Isolation, Identification And Mechanism Of Arbuscular Mycorrhizal Fungi Against Apple Replant Disease

Apple replant disease(ARD)restrict the healthy and sustainable development of China's apple industry.It is an ideal way to prevent and control ARD by seeking green and efficient prevention and control measures.Biological control of apple replant disease is one of the important ways.This experiment explored the correlation between the severity of apple replant diseases and the diversity of arbuscular mycorrhizal fungi(AMF)in 27 perennial orchards,screened a dominant AMF strain,verified its effect on preventing and controlling apple replant disease,and analyzed the physiological and molecular mechanism of apple rootstocks inoculated with AMF against Fusarium solani infection.1.To explore the correlation between the ARD severity in perennial orchard soil and the AMF diversity.A linear regression indicated that the AMF Shannon index were significantly negatively correlated with the dry mass inhibition rate(P<0.05).Redundancy analysis and canonical correspondence analysis results showed that available phosphorus and total nitrogen were the main negative drivers of AMF community.The structural equation model(SEM)results showed that the contents of soil p H,available phosphorus,total nitrogen and phosphorus in 27soil samples indirectly affected the dry weight inhibition rate of replanted apple seedlings by affecting the diversity of AMF.These results suggest that soil properties can indirectly affect the severity of ARD by changing the diversity and community structure of AMF in perennial apple orchard soil.2.The physiological effects of inoculating indigenous arbuscular mycorrhizal fungi(AMF)in soil to improve the resistance of apple rootstocks were studied in greenhouse condition.The results showed that under continuous cropping conditions,inoculation of indigenous AMF could increase plant biomass and promote root growth.The activities of antioxidant and defense related enzymes in plants increased significantly,including superoxide dismutase,peroxidase,catalase,ascorbate peroxidase,phenylalanine ammonia lyase and polyphenol oxidase.In the roots of seedlings,H₂O₂ content and·O₂^-yield decreased,and the degree of lipid oxidation decreased.Native AMF treatment also induced chitinase and?-1,3-glucanase activity.This showed that under replanting conditions,inoculation of indigenous AMF could enhance the resistance of apple rootstocks by regulating the level of root reactive oxygen species and antioxidant system.3.The spores of soil arbuscular mycorrhizal fungi were isolated by wet screening method,and then enriched and cultured by clover enrichment method.According to its morphological characteristics and phylogenetic analysis based on 18S r DNA gene,it was identified as Paraglomus sp.,named SW1.At present,SW1 strain has been preserved in the China General Microbiological Culture Collection Center(CGMCC),and the deposit number is CGMCC No.20744.4.The control effect of high-efficiency AMF on apple replant disease was verified.The results of pot experiment showed that the inoculation of SW1 could significantly promote the growth of Malus hupehensis Rehd.seedlings and effectively improve the replanting soil environment.Compared with the control,SW1 inoculation can significantly increase the plant biomass of M.hupehensis Rehd.seedlings,promote root growth,improve the defense related enzyme activities in the plant root,significantly reduce the total amount of fungi,the copy numbers of Fusarium,promote the reproduction of AMF in the replanting soil,and change the microbial community structure in the replanting soil.The results of field experiment showed that inoculation with SW1 promoted the growth of replanted young apple trees in replanting orchard,and changed the replanting soil environment and microbial community structure.SW1inoculation treatment significantly increased the biomass of replanting young apple trees in Yiyuan,Qixia and Laizhou.The plant height of replanting young apple trees in the three places was increased by 16.3%,15.4%and 15.7%respectively,compared with the replanting control.SW1 inoculation treatment effectively increased the amounts of bacteria,fungi and actinomycetes in the replanting soil,reduced the copies number of Fusarium spp.T-RFLP result showed that SW1 inoculation improved the diversity of soil fungi and arbuscular mycorrhizal fungi,and changed the microbial community structure in replanting soil.Spearman correlation analysis showed that Plant growth status was significantly positively correlated with bacteria,actinomyces,urease activity,catalase activity,p H,organic matter content,and significantly negatively correlated with copy number of F.proliferatum,F.moniliforme,F.solani.5.The defense mechanism of apple stock with SW1 inoculated against F.solani was analyzed.Infection with F.solani significantly reduced the dry weight of apple roots,and the roots of mycorrhizal apple plants were less damaged when the plants were infected with F.solani.They also had enhanced activity of antioxidant enzymes and a reduction in the oxidation of membrane lipids.The transcriptional profiles of mycorrhizal and non-mycorrhizal plants infected by F.solani were compared using RNA-Seq.A total of 1,839 differentially expressed genes(DEGs)were obtained after mycorrhizal and non-mycorrhizal apple plants were infected with F.solani.A gene ontogeny(GO)analysis showed that most DEGs were involved in the binding of ADP and calcium ions.In addition,the enrichment observed from an analysis with the Kyoto Encyclopedia of Genes and Genomes(KEGG)primarily involved plant-pathogen interaction and Glycolysis/Gluconeogenesis,the mitogen-activated protein kinase(MAPK)signalling pathway,and plant hormone signal transduction.Based on a Map Man analysis,an enormous number of DEGs were involved in the response of mycorrhizal plants to stress.6.Md WRKY40(MD15G1039500)was screened inoculated with Paraglomus sp.SW1apple rootstock against F.solani infection.The results show that Md WRKY40 belongs to the WRKY IIa group based on the WRKY domain and zinc finger motif features.Subcellular localization analysis Md WRKY40 was localized in the nucleus.Overexpression of the Md WRKY40 gene in apple callus decreased in F.solani,suggesting that overexpression of Md WRKY40 apple callus can significantly improve resistance to F.solani.?-1,3-Glucanase gene(Md GLU)was significantly upregulated in apple callus overexpressing Md WRKY40 by F.solani infected.Yeast single-hybrid and electrophoretic mobility assays demonstrated Md WRKY40 was bound to Md GLU promoter.In conclusion,Md WRKY40 promotes the expression of?-1,3-glucanase gene by binding to its promoter,so as to improve the resistance of apple rootstocks to F.solani infection.