The main aim of the project was to investigate signalling and other biochemical interactions, mediated by saponins and other substances, between roots of plants, soilborne fungi (including plant pathogens) and rhizosphere bacteria. The objectives were to contribute to an understanding of microbial population dynamics and plant disease, and to the role of saponin detoxification as a virulence mechanism of fungi.
Gaeumannomyces graminis var. avenae (Gga) can detoxify the fungitoxic saponin avenacin, which is found in oat roots, through the action of the enzyme avencinase, an essential virulence determinant. Avenacin was shown to be released from roots into the rhizosphere at biologically effective levels. A collection of rhizosphere fungi has been established from cereal roots; those from oats show much higher resistance to avenacin. Resistance is necessary but not sufficient for pathogenicity. A quantitative PCR proceedure has been devised to specifically measure Gga biomass in the rhizosphere and in infected roots. Mutagenesis of the oat species Avena strigosa has yielded mutant lines deficient in avenacin for experimental studies of the role of avenacin in determining dynamics of microbial populations.
Populations of bacteria, particularly fluorescent pseudomonads, which are adapted to the oat rhizosphere have been characterised using SDS-PAGE and FAME analysis. 28 clonal groups of these bacteria were predominant, and population dynamics and community structures were studied throughout the growing season of field grown plants. The growth of certain bacterial isolates was affected by avenacin, which is usually thought to affect only eukaryotes. Laboratory microcosm studies were performed with wild type and avenacin-deficient oat plants to study the influence of avenacin on bacterial and fungal population dynamics and disease development. Future work will be aimed at relating the results to the selective effects on microbes noted in field grown cereals.
Circumstantial evidence suggests that the saponin tomatine plays a role in resistance of tomato to some fungi. A tomatine-detoxifying enzyme has been purified from Fusarium oxysporum f.sp. lycopersici (Fol) and characterised. Similar enzymes have been found in other fusaria which are not tomato pathogens. A Fol cDNA clone has been isolated which hybridises with the Gga avenacinase gene and is tomatine-inducible, although it appears not to be related to the tomatinase gene. Other approaches to clone the tomatinase gene using amino acid sequence data and immunodetection are being used. This will permit targeted disruption of the tomatinase genes and hence evaluation of the role of tomatinase in pathogenicity of the fungi.
Five classes of reporter gene fusion mutants of a rhizosphere strain of Pseudomonas fluorescens have been isolated which show transcriptional down-regulation by signal metabolites from the fungus Pythium ultimum. One of the genes is involved in nitrogen metabolism (glutamate synthase) and in two other cases the mutations lie in ribosomal RNA operons. The fungal signalling molecule in one case is believed to be a protein or peptide. Aspergillus and Penicillium species have also been shown to affect gene expression in the mutants.
MAJOR SCIENTIFIC BREAKTHROUGHS:
The saponin avenacin has been shown to accumulate in the oat rhizosphere and to influence the composition of the fungal microflora. The bacterial microflora of oat roots has been characterised revealing characteristic communities including some avenacin-sensitive strains. A specific and sensitive PCR procedure enables the development of Gga in the rhizosphere and roots to be measured, and application of this and other analytical methods has permitted the dynamics of root fungal and bacterial populations to be measured. The tomatinase of Fol has been purified and shown to be much smaller than saponin-detoxifying enzymes previously characterised from Gaeumannomyces and Septoria. It may therefore represent a new family of these enzymes. Five genes of Pseudomonas fluorescens have been found which show reduced expression in the presence of metabolites of Pythium ultimum and other fungi, the first report of a negative effect on gene expression in a rhizosphere bacterium in response to a fungal plant pathogen.
Funding SchemeCSC - Cost-sharing contracts
OX1 3SR Oxford