Modulation of the synthesis of the main preformed antifungal compound as abasis for the prevention of postharvest disease of C. gloeosporioides in avocado fruits

The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned , The most important pathological factor limiting fruit life after harvest in subtropical fruits are quiescent infections of anthracnose caused by Colletotrichum gloeosporioides. Prusky and Keen elucidated the mechanism of resistance in avocado fruits to quiescent infections of C. gloeosporioides and determined that the major biocide involved is the preformed compound,1-acetoxy-2-hydroxy-4-oxo-heneicosa-13, 15 diene. Two possibilities exist for maintaining fungitoxic levels of antifungal compounds in the tissue of ripening fruits: (i). Prevention of catabolism (ii). Induction of synthesis. Previous work has demonstrated that increased fruit susceptibility after fruit harvest occurs through diene catabolism mediated by oxidation of the antifungal compound by the enzyme lipoxygenase. Levels of a non-specific inhibitor, epicatechin, in turn, regulate activity of lipoxygenase, present in the peel of unripe but not ripe fruit. In this proposal, we examined the possibility of exploiting induced synthesis of the antifungal compound for the study of the synthetic pathway. The general objective of the present research was to study the mechanism of biosynthesis of natural antifungal compounds in order to regulate the process of resistance to postharvest diseases in ripening avocado fruits. The specific objectives of the research were: 1. To localize synthesis of the antifungal diene and modulate the process by biotic or a biotic elicitors. 2. To determine the relation between synthesis of the diene and accumulation in the peel and fruit resistance to decay 3. To characterize the biosynthetic pathway and the diene and the genes involved. The analysis of the antifungal compounds in avocado resulted in the detection of a new antifungal compound (E, Z, Z)-1-acetoxy-2-hydroxy-4-oxo- heneicosa-5, 12,15-triene. This new compound was shown to inhibit spore germination of C. gloeosporioides similarly as the antifungal diene. We had localized one of the biosynthetic places of these antifungal compounds in specialized idioblast cells (oil cells) in the mesocarp that can be easily enhanced by elicitors as ethylene. Results have also suggested that the antifungal compounds can be "exported" from the mesocarp to the pericarp where its main activity takes place. The search for the biosynthesis of antifungal compounds and the genes involved took two directions i. direct search for specific genes involved in the synthesis of the diene and ii. Indirect selection of genes using the differential display library. We have cloned D9 and D12 desaturase, a protein kinase and a elongase that their transcriptional activation is significantly enhanced during the enhanced synthesis of the antifungal diene. Although we are far away from a complete elucidation of the synthesis of the antifungal compound we have stepped forward determining some of the key steps that might be involved in its synthesis.