In situ proteome study of Pichia anomala strain K, an antagonist of the apple pathogen Botrytis cinerea

Postharvest fungal pathogens, mainly Botrytis cinerea, Penicillum expansum and Gloeosporium spp., annually cause 5-20% losses of fruit. Control measures against these diseases include chemical fungicide applications, but the development of resistant fungal explains the growing interest for biological control. Pichia anomala strain K was previously identified as an efficient antagonist of pathogens on apples. Indeed, the percentage of protection of P.anomala against B.cinerea reached from 90 to 100% on apple wounds according to the experimental conditions. Microbiological, biochemical and molecular approaches demonstrated the implication of exo-β-1,3-glucanases in the mechanism of action of P.anomala. However, study of these mechanisms could be improved by observations under natural infection conditions in order to take into account the tripartite interactions host/antagonist/pathogen. The proteomic tool allowed an overview of process implicated in the antagonism against B.cinerea in such conditions.
One 50mm wound per apple were covered by a membrane and inoculated by a P.anomala suspension then by B.cinerea or not. Samples were collected during the exponential and stationary phase to identify the early and later responses to the presence of B.cinerea. After extraction, proteins were separated on 2DE gels. Spots influenced by the presence of B.cinerea in exponential and stationary phases were identified by MALDI-ToF.
One hundred five and sixty spots of proteins were influenced by the presence of B.cinerea in exponential and stationary phase respectively. In exponential phase, influenced proteins were implicated in the different steps of the proteins biosynthesis: amino acid synthesis, translation or mRNA maturation and in energy synthesis. On the other hand, in stationary phase, influenced proteins were mainly implicated in energy metabolic pathway: glycolysis, alcoholic fermentation or gluconeogenesis. The presence of B.cinerea seemed to induce a slowdown in the metabolism of P.anomala without influenced its growth. Further studies have to be realized to understand the complexity of these modes of action.