On the nature, symptoms and genetic diversity of Monilinia isolates and their viruses in Western Australia

Monilinia species occur in stone fruit (Prunus species) production areas in many parts of the world, where they cause the serious disease brown rot. Incursion into Western Australia (W.A.) by Monilinia fructicola was declared in 1997, and along with Monilinia laxa has subsequent spread throughout the state has cost the stone fruit industry millions of dollars in lost production and the cost of fungicidal sprays. Until this study, no studies had been made in W.A. of Monilinia species identity, genetic diversity, distribution, virulence, fungicide tolerance or presence of mycoviruses. This project aimed to address these knowledge gaps. A collection of Monilinia isolates was made across the major stone fruit production regions in W.A. , and the fungal species were identified. Both species were present, but they were not evenly distributed between and within production regions, indicating the main agent of spread is people. High levels of genetic diversity have been reported for WA population, and both pathogens are not recently introduced in this state. ISSR (Inter Simple Sequence Repeat) markers were used to identify intra-specific diversity in populations, and compare modern populations with the strains that first invaded W.A. This study revealed that the original incursive strain of M. fructicola has probably become extinct, replaced by new genotypes. Conversely the incursive strain of M. laxa remains widely distributed within the state. New strains appear to have subsequently invaded W.A. and pose ongoing serious threats to the industry. Although commercial stone fruit growers routinely spray their crops with fungicides to control brown rot, small scale and ‘organic’ growers may not. A study was done to assay the relative tolerance to three fungicides of Monilinia isolates collected from sprayed and unsprayed trees. No resistant isolates have been found in WA. Sprayed isolates exhibited a significantly greater range of responses to fungicides than unsprayed isolates, indicating that there is strong positive selection for tolerance in the orchards that routinely apply fungicides. We found there were significant differences between M. fructicola and M. laxa isolates in average tolerance to propiconizole-based fungicides. Assays for virulence revealed the existence of highly virulent, moderately virulent and avirulent strains of both species, and these occurred in both sprayed and unsprayed orchards. On nutrient agar plates, M. laxa isolates presented as four distinct morphologies, while M. fructicola colonies were very similar in appearance across all isolates tested. A survey of 28 isolates of both species revealed three mycoviruses co-infecting three M. laxa isolates and one M. fructicola isolate. The complete or partial sequences of one isolate each of all three viruses was obtained. One M. fructicola isolate was co-infected with all three viruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). To test the influence of these viruses on fungal pathogenicity, several methods were applied to cure isolate M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. Morphology and virulence assays were carried out to determine how mycoviruses influence growth of the fungal host. Surprisingly, growth of fungal mycelia was promoted by the presence of three viruses when they were cultured on nutrient agar medium in vitro, but did not influence fungal virulence after inoculation to fruits of sweet cherry.