Characterization Of The Vinclozolin-resistant Mutants Of Coniothyrium Minitans And Their Potential In Biocontrol And Ecological Studies

Sclerotinia sclerotiorum (Lib.) de Bary is an important parthogen, responsible for leaf blight, stem rot and pod rot of oil seed rape(Brassica napus L.), resulting in great losses to this crop, especially in the canola-growing areas along the Yangtze River. In recent years, biocontrol of sclerotinia diseases using the mycoparasite Coniothyrium minitans has attracted attention of many researchers. We hypothesized that maintaining high level of C. minitans population and integration of fungicides with C. minitans may improve the control efficacy of C. minitans. A study was initiated to mutate C. minitans to be resistant to the fungicide vinclozolin and to evaluate the possibility of some mutants as biocontrol agents and as a tool for monitoring the population dynamics of C. minitans.Four mutants resistant to vinclozolin were isolated by incubation of the wild type strain Chy-l(conidia or mycelia) on potato dextrose agar(PDA) amended with the fungicide. The trait of the fungicide resistance was proved to be genetically stable as the 8th generation progenies of these isolates were still resistant to vinclozolin. The effective concentration(EC50) of Chy-1 was 1.1 ug a.i./mL for mycelial growth and 140.0 ug a.i./mL for conidial germination, whereas EC50 of the stable mutants was 2219.1-2683.9 ug a.i./mL for mycelial growth and 609.3-931.3 ug a.i./mL for conidial germination.The growth rate and germination rate mutants were reduced compared to the wild type strain. However, there was no significant difference for these strains in infection of sclerotia of S. sclerotiorum. Some changes in production chitinase, amylase and proteinase were observed in these mutants.Field experiments in 2003 and 2004 showed that C. minitans + low dosage of fungicides(carbendezium and vinclozolin) could significantly suppress sclerotinia stem rot. In the 2003 trial, sclerotia formed in diseased canola plants in the plots treated with C. minitans and C. minitans + carbendezium were parasitized by C. minitans by 46.0% and 45.3%, respectively.A selective medium containing penicillin at 500 ug a.i./mL(a bacterial inhibitor), streptomycin at 500 ug a.i./mL(another bacterial inhibitor) and vinclozolin also at 500 ug a.i./mL(a fungal inhibitor) was formulated based on the ingredients of potato dextroseagar. Using this medium, the survival of C. minitans in soil and on flower petals of canola was tracked. The results showed that the population density of C. minitans was decreased with time. Uv-irradiation from the sun and water wash from raining were the factors responsible for the decrease of vital C minitans.