Residual impact of the biocontrol inoculant Pseudomonas fluorescens F113 on the resident population of rhizobia nodulating a red clover rotation crop.

A field trial was previously conducted in which sugarbeet seeds were either untreated, inoculated with the biocontrol strain Pseudomonas fluorescens F113Rif, or treated with chemical fungicides. Following harvest of sugarbeet, the field site was sown with uninoculated red clover. The aim of this study was to assess the residual impact of the microbial inoculant (and the fungicide treatment) on the diversity of resident rhizobia nodulating the red clover rotation crop. The percentage of nodules yielding rhizobial isolates after surface disinfection was 67% in the control and 70% in the P. fluorescens F113Rif treatment, but only 23% in the chemical treatment. Isolates were characterized by RAPD analysis. The main RAPD cluster (arbitrarily defined at 70% similarity) was prevalent in all three treatments. In addition, the distribution of RAPD clusters followed a log series model, regardless of the treatment applied, indicating that neither the microbial inoculant nor the fungicide treatment had caused a strong perturbation of the rhizobial population. When the P. fluorescens F113Rif and control treatments were compared using diversity indices, however, it appeared that the genetic diversity of rhizobia was significantly less in the inoculated treatment. The percentage of rhizobia sensitive to 2,4-diacetylphloroglucinol (Phl; the antimicrobial metabolite produced by P. fluorescens F113Rif) fluctuated according to field site heterogeneity, and treatments had no effect on this percentage. Yet, the proportion of Phl-sensitive isolates in the main RAPD cluster was lower in the P. fluorescens F113Rif treatment compared with the control, raising the possibility that the residual impact of the inoculant could have been partly mediated by production of Phl. This impact on the rhizobial population took place without affecting the functioning of the Rhizobium-clover symbiosis.

Reverse transcription-PCR analysis of the regulation of ethylbenzene dioxygenase gene expression in Pseudomonas fluorescens CA-4.

Pseudomonas fluorescens strain CA-4 is a bioreactor isolate previously characterised by the presence of a side chain oxidation pathway for ethylbenzene breakdown. In this report a second pathway involving ethylbenzene ring dioxygenation has been identified in this strain. We examine here second substrate inhibition of the genes encoding the initial enzymes of this pathway, using reverse transcription (RT)-PCR. The genes of the ring-dioxygenation have been cloned and sequenced. They exhibit near identity to the gene clusters encoding the aromatic ring dioxygenase enzymes of two previously described isopropyl degrading strains, Pseudomonas sp. strain JR1 and P. fluorescens IP01. This dioxygenase pathway appears to be the major pathway for ethylbenzene degradation in this strain. The expression of these genes appears to be affected by the presence of second carbon substrates. Using RT-PCR we demonstrate that the negative effect of glutamate present in the growth medium together with ethylbenzene on the rate of ethylbenzene metabolism is mediated at the transcriptional level on the ethylbenzene dioxygenase genes.

Ethylbenzene degradation by Pseudomonas fluorescens strain CA-4.

Pseudomonas fluorescens strain CA-4 is a bioreactor isolate capable of ethylbenzene degradation. Transposon mutagenesis and enzyme assays have been performed which allow us to propose the ethylbenzene degradative pathway in operation in this strain. Ethylbenzene is initially converted to 2-phenylethanol. This is degraded to phenylacetaldehyde and then to phenylacetic acid. The major inducer of the pathway is ethylbenzene itself. The pathway is regulated by the presence of non-aromatic carbon sources. Oxidation of ethylbenzene is repressed by glutamate, but not by citrate or glucose. A clone from a chromosomal library has been found to complement a mutant deficient in the ability to convert ethylbenzene to 2-phenylethanol.