Incidence of the cblA major subunit pilin gene amongst Burkholderia species.

PCR-amplification has been used to screen 75 isolates of the Burkholderia cepacia complex for the cblA pilin gene. PCR-amplified products of the correct size (664 bp) were cloned and sequenced and the sequences compared. Apart from in the control, epidemic cystic fibrosis (CF)-associated B. cepacia lineage we also identified, for the first time, cblA genes in a unique, non-CF clinical isolate from France and a plant (onion) pathogenic isolate from Italy. The sequence of the cblA gene amplified from the clinical isolate was more diverged from the epidemic lineage than that amplified from the onion pathogenic isolate.

Development of a species-specific recA-based PCR test for Burkholderia fungorum.

The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B. fungorum. Previous analysis of several novel rhizospheric, environmental isolates belonging to the B. cepacia complex suggested they may be closely related to B. fungorum. To discover any relationship between these isolates and B. fungorum we set out to clone and sequence a portion of the B. fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay. Using a similar procedure we extended the recA-based PCR assay to identify B. sacchari and B. caledonica, two additional soil-inhabiting Burkholderia spp.

Variation among strains of Xanthomonas campestris pv. vasculorum from Mauritius and other countries based on fatty acid analysis.

Fatty acid profiling was used to study variation amongst strains of Xanthomonas campestris pv. vasculorum (Xcv). They could be divided into five groups using cellular fatty acid profiles. Group A strains represent a new and little known taxon and all came from plants of broom bamboo (Thysanolaena maxima) from Mauritius. Group B strains included the Xcv pathotype reference strain and were from palms, broom bamboo and sugarcane from Mauritius, Reunion and Australia. Group C contained southern African and Malagasy strains from sugarcane and maize, together with X. campestris pv. holcicola strain. No Mascarene strains fell into this group. Group D strains isolated from sugarcane, maize and royal palm (Roystonea regia) were from Mauritius and Reunion, the earliest known strains coming from Réunion. These groups represented in the Mascarene Islands possibly belong to three different Xanthomonas species. A further Group E comprised one Xcv strain (NCPPB 182) from Puerto Rico, one X. vasicola pv. holcicola strain plus 6 other unclassified Xanthomonas strains causing red stripe disease symptoms in sugarcane. Three of these groups occur on Mauritius and two occur on Réunion. Group B strains originally caused serious problems in noble canes. As resistant interspecific hybrids were introduced, group D strains appeared in Mauritius possibly being introduced from Reunion but having similar host ranges within the Gramineae and Palmae. The findings that 3 of these groups (A, B, D) can cause gumming disease in a grass species (T. maxima) and that 2 of them (B, D) also cause gumming disease in sugar cane (Gramineae) and palms (Palmae) is unusual.

Classification, nomenclature, and plant pathogenic bacteria - a clarification.

ABSTRACT In a recent Letter to the Editor of Phytopathology, proposals were made for endorsement and for rejection of selected names of plant pathogenic Pseudomonas spp. and Xanthomonas spp. We believe that support for, and rejection of, several names was based on misconceptions concerning the Approved Lists of Bacterial Names and entails misinterpretations of several Rules of the International Code of Nomenclature of Bacteria. This letter aims to clarify those misconceptions and misinterpretations.

Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR.

AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR. CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.

Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat.

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).

Diversity of Burkholderia isolates from woodland rhizosphere environments.

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.

Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay.

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon.

AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.