Unraveling the Tritrophic interactions between fruit host-pathogen-antagonist in the post-harvest environment

Abstract

Fruits and vegetables post-harvest losses due to the attack of several fungal pathogens can reach levels of up to 50% depending on the commodity. Besides the economic losses, some of these fungi may represent a potential health risk because of mycotoxin production. To control post-harvest diseases of fruits, synthetic fungicides, when permitted, are the primary means used. However, the emergence of fungicide-resistant strains and the willingness to use safer and eco- friendly treatments, have generated interest in the development of alternative non-chemical methods to reduce post-harvest losses. Biological control using microbial antagonists has emerged as one of the most promising alternatives to fungicides, either alone or as part of an integrated pest management. The antagonistic activity of biocontrol agents has been widely demonstrated, however the mechanisms of action of most biocontrol agents of post-harvest diseases has not yet been fully understood which is important to know for a successful implementation of post-harvest biocontrol technology. Therefore a part of my PhD focused on this aspect; we have successfully cloned, expressed in P. pastoris and confirmed the involve- ment of a novel endochitinase gene MfChi and the protease gene ALP5 in the biocontrol activity of the antagonistic yeasts Metschnikowia fructicola AP47 and Aureobasidium pullulans PL5 successively. Our work proved that the alkaline serine protease ALP5 and the endochitinase MfChi could be developed as post-harvest treatments with antimicrobial activity for fruits undergoing a short shelf life to control Botrytis cinerea, Monilinia fructicola and Monilinia laxa on pome and stone fruits. Moreover, besides developing biocontrol agents as alternative approaches to chemicals to control the pathogens, we should also be familiar with the decaying agents, such as their nature and the molecular basis of the infection. Due to recent technological advances, sequencing has revolutionized biological research and has become the forefront of bio- logical experimentation in the last decade. Therefore, we have sequenced for the first time Penicillium griseofulvum PG3 which is associated with blue mould decay, the most important post-harvest disease of pome fruit worldwide. The fungal pathogen has the ability to simultaneously produce both detrimental and beneficial Secondary Metabolites (SM). Our work aimed to analyse some important SM clusters present in the studied strain in order to gain insight into SM synthesis in P. griseofulvum. Genome-wide analysis of PG3 genes revealed a complete putative gene cluster for patulin biosynthesis, and partial griseofulvin and roquefortine C clusters. Besides the bioinformatics analysis of these gene clusters, we quantified the SM production in vitro and during disease development on apple. Furthermore, the SM detected in infected apple were examined by studying the expression kinetics of their key genes under controlled conditions. In addition, we found additional SM clusters in PG3, including those potentially responsible for the synthesis of penicillin and cyclopiazonic acid. These findings provide relevant information to understand the molecular basis of SM biosynthesis in P. griseofulvum and this resource will allow further research directed to the overexpression or blocking of specific SM synthesis, to assess its potential in terms of biotechnological applications for beneficial SM, such as griseofulvin.