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.     

Population size,distribution, and symbiotic characteristics of indigenous Bradyrhizobium spp. that nodulate TGx soybean genotypes in Africa

Soils of many potential soybean fields in Africa are characterized by low levels of biological nitrogen fixation (BNF) activities and often cannot support high soybean yields without addition of inorganic N fertilizers or external application of soybean rhizobia. The most probable number (MPN) technique was used to determine the bradyrhizobial populations that nodulate TGx soybean genotypes (a cross between nonpromiscuous North American soybean genotypes and promiscuous Asian soybean genotypes), cowpea or North American soybean cv. Clark IV, in soils from 65 sites in 9 African countries. The symbiotic effectiveness of isolates from these soils was compared to that of Bradyrhizobium japonicum strain USDA110. The bradyrhizobial population sizes ranged from 0 to 10⁴ cells g⁻¹ soil. Bradyrhizobium sp. (TGx) populations were detected in 72% and B. japonicum (Clark) in 37% of the soil samples. Bradyrhizobium sp. (TGx) populations were generally low, and significantly less than that of the cowpea bradyrhizobial populations in 57% of the samples. Population sizes of less than 10 cells g⁻¹ soil were common as these were detected in at least 43% of the soil samples. B. japonicum (Clark) occurred in higher population densities in research sites compared to farmers’ fields. Bradyrhizobium sp. (TGx) populations were highly correlated with biotic but not abiotic factors. The frequent incidence of low Bradyrhizobium sp. (TGx) populations is unlikely to support optimum BNF enough for high soybean yields while the presence of B. japonicum (Clark) in research fields has the potential to compromise the selection pressure anticipated from the indigenous Bradyrhizobium spp. (Vigna) populations. Bradyrhizobium isolates could be placed in four symbiotic phenotype groups based on their effectiveness on a TGx soybean genotype and the North American cultivar Clark IV. Symbiotic phenotype group II isolates were as effective as B. japonicum strain USDA110 on both soybean genotypes while isolates of group IV were effective on the TGx soybean genotype but not on the Clark IV. The group IV isolates represent a unique subgroup of indigenous bradyrhizobia that can sustain high soybean yields when available in sufficient population densities.     

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.     

Effects of local environmental variables and geographical location on the genetic diversity and composition of Rhizobium leguminosarum nodulating Vicia cracca populations

Different legume populations are known to accommodate different genotypes of Rhizobium leguminosarum. However, in contrast to interspecific diversity and composition, very little is known regarding which environmental factors drive the genetic diversity and genetic composition of a single Rhizobium species. Based on chromosomal and plasmid genes, we quantified the genetic diversity and compositional differences of R. leguminosarum biovar viciae genotypes associated with twenty-four different Vicia cracca populations across a wide environmental and geographical range. Long-term soil nitrogen availability had a positive effect on chromosomal and plasmid diversity, whereas salinity had a negative effect on chromosomal diversity. Soil pH and geographic distance were the main factors driving compositional differences among populations. In contrast to differences in chromosomal composition, differences in the symbiotic plasmid composition were primarily related to geographic distance or unmeasured related environmental factors (e.g. host plant genetic differentiation). We propose different hypotheses to explain how long-term soil nitrogen availability affects rhizobial genetic diversity. Furthermore, our findings demonstrate that ecological processes that are known to operate at the interspecific level do not necessarily result in the same patterns at the intraspecific level.     

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.     

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.     

Heavy metal effects on soil populations and heavy metal tolerance of Rhizobium meliloti,nodulation, and growth of alfalfa

Effects of heavy metals on rhizobia and the symbiotic association with leguminous hosts are currently unclear. To investigate this problem, we examined Rhizobium meliloti (microsymbiont) and alfalfa (Medicago sativa) (macrosymbiont) collected from soils contaminated with varying concentrations of heavy metals (varying distances from a Zn smelter operating 90 yr.). Soil populations of R. meliloti were not correlated with metal concentrations in soil. The lowest rhizobial population was found in the soil with the highest extractable metal concentrations, but the highest populations were found in soil which was moderately contaminated. A greenhouse study in which alfalfa was grown in the same soils showed no significant trend for nodulation or nitrogenase activity of roots. Highest nodule number and nitrogenase activity were observed in those soils which had the lowest population of R. meliloti. When the heavy metal Minimum Inhibitory Concentration (MIC) of individual isolates was examined, no correlation was found between the MIC and soil metal concentration (total, or water or 0.01 M Ca(NO₃)₂ extractable).These results indicate that even in highly contaminated soils, metal activity was not high enough to exert an antagonistic influence on the soil rhizobial population or the symbiotic association between alfalfa and R. meliloti.     

Effects of soil application of olive mill wastewaters on the structure and function of the community of arbuscular mycorrhizal fungi

Recycling of olive mill wastewaters (OMW) into agricultural soils is a controversial issue since benefits to soil fertility should counterbalance potential short-term toxicity effects. We investigated the short-term effects of OMW on the soil-plant system, regarding the diversity, structure and root colonization capacity of arbuscular mycorrhizal (AM) fungi and the respective growth response of Vicia faba L, commonly used as green manure in olive-tree plantations. A compartmentalized pot system was used that allowed the establishment of an AM fungal community in one compartment (feeder) and the application of three OMW dose levels in an adjacent second compartment (receiver). At 0, 10, and 30 days after OMW treatment (DAT), V. faba pre-germinated seeds were seeded in the receiver compartment. At harvest, shoot and root dry weights, AM fungal root colonization, soil hyphal length and P availability were recorded in the receiver compartment. In addition, OMW effects on AM fungal diversity in plant roots were studied by DGGE. A transient effect of OMW application was observed; plant growth and AM fungal colonization were initially inhibited, whereas soil hyphal length was stimulated, but in most cases differences were absent when seeding was performed 30 DAT. Similarly, changes induced in the structure of the root AM fungal community were of transient nature. Cloning and sequencing of all the major DGGE bands showed that roots were colonized by Glomus spp. The transient effects of OMW on the structure and function of AM fungi could be attributed to OMW-derived phytoxicity to V. faba plants or to an indirect effect via alteration of soil nutritional status. The high OMW dose significantly increased soil P availability in the presence of AM fungi, suggesting efficient involvement of AM fungi in organic-P minerilization. Overall our results indicate that soil application of OMW would cause transient changes in the AM fungal colonization of V. faba plants, which, would not impair their long-term plant growth promoting ability.     

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.     

Low levels of genetic variation among southern peripheral populations of the threatened herb, Leontice microrhyncha (Berberidaceae) in Korea

Levels of genetic variation and intrapopulation genetic structures of Leontice microrhyncha S. Moore (Berberidaceae) were assessed for six populations in South Korea, representing the southern most range of a species found in Northeast China and the Korean peninsula. Detected genetic diversity (H_es) was very low (0.024) and F_IS values showed large heterozygote deficiencies. The small percentage of polymorphic loci and numbers of alleles per locus suggest that L. microrhyncha has a history of severe or long-lasting population bottlenecks that have eroded genetic diversity. This study suggests that the Korean population appears to consist of two historically isolated and independently evolving populations. It seems likely that these groups have been isolated and unstable for a significant period of time. However, the effects of recent habitat fragmentation on the historically disjunct and fragmented population system found in L. microrhyncha were not those predicted from the lack of significant relationships between population-level patterns of genetic variation and population sizes. Most non-unique genotypes were shared by most individuals and the lower level of diversity, high levels of inbreeding and population differentiation as well as high rate of seed production indicated that this species is autogamous and self-compatible and probably largely selfing. Therefore, to preserve extant genetic variation, all populations must be protected across the small geographic range of the species to retain both allelic and genotypic diversity.     

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.     

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.     

Comparative efficieny of Rhizobium/Bradyrhizobium spp. strains in nodulating Cajanus cajan in relation to characteristic metabolic enzyme activities

The comparative symbiotic properties of Rhizobium spp. and Bradyrhizobium spp. strains infecting pigeon pea were evaluated. Bradyrhizobium strains (Cajanus) were found to be superior to Rhizobium strains (Cajanus) and the superiority was ascertained to be due to the higher enzyme activity of the tricarboxylic acid (TCA) cycle in comparison to Rhizobium spp. strains. Moreover, metabolic superiority or rapid growth rate does not necessarily correlate with symbiotic effectiveness. The symbiotic performance of isolates varied with the host cultivar. The dry matter accumulation could be correlated with the total acetylene reduction activities rather than nodule number or nodule fresh weight per plant. Received: 3 March 1993     

Host status of 10 fungal isolates for two nematode species, Filenchus misellus and Aphelenchus avenae

The classification of nematodes in the family Tylenchidae into plant parasites, plant associates or fungal-feeders for community analyses, have been much discussed by nematode ecologists. For an appropriate classification, fungal-feeding habits in the family need to be studied. To evaluate the host status of 10 fungal isolates for Filenchus misellus (Tylenchidae) and Aphelenchus avenae (Aphelenchida, Aphelenchidae), population growth rates, body length and width and sex ratios of the nematodes were measured after 40-day culture on fungal colonies at 25 °C. For F. misellus, the fungi determined as good hosts were two Basidiomycota fungi (Agaricus bisporus, Coprinus cinereus), three Ascomycota fungi (Chaetomium cochlioides, Chaetomium funicola, Chaetomium globosum) and a plant-pathogenic fungus (Rhizoctonia solani) on the basis of nematode population growth rate and female body length. Interestingly Pleurotus ostreatus, known as a predaceous fungus for the other nematodes, was also a good host for F. misellus. While, for A. avenae, good hosts were four plant-pathogenic fungi (Fusarium oxysporum f. sp. conglutinans, F. oxysporum f. sp. cucumerinum, Pythium ultimum, R. solani) and A. bisporus. A. avenae was trapped and preyed upon by Pleurotus hyphae. In F. misellus, males were 7-21% of adults, but the ratio did not correlate significantly with the population growth rate. In A. avenae, no male occurred. Differences in habitat preference between Filenchus and Aphelenchus were explained on the basis of the host status and habitat preferences of the tested fungi.     

Molecular markers and a sequence deletion in intron 2 of the putative partial homologue of LEAFY reveal geographical structure to genetic diversity in the acutely threatened legume genus Clianthus

Clianthus is an acutely threatened, bird-pollinated genus endemic to New Zealand, represented in the wild by only one population of C. puniceus and 11 populations of C. maximus, each with very few individuals (typically <10 per population). A limited number of named Clianthus cultivars of indeterminate origin are commonly grown as ornamentals. Genomic DNA from individual Clianthus plants was extracted for genetic diversity analysis using a range of molecular markers, including amplified fragment length polymorphism (AFLP). Data were analysed by the unweighted pair-group method with arithmetic averaging (UPGMA), the generation of Neighbor-Joining trees, and analyses of molecular variance (AMOVA). Genetic distance between wild populations of C. maximus was highly correlated with geographical distance between populations. Sequencing of intron 2 of a putative partial homologue of the floral meristem identity gene LEAFY (CmLFY) revealed a 7 bp deletion that was exhibited homozygously in the more northern populations of C. maximus, and in all individuals tested from the sole population of C. puniceus. This deletion was not exhibited in more southern populations of C. maximus. Further, one geographically intermediate population contained some plants that were heterozygous for the deletion. Parallel analyses of cultivated Clianthus genotypes, more than half of which were also homozygous for the 7 bp deletion, showed that these were not representative of the broad, but threatened, diversity remaining in the wild. It is argued that wild populations of C. maximus are unlikely to have arisen from the escape of plants from cultivation. Conservation effort should focus on the protection and study of the extant plants in these wild populations, rather than on the introduction of disturbance regimes to uncover potential seed banks.     

Conservation implications of the distribution of genetic diversity at different scales: a case study using the marsh fritillary butterfly (Euphydryas aurinia)

In the UK, Euphydryas aurinia exists in fragmented habitat patches, and undergoes population fluctuations as a result of a larval parasitoid. Its range is declining in the UK and conservation is thought to require a landscape approach since populations spread over large areas in some years and contract to core breeding patches in others. We examined populations at a range of geographic scales using allozyme electrophoresis to look for evidence of gene flow and differences in genetic diversity among populations. Nationally, our F_ST value was 0.1542 but between population groups within the suspected colonisation range of the butterfly (ca. 20 km), F_ST values were not significantly different from zero. Genetic diversity in terms of number of alleles and heterozygosity was reasonably high in natural populations (H_e=0.267) but low in an introduced, isolated population. We infer that migration between closely spaced subpopulations (in a metapopulation) maintains a high genetic effective population size (large number of individuals in a population that contribute genes to the next generation) which offsets any local reductions in population numbers due to stochastic extinctions or parasitoid effects. We therefore conclude that effective conservation of the species must seek to provide networks of suitable habitat for groups of subpopulations, rather than maintaining habitat for isolated populations.     

Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

The symbiotic nitrogen-fixing soil bacterium, Sinorhizobium meliloti, is well known for its ability to interact with the leguminous plant Medicago sativa L. It has, however, not been reported that this species possesses the capability to degrade toxic nitroaromatic compounds, such as 2,4-dinitrotoluene (DNT) which is commonly associated with the degradation of the explosive trinitrotoluene (TNT). In this study, the pJS1 DNT-biodegradative plasmid was genetically transferred to S. meliloti strain USDA 1936, which was confirmed by plasmid profile analysis. Several standard analytical and chemical tests including high performance liquid chromatography (HPLC), nitrite (NO₂) release assays, rhizosphere population and plant greenhouse studies were conducted to test the ability of S. meliloti to degrade 2,4-DNT. The possible presence of 2,4-DNT remaining in the treated soil was tested, and no 2,4-DNT had been absorbed by the soil. The pJS1-carrying recombinant strain DHK1 produced ‘ARC’ alfalfa plants that were almost 2-fold higher in shoot dry weight than that produced by the parent strain on soil containing 0.14 mM 2,4-DNT. The transconjugant strain DHK1 reduced significantly one-third more 2,4-DNT in both 0.14 and 0.28 mM contaminated soil, and in 0.55 mM contaminated soil it degraded 94% of the 2,4-DNT present. In liquid cultures, however, only about 4% reduction in 2,4-DNT concentrations was obtained in 10 days. We interpret the results as clearly establishing that genetic modification was successfully used, for the first time, to improve the capability of the symbiotic nitrogen-fixing soil bacterium S. meliloti DHK1 to bioremediate in situ 2,4-DNT-contaminated soil in the presence of alfalfa plants.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.     

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.     

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