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ANTI-FUNGAL METABOLITES PRODUCED BY PSEUDOMONAS FLUORESCENS
FD6 AND ACTIVITY AGAINST THE PLANT PATHOGENIC BOTRYTIS CINEREA,
PYTHIUM AND RHIZOCTONIA SO
Q.X. Zhang AB , L. Q. Zhang C and P.R. Harvey A
A
CSIRO Ecosystem Sciences & Sustainable Agriculture Research Flagship (SAF), Waite Campus, PMB2, Glen Osmond, 5064, SA
B
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, Jiangsu, P.R. China.
Email: Qingxia.Zhang@csiro.au
C
College of Agriculture and Biotechnology, China Agricultural University, Beijing 100094, P.R. China
ABSTRACT. . The rhizosphere bacteria Pseudomonas fluorescens FD6 showed significant in vitro growth inhibition of
plant pathogenic Pythium, Rhizoctonia solani and Botrytis cinerea. Potential mechanisms of fungal inhibition by were
investigated by PCR screening for antibiotic biosynthesis genes, thin layer chromatography (TLC) of the antibiotics and antifungal
activity via TLC-bioautography. Ps. fluorescens FD6 produced the antibiotics pyrrolnitrin (prn), 2, 4-
diacetylphloroglucinol (phl), pyoluterorin (plt), but not phenazine-1-carboxylic acid (pca). Only prn directly inhibited spore
germination and mycelial growth of B. cinerea. AFLP analyses of FD6 genomic DNA has identified 4 strain-specific
fragments with potential to be developed as qPCR markers to quantify environmental competence of this disease suppressive
inoculants.
INTRODUCTION
Plant-beneficial Pseudomonas spp. are a major group of
rhizobacteria widely dispersed in agricultural soils.
Pseudomonas spp. have been reported to promote plant
growth directly, via the production of phytohormones and
indirectly, via suppressing plant pathogenic fungi.
Pseudomonas strains can synthesize the antifungal
metabolites pyrrolnitrin (prn), 2, 4-diacetylphloroglucinol
(phl), pyoluterorin (plt) and phenazine (PCA). These
antibiotics inhibit fungal growth and are important
mechanisms of disease suppression. Ps. fluorescens FD6
was isolated from a canola rhizosphere in Fujian Province
(China) and showed significant in vitro growth inhibition of
the soil-borne pathogens Rhizoctonia solani and Pythium
aphanidermatum and the foliar pathogen B. cinerea.
Research was undertaken to characterise the production of
antifungal metabolites by FD6 and their relationship to
disease suppressive efficacy. AFLP is being undertaken to
differentiate FD6 from related Pseudomonas strains and
quantify environmental competence of the strain.
MATERIALS AND METHODS
DNA extractions and PCR screening of Ps. fluorescens FD6
for the presence of antibiotic biosynthesis genes prn, phl, plt
and PCA was as described previously (1). Thin layer
chromatography (TLC) was used for qualitative analyses of
antibiotic production and TLC-bioautography was used to
test the inhibitory activities of antibiotics against B. cinerea
(2). AFLP analyses of FD6 and related Pseudomonas strains
were as described previously (3).
RESULTS AND DISCUSSION
PCR screening of Ps. fluorescens FD6 with primers for
antibiotic biosynthesis genes indicted the presence of the
prn, phl and plt pathways. The target gene for PCA
biosynthesis was not detected. TLC of commercially
available phl, prn and plt standards in comparison with FD6
culture extracts confirmed that this antagonistic bacteria can
produce all 3 of these antifungal metabolites. TLC bioautography
results using purified and FD6-derived prn
indicated that the both compounds were inhibitory to
germination of B. cinerea conidia and mycelia growth. In
contrast, phl and plt standards and FD6 extracts did not
inhibit growth of B. cinerea. Therefore, the antibiotic prn
significantly inhibits fungal growth and may be an important
mechanism by which Ps. fluorescens FD6 suppresses fungal
root diseases.
AFLP analysis resolved unique banding profiles for FD6
in comparison with related Pseudomonas strains. Four FD6-
specific fragments were purified, cloned and sequenced.
Comparison with GenBank indicated high homologies to P.
fluorescens Pf5 DNA, specifically genes for the RebB
protein, a DNA-binding response regulator, a putative
dihydroortase and a conserved hypothetical protein.
Sequences of these 4 FD6-specific fragments are being used
to design qPCR primers for quantitative detection and
monitoring of this disease suppressive bacterium following
plant and soil inoculation.
ACKNOWLEDGEMENTS
This study was supported through the NSFC (NSF
31000875) and CSC programs of China and CSIRO SAF.
REFERENCES
1. Raaijmakers J.M, Weller D.M. et al. (1997). Applied and
Environmental Microbiology 63: 881-887.
2. Costa R., van Aarle I.M., et al. (2009). Environmental
Microbiology 11: 159-175.
3. Gomez, D., Evans, K.J., Harvey, P.R. et al (2006).
Mycological Research, 110: 423-430.
7th Australasian Soilborne Diseases Symposium 43