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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)

Eight Frankia-Gymnostoma nitrogen-fixing symbioses endemic to New Caledonia were studied using a range of techniques including molecular typing of Frankia , infectivity/effectivity and host spectrum of the isolates. The study was conducted on 128 Frankia isolates from the eight Gymnostoma species. The RFLP analysis of the rRNA 16S-23S ITS allowed us to cluster the strains in four groups. Symbiotic characterization of the four groups failed to reveal any host specificity both in term of infectivity or effectivity. The characteristics of the strains were those of Eleagnaceae -infective Frankia strains. Co-inoculation of two Gymnostoma species with mixtures of strains evidenced 11 % of mixed RFLP profiles. In such controlled conditions, this was interpreted as co-infections. This work evidenced original features of these Gymnostoma isolates: (i) they were closer to Eleagnaceae -infective Frankia than Casuarina -infective Frankia ; (ii) closer to atypical Frankia from Casuarina ; (iii) poorly host-specific; and (iv) they had a tendency of co-infection. They could represent a primitive group of Frankia able to survive in a wide range of habitats.

The Anopheles genus is probably one of the best studied genera among insects of medical importance. Of more than 500 species currently listed in the world about sixty species are vectors of malaria agents and about thirty species are responsible for most of the transmission [1-3]. The important epidemiological role of anopheline species has motivated many studies of taxonomy and systematics with traditional tools. With the advent of molecular tools, the development of informatics databases and new mathematical concepts on shape, characteriz‐ ing insects has become more and more accurate. Molecular tools based on the nucleotide polymorphism of DNA have allowed the identification of cryptic diversity, confirming and refining previous findings suggesting the existence of many species groups or complexes of sibling species. The development of international genetic sequence database collabora‐ tionshttp://www.insdc.org, http://www.barcodinglife.com/ allowed the use of reference sequences for species identification. Although not well developed, the same need for infor‐ matic databases arose for traditional taxonomy.

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.

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is a widespread disease that affects cassava (Manihot esculenta Crantz). Studies on the pathogen population structure, pathogen diagnosis, identification and expression of plant genes involved in resistance have been carried out. Different molecular techniques were developed to assess the genetic diversity among the Xampopulations. Characterization of Xam population dynamics over time had enable us to determine the different factors that are associated with resistance breakdown and those that influence the genetic structure or virulence phenotypes of the pathogen's population. Methods for detecting the pathogen in vegetative planting materials and true seeds were developed and contributed to reduce the impact of the disease. To better understand the genetics of resistance a quantitative trait loci (QTLs) approach was developed. Using a PCR-based strategy with degenerate primers we isolated two resistance gene candidates in cassava. We also characterized a region of a chromosome rich in R-gene like sequence. In this review we also report the main results obtained by transcript profiling methodologies, cDNA-AFLP and ESTs developed by the authors to characterize the genes involved in disease resistance. All together these techniques allowed the identification of molecular markers either associated to CBB resistance or that may represent putative genes involved in disease resistance. This article reviews current knowledge on the molecular cassava-Xam interactions.

The diversity of Bradyrhizobium strains nodulating three cowpea (Vigna unguiculata L. Walp.) cultivars in favorable and water-limited conditions occuring at flowering was analysed. PCR- Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of 16S-23S rDNA intergenic spacer region (IGS) directly applied on 85 crushed nodules distinguished four genetic profiles, IGS types I, II, III and IV. The distribution of these IGS types according to water conditions and cowpea cultivars (B-21, TN 88-63 and Mouride) showed that nodulating strains appeared more diverse in water-limited condition. More than three quarters of prospected nodules presented the IGS type I. They were formed on all three cultivars and in both water conditions. Only a small part of nodules was distributed between the IGS type II, III and IV. Nodules showing the IGS types II and III were found mainly in limited conditions on TN 88-63 and Mouride cultivars, whereas nodules presenting the IGS type IV were collected only from cultivars B-21 and Mouride, in both water conditions. Strains corresponding to the different profiles were isolated. The phylogenetic analysis of 16S rRNA gene sequences showed that they belong to the genus, Bradyrhizobium. The sequence analysis of 16S-23S rDNA IGS revealed that the strains exhibiting IGS types II, III and IV were closely related to some Faidherbia albida isolates from Senegal. IGS type II can be assigned with at least 98% similarity to Bradyrhizobium genospecies IV. IGS types III and IV showed more than 96% similarity with genospecies VII and could belong to the same genospecies. IGS type I, the most frequent, exhibits low IGS similarity with reported sequences in the databases, and could represent a new genospecies. (African Journal of Biotechnology: 2003 2(1): 13-23)

Rice progenies used for the construction of genetic maps permit exhaustive identification and characterization of resistance genes present in their parental cultivars. We inoculated a rice progeny derived from the cross IR64 x Azucena with different Magnaporthe grisea isolates that showed differential responses on the parental cultivars. By QTL mapping, nine unlinked loci conferring resistance to each isolate were identified and named Pi-24( t) to Pi-32( t). They could correspond to nine specific resistance genes. Five of these resistance loci (RLs) were mapped at chromosomal locations where no resistance gene was previously reported, defining new resistance genes. Using degenerate primers of the NBS (nucleotide binding site) motif found in many resistance genes, two resistance gene analogues (RGAs) IR86 and IR14 were identified and mapped closely to two blast RLs (resistance identified in this study, i.e. Pi-29(t) and Pi-30(t) respectively). These two RLs may correspond to the Pi-11 and Pi-a blast resistance genes previously identified. Moreover, the ir86 and ir14 genes have been identified "in silico" on the indica rice cultivar 93-11, recently sequenced by Chinese researchers. Both genes encodes NBS-LRR-like proteins that are characteristics of plant-disease resistance genes.

Eighteen strains of anaerobic thiosulfate-reducing bacteria unable to use sulfate as electron acceptor (TSRnSR) were isolated from four ricefield soils originating from France and the Philippines, using peptides as energy sources, H2 as electron donor, thiosulfate as electron acceptors, and four enrichment methods to vary the selection pressure. They were strict proteolytic asaccharolytic anaerobes producing H2S when grown on thiosulfate + H2. They exhibited the same RFLP profile (11 restriction enzymes tested). Partial sequencing of the 16S rDNA showed that they belonged to the genus Clostridium and were phylogenetically related to C. subterminale. DNA-DNA hybridization of a representative strain with the closest C. subterminale strain (DSM 6970T) yielded a value of 68.9%. Previous counts of TSRnSR in ricefield soils, their identification as Clostridium strains, and the known ubiquity of this genus in such soils indicate that TSRnSR may play a significant role in S cycling in some wetland soils. (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)

An interspecific partial genetic linkage map of #Coffea$ sp. based on 62 backcross hybrids is presented. F1 hybrids were generated by a cross between the wild #C. pseudozanguebariae$ and the anciently cultivated #C. liberica$ var. #dewevrei$ (DEW) ; progeny were then derived from a backcross between F1 hybrid and DEW. The map construction consisted of a two-step strategy using 5.5 and 3.1 LOD scores revealed by simulation file. The map consisted of 181 loci : 167 amplified fragment length polymorphism (AFLP) and 13 radom fragment length polymorphism (RFLP) loci. The markers were assembled into 14 linkage groups, each with 4-31 markers covering 1,144 cM. Segregation distorsion was observed for 30% of all loci, in particular 3:1 and 1:3 ratios equally favouring each of the two parents. The existence of such ratios suggests genetic conversions events. This map also represents an initial step towards the detection of quantitative trait loci. (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)

One of the enduring problems in the epidemiology of sleeping sickness is that there are 3 morphologically indistinguishable subspecies of Trypanosoma brucei involved in a complex transmission cycle between humans, tsetse and reservoir hosts. Two subspecies, T. b. gambiense and T. b. rhodesiense, are infective to man and cause gambian and rhodesian sleeping sickness, respectively. The third subspecies T. b. brucei cannot by definition infect humans, but coexists with the other trypanosomes in reservoir hosts and vectors.

La NouvelleCalédonie possède une forte concentration en Casuarinaceae endémiques. On y dénombre huit espèces de #Gymnostoma# et une espèce de #Casuarina : C. collina#. Vingt-six souches de #Frankia# ont été isolées de nodules de #C. collina# et étudiées par diverses techniques incluant l'infectivité, l'effectivité, le spectre d'hôte et la PCR/RFLP de l'ITS 16S-23S. Tous ces #Frankia# présentent les mêmes caractéristiques que celles décrites chez les souches atypiques de #Casuarina# spp. Ce travail a mis en évidence, chez les #Frankia# de #C. collina#, les caractéristiques originales suivantes : (i) ils sont localisés à l'intérieur des nodules ; (ii) ils appartiennent au groupe des #Frankia# infectifs sur #Elaeagnus#;(iii) ils se regroupent dans quatre groupes ITS déjà décrits chez #Gymnostoma#. En fait, il est très probable que les souches atypiques de #C. collina# et les #Frankia# de #Gymnostoma# soient les mêmes. On peut formuler l'hypothèse que les #Frankia# de #Gymnostoma# pourraient utiliser #C. collina# comme alternative en absence de leur hôte naturel ou quand les conditions écologiques sont favorables à #C. collina#.

La réaction en chaîne des polymérases/polymorphisme des fragments de restriction (PCR-RFLP) a été utilisée pour séparer des isolats du nématode #Bursaphelenchus$. Les isolats de #B. xylophilus$ examinés provenaient du Japon, des USA, de Chine et du Canada, et ceux de #B. mucronatus$ du Japon, de Chine et de la France. L'ADN ribosomal contenant le gène 5.8S, les segments de transciption interne 1 et 2, et les segments partiels des gènes 18S et 28S ont été amplifiés par PCR. La digestion des produits amplifiés provenant de chaque isolat à l'aide de douze endonucléases de restriction et l'examen des données en RFLP qui en découlent révèlent, par une analyse en grappe, une séparation significative entre #B. xylophilus$ et #B. mucronatus$. Parmi les isolats de #B. xylophilus$ examinés, les isolats pathogènes du Japon, ceux de Chine et des USA étaient tous identiques, tandis que les isloats non pathogènes du Japon étaient légèrement distincts et que ceux du Canada formaient une grappe séparée. Parmi les isolats de #B. mucronatus$, deux isolats provenant du Japon étaient très semblables ; de même un autre isolat du Japon et un isolat de Chine étaient identiques. Les données provenant des séquences d'ADN montrent 98 différences (substitution nucléotidiques ou séparations) dans les 884 paires de bases examinées chez les isolats de #B. xylophilus$ et #B. mucronatus$. Les données provenant des séquences d'ADN chez #Aphelenchus avenae$ et #Aphelenchoides fragariae$ diffèrent non seulement de celles des #Bursaphelenchus$ mais aussi entre elles. Afin de préciser les relations phylogéniques de ces espèces, les données séquentielles du gène 5.8S provenant de l'ADN ribosomal ont été examinées... (D'après résume d'auteur)

Resistance to antifolate antimalarial drugs (cycloguanil, a biologically active metabolite of proguanil, and pyrimethamine) is associated with a Ser- to Asn-108 point mutation in the dihydrofolate reductase thymidylate synthase gene of #Plasmodium falciparum$. The frequency of this mutation was studied in 127 clinical isolates obtained in Yaounde, Cameroon using a simple and rapid molecular technique based on the polymerase chain reaction and restriction fragment lengh polymorphism. Of the 127 isolates, pure wild-type Ser-108 codon, pure mutant-type Asn-108 codon, and mixed codons were observed in 66, 55, and six parasites, respectively. The proportion of antifolate-resistant, pure mutant-type codon, with respect to pure wild-type or mixed alleles, was 43% (55 to 127). The results of the molecular assay were compared with those of semimicro isotopic in vitro assay in 34 isolates. All 17 pure Ser-108 isolates and two isolates with mixed alleles were sensitive to both pyrimethamine (50% inhibitory concentration [IC50] < 100 nM) and cycloguanil (IC50 < 50 nM). Fourteen of 15 isolates with the mutant-type Asn-108 codon were resistant to pyrimethamine and cycloguanil. One isolate with Asn-108 showed a slightly elevated pyrimethamine IC50 (78 nM), which was within the sensitive range. This study provides further evidence that antifolate-resistant #P. falciparum$ isolates are already present in Yaounde, Cameroon. (Résumé d'auteur)

L'analyse du polymorphisme de longueur des fragments de restriction d'ADN amplifie (PCR-RFLP) presente de nombreux avantages pour la caracterisation de la diversite microbienne: relative rapidite, faible cout, possibilite d'application a l'ensemlble ries taxons bacteriens, bonne reproductibilite, possibilite d'analyse simultanee d'un grand nombre de souches, interet au niveau taxonomique. Afin d'optimiser sort usage, nous avons cherche a determiner combien d'enzymes et lesquels etaient le plus approprie, en effectuant une analyse systematique par ordinateur de la capacite de 25 enzymes de restriction differentes a generer par PCR-RFLP, seules ou en combinaison, (les profils de digestion permettant la distinction d'especes proches taxonomiquement. La sous-unite 16S de l'ARN ribosomal (rrs; pour ribosomal RNA small subunit) a ete choisie pour simuler ces PCR-RFLP: elle correspond a la region la plus frequemment etudiee et permet en general une bonne discrimination au niveau de l'espece; de plus, de nombreuses sequences rrs sont disponibles dans les banques de donnees. L'etude a ete focalisee sur les rhizobia qui representent un bon modele de genres differents, entremeles avec d'autres genres tres proches taxonomiquement, et parce que leur importance en agriculture induit la necessite d'outils performants pour la caracterisation de grands nombres de nouveaux isolats. La digestion de 24 sequences rrs correspondant aux souches-types de differentes especes a ete simulee par ordinateur. Les profils de digestion obtenus pour les differentes sequences ont ete compares deux par deux afin de determiner le nombre de profils distincts generes par chaque enzyme individuellement. ou par les combinaisons systematiques de 2, 3, 4, 5, 6 ou 7 enzymes. Differentes combinaisons de 3, 4 ou 5 enzymes permettent la distinction entre les especes. La frequence a laquelle les differentes enzymes sont trouvees dans les i:ounfiinaisons discrimimantes ne correspond pas completement a leur pouvoir de discrimation individuel, mais a la complementarite qu'elles presentent lorsqu'ellas sont analysees ensemble. La combination des profils obtenus avec six enzymes, choisies parmi les sept puis frequentes dans les combinaisons discriminantes, permet en moyenne de distinguer plus de 99,5%, des sequences etudiees, quelle que soit la similarite entre ces sequences. L'utilisation d'au moins six de ces sept enzymes est suggeree afin d'assurer une bonne distinction des especes de rhizobia, connues et inconnues.

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.

Neptunia oleracea is an annual aquatic stem-nodulated legume indigeneous of waterlogged places in Senegal. Bacterial isolates from nodules of N. oleracea can infect Medicago sativa roots but the nodules are not efficient. No information is available on the taxonomy of Neptunia specific bacteria.

It has been postulated that chloroquine resistance may be associated with a single point mutation at codon 86 of the #Plasmodium falciparum$ multidrug resistance 1 (pfmdr 1) gene. Using a simple and rapid molecular technique involving polymerase chain reaction and restriction fragment length polymorphism, the frequency of the Asn-to-Tyr mutation associated with chloroquine resistance was established among 129 clinical isolates obtained from indigenous patients in Yaoundé, Cameroon. The results showed that 110 of 129 isolates display a mutant codon. The other clinical isolates had either a pure wild-type Asn-86 codon (n = 12) or mixed Asn/Tyr alleles (n = 7). In vitro drug assays were performed to compare the genotype and phenotype in 102 clinical isolates. Of these isolates, 86 displayed pure Tyr-86 mutant codon ; 48 (56%) mutant isolates were chloroquine-resistant (50% inhibitory concentration [IC50] > 100 nM), as expected, but 38 (44%) mutant isolates were chloroquine-sensitive (IC50 < 100 nM). Three chloroquine-resistant isolates and seven chloroquine-sensitive parasites carried a wild-type Asn-86 codon. Mixed alleles were found in six isolates (four chloroquine-sensitive and two chloroquine-resistant isolates). Our results did not confirm previous observations on the possible association between chloroquine resistance phenotype and genotype based on the pfmdr 1 gene. (Résumé d'auteur)

We analysed the genetic differentiation among 17 natural populations of the Nile tilapia Oreochromis niloticus (Linnaeus, 1758) using allozymes and restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA). The populations studied, from the River Senegal to Lake Tana and from Lake Manzalla to Lake Baringo, represent all subspecies which have been previously described. Sixteen variable nuclear loci showed that these populations can be clustered in three groups: (1) West African populations (Senegal, Niger, Volta and Chad drainages), (2) Ethiopian Rift Valley populations (Lakes Awasa, Ziway, Koka and the Awash River) and (3) Nile drainage (Manzalla, Cairo, Lake Edward) and Kenyan Rift Valley populations (Lakes Turkana, Baringo and River Suguta). Nine different mtDNA haplotypes were found in the RFLP analysis of a 1 kb portion of the D-loop region. The network obtained showed that there are three geographically distinct groups; all West African populations and O. aureus are clustered, the two Ethiopian Rift Valley populations are distinct and between these two groups are the Kenyan and Ugandan Rift Valley populations. Nile populations show affinities both with West African populations and with specimens from Lakes Tana and Turkana. Taxonomic and biogeographical implications of these results are discussed.

From 1989 to 1992, a longitudinal study of the relationships between different tick species and domestic ungulates in the transmission and amplification of Crimean-Congo hemorrhagic fever (CCHF) virus was undertaken in the Bandia area in Senegal where the presence of the virus had been reported previously. An epizootic occurred in 1991-1992 and 22 strains of CCHF virus were isolated from Hyalomma marginatum rufipes Koch, Amblyomma variegatum (F.), Rhipicephalus guilhoni Morel & Vassiliades, and R. evertsi evertsi Neumann ticks collected from cattle and goats. No human cases were reported. Transmission of CCHF virus in the area involves a complicated pattern including many tick species and hosts. Amplicons of the S fragment (536 bp) of the CCHF genome of 12 isolates from the study were obtained by polymerase chain reaction and analyzed by restriction-length fragment polymorphism. Three different genotypes of CCHF virus were identified and present during the epizootic. One genotype was recovered from A. variegatum, R. guilhoni, and R. e. evertsi and 2 genotypes were isolated from H. m. rufipes.

The taxonomy of the #Culex pipiens$ complex remains a controversial issue in mosquito systematics. Based on morphologic characters, 2 allopatric taxa are recognized, namely #Cx. pipiens$ (including the form "#molestus$") in temperate areas and #Cx. quinquefasciatus$ in tropical areas. Here we report on variability at nucleotide level of an acetylcholinesterase gene in several strains and natural populations of this species complex. Few polymorphisms were found in coding regions within a subspecies but many polymorphisms were observed between subspecies in noncoding regions. We describe a method based on a restriction enzyme polymorphism in polmerase chain reaction-amplified DNA, in which the presence or absence of one restriction site discriminates #Cx. pipiens$, #Cx. quinquefasciatus$, and their hybrids. This technique reliably discriminates mosquitoes from more than 30 worldwide strains or populations. Polymerase chain reaction amplification of specific alleles may also be a useful tool for characterizing specific alleles of each sibling taxon. (Résumé d'auteur)

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)

The African Anopheles funestus and the Asian An. minimus groups are closely related and are probably considered distinct only because of their geographic separation. This study aimed at improving two identification methods based on polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) already devel- oped for either group. Each PCR-RFLP, either on the internal transcribed spacer 2 (ITS2) for the An. minimus group, and domain 3 (D3) for the An. funestus group, was applied to the other group for the standardization of one identifi- cation method applicable on both continents. The ITS2 fragment digested by Bsi ZI showed the highest diagnostic power. This assay allowed the discrimination of at least 13 Anopheles species within the subgenus Cellia from two continents (Africa and Asia), among which are five major malaria vectors. Moreover, digestion of the D3 with Msp I showed intragenomic variations within An. funestus populations. Two types of D3 copies (M and W) occurred in specimens from southern Africa. The populations from West-Central Africa presented only type W and East-Malagasy populations exhibited type M. Since An. funestus shows a great capacity of adaptation, these molecular variations, along with behavioral and ecologic ones, reinforce the hypothesis of a species complex that will need to be further investigated.