and nodC genes showed that the nodulation by Rhizobium etli was severely inhibited. The in vitro analysis of salt tolerance indicated that drought stress favoured nodulation by salt-tolerant strains. In the second experiment, the effect of water deficiency was studied on sterilized sand using Rhizobium tropici CIAT899^T and Ensifer meliloti bv. mediterranense 4H41 as inoculants. The results showed that strain 4H41, which is the more salt tolerant, was more competitive and more effective under water deficiency than strain CIAT899^T. In the third experiment, the strain 4H41 was used to inoculate four fields. A significant increase in nodule number, shoot dry weight and grain yield was observed even in the non-irrigated soils. This work constitutes the first report of a strain enhancing the growth and the grain yield of common bean under water deficiency.     

Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

Characterization of rhizobia from <Emphasis Type="Italic">Sesbania</Emphasis> species native to seasonally wetland areas in Uruguay

Naturally growing Sesbania species with tolerance to unfavourable habitats are widely distributed in non-cultivated seasonally wetland areas in Uruguay. We investigated the relative abundance, diversity and symbiotic efficiency of Sesbania punicea and S. virgata rhizobia in three ecologically different undisturbed and water-logged sites in Uruguay. Numbers of native-soil rhizobia infective on S. punicea or S. virgata were low, with higher numbers associated with the presence of S. virgata. Plants of S. virgata inoculated with soil suspension showed aerial and nodule biomass greater than that obtained with S. punicea. The rhizobia nodulating Sesbania species in water-logged lands in different regions of Uruguay were diverse differing in growth rates, acid production, growth at 39°C and in LB medium, host range and symbiotic efficiency. Seventeen representative strains clustered into four groups on the basis of phenotypic characteristics, ARDRA and DNA fingerprinting (GTG₅-PCR). Partial sequence of 16S rRNA from eight of these strains classified them into at least two genera with four species: Azorhizobium doebereinerae, Rhizobium sp. related to R. etli and two different Rhizobium sp.-Agrobacterium. Our results confirm the presence of the specie Azorhizobium doebereinerae as microsymbionts of S. virgata in South America. No strain of Rhizobium etli has previously been reported as a microsymbiont of Sesbania, though R. etli like organisms have also been recovered from Dalea purpurea and Desmanthus illinoensis. Significant increases in dry matter production were obtained with S. virgata plants inoculated with selected rhizobial strains under growth chamber conditions.     

Phylogeny and nodulation signal molecule of rhizobial populations able to nodulate common beans—other than the predominant species Rhizobium etli—present in soils from the northwest of Argentina

We examined the bean rhizobia community other than the predominant species Rhizobium etli present in soils of a region that is part of the range occupied by the host in Northwest Argentina, which showed Rep and 16S rDNA RFLP polymorphism. Two populations represented by isolates T29N3L and T44N22P were found to be distinct chromosomal genotypes and closely related to species Rhizobium tropici and Agrobacterium rhizogenes. Their symbiotic genes were analyzed and found to cluster with those from R. tropici as well as with rhizobia isolated from leguminous trees. Three nodulation metabolites produced by T44N22P were detected which are tetra- and pentameric chitocompounds, N-methylated, O-carbamoylated, and N-substituted either by a C_18:0 or C_18:1 acyl chain at their non-reducing end, and all them sulphated at the reducing end. Isolates T29N3L and T44N22P exhibited broad host range but unlike T29N3L, only T44N22P was able to efficiently nodulate Medicago truncatula.