Effect And Mechanism Of Ochroconis Guangxiensis Strain X22 Inducing Resistance To Banana Fusarium Wilt

Banana Fusarium wilt is a soilborne disease caused by Fusarium oxysporum f.sp.cubense（Fo C）,which is a serious threat to the development of banana industry.Due to the unsatisfactory effect of chemical control,and the difficulty in combining disease resistance,high yield and quality in breeding,environmentally friendly biological control has always been a research hotspot for scholars.Dark septate endophytes（DSEs）are a class of plant symbiotic fungi that colonize the plant roots without causing obvious pathological feature of the host.DSEs have the functions of promoting the growth of host plants,improving the host stress resistance and enhancing the host disease resistance.The DSE strain Ochroconis guangxiensis X22 is a new species of fungus isolated and screened from the root of banana by our research group.In this study,Ochroconis guangxiensis X22-banana was used as the research object to explore the effects and mechanism of strain X22 on banana Fusarium wilt,and to provide a theoretical basis for DSE fungi in the biological control of banana Fusarium wilt.The main findings are as follows:（1）Under indoor conditions,the dish symbiosis system of strain X22 and banana tissue culture seedlings was established.It was found that strain X22 could colonize the roots and stems of banana plants,and the typical colonization characteristics of DSE such as dark and separated hyphae could be observed in the roots.The plant height,fresh weight and dry weight of X22-banana symbiont increased by 17.68%,33.33%and 37.50%respectively compared with CK.At the same time,the contents of lignin,phenylalanine ammonialyase and chitinase in the symbiont were significantly increased by 34.49%,30.14%and 28.60%compared with CK respectively.The control effect of X22-banana symbionts on Fusarium wilt was 58.93%.The contents of phenylalanine ammonialyase and chitinase in banana seedlings inoculated with strain X22 and pathogen Foc4 were significantly increased by 41.61%and 4.24%,and the content of lignin was significantly increased by 40.36%compared with those inoculated with Foc4 alone.（2）In the pot experiment,the plant height,stem diameter and fresh weight of banana seedlings inoculated with strain X22 were 1.10,1.14 and 1.24 times that of the control,respectively.After inoculation with strain X22 and Foc4,the control effect of strain X22 on banana Fusarium wilt was 49.43%.Compared with Foc4 alone,the plant height,stem diameter and fresh weight increased by 35.92%,4.13%and 8.24%respectively.Meanwhile,the contents of lignin,phenylalanine ammonialyase,chitinase and malondialdehyde of the plant were also significantly increased.Different trends were observed from day 7 to day 28 after Foc4 inoculation.（3）The genetic transformation system of O.guangxiensis X22 was successfully established by PEG-CaCl₂-mediated protoplast transformation.The optimal conditions for the preparation of strain X22 protoplasts were obtained by screening and enzymatic hydrolysis of 1%Lysing enzyme,Drisease and Snailase for 3 h.At this time,the maximum yield could reach 1.4×10⁷cfu/ml.The plasmid p CHMC containing the red fluorescent protein gene was transferred into the protoplasts of strain X22,and the transformants with strong fluorescence stability RFP-X22 were screened.There were no significant differences between the transformants and wild-type strain X22 in terms of colony morphology,mycelium growth rate and biocontrol of banana Fusarium wilt.After strain RFP-X22 was inoculated into banana seedlings,it could be observed by fluorescence microscope that strain RFP-X22 colonized in tissue cells of banana seedling roots and produced stable red fluorescence.（4）Polysaccharide,oligosaccharide and activator protein were extracted from mycelium of strain X22,and their effects on banana seedling growth and resistance to banana Fusarium wilt were evaluated by hydroponic experiment.The results showed that the plant height,root length and fresh weight of banana seedlings treated with polysaccharide were significantly increased by 16.88%,57.83%and 3.93%respectively compared with the control.The control effect of oligosaccharide on Fusarium wilt of banana seedlings was the most obvious,and their control effects of above-ground part and corm were 46.24%and 54.73%respectively.The contents of superoxide dismutase（SOD）,peroxidase（POD）,phenylalanine ammonialyase（PAL）and polyphenol oxidase（PPO）in the leaves of the polysaccharide treated banana seedlings inoculated with Foc4 were significantly higher than those of the control within 9 days.（5）In this study,transcriptome analysis of banana plants with four treatments（X22,Foc4,CK,X22+Foc4）was carried out.The results showed that there were 4822differentially expressed genes（DEGs）in the Foc4 treatment group compared with CK.Among them,2369 were up-regulated and 2453 were down-regulated.Compared with CK,there were 2776 DEGs in the inoculated strain X22 treatment group,among which1681 DEGs were up-regulated and 1095 DEGs were down-regulated.Compared with CK,there were 3801 DEGs in the X22+Foc4 treatment group,of which 2150 were up-regulated and 1651 were down-regulated.Compared with Foc4-treated banana seedlings,banana seedlings inoculated with X22+Foc4 can induce the expression of resistance related genes,regulate the metabolic pathways such as phenylpropene biosynthesis,plant-pathogen interaction and plant hormone signal transduction,and form a series of defense mechanisms,thus improving the disease resistance of plants.