interactions and signalling between phytopathogenc fungi, rhizobacteria
and plant roots with particular reference to saponins: implications for
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
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.
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.
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.
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
Physiological and Molecular Plant Pathology, 50, 1997,
Fedi S., Tola E., Moenne-Loccoz Y., Dowling D., Smith L., OGara
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.
December 1994 June 1997
The Sainsbury Laboratory
Natural Environment Research Council (NERC)
University College Cork (IE)
M. Ruiz Rubio
Universidad de Cordoba (ES)