Project summary

Discovery of antimicrobial natural products made by Trichoderma spp. and study of their biosynthesis

Erwinia mallotivora, as many members of the genus, is a plant pathogen which causes papaya dieback disease, the most important cause of yield loss in Malaysia and other East Asian countries. The disease can be spread via both biotic and abiotic factors such as insects of rainfall. Symptoms include water-soaked lesions on stems and leaves, spots on leaves, opening the plant for further infections and eventually death of the affected plant.

Biocontrol is the most promising strategy to explore as others such as crop management, chemical treatment or the use of resistant plants are either not manageable or not available. My project focuses on three strains of Trichoderma koningiopsis which have been isolated and identified as being active against E. mallotivora. My aim is to characterise specialised metabolites from those three Trichoderma strains showing bioactivity against E. mallotivora.

So far, results show that crude extracts of all three strains have antibacterial activity with one showing additional antifungal activity. Citric acid was identified as one of the antibacterial compounds active against Gram negative bacteria and E. mallotivora specifically. Other antimicrobial compounds have been isolated using different cultivation strategies (carbon sources, solid/liquid cultivation methods, epigenetic manipulation) and their structures are being elucidated using NMR spectroscopy.

In addition, the genomes of the three Trichoderma strains have been sequenced and PKS and NRPS biosynthetic gene clusters (BGC) have been identified through AntiSMASH. Clusters of interest have been selected for their unusual structures and will be targeted for knock-outs. A transformation method for the environmental strains of T. koningiopsis is being developed and CRISPR/Cas9 knockouts will be created to investigate the BGCs producing the antimicrobial compounds.

Key organisms involved in papaya dieback disease and its biocontrol. Symptoms of papaya dieback disease induced by Erwinia mallotivora infections are shown on A, B and C. Pictures were provided by Noriha Binti Mat Amin from the Malaysian Agricultural Research and Development Institute (MARDI). (A) Bent water-soaked stems leading to the death of the plant, (B) Necrotic lesion on the leaf, (C) Water-soaked lesion on the infected stem. The red arrows indicate the infected regions showing symptoms. (D) Transmission electron microscope image of the strain of E. mallotivora isolated from dieback-infected papaya tree. The three strains of T. koningiopsis RA3a (E), RA5 (F) and RA6 (G) showed antibacterial activity against E. mallotivora.