Biological control of Botrytis cinerea in lettuce & strawberry crops

Two preliminary biological control screens were developed, using lettuce & strawberry as model systems, to test potential biocontrol micro-organisms for their ability to control Botrytis cinerea, the causal agent of grey mould. The first screen evaluated a range of saprophytic yeasts, bacteria & fungi for their ability to inhibit Botrytis lesions on whole lettuce plants. In a controlled environment (18 -22°C, 85-90% relative humidity & 12 h photoperiod), lettuce leaves were spray inoculated with antagonists (10⁷ fungal spores mL⁻¹ or 10⁸ bacterial cells mL⁻¹), allowed to dry & challenged by spray inoculation with the pathogen (10⁶ conidia mL⁻¹). After 7 days Pseudomonas fluorescens LU1001 & LU1003, P. synxantha LU1004 & P. veronii LU1002 had significantly reduced lesion areas by an average of 79% on whole lettuce plants. An Ulocladium sp. LU865 & Epicoccum purpurascens LU148 reduced lesion areas by 94% & 78%, respectively. The second screen evaluated selected saprophytic bacteria & filamentous fungi, with previously identified antagonistic properties (against a range of plant pathogens), for the ability to inhibit B. cinerea on detached strawberry tissues & whole strawberry plants. The most successful isolates, E. purpurascens LU148 & Trichoderma atroviride LU132, inhibited lesion development by up to 32% & suppressed sporulation on detached strawberry leaves by up to 87%. They were further evaluated on detached strawberry flowers & whole strawberry plants in a controlled environment (18°C & 85-95% relative humidity). The most consistently effective antagonist was T. atroviride LU132, which inhibited B. cinerea in all experiments, significantly (P≤0.05) suppressing B. cinerea sporulation on detached strawberry flowers & leaf discs removed from inoculated whole plants by 100% & 81%, respectively. This was similar to the level of disease suppression given by the commercial biocontrol product, Prestop & the fungicide, fenhexamid. The mechanism of action of T. atroviride LU132 was investigated. Competition for glucose & sucrose occurred on detached strawberry leaf pieces & on polytetrafluoroethylene (PTFE) membranes, significantly inhibiting B. cinerea germ tube length at low sugar concentrations. The isolate also produced non-volatile inhibitory substances in minimal medium that inhibited B. cinerea germ tube elongation & in dual culture, T. atroviride LU132 was found to directly parasitize B. cinerea hyphae leading to cell collapse. The biological activity of T. atroviride LU132 against the grey mould pathogen was investigated on three cultivars of strawberry under field conditions. The study also examined the effect of commonly used botryticides on germination & growth of T. atroviride LU132 to allow selection of a fungicide for mixed application. The fungicide fenhexamid significantly increased conidial germination frequency by up to 34% & was later used in mixed tank applications. The field trials demonstrated that treatment with T. atroviride LU132, fenhexamid, & the tank-mix of both could all equally suppress B. cinerea sporulation, significantly, on strawberry leaves & stamens by 42-100%. These treatments increased yield of strawberry fruit by up to 36% but they did not reduce disease incidence in the fruit in a 7 day ambient storage trial. This research has identified T. atroviride LU132 as a successful biological control agent (BCA) of B. cinerea in strawberry. The more obvious mechanisms of action such as antibiosis, competition for nutrients & mycoparasitism have been investigated & the use of the BCA within an integrated management strategy, with the fungicide fenhexamid, has been successfully implemented. Future work will focus on developing a suitable formulation for the BCA in order to increase its efficacy under field conditions.