Study Of The Active Ingredients From Diaporthe Sp. HT-79 Against Xanthomonas Citri Subsp. Citri

Citrus is the world’s one of the most valuable fruits and is also the economic pillar industry of the Jiangxi Ganan region in China,but some pests and diseases seriously hinder the development of the citrus industry,such as citrus canker.Citrus ulcer disease is a bacterial disease,the onset of serious citrus fruit trees will cause leaf drops,flower drops,and even fruit drops.The chemical agents currently used to control this disease are mainly copper formulations,which have good control efficacy but also raise a series of health concerns.The secondary metabolites of plant endophytes are an important source for the development of natural control agents.In this paper,the active fungus of Diaporthe sp.HT-79 was used as a research object,and the secondary metabolites of this strain were isolated and purified by column chromatography,GC-MS technique,and MIC test,and the active monomer compounds were screened and the active molecules were selected for a series of antimicrobial mechanism studies.The main results are as follows.1、The MIC of each organic phase extracted from the fermentation crude extracts of the genus Diaporthe HT-79 was determined,and the results showed that the petroleum ether phase had the best antibacterial effect with MIC value of 0.625 g/L,followed by the ethyl acetate phase with MIC value of 1.25 g/L,so the subsequent study of the active molecules in the petroleum ether phase was carried out first.The results of GC-MS experiments showed that linoleic acid played the main antibacterial role in the petroleum ether phase with MIC value of 0.078125 g/L,followed by oleic acid with MIC value of 0.15625 g/L.The subsequent study focused on a series of antibacterial mechanisms of linoleic acid.2、The results of growth curve experiments showed that linoleic acid had an inhibitory effect on the growth of the pathogens,and the greater the concentration of application,the clearer the inhibitory effect;when measuring the changes of reducing sugar in the bacterial solution,it was found that linoleic acid affected the ability of the pathogens to take up carbon sources;In the extracellular protein leakage experiment,it was found that the measured extracellular protein content was significantly higher than that of the control group as the concentration of linoleic acid was increased and the duration of action was prolonged,which led to the speculation that linoleic acid disrupts the cell membrane of the pathogen and causes the protein contents of the bacterium to flow out;Scanning electron microscopy experiments also confirmed the speculation that the morphology of linoleic acid-treated bacteria changed significantly,with depression,deformation and breakage on the surface of the bacteria,while the control group remained relatively intact;meanwhile,ROS experiments showed that linoleic acid stimulated the production of excessive ROS in the bacteria,causing oxidative damage to the cell membrane and important structures of the bacteria,which led to irreversible damage and death of the bacteria.Finally,in the experiment of linoleic acid spraying on susceptible leaves,the susceptible leaves treated with linoleic acid showed cavities and condensation around the spots,and the spots no longer expanded,while the control group developed normally,with typical characteristics of citrus ulcer disease.3、Six compounds,including steroids,alkaloids,and cyclic peptides,were also isolated from the petroleum ether phase,of which compounds 4 and 6 were new compounds.The MIC experiments were used to screen the activity of these 6 monomeric compounds,and the results showed that compounds 4 had a good antibacterial effects with MIC values of 0.015625 g/L.And11 compounds were isolated and identified in the ethyl acetate phase,containing steroids,nitrogencontaining heterocycles,phenolic acids,pyranoids and some characteristic compounds of this genus of fungi,of which compounds 11 and 12 were new.the MIC experiments showed that the best antibacterial effect was obtained for compound 11 with MIC value of 0.015625 g/L,followed by compounds 14 with MIC values of 0.0625 g/L,and compound 12 with MIC value of 0.125 g/L,compound 8、12 both with MIC value of 0.25 g/L.In this paper,we searched for natural active molecules against citrus bacterial canker from secondary metabolites of the genus Diaporthe to provide a theoretical basis for developing green pesticides against citrus bacterial canker.