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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Chemical interactions and signalling between phytopathogenc fungi, rhizobacteria and plant roots with particular reference to saponins: implications for disease

Background and objectives

The fungus Gaeumannomyces graminis var. avenae (Gga) can detoxify the fungitoxic saponin avenacin, found in oat roots, through the action of the enzyme avenacinase, an essential virulence determinant. Avenacin is released from roots into the rhizosphere at biologically effective levels. Rhizosphere fungi were collected from a range of cereal roots and it was found that oats show the highest resistance to avenacin. The main aim of this study was to investigate signalling and other biochemical interactions mediated by saponins and other substances, between plant roots, soilborne fungi (including plant pathogens) and rhizosphere bacteria. This study contributed to the understanding of microbial population dynamics, plant disease and to the role of saponin detoxification as a virulence mechanism of fungi.


Approach and methodology

We devised a quantitative PCR procedure to specifically measure Gga biomass in the rhizosphere and infected roots. Mutagenesis of the oat species, Avena strigosa, yielded mutant lines deficient in avenacin which we used to study the role of avenacin in determining microbial population dynamics.

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 on disease development.

image Oats.



Main findings and outcome

SDS-PAGE and FAME analysis of bacteria adapted to the oat rhizosphere, particularly fluorescent pseudomonads, revealed 28 predominant clonal groups. The 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.

Circumstantial evidence suggests that the saponin, tomatine plays a role in resistance of tomato plants to some fungi. A tomatine-detoxifying enzyme was purified from Fusarium oxysporum f.sp. lycopersici (Fol) and was characterised. Similar enzymes were found in other fusaria which are not tomato pathogens. A Fol cDNA clone, that hybridises with the Gga avenacinase gene and is tomatine-inducible, was isolated. It appears not to be related to the tomatinase gene. We are currently using amino acid sequence data and immunodetection to clone the tomatinase gene. 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, that show transcriptional down-regulation by signal metabolites from the fungus Pythium ultimum, were isolated. 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 were also shown to affect gene expression in mutants.

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Conclusions

The saponin avenacin was shown to accumulate in the oat rhizosphere and to influence the composition of the fungal microflora. Characterisation of the bacterial microflora of oat roots revealed characteristic communities including some avenacin-sensitive strains. A specific and sensitive PCR procedure enabled the development of Gga in the rhizosphere and roots to be measured. Application of this and other analytical methods permitted the measurement of the dynamics of root fungal and bacterial populations. The tomatinase of Fol was purified and shown to be much smaller than saponin-detoxifying enzymes from Gaeumannomyces and Septoria. Fol may, therefore, represent a new family of these enzymes. Five Pseudomonas fluorescens genes were found that show reduced expression in the presence of Pythium ultimum and other fungi. This is the first report of a negative effect on gene expression in a rhizosphere bacterium in response to a fungal plant pathogen.


Major publications

Osbourn A., Bowyer P., Lunness P., Clarke B., Daniels M., “Fungal pathogens of oat roots and tomato leaves employ closely related enzymes to detoxify different host plant saponins”.
Molecular Plant-Microbe Interactions,
8, 1995, pp. 971-978.

Osbourn A., “Saponins and plant defence – a soap story”.
Trends in Plant Science,
1, 1996, pp. 4-9.

Lairini K., Pérez-Espinoza A., Pineda M., Ruiz-Rubio M., “Purification and characterization of tomatinase from Fusarium oxysporum f. sp. lycopersici".
Applied and Environmental Microbiology,
62, 1996, pp. 1604-1609.

Lairini K., Pérez-Espinoza A., Ruiz-Rubio M., “Tomatinase induction in formae speciales of Fusarium oxysporum not pathogenic to tomato”.
Physiological and Molecular Plant Pathology, 50, 1997, pp. 37-52.

Fedi S., Tola E., Moenne-Loccoz Y., Dowling D., Smith L., O’Gara F., “Evidence for signalling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113 resulting in altered ecological fitness”.
Applied and Environmental Microbiology,
63, 1997, pp. 4261-4266.
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imageResearch project
 

Contract number
BIO2-CT94-3001

Period
December 1994 – June 1997

Coordinator
M.J. Daniels
The Sainsbury Laboratory
Norwich (UK)

 
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Partners


M.J. Bailey
Natural Environment Research Council (NERC)
Oxford (UK)

F. O’Gara
University College Cork (IE)

M. Ruiz Rubio
Universidad de Cordoba (ES)

 
 
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