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.     

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.     

New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

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.     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

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.     

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.     

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.     

Rhizobia phylogenetically related to common bean symbionts Rhizobium giardinii and Rhizobium tropici isolated from peanut nodules in Central Argentina

Our previous studies of the native rhizobial population associated with peanut nodules in the Córdoba soils of Argentina revealed that this population is highly diverse and includes slow- and fast-growing isolates. The native fast-growing isolates NCHA22 and NET30 were selected on the basis of their plant growth promoting properties and their chromosomal genotypes were determined by 16S rDNA sequencing. NCHA22 and NET30 16S rDNA alleles were found to cluster with those of Rhizobium tropici group IIB and Rhizobium giardinii bv. giardinii strain H152, respectively. We have now characterized these isolates by analyzing the glnA and nifH genes to clarify their taxonomic position. These studies confirmed that fast-growing isolates belonging to species earlier described as bean symbionts were obtained from nodules of a leguminous plant that has been described as efficiently nodulated exclusively by slow-growing rhizobial strains.     

Polyphasic characteristics of bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China

Peanuts (Arachis hypogaea L.) were introduced to China about 500 years ago. However, the diversity of Rhizobial strains in China that can nodulate peanut was poorly understand. Diversity and phylogeny of 50 slow-growing strains, isolated from root nodules of peanut in different geographical regions of China, were studied using polyphasic techniques. All stains were clustered by phenotypic tests into two distinct groups: Group I: 16S rRNA RFLP genotype 3, and Group II, which divided into 16S rRNA RFLP genotypes 1 and 2. Genotype 1 shares the same genotype with USDA110, USDA122 and USDA127 of Bradyrhizobium japonicum, and genotype 2 solely consisted of extra-slow growing bradyrhizobia isolated from Hongan, China. Results of 16S rRNA sequencing revealed that peanut bradyrhizobia were phylogenetically related to B. japonicum and their sequence divergence was less than 1.1%. Based upon the size of the internally transcribed spacer (ITS) between the16S and 23S RNA genes, strains were classified into ITS-I, ITS-II and ITS-III genotypes. Strains could be further divided into sub-clusters IA, IB, IIa, IIb and IIc five sub-clusters through ITS PCR-RFLP and repetitive extragenic palindromic PCR (REP-PCR) analysis. Host specificity test revealed that all peanut bradyrhizobia tested nodulated Phaseolus vulgaris and strains of clusters IIb and IIc nodulated Glycine soja efficiently. Bradyrhizobia isolated from peanut were related, but still exhibited phylogenetical divergence with B. japonicum.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

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.     

The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

For optimum production, the use of commercial rhizobial inoculant on pea (Pisum sativum L.) at seeding is necessary in the absence of compatible rhizobial strains or when rhizobial soil populations are low or symbiotically ineffective. Multiple site experiments were conducted to characterize the abundance and effectiveness of resident populations of Rhizobium leguminosarum bv. viciae (Rlv) in eastern Canadian prairie soils. A survey of 20 sites across a broad geographical range of southern Manitoba was carried out in 1998 and was followed by more intensive study of five of the sites in 1999 and 2000. Appreciable nodulation of uninoculated pea was observed at all sites which had previously grown inoculated pea. However, uninoculated pea grown at two sites, which had not previously grown pea, had negligible nodulation. Likewise, wild Lathyrus sp. and Vicia sp. plants collected from uncultivated areas adjacent to agricultural sites were poorly nodulated. In the more intensively studied sites, there was a tendency towards higher nodulation in pea plants receiving commercial inoculant containing Rlv strain PBC108 across all site-years (e.g., 4.7% in nodulation and 22% in nodule mass), but the effect was significant at only 2 of 10 site-years. Despite a relatively high range of soil pH (6-8), regression analysis indicated that decreasing soil pH resulted in lower nodulation rates. Likewise, electrical conductivity (EC) was correlated to nodulation levels, however the effect of EC was likely more indicative of the influence of soil texture and organic matter than salinity. As with nodulation, commercial inoculation tended to increase above-ground dry matter (DM) and fixed-N (estimated by the difference method) at the early pod-filling stage, but again the effects were significant at only 2 of 10 site-years. Specifically, above-ground DM and fixed-N levels were up to 29 and 51% greater, respectively, in inoculated compared to non-inoculated treatments at these sites. Addition of N-fertilizer at a rate of 100 kg N ha⁻¹ decreased nodulation at almost all site-years (by as much as 70% at one site), but rarely resulted in increases in above-ground DM compared to inoculated plots. The study indicates for the first time that populations of infective, and generally effective strains of Rlv occur broadly in agricultural soils across the eastern Canadian prairie, but that there is a tendency for increased symbiotic efficiency with the use of commercial inoculant.     

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.     

Rhizobia nodulating African Acacia spp. and Sesbania sesban trees in southern Ethiopian soils are metabolically and genomically diverse

The diversity of 110 rhizobial strains isolated from Acacia abyssinica, A. seyal, A. tortilis, Faidherbia albida, Sesbania sesban, Phaseolus vulgaris, and Vigna unguiculata grown in soils across diverse agro-ecological zones in southern Ethiopia was assessed using the Biolog™ system and amplified fragment length polymorphism (AFLP) fingerprinting technique. By cluster analysis of the metabolic and genomic fingerprints, the test strains were grouped into 13 Biolog and 11 AFLP clusters. Twenty-two strains in the Biolog method and 15 strains in the AFLP analysis were linked to eight and four reference species, respectively, out of the 28 included in the study. Most of the test strains (more than 80% of 110) were not related to any of the reference species by both methods. Forty-six test strains (42% of 110) were grouped into seven corresponding Biolog and AFLP clusters, suggesting that these groups represented the same strains, or in some cases clonal descendants of the same organisms. In contrast to the strains from S. sesban, isolates from Acacia spp. were represented in several Biolog and AFLP clusters indicating the promiscuous nature of the latter and widespread occurrence of compatible rhizobia in most of the soil sampling locations. The results showed that indigenous rhizobia nodulating native woody species in Ethiopian soils constituted metabolically and genomically diverse groups that are not linked to reference species.     

Distribution of Aspergillus section Flavi in soils of maize fields in three agroecological zones of Nigeria

Fungal communities in soils of Nigerian maize fields were examined to determine distributions of aflatoxin-producing fungi and to identify endemic atoxigenic strains of potential value as biological control agents for limiting aflatoxin contamination in West African crops. Over 1000 isolates belonging to Aspergillus section Flavi were collected from soil of 55 Nigerian maize fields located in three agroecological zones by dilution plating onto modified Rose Bengal agar. The most common member of Aspergillus section Flavi (85% of isolates) was the A. flavus L-strain followed by the unnamed taxon known as strain S_BG (8%), A. tamarii (6%) and A. parasiticus (1%). Highest incidence of S_BG was in Zaria district, and lowest was in Ogbomosho and Ado-Ekiti districts. Only 44% of 492 A. flavus isolates produced aflatoxins in liquid fermentation (limit of detection 5 ng g⁻¹). Thirty-two percent of the A. flavus isolates produced >1 μg g⁻¹ total aflatoxins but no A. flavus isolate produced G aflatoxins. When the agroecological zones were compared, significantly (P < 0.05) greater proportions of aflatoxigenic A. flavus isolates were found in the Northern Guinea Savannah (61%) than in Southern Guinea Savannah (31%). The Derived Savannah was intermediate between the other two agroecological zones. Each of the regions had atoxigenic strains of potential value as biological control agents. All S_BG and A. parasiticus isolates produced both B and G aflatoxins and greater than 300 μg g⁻¹ total aflatoxins. S_BG and A. parasiticus isolates were the greatest contributors to the aflatoxin-producing potential of fungal communities in regions where these isolates occurred.     

Conserving marginal populations of the food plant (Impatiens noli-tangere) of an endangered moth (Eustroma reticulatum) in a changing climate

Impatiens noli-tangere is scarce in the UK and probably only native to the Lake District and Wales. It is the sole food plant for the endangered moth Eustroma reticulatum. Significant annual fluctuations in the size of I. noli-tangere populations endanger the continued presence of E. reticulatum in the UK. In this study, variation in population size was monitored across native populations of I. noli-tangere in the English Lake District and Wales. In 1998, there was a crash in the population size of all metapopulations in the Lake District but not of those found in Wales. A molecular survey of the genetic affinities of samples in 1999 from both regions and a reference population from Switzerland was performed using AFLP and ISSR analyses. The consensus UPGMA dendrogram and a PCO scatter plot revealed clear differentiation between the populations of I. noli-tangere in Wales and those in the Lake District. Most of the genetic variation in the UK (H_T=0.064) was partitioned between (G_ST=0.455) rather than within (H_S=0.034) regions, inferring little gene flow occurs between regions. There was similar bias towards differentiation between metapopulations in Wales, again consistent with low levels of interpopulation gene flow. This contrasts with far lower levels of differentiation in the Lake District which suggests modest rates of gene flow may occur between populations. It is concluded that in the event of local extinction of sites or populations, reintroductions should be restricted to samples collected from the same region. We then surveyed climatic variables to identify those most likely to cause local extinctions. Climatic correlates of population size were sought from two Lake District metapopulations situated close to a meteorological station. A combination of three climatic variables common to both sites explained 81-84% of the variation in plant number between 1990 and 2001. Projected trends for these climatic variables were used in a Monte Carlo simulation which suggested an increased risk of I. noli-tangere population crashes by 2050 at Coniston Water, but not at Derwentwater. Implications of these findings for practical conservation strategies are explored.     

Lotononis angolensis forms nitrogen fixing, lupinoid nodules with phylogenetically unique, fast-growing, pink-pigmented bacteria, which do not nodulate L. bainesii or L. listii

Root-nodule bacteria that nodulate the legume genus Lotononis are being investigated to develop new forage species for agriculture. Bacteria isolated from nodules of Lotononis angolensis were fast-growing, highly mucoid and pink-pigmented, and on the basis of 16S rRNA phylogeny <94% related to other genera in the Alphaproteobacteria. Root-nodule bacteria isolated from other Lotononis species (L. bainesii, L. solitudinis and L. listii) resembled the more common dry, slow-growing, pink-pigmented rhizobia previously described for L. bainesii. These isolates could be attributed to the Methylobacterium genus, although not to the type species Methylobacterium nodulans. Further differences were uncovered with nodulation studies revealing that nodule isolates from L. angolensis were effective at nitrogen fixation on their host plant, but could nodulate neither L. bainesii nor L. listii. Reciprocal tests showed isolates from L. bainesii, L. listii and L. solitudinis were incapable of nodulating L. angolensis effectively. Nodule morphology for L. bainesii, L. angolensis and L. listii was characteristically lupinoid, with little structural divergence between the species, and with nodules eventually enclosing the entire root.     

Symbiotic relationships and root nodule ultrastructure of the pasture legume Biserrula pelecinus L.—a new legume in agriculture

Biserrula pelecinus is a pasture legume species new to Australian agriculture. The potential N benefit from B. pelecinus pastures in agricultural systems may not be realised if its symbiotic interactions with Mesorhizobium spp. are not well understood. This study evaluated the symbiotic interactions of four strains of Biserrula root-nodule bacteria (WSM1271, WSM1283, WSM1284, WSM1497) with four genotypes of B. pelecinus (cv. Casbah, 93GRC4, 93ITA33, IFBI1) and with a range of related legumes, including species known to be nodulated by strains of Mesorhizobium loti and other Mesorhizobium spp. Structures of root nodules were studied using light and electron microscopy enabling the ultrastructure of effective and ineffective nodules to be compared. B. pelecinus always formed typical indeterminate, finger-like nodules. The number of bacteroids inside symbiosomes varied between host×strain combinations, however, nodules formed by ineffective associations had well developed peribacteroid membranes and abundant bacteroids. Considerable variation was found in N₂-fixing effectiveness of strains isolated from B. pelecinus on the four B. pelecinus genotypes. Strains WSM1271, WSM1284 and WSM1497 nodulated Astragalus membranaceus, only strains WSM1284 and WSM1497 nodulated Astragalus adsurgens. Strain WSM1284 also nodulated Dorycnium rectum, Dorycnium hirsutum, Glycyrrhiza uralensis, Leucaena leucocephala, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus and Ornithopus sativus. The four strains from B. pelecinus did not nodulate Amorpha fruticosa, Astragalus sinicus, Cicer arietinum, Hedysarum spinosissimum, Lotus parviflorus, Macroptilium atropurpureum or Trifolium lupinaster. M. loti strain SU343 nodulated all four genotypes of B. pelecinus. However, M. loti strain CC829 only nodulated B. pelecinus genotypes 93ITA33 and IFBI1 and the nodules were ineffective. The root nodule isolates from H. spinosissimum (E13 and H4) nodulated B. pelecinus cv. Casbah whereas the commercial inoculant strain for Cicer (CC1192) could not nodulate any genotype of B. pelecinus. These results indicate that strains WSM1271, WSM1283 and WSM1497 isolated originally from B. pelecinus have a specific host range while strain WSM1284 is promiscuous in its capacity to nodulate with a broad range of related species. As B. pelecinus can be nodulated by Mesorhizobium spp. from other agricultural legumes, particularly Lotus, there is an opportunity to utilise this trait in cultivar development.