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Fifty-eight new isolates were obtained from root nodules of common bean (#Phaseolus vulgaris$) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 #Rhizobium$ isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to #Rhizobium tropici$ type B and #Rhizobium etli$, respectively. By MLEE #R. etli$ and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains). (Résumé d'auteur)

SDS-PAGE of total bacterial proteins was applied to the classification of 25 Sudanese and five Kenyan strains isolated from the root nodules of #Acacia senegal$ and #Prosopis chilensis$. Twenty strains were also studied by multilocus enzyme electrophoresis (MLEE) and the whole 16S rRNA gene was sequenced from two strains representing the two major clusters. These results, together with the previuously reported numerical taxonomy analysis, pulsed-field gel electrophoresis studies, DNA-DNA dot-blot hybridisation, genomic fingerprinting using repetitive sequence-basec PCR, DNA base composition analysis, DNA-DNA reassociation analysis, partial sequencing of the 16S rRNA gene and RFLP analysis of the amplified 16S rRNA gene, showed that all 30 strains belong to the genus #Sinorhizobium$. Two of the strains grouped with #Sinorhizobium saheli$ and seven with #Sinorhizobium terangae$, while the rest did not cluster with any of the established species. The majority of the strains formed two phenotypically and genotypically distinct groups and we therefore propose that these strains should be classified as two new species, #Sinorhizobium arboris$ sp. nov. and #Sinorhizobium kostiense$ sp. nov. (Résumé d'auteur)

Polyploids in Tripsacum, a wild relative of maize, reproduce through the diplosporous type of apomixis, an asexual mode of reproduction through seeds. Diplosporous apomixis involves both the failure of meiosis and the parthenogenetic development of the unreduced gametes, resulting in progenies that are exact genetic copies of the mother plant. Apomixis is believed to be controlled by one single dominant allele, responsible for the whole developmental process. Construction of a linkage map for the chromosome controlling diplosporous apomixis in Tripsacum was carried out in both tetraploid-apomictic and diploid-sexual Tripsacum species using maize restriction fragment length polymorphism (RFLP) probes. A high level of collinearity was observed between the Tripsacum chromosome carrying the control of apomixis and a duplicated segment in the maize genome. In the apomictic tetraploid, there was a strong restriction to recombination, as compared to the corresponding genomic segment in sexual plants and maize. This suggests that apomixis, although inherited as a single Mendelian allele, might really be controlled by a cluster of linked loci. The analysis also revealed the tetrasomic nature of the inheritance of the chromosomal segment controlling apomixis, which contradicts the usually accepted hypothesis of an allopolyploid origin of apomictic species. The implications of these data for the transfer of apomixis into cultivated crops are discussed, and a new approach to studying the genetics of apomixis, based on comparative mapping, is proposed.

Two indigenous bradyrhizobia strains displaying different natural behaviours towards water regime (strain ORS 3257, nodulating more frequently in favourable-water conditions and strain ORS 3260, in limited-water conditions) were studied for their competitivity for nodulation of cowpea (Mouride cultivar) under favourable and limited water conditions in non-sterile soil. The nodule occupancy was studied by PCR-RFLP analysis. Both strains showed good competition with other indigenous rhizobia populations under favourable- and limited-water conditions. Competition between the inoculated strains in the mixture varied between water regimes. In non-limited-water conditions, strain ORS 3257 was the best competitor, whereas in limited-water conditions, strain ORS 3260 was the best competitor. Results indicated that screening of strains according to their environmental origin could ensure successful rhizobia Inoculation.

International audience; Cassava suffers from bacterial blight attack in all growing regions. Control by resistance is unstable due to high genotype–environment interactions. Identifying genes for resistance to African strains of Xanthomonas axonopodis pv. manihotis can support breeding efforts. Five F1 cassava genotypes deriving from the male parent ‘CM2177-2’ and the female parent ‘TMS30572’ were used to produce 111 individuals by backcrossing to the female parent. In all, 16 genotypes among the mapping population were resistant to stem inoculation by four strains of X. axonopodis pv. manihotis from different locations in Africa, and 19 groups with differential reactions to the four strains were identified, suggesting that the strains represent different pathotypes. Four genotypes were resistant to leaf inoculation, and three were resistant to both stem and leaf inoculations. Genotypes with susceptible, moderately resistant, and resistant reactions after leaf and stem inoculation partly differed in their reactions on leaves and stems. Based on the genetic map of cassava, single-marker regression analysis of disease severity after stem-puncture inoculation was performed. Eleven markers were identified, explaining between 16 and 33.3% of phenotypic variance of area under disease progress curve. Five markers on three and one linkage groups from the female- and malederived framework of family CM8820, respectively, seem to be weakly associated with resistance to four strains of X. axonopodis pv. manihotis. Based on the segregation of alleles from the female of family CM8873, one marker was significantly associated with resistance to two X. axonopodis pv. manihotis strains, GSPB2506 and GSPB2511, whereas five markers were not linked to any linkage group. The quantitative trait loci (QTL) mapping results also suggest that the four African strains belong to four different pathotypes. The identified pathotypes should be useful for screening for resistance, and the QTL and markers will support breeding for resistance.

A direct comparison of the genetic linkage maps of sorghum and rice is proposed. It is based on the mapping of a common set of 123 RFLP probes scattered on the genomes of both species. For each species a composite map was established by merging two individual maps comprising many common loci. This enabled us to confirm the global correspondence scheme that had previously been established between the chromosomes of sorghum and rice. It also provided a more detailed insight into the conservation of synteny and colinearity : 69% of the loci mapped on a given rice chromosome mapped to the corresponding homoeologous chromosome in sorghum ; among them, 84% formed a colinear arrangement between the two species. Local inversions and translocations were detected. (Résumé d'auteur)

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for #Mycobacterium tuberculosis$ typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the #M. tuberculosis$ genome. MIRU-VNTR profiles of 72 different #M. tuberculosis$ isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIR-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all #M. tuberculosis$ isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of #M. tuberculosis$. (Résumé d'auteur)

Cassava bacterial blight (CBB), caused by #Xanthomonas axonopodis$ pv. #manihotis$ (#Xam$), is a major disease of cassava (#manihot esculenta$ Crantz) in Africa and South America. Planting resistant varieties is the preferred method of disease control. Recent genetic mapping of an F1 cross (TMS 30572 X CM 2177-2) led to the construction of the first molecular genetic map of cassava. To better understand the genetics of resistance to CBB, we evaluated individuals of the F1 cross for CBB resistance by controlled greenhouse inoculations and visually assessed symptoms on days 7, 15, and 30 days after inoculation, using a scale where 0 = no disease and 5 = maximum susceptibility. Five #Xam$ strains were used : CIO-84, CIO-1, CIO-136, CIO-295 and ORST X-27. Area under the disease progress curve (AUDPC) was used as a quantitative measure of resistance in QTL analysis by single-marker regression. Based on the AUDPC values, eight QTLs (Quantitative trait loci), located on linkage groups B, D, L, N, and X of the female-derived framework map, were found to explain 9-20% of the phenotypic variance of the crop's response to the #Xam$ strains. With the male-derived framework map, four QTLs on linkage groups G and C explained 10.27-27.1% of the variance. A scheme to confirm the usefulness of these markers in evaluating segregating populations for resistance to CBB is proposed. (Résumé d'auteur)

At the present time, the precise taxonomial status of many strains classified as #Bradyrhizobium$ still remains unclear. There is a need to develop a reliable grouping method, specifying the genetic relationships between these strains. Phenotypic methods as auxanography or protein profiling did not prove valuable for classification of bradyrhizobia and were not in good agreement with phylogenetic data. The purpose of this work is to develop a strategy to analyse the diversity of bradyrhizobia and to identify genomic groups among our collection of strains isolated from 9 small legume species in Senegal. #B. japonicum$, #B. elkanii$ and representatives of previously described groups by Moreira et al. (1993) and Dupuy et al. (1994) were included as references. Bacterial diversity was assessed by two different techniques, PCR-RFLP analysis of the IGS region between 16S and 23S rDNA (IGS) and the AFLP technique. Groupings of strains obtained by the two methodologies will be presented and compared. 16S rDNA PCR-RFLP analysis of strains representative of IGS clusters from small legumes was also performed. (Résumé d'auteur)

We isolated 71 slow-growing bacterial strains from nodules of 27 native leguminous plants species in Senegal (West Africa) belonging to the genera #Abrus$, #Alysicarpus$, #Bryaspis$, #Chamaecrista$, #Cassia$, #Crotalaria$, #Desmodium$, #Eriosema$, #Indigofera$, #Moghania$, #Rhynchosia$, #Sesbania$, #Tephrosia$, and #Zornia$ playing an ecological role and having agronomic potential in arid regions. The isolates were charaterised by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 165 rDNA and comparative SDS-PAGE of whole-cell proteins ; reference strains of the different known rhizobial species and groups were included as references. We conclude that these nodule isolates are diverse, and form several phylogenetic subgroups inside #Bradyrhizobium$. Nodulation tests performed on 5 plant species demonstrated host specificity among the strains studied. (Résumé d'auteur)

Analyses of DNA polymorphism and virulence variation were used to evaluate the population structure of Xanthomonas axonopodis pv. manihotis, the pathogen causing cassava bacterial blight in Colombia. We collected strains from the major cassava-growing regions which can be grouped into different edaphoclimatic zones (ECZs) according to environmental conditions, production constraints, and economic parameters. DNA polymorphism was assessed by a restriction fragment length polymorphism analysis, using an X. axonopodis pv. manihotis plasmid DNA sequence (pthB) as a probe to evaluate the genetic relatedness among 189 Colombian strains. The sampling intensity permitted the estimation of genetic differentiation within and among ECZs, sites, and fields and even within an individual plant. A multiple correspondence analysis indicated that the Colombian X. axonopodis pv. manihotis population showed a high degree of diversity relative to X. axonopodis pv. manihotis populations studied previously, and the entire collection was grouped into seven clusters. A general correlation was observed between the clusters and the geographical origin of the strains, as each cluster was largely composed of strains from the same ECZ. Representative strains, identified with pthB, were further characterized by ribotyping, hybridization to two repetitive genomic probes (pBS6 and pBS8), and restriction analysis of plasmid contents to evaluate the complementarity of these markers. Virulence variation was observed within the Colombian collection. Strains of different aggressiveness were found in all ecological zones, but no correlation between virulence variation and DNA polymorphism was observed. The genetic and virulence analyses contribute to understanding the X. axonopodis pv. manihotis population structure in Colombia.

The biodiversity of nitrogen-fixing organisms is huge. Taxonomic and phylogenetic research is needed to structure this diversity, to facilitate communication among scientists, and to increase our understanding of the evolution and biology of diazotrophs. Molecular tools for taxonomic and biodiversity studies of diazotrophic rhizobia, frankiae, cyanobacteria and bacilli are presented in sections 2 to 5. Sections 6 to 9 focus on problems with genus and species assignment.

Plasmids were recovered by an alkaline procedure from six symbiotically effective strains of Rhizobium meliloti of diverse geographical origin, reported to harbour only one middle-size large plasmid (ranging from 89 to 143 Megadaltons). Each purified plasmid was digested with eight restriction endonucleases; cleavage patterns were very complex: only KpnI and XbaI gave a limited number of bands. Fingerprints were very different, whatever the restriction enzyme or the geographical origin of the strains. However, Southern DNA-DNA hybridizations revealed that the plasmids showed homologous sequences having a high thermal stability. We gave evidence that some of these sequences are common to all the plasmids of R. meliloti. The biological function of these common sequences is unknown. Hybridization with cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae had demonstrated that nif genes were not located on the middle -- size plasmids of R. meliloti studied in this paper.

Fifty-one fast growing rhizobial strains isolated from root nodules of #Acacia senegal$ and #Prosopis chilensis$ in Sudan and Kenya were divided into DNA homology groups using non-radioactive DNA-DNA dot-blot hybridisation. #Rhizobium leguminosarum$ , #R. galegae$, #R. tropici$, #Mesorhizobium loti$, #Sinorhizobium fredii$, #S. meliloti$ used in numerical taxonomy were included in the hybridisation experiments as reference strains and, at a later strage #S. saheli$ and #S. terangae$. Scores given to the intensities of dots detected in the hybridisation experiments were used in principal component analysis, which clustered the majority of the tree rhizobia in two separate DNA-homology groups. The 51 strains were also analysed by genomic fingerprinting using the repetitive sequence-based polymerase chain reaction (rep-PCR) method with REP, ERIC, BOX and GTG5 primers. The resulting genomic fingerprints were compared with strains representing 15 rhizobial species. The relationship of 17 Sudanese strains to established sinorhizobial species was examined using the optical renaturation rates method and the G+C content of nine strains was determined. Results from dot-blot hybridisation and rep-PCR experiments were found to be in close agreement with each other and with the results obtained from spectrophotometric reassociation analysis. We suggest that rep-PCR fingerprinting can be used as a first and dot-blot hybridisation as a second rapid and dependable genomic screening method to classify new rhizobial isolates of unknown taxonomic status and to choose the representative strains for the more laborious DNA-DNA reassociation experiments. (Résumé d'auteur)

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated #Aeschynomene$ species #A. elaphroxylon$, #A. uniflora$, or #A. schimperi$ and 69 root or stem nodule isolates from the stem-nodulated #Aeschynomene$ species #A. afraspera$, #A. ciliata$, #A. indica$, #A. nilotica$, #A. sensitiva$, and #A. tambacoundensis$ from various places in Senegal. These isolates, together with 45 previous isolates from various #Aeschynomene$ species, were studied for host-specific nodulation within the genus #Aeschynomene$, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732-734, 1985). The whole collection of #Aeschynomene$ nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high pressure liquid chromatography, and thin layer chromatography analyses. The presence of puf genes in photosynthetic #Aeschynomene$ isolates was evidenced both by Southern hybridization with a #Rhodobacter capsulatus$ photosynthetic gene probe and by DNA amplificaton with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 #Aeschynomene$ isolates, including strain BTAi1, and 19 reference strains from #Bradyrhizobium japonicum$, #Bradyrhizobium elkani$, and other #Bradyrhizobium$ sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic #Aeschynomene$ nodule isolates share the ability to nodulate particular stem-modulated species and form a separate subbranch on the #Bradyrhizobium$ rRNA, distinct from #B. japonicum$ and #B. elkanii$. (Résumé d'auteur)