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pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and other strains of X. campestris associated with Citrus spp. as templates in the PCR. Four primer pairs directed the amplification of target DNA from X. campestris pv. citri strains but not from strains of X. campestris associated with a different disease, citrus bacterial spot. Primer pair 2-3 directed the specific amplification of target DNA from pathotype A but not other pathotypes of X. campestris pv. citri. A pH 9.0 buffer that contained 1% Triton X-100 and 0.1% gelatin was absolutely required for the successful amplification of the target DNA, which was 61% G+C. Limits of detection after amplification and gel electrophoresis were 25 pg of purified target DNA and about 10 cells when Southern blots were made after gel electrophoresis and probed with biotinylated pFL1. This level of detection represents an increase in sensitivity of about 100-fold over that of dot blotting with the same hybridization probe. PCR products of the expected sizes were amplified from DNA extracted from 7-month-old lesions from which viable bacteria could not be isolated. These products were confirmed to be specific for X. campestris pv. citri by Southern blotting. This PCR-based detection protocol will be a useful addition to current methods of detection of this pathogen, which is currently the target of international quarantine measures.

International audience; A total of 26 strains, including 15 strains isolated from garlic plants with the typical symptoms of 'Café au lait' disease and 11 strains isolated from diseased or healthy rice seeds and sheaths infested by Pseudomonas fuscovaginae, were compared with 70 type or reference strains of oxidase-positive pathogenic or non-pathogenic fluorescent pseudomonads. The strains were characterized by using a polyphasic taxonomic approach. Numerical taxonomy of phenotypic characteristics showed that the garlic and rice strains were related to each other. However, they clustered into separate phenons, distinct from those of the other strains tested, and were different in several nutritional tests. On the basis of DNA-DNA hybridization, the garlic and rice strains constituted two distinct DNA hybridization groups, indicating that they belonged to separate species. The two groups of strains were also well differentiated by siderotyping. Garlic strains were pathogenic to garlic plants and either weakly pathogenic or non-pathogenic on rice; rice strains were either weakly pathogenic or non-pathogenic on rice and non-pathogenic on garlic. A phylogenetic analysis of 16S rRNA gene sequences confirmed that the two groups of strains belonged to the y-Proteobacteria and to the genus Pseudomonas. The names Pseudomonas salomonii sp. nov. and Pseudomonas palleroniana sp. nov. are respectively proposed for the garlic strains and the rice strains. The type strains are P. salomonii CFBP 2022(T) ( = ICMP 14252(T) = NCPPB 4277(T)) and P. palleroniana CFBP 4389(T) (= ICMP 14253(T) = NCPPB 4278(T))

Nine phylogenetic oligonucleotide probes were used to describe at the genus level the microbial community responsible for the spontaneous fermentation of maize, leading to the reproduction of Mexican pozol. Ribosomal RNAs of specific groupds and genera, in particular, lactic acid bacteria, were quantified using a culture-independent approach. In the early stage of the fermentation, #Lactococcus$ and #Leuconostoc$ appeared to be the dominant genera. A contrario, these represented minor genera at the end of the fermentation when #Lactobacillus$ dominated the process. In addition, eukaryotes seemed to play a significant role throughout the fermentation and enterobacteria could be detected by this method. (Résumé d'auteur)

Le cocotier est affecté par de nombreux ravageurs et microbes particulièrement en Amérique latine et dans les Caraïbes. Pour identifier les maladies, l'observation des symptômes est la première étape, l'étape suivante étant d'isoler le pathogène suspecté par culture #in vitro# ou purification, de le caractériser et de prouver sa pathogénicité. L'approche conventionnelle, peu coûteuse en général, est l'étape logique après la symptomatologie. Si l'on suspecte une origine bactérienne ou fongique, l'isolement et la culture #in vitro# peuvent être complétés par des tests sérologiques ou une observation au microscope. Dans le cas de virus ou phytoplasmes, la microscopie électronique doit être utilisée, mais on peut également identifier un virus donné par des techniques sérologiques. Ces méthodes conventionnelles, satisfaisantes pour le diagnostic, ne sont pas assez spécifiques pour les études épidémiologiques ou les études de la variabilité au niveau des espèces ou des souches. L'avantage de l'approche moléculaire est d'utiliser toute l'information génétique concernant le pathogène. Il y a plusieurs techniques pour cette approche : l'hybridation moléculaire, l'amplification en chaîne par polymérase(ACP), l'ACP couplée avec l'immunocapture. Ces techniques plus sensibles sont généralement plus coûteuses et plus longues pour l'obtention des résultats

The kinetoplast DNA of 27 plant trypanosomatid stocks (22 intraphloemic Phytomonas associated with coconut Hartrot, oil palm Marchitez, or decay of Alpinia purpurata, 3 Phytomonas isolates from latex, and 2 fruit trypanosomatids) was studied. Four classes of minicircle sizes were obtained: 1.6 and 1.8 kb for isolates originating from coconut palms, oil palms, Alpinia purpurata, and fruits; 850 bp and 2.8 kb for isolates from latex. Restriction endonuclease analysis showed that for almost all of the isolates minicircles were heterogeneous in base sequence. Cross-hybridization experiments were performed by Southern blot. A high sequence similarity only occurred between isolates of the same class of minicircle sizes, except for the 1.6-kb class, in which isolates from fruits and isolates originating from diseased coconut belonged to two hybridization groups. Moreover, the intraphloemic isolates that belonged to two classes, 1.6 and 1.8 kb, showed sequence similarity. These data confirm the existence of at least two groups of intraphloemic trypanosomatids associated with wilts, several groups of latex trypanosomatids, and one group of fruit trypanosomatids and could help in the classification of the lower trypanosomatids.

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.

Plasmid DNA was isolated from 54 strains of Xanthomonas campestris pv. citri, associated with different forms of citrus bacterial canker disease (CBCD). The number of plasmids per strain varied from one to five. A total of 24 plasmid bands with sizes from 7 to 100 kilobases (kb) were identified. Strains that had identical plasmid profiles were generally associated with the same form of CBCD. After digesting the plasmid DNA with each of three restriction endonucleases, 87 fragments with different sizes from about 1 to 30 kb were visualized (...)

Double-stranded RNAs were isolated from plants infected with five different isolates of apple chlorotic leaf spot virus (ACLSV). Analysis by PAGE and by Northern blot hybridization showed that six major species of viral dsRNA of approximately 7.5, 6.4, 5.4, 2.2, 1.1 and 1.0 kbp can be detected in infected plants, irrespective of the ACLSV isolate used. In addition to the dsRNA of 7.5 kbp corresponding to the full-length genome, the size and position on the genome of the 2.2 and 1.1 kbp species indicate that these are very probably double-stranded forms of subgenomic RNAs allowing the expression of the internal open reading frames coding respectively for the ACLSV 50K and coat proteins. The subgenomic messenger for the coat protein was indeed detected in total RNA preparations from infected plants. Surprisingly, the two most abundant dsRNA species, of 6.4 and 5.4 kbp, were found to be 5'-coterminal with the genomic RNA. A model for the expression of the genome of ACLSV and for the production of the molecules 5'-coterminal with the genomic RNA is presented.