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

番木瓜(Carica Papaya L.)由欧文氏杆菌感染引起的番木瓜枯萎病正在摧毁世界各地的种植园，并导致植物死亡。生物防治措施是最有希望探索的，因为其他战略，如作物管理、化学处理或使用抗性植物要么无法管理，要么不可用。来自木霉属的三个菌株已被鉴定为对木霉菌有活性。本项目的目的是从这三个木霉菌株中提纯和鉴定具有抗麦芽窄食杆菌生物活性的次生代谢物。要做到这一点，第一步将是使用高效液相色谱法纯化所有三种菌株产生的所有主要代谢物，并测试它们对革兰氏阴性菌(如大肠杆菌和嗜麦芽孢杆菌)的活性。核磁共振波谱实验，然后将对几个代谢产物显示出较强的抗菌活性，以确定他们的特点和阐明他们的结构。一旦确定了特征，将对这三种菌株的基因组进行探索，以确定负责其生产的候选BGC。这将是便利的，因为已经对BGC进行了抗SMASH检测。在鉴定BGC后，将利用CRISPR/Cas9缺失或异源表达，并结合LC/MS分析和核磁共振光谱实验来阐明存在于簇中的每个基因的作用。

Papaya (Carica papaya L.) plantations around the world are being devastated by papaya dieback disease caused by Erwinia mallotivora infections and lead to plant death. Biocontrol measures are the most promising to explore as other strategies such as crop management, chemical treatment or the use of resistant plants are either not manageable or not available. Three strains from the genus Trichoderma have been identified as active against E. mallotivora. This project has for aim to purify and characterise secondary metabolites from those three Trichoderma strains showing bioactivity against E. mallotivora. To do so, the first step will be to purify all major metabolites made by all three strains using HPLC and testing their activity on Gram-negative bacteria, such as Escherichia coli and E. mallotivora. NMR spectroscopy experiments will then be performed on a few metabolites which show strong antimicrobial activity against E. mallotivora to characterise them and elucidate their structure. Once characterised, the genomes of the three strains will be explored to identify candidate BGCs responsible for their production. This will be facilitated as AntiSMASH detection of BGCs has already been performed. After identification of the BGCs, work with CRISPR/Cas9 deletions or heterologous expression coupled with LC/MS analysis and NMR spectroscopy experiments will be performed to elucidate the role of each gene present in the cluster.