A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia genomovar III, a genomic species associated with "cepacia syndrome" in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B. cepacia complex consists of two independent phylogenetic lineages.In a survey of nonnative plant rhizosphere bacteria conducted in La Côte Saint André (France) with maize and in Kapunda (South Australia, Australia) with wheat, high levels of two groups of Burkholderia strains were found. The first group was characterized by using a polyphasic approach and formed a new taxon, Burkholderia graminis (15). Strains of the second group (designated phenon B) were found to be closely related to the Burkholderia cepacia complex; large numbers of these strains were present on roots, and more recently, new isolates were also obtained from inside the tissues of wheat and lupine in Kapunda (Table 1). Here, characterization of this taxonomic group was revisited by including reference strains of the B. cepacia complex in DNA-DNA hybridization, whole-cell protein electrophoretic, and 16S ribosomal DNA (rDNA) sequence analyses.Total DNA-DNA hybridization analyses were performed by using two methods, one involving tritiated reference DNAs (Table 2) and one involving photobiotin-labeled probes (Table 3). In a preliminary study, the two methods showed good correlation. For instance, the levels of hybridization of strain AUS 27 DNA with DNA of strain LMG 12614 were 65% when tritiated DNA was used and 63% when photobiotin-labeled DNA was used.In the first experiments we used tritiated reference DNAs of eight isolates, including two rhizosphere isolates (AUS 27 and C3B1M), one recent cystic fibrosis isolate (1-36) T ), and three recent cystic fibrosis isolates (strains 751, 1-36, and 1-47) were hybridized with these radioactively labeled DNAs. When hybridized with labeled DNA of strain AUS 27, all rhizosphere isolates except m35b showed levels of DNA-DNA hybridization greater than 65% and differences in melting temperatures (⌬T m values) less than 5°C, indicating that they belong to the same genomic species (12). When they were hybridized with labeled DNA of strain C3B1M, slightly lower values (as low as 61%) were obtained, indicating a certain degree of genomic heterogeneity in this species. Strain m35b showed significant but low levels of hybridization (40 to 48%) with all reference strains and thus does not belong to any of the genomovars examined. The possibility that this strain could belong to Burkholderia stabilis was not eliminated and will be tested further. B. cepacia genomovar III reference strains exhibited levels of hybridization of 58 to 76% with labeled DNA of strain AUS 27, indicating that the rhizosphere isola...

In a survey of soil and wheat or maize rhizoplane bacteria isolated using a medium containing azelaic acid and tryptamine as sole carbon and nitrogen sources, respectively, a large proportion of Burkholderia-l i ke bacteria were found. Among them, a homogeneous group of strains was identifiable based on phenotypic properties, fatty acid composition, DNA-DNA hybridizations and 16s rDNA sequences. According to molecular data, this group belongs to the genus Burkholderia but its weak similarity to previously described species suggests that it belongs to a novel species. Closest 16s rDNA phylogenetic neighbours of this species are Burkholderia caryophylli and two previously named Pseudomonas species which clearly appear to be part of the Burkholderia genus and were thus named Burkholderia glathei comb. nov. ), where wheat is grown either continuously or in rotation with a lupin-based pasture; the soil is an alphisol. Walpeup is an experimental wheat-growing station, situated in Victoria (Australia), on a very poor sandy soil, in a fixed sand dune system. Soil samples of the two Australian stations have been collected and used for growing wheat (cv. Spear) in pots under glasshouse conditions (three plants per pot containing 1.5 kg soil). After 3-4 weeks, wheat plants were harvested and used to isolate bacteria from their rhizoplane, as above. A few strains were isolated directly on PCAT medium from salt-affected and hydrophobic soils near Adelaide. Also included in Table 1 are 18 reference strains of Burkholderia, Pseudomonas, Ralstonia and Alcaligenes. Among the eleven type strains of Burkholderia species, only type strains of B. mallei and B. pseudomallei were not grown in this laboratory.Biochemical characterization. All tests were performed at 28 "C. The Biolog GN system was used as recommended by the manufacturer to test the oxidation of 95 carbon substrates. Results were read automatically with a spectrophotometer after 24 or 48 h incubation at 28 "C. To test the reproducibility of the method, eight isolates were run in duplicate. Numerical analysis of the results was made using the G N Microlog 2N software which calculates Microlog distances derived from the number of differences between strains. This software also permits clustering analysis using the UPGMA (unweighted mean pair group method) algorithm of Sneath & Sokal(44).Carbon substrate assimilation tests were performed using auxanographic API 50CH strips (bioMCrieux) as recommended by the manufacturer. Nine isolates were tested in duplicate. Numerical analysis was performed on data obtained after 7 d incubation. Interstrain distances were calculated using the coefficient of Dice and a phenogram was built using UPGMA.The API 20NE microtube system (bioMCrieux) was used as a standardized method to test oxidase activity, nitrate reduction, gelatin and aesculin hydrolysis, glucose fermentation, arginine dihydrolase activity and production of indole, P-galactosidase and urease.MIDI-FAME. The MIDI-FAME technique is based on the conversion of fatty acids to met...

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