Burkholderia tropica sp. nov., a novel nitrogen-fixing, plant-associated bacterium In an ecological survey of nitrogen-fixing bacteria isolated from the rhizosphere and as endophytes of sugarcane, maize and teosinte plants in Brazil, Mexico and South Africa, a new phylogenetically homogeneous group of N 2 -fixing bacteria was identified within the genus Burkholderia. This polyphasic taxonomic study included microscopic and colony morphology, API 20NE tests and growth on different culture media at different pH and temperatures, as well as carbon source assimilation tests and whole-cell protein pattern analysis. Analysis of 16S rRNA gene sequences showed 99?2-99?9 % similarity within the novel species and 97?2 % similarity to the closest related species, Burkholderia sacchari. The novel species was composed of four distinct amplified 16S rDNA restriction analysis groups. The DNA-DNA reassociation values within the novel species were greater than 70 % and less than 42 % for the closest related species, B. sacchari. Based on these results and on many phenotypic characteristics, a novel N 2 -fixing species is proposed for the genus Burkholderia, Burkholderia tropica sp. nov., with the type strain Ppe8 T (=ATCC BAA-831

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...

During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some "endophytes" were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using (15)N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.

During a survey of nitrogen-fixing Burkholderia associated with sugarcane in Tamil Nadu, some endophytes were isolated on PCAT medium. Isolation was based on the use of the selective PCAT medium. Four isolates were studied, all belonging to the genus Burkholderia. One of them, MG43 was consistently more active in reducing acetylene and was identified as Burkholderia vietnamiensis. This isolate was used to inoculate micro-propagated sugarcane plantlets in a comparison with two other diazoptrophs, viz. Gluconacetobacter diazotrophicus T and Herbaspirillum seropedicae T . Inoculated plants and uninoculated controls were used in a pot experiment followed by two field experiments under different rates of nitrogen fertilisers. MG43 and G. diazotrophicus performed best in sugarcane, their natural host. Biomass increase due to MG43 inoculation reached 20% in the field. Inoculated plants were heavily colonised by the inoculated bacterium (up to 115,000 CFU g )1 root fresh weight). Inoculation by a combinaison of the three strains performed less well than inoculation by a single MG43 suspension. Ecological implications are discussed, as well as the potential of these bacteria to provide a feasible alternative to higher N fertilisers rates in a low input and long term sustainable rural economy.

In the frame of a survey of potentially endophytic N2-fixing Burkholderia associated with maize in Mexico, its country of origin, the soil of an indigenous maize field near Oaxaca was studied. Under laboratory conditions, plant seedlings of two ancient maize varieties were used as a trap to select endophyte candidates from the soil sample. Among the N2 fixers isolated from inside plant tissues and able to grow on PCAT medium, the most abundant isolates belonged to genus Burkholderia (API 20NE, rrs sequences). Representative isolates obtained from roots and shoots of different plants appeared identical (rrs and nifH RFLP), showing that they were closely related. In addition, their 16S rDNA sequences differed from described Burkholderia species and, phylogenetically, they constituted a separate deep-branching new lineage in genus Burkholderia. This indicated that these isolates probably constituted a new species. An inoculation experiment confirmed that these N2-fixing Burkholderia isolates could densely colonize the plant tissues of maize. More isolates of this group were subsequently obtained from field-grown maize and teosinte plants. It was hypothesized that strains of this species had developed a sort of primitive symbiosis with one of their host plants, teosinte, which persisted during the domestication of teosinte into maize.

When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10 2 -to 10 4 -fold decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism Burkholderia vietnamiensis TVV75. This artifact could not be eliminated simply by rinsing the seeds, even thoroughly, with sterile distilled water. When growth resumed, a significant increase in the frequency of rifampin-and nalidixic acid-resistant mutants in the population was observed compared to the control without seeds. This phenomenon was a specific effect of hypochlorite; it was not observed with hydrogen peroxide alone. It was also not observed when the effect of hypochlorite was counteracted by sodium thiosulfate. We hypothesized that the hypochlorite used for disinfection reacted with the rice seed surface, forming a chlorine cover which was not removed by rinsing and generated mutagenic chloramines. We studied a set of rifampin-and nalidixic acid-resistant mutants obtained after seed surface sterilization. The corresponding rpoB and gyrA genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidixic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial bacterial colonization of a plant is studied.The use of hypochlorite salts for disinfection dates back to the mid-18th century. Since that time, chlorination has been the most widely used bactericidal treatment for conventional disinfection of municipal drinking water for prevention of epidemic diseases such as cholera and typhoid, and it is still the most widely used method for disinfecting water (18). Hypochlorite is also routinely used as a sanitizer for domestic uses, as well as in food-processing plants to remove surface contaminants which can alter food quality or lead to food-borne diseases (2,3,23,29).Hypochlorite is known to be a very effective to killer of bacteria; even micromolar concentrations are enough to reduce bacterial populations significantly (27). However, little is known about the exact mechanisms of bacterial killing by this sanitizer. When diluted in water, the hypochlorite salts used [NaOCl, Ca(OCl) 2 , LiOCl, and KOCl] lead to formation of HOCl, whose concentration is correlated with bactericidal activity (27). Bacterial killing by HOCl may be due at least in part to lethal DNA damage (13, 42). However, HOCl itself is so reactive that it is unlikely to penetrate cells and reach the DNA; rather, it seems that the bactericidal activity is due to formation of secondary products, as hypochlorous acid reacts avidly with a wide variety of subcellular compounds (membranes, proteins, etc.) (10, 18). In particular, HOCl reacts with NH 4 ϩ and organic amines to form highly toxic chloramines, which also are strong oxidizing and chlorinating compounds and could be the actual killing agents. These chloramin...

Representative N2-fixing bacteria have been isolated from the rhizosphere of rice using the "spermosphere model." They have been characterized using conventional biochemical tests, DNA composition, and DNA–rRNA hybridization studies. In addition to a large number of Enterobacteriaceae, the most commonly encountered isolates belong to the genus Azospirillum and to Pseudomonas paucimobilis, a taxon related to Flavobacterium capsulatum. A nonmotile Azospirillum has been found.