Frequency and biodiversity of 2,4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth.

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.

Polyphasic approach to the characterisation of marine luminous bacteria.

Fifteen luminous bacterial strains were isolated from the Tyrrhenian Sea coastal waters off northeastern Sicily and characterised by a combination of phenotypic and molecular tests in order to identify them and to determine their intraspecific genetic variability. Five luminous type strains, Vibrio splendidus NCIMB 1, V. harveyi NCIMB 1280, V. fischeri NCIMB 1281, V. orientalis NCIMB 2195 and Photobacterium leiognathi NCIMB 2193, were used as reference. On the basis of their phenotypic characters, the isolates were assigned to the family Vibrionaceae and all were related to the V. harveyi reference strain. Amplified 16S ribosomal DNA restriction analysis (ARDRA) enabled the strains to be subdivided into three groups, two of which exhibited the same restriction pattern as the two reference strains, V. harveyi and V. splendidus. Comparison of the full 16S rDNA sequence and of a 100-bp highly variable 16S rDNA region (selected as a 'signature' sequence for the luminous bacteria) confirmed ARDRA data and suggested that the strains of the third group could be considered a subspecies of V. harveyi or a tyrrhenian biovar, different from the other reference strains whose 16S rDNAs have already been sequenced. Random amplified polymorphic DNA (RAPD) fingerprinting and analysis of plasmid content suggested a high degree of intraspecific genetic variability within the largest ARDRA group. Data obtained suggest that the ARDRA method and the sequencing of the 16S rDNA signature region could be a powerful tool for a rapid identification of marine luminous bacteria.

Isolation and characterisation of a new antagonistic Burkholderia strain from the rhizosphere of healthy tomato plants.

A new Burkholderia strain (PVFi5A) which exhibits antagonism towards many bacterial and fungal plant pathogens has been partially characterised. This strain was isolated from the rhizosphere of tomato plants and was referred to the Burkholderia cepacia complex on the basis of cultural, morphological and biochemical characteristics, including determination of the 16S ribosomal DNA sequence and fatty acid profile. Strain PVFi5A is a Gram-negative, aerobic bacterium, oxidase- and catalase-positive, motile with a polar tuft of flagella, able to grow on a variety of media without producing diffusible pigments; it is avirulent to onion, able to grow at 41 degrees C and resistant to several antibiotic substances. Its fatty acid profile contains the hydroxy acids 18:1 20H, 14:0 3OH and 16:0 3OH, but not the hydroxy acids 16:0 2OH. The antagonistic activity of strain PVFi5A is due to its production of various, as yet unidentified, antimicrobial compounds, one or more of which may differ from those reported previously for certain 'B. cepacia' strains. The ability of PVFi5A to suppress the growth of important bacterial and fungal phytopathogens makes this strain a potential biocontrol agent.

Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages.

A Burkholderia cepacia population naturally occurring in the rhizosphere of Zea mays was investigated in order to assess the degree of root association and microbial biodiversity at five stages of plant growth. The bacterial strains isolated on semiselective PCAT medium were mostly assigned to the species B. cepacia by an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (16S rDNA) (ARDRA) with the enzyme AluI. Partial 16S rDNA nucleotide sequences of some randomly chosen isolates confirmed the ARDRA results. Throughout the study, B. cepacia was strictly associated with maize roots, ranging from 0.6 to 3.6% of the total cultivable microflora. Biodiversity among 83 B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique with two 10-mer primers. An analysis of RAPD patterns by the analysis of molecular variance method revealed a high level of intraspecific genetic diversity in this B. cepacia population. Moreover, the genetic diversity was related to divergences among maize root samplings, with microbial genetic variability markedly higher in the first stages of plant growth; in other words, the biodiversity of this rhizosphere bacterial population decreased over time.

Molecular characterization of an n-alkane-degrading bacterial community and identification of a new species, Acinetobacter venetianus.

Twenty-five bacterial strains isolated from the Venice lagoon and implicated in the degradation of n-alkanes, n-alkanols, n-alkanals and n-alkanoates were characterized in molecular and physiological terms. The isolates were grouped by amplified ribosomal DNA restriction analysis (ARDRA) into seven clusters, corresponding to seven species, six of which were identified on the basis of 16S rDNA sequencing. Genetic variability among strains was shown by random amplified polymorphic DNA (RAPD). Only strains of the new species Acinetobacter venetianus grew with n-alkanes (C10, C14 and C20) and their respective oxidation products as sole carbon sources. Strains of the other three species identified thrived on n-alkane oxidation products (n-alkanols, n-alkanals, n-alkanoates). The other three species were not able to grow on any of the substrates tested. Analysis of plasmid content showed that only A. venetianus strains harboured plasmids. These plasmids contained sequences homologous to the Pseudomonas oleovorans alkBFGH genes.