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.Ralstonia solanacearum (Smith) (30) is the agent of bacterial wilt, infecting over 450 plant species, including many economically important crops (12). This species has been subclassified into biovars based on biochemical tests and host-dependent races. Biovar 2A (equivalent to race 3) is adapted to temperate climates, has a narrow host range, and is responsible for recent outbreaks of potato brown rot disease in several countries of Western Europe and elsewhere worldwide (13,27). Although other biovars can also infect potatoes, biovar 2A is the most destructive phenotype in temperate areas.R. solanacearum is listed as a quarantine organism in the European Union (EU) (2), where new legislation has been introduced to control and eradicate the organism (3). Latent infections in seed potato tubers (6) have lead to the spread of the organism, both locally and internationally, and effective control of brown rot is dependent on the reliability of detection of the pathogen at this latent stage. For practical purposes, a detection assay is required which is rapid, specific, and sensitive to levels lower than those occurring in naturally infected potatoes and should be applicable to a crude sample of the specimen of interest (25). Serological techniques such as immunofluorescence (IF) microscopy, the enzyme-linked immunosorbent assay (ELISA) (10,15,21), and molecular techniques involving the PCR (9, 24) have been described for detection of R. solanacearum. An EU control directive (3) allows for a variety of detection methods to be employed. Briefly, a primary screening test (i.e., IF and/or selective isolation) is conducted with extracts from vascular tissue sampled from 200 tubers per 25-tonne lot. To confirm th...

Previously, we have produced a phylogeny of species type strains from the plant-pathogenic genus Xanthomonas based on gyrB sequences. To evaluate this locus further for species and infraspecies identification, we sequenced an additional 203 strains comprising all the pathovar reference strains (which have defined plant hosts), 67 poorly characterized pathovars, currently classified as Xanthomonas campestris, and 59 unidentified xanthomonads. The well-characterized pathovars grouped either in clades containing their respective species type strain or in clades containing species related to Xanthomonas axonopodis. The Xanthomonas euvesicatoria, Xanthomonas perforans and Xanthomonas alfalfae species complex, Xanthomonas fuscans and Xanthomonas citri were discriminated as X. axonopodis-related clades and comprised a large proportion of unidentified strains as well as 80 pathovars representing all the X. axonopodis pathovars and many poorly characterized pathovars, greatly increasing the plant host ranges of the constituent species. Most xanthomonads from these three large clades were isolated from a taxonomically diverse range of plant hosts, including many weed species, from field systems in India, suggesting that these lineages became established and diversified in agricultural areas in this region. The majority of these xanthomonads had minimal sequence diversity, consistent with rapid and highly extensive pathovar diversification that has occurred in relatively recent times. Low-intensity farming practices may have provided conditions conducive to pathovar development, and evidence for pathovar diversification within other regional angiosperm floras is discussed. The gyrB locus was sufficiently discriminating to identify diversity within many species. Seven branches or clades were sufficiently distinct to be considered as potential novel species. This study has provided a comprehensive xanthomonad classification framework and has firmly established gyrB sequencing as a rapid and efficient identification tool.

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel‐end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 106–7.4 × 107 colony‐forming units per ml. Following concentration by high‐speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi‐selective media, enzyme‐linked immunosorbent assay (ELISA), indirect immunofluorescent‐antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre‐enrichment of samples in semi‐selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi‐selective medium and tomato bioassay could detect fewer than 104 cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.

Approximately 500 fatty acid profiles were prepared for 340 strains of plant-pathogenic and other bacteria currently or recently classified in the genus Pseudomonas Migula 1984. Strains representing some infraspecific taxa were included. The fatty acid profiles were stable and reproducible provided that cultural and chemical techniques were standardized. The 2-and 3-hydroxy fatty acids were found to be useful in grouping strains into six major groups, several of which were further differentiated into subgroups. Group 1 contained strains of the following species and subspecies: Pseudomonas aeruginosa, P. agarici, P. asplenii, P. aureofaciens, P. caricapapayae, P. chlororaphis, P. cichorii, P. Jicuserectae, P. jfuorescens, P. jbscovaginae, "P. gingeri," P. marginalis, P. meliae, P. putida, 4LP. reactans," P. syringae, P. t o h s i i , and P. viridijfava (subgroup la); P. corrugata (subgroup lb); P. rubrisubalbicans (subgroup lc); P. alcaligenes, P. pseudoalcaligenes subsp. pseudoalcaligenes, and P. stutzeri (subgroup Id); P. amygdali (subgroup le); and P. cattleyae NCPPB 1874

Fatty acid profiles of 773 strains representing 25 taxa of plant pathogenic and related saprophytic bacteria were compared with two commercially available broad-spectrum libraries and one self-generated library based primarily on cultures from the National ,Collection of Plant Pathogenic Bacteria. T h e accuracy of identification at specific level was often loo%, although for some closely related species and infraspecific taxa accuracy was sometimes significantly less than this. T h e accuracy of identification of Xanthomonas campestris pathovars was much better than for Pseudomonas syringae pathovars. Almost all identifications were made within 24-48 h.Standardization of cultural conditions was essential. Hydroxy fatty acids were of great taxonomic value in classification of Gram-negative bacteria. Improved library development and standardization of cultural and analytical techniques will further increase the accuracy of identification. Fatty acid profiling offers a valuable rapid, accurate method for identification of many bacteria.