Basic Research And Application Of Key Prevention Technologies For Fusarium Wilt Of Bitter Gourd

Fusarium wilt caused by F.oxysporum is the most harmful plant disease in the world,and loss caused by F.oxysporum is increasing year by year.F.oxysporum is a facultative saprophytic fungus that can propagate not only in the plant but also in the plant residues.After the host has been infected,even if the plant has no obvious external symptoms,F.oxysporum grew and reproduced a large number of conidia,while it can still propagate in plant debris and produce chlamydospore after the host plant has died.The plant detritus facilitates the colonization and durability of F.oxysporum in the soil,once it successfully colonized in the soil,it is almost impossible to remove it.The purpose of this research is to screen an effective control measure to prevent the occurrence of Fusarium wilt.In this study,the disease nursery of bitter gourd wilt was prepared by artificial inoculation of F.oxysporum,and the control efficacy of resistant varieties,soil fumigation,and biological control on bitter gourd wilt were evaluated.The effects of F.oxysporum invasion on soil micro-ecosystem,soil fumigation and microbial agents on F.oxysporum population in the soil,the resistance mechanism of resistant bitter gourd to Fusarium wilt and the mechanism of microbial agent improving host plant resistance to Fusarium wilt were investigated using metagenomic technology,high-throughput sequencing,and transcriptome sequencing.1Isolation and identification of pathogens of bitter gourd wilt in ShandongIn this study,7 pathogen strains of bitter gourd wilt were isolated from the areas that high incidence of bitter gourd wilt,such as Shouguang and Feicheng in Shandong Province.Characteristics of the colony and conidia morphology,18 S r DNA sequence,and symptoms of bitter gourd wilt showed that they were pathogenic strains of bitter gourd wilt.Through genetic identification of 26 F.oxysporum f.sp.momordicae strains from 6 regions in China,the results showed that the Shandong F.oxysporum f.sp.momodicae was closest to Guangxi F.oxysporum f.sp.momodicae.2The artificial disease nursery of bitter gourd Fusarium wilt and distribution characteristics of FusariumWe successfully established the artificial disease nursery of bitter gourd Fusarium wilt by artificially adding F.oxysporum in 2015.The incidence of Fusarium wilt in the artificial disease nursery reached more than 60% and the disease index reached more than 50 after 45 days of colonization of bitter gourd in 2017-2019,In the artificial disease nursery,the relative abundance of F.oxysporum increased significantly in the soil,while the relative abundance of other species of Fusarium such as F.solani and F.fujikuroi did not change.At the same time,F.oxysporum mainly concentrated in plant residues and 0?10 cm depth of soil cultivation layer3 The effect of F.oxysporum invasion on soil micro-ecosystemIn this study,the characteristics of the microbial community in bitter gourd Fusarium wilt were analyzed by high-throughput sequencing and metagenomic technology.The results showed that F.oxysporum invasion caused dramatic changes in soil microbial community structure and soil enzyme activity.F.oxysporum significantly decreased the relative abundance of dominant microorganisms,such as Bradyrhizobium,Streptomyces,and Luteitalea,while increased the relative abundance of non-dominant microorganisms in Proteobacteria,Actinobacteria,Gemmatimonadetes,Chloroflexi and Ascomycetes,and they involved in the inhibition of growth of F.oxysporum.At the same time,F.oxysporum invasion significantly decreased the activities of FDA hydrolysis and polyphenol oxidase in soil,and significantly increased the content of soil organic matter.It can be thought that F.oxysporum affected the structure and function of the soil microbial community by regulating the utilization of plant residues by other microorganisms.4Control efficacy of soil fumigation on the Fusarium wiltSoil fumigation can significantly reduce the disaster of Fusarium wilt,and the control effect on bitter gourd wilt is more than 70%(the second year after soil fumigation).The abundance of F.oxysporum in 5?10 cm topsoil was significantly reduced by soil fumigation with metham-sodium,but had little influence on the abundance of F.oxysporum in soil below20 cm.At the same time,the population abundance of F.oxysporum increased even without host plants after soil fumigation.5Isolation and screening of biocontrol microorganismsMicroorganisms such as Streptomyces and Bacillus play an important role in the interaction between plants and microorganisms,and they can improve resistance to plant disease and promote plant growth.In Fusarium pathogenic soil,the relative abundance of beneficial microorganisms such as Streptomyces,Bacillus,Trichoderma,Aspergillus,and Penicillium increased,which may be involved in the inhibition of the growth of F.oxysporum.For this reason,biocontrol microbes were isolated from the Fusarium pathogenic soil.Highly effective biocontrol agents could be obtained.Several strains of Bacillus and Penicillium which have inhibition to F.oxysporum were isolated from pathogenic soil and plant rhizosphere.The results showed that Penicillium strain Q2 could reduce the disaster of various plant diseases such as Fusarium wilt in the greenhouse or field.We identified it as Talaromyces purpureogenus by morphological,biological,and DNA molecular information.The asexual type of strain Q2 is Penicillium purpureogenum.6The biocontrol potential of strain Q2T.purpureogenus strain Q2 is a beneficial microorganism isolated from the rhizosphere soil of healthy cucumber plants in the high incidence area of Fusarium wilt.It has a good control effect on a variety of soil-borne diseases,such as Fusarium wilt of bitter gourd,potato stem canker,black shank of tobacco,and black root rot of tobacco in the greenhouse or field.Its biocontrol efficacy on bitter gourd wilt was more than 50% in the greenhouse and field.The results of transcriptome sequencing showed that strain Q2 could inhibit the growth and reproduction of F.oxysporum by secreting cell wall degrading enzymes and secondary metabolites.The target site where strain Q2 inhibits F.oxysporum may be cell wall and cell membrane.Simultaneously,strain Q2 could enhance plant resistance to Fusarium wilt by inducing the expression of plant lignin and its biosynthesis-related genes.The results of highthroughput sequencing and metagenomic sequencing showed that strain Q2 could colonize stably in soil and inhibit the population recovery of F.oxysporum by directly interacting and indirectly promote the growth of beneficial microorganisms such as Actinobacteria and Gemmatimonadetes.7Evaluation of resistance of Momordica charantia resources to F.oxysporum strain SG-15In this study,the resistance of the bitter gourd including 6 inbred lines and 4 hybrid firstgeneration was evaluated.It was found that the highly resistant cultivar ‘1516' and its parents‘2012-5?' and ‘09-3-55' showed only slight Fusarium wilt with disease indices less than 15.Based on transcriptome sequencing,lignin and its synthesis-related genes were involved in the resistance of Momordica charantia against F.oxysporum.Lignin content,PAL and POD enzyme activities and related gene expression levels in resistant materials were higher than those in susceptible materials.At the same time,we found that the number of F.oxysporum in the root of resistant bitter gourd 09-3-55 was significantly lower than that of susceptible bitter gourd 9208.Through more than five years of research,we found that reducing the number of F.oxysporum in plant rhizosphere soil and decreasing the colonization number of F.oxysporum in the root of the host is the main strategy for effective prevention of Fusarium wilt.This research shows that the comprehensive control measures including biological control technology,soil fumigation technology,disease resistant varieties and so on can effectively reduce the number of F.oxysporum in soil and host root tissue,and effectively reduce the disaster of Fusarium wilt.