| Apple replant disease（ARD）has become a serious threat to the sustainable development of apples in the region.It is caused by the buildup and long-term survival of soilborne necrotrophic fungi and oomycetes.However,the manner in which the fungal community and functional groups are affected by continuous apple cropping remains and the main pathogenic fungi that cause ARD unclear.In the present study,we selected 16 replanting orchards from the Northwest Loess region and Around Bohai Gulf.Diseased roots and rhizosphere soils from healthy apple trees and trees showing ARD symptoms were sampled at random.Fungal communities in healthy（HRS）and diseased apple tree（DRS）rhizosphere soil samples and screened for ARD-associated microbial taxa using fungal ITS（Internal transcribed spacer）quantitative sequencing and screen microbial taxa related to the ARD,and isolated and identified ARD pathogenic fungi specialized type.Further,the potential pathogenic factors of pathogenic fungi and the defense mechanism of apple root system were determined through genome-wide and transcriptome analysis.Finally,the endophytic bacteria that had antagonistic effects on them were screened,and its control effect on ARD and the main findings are as follows:1.Fusarium spp.is the leading pathogenic fungus causing ARD in China.The predominant phyla were Ascomycota in all soil samples,and the abundance and Simpson index of the fungal community in the DRS were significantly higher than that of HRS.Cluster and FUNGuild database analysis of the fungal communities highlight the significant compositional and functional differences in microbial communities between DRS and HRS.Most of the fungi isolated in the DRS were plant pathogens dominated by Fusarium（Ascomycota,Nectriaceae）,and were also the main dominant fungal genus detected in the DRS.By contrast,genera,such as Mortierella,Chaetomium,and Acremonium showed higher abundance in HRS.To verify the sequencing results,most of Fusarium were isolated and verified as pathogens,among which F.proliferatum,F.oxysporum,and F.solani have the highest disease intensity.The soil properties（pH,organic matter,available N,and available P）and phenolic acid（syringate,p-hydroxybenzoic acid,and phloridin）had the most significant effect on fungal taxa.2.Fusarium was further confirmed to be the main pathogen leading to the occurrence of ARD in China,through pathogenicity assays.A pathogenicity assay was carried out by the dip-and-cut technique using different host plants,it was found that strain MR5 showed strong pathogenicity to apple rootstocks.Diseased seedlings specifically exhibited chlorosis of the leaves,browning from the edge of the leaf followed by rolling and yellowing of the leaves,resulting in wilting and eventually death.Strain MR5 was preliminarily identified as F.proliferatum,according to the morphological and cultural characteristics.A maximum likelihood（ML）analysis of identities based on six gene sequences（ITS,TUB2,IGS,mt SSU r DNA,RPB2,and the TEF gene）alignments between the strain MR5 and other strains showed 99% to 100% homology with F.proliferatum.Based on our test results,strain MR5 can be identified as F.proliferatum f.sp.malus domestica（abbreviated hereafter as Fpmd MR5）.3.Here,we obtained the whole-genome sequence of the highly virulent Fpmd MR5 using the Illumina PE150 platform.A genome-wide length of 44.22 Mb and N50 value of865,990 bp.The overall G+C content of the strain MR5 assembly was 48.8%,and the genome coverage was 100×.A total of 9,047 protein-coding genes were annotated,and 98.95% were assigned at least one functional annotation.The genes involved in cytopathicity,carbohydrateactive proteins,cell-secreted proteins,and synthetic gene clusters of secondary metabolites were identified.A total of 70 secreted proteins,593 carbohydrate-active enzymes,and 304 secondary metabolite synthesis clusters were predicted and annotated,suggesting that they are likely involved in pathogenicity and host-pathogen interactions.We also predicted 260 genes that might be related to specific virulence factors,which were mainly related to fungal vegetative growth and the production of cell wall-degrading enzymes.Comparative analysis of F.proliferatum revealed that they shared a total of 6,366 common clusters with Fpmd MR5 showing higher similarity with Fp＿A8.Laboratory tests revealed that Fpmd MR5 can produce Fusarium mycotoxins（deoxynivalenol,zearalenone,T-2 toxin,and fumonisin）,pectinase and cellulase and was sensitive to two fungicides: flusilazole and bromothalonil.4.A total of approximately 518.1 million high-quality reads were generated by means of the RNA sequencing（RNA-seq）method.Comparative analysis between the mock inoculated and Fpmd MR5 infected revealed 28,196 significantly differentially expressed genes（DEGs）,including 14,572 up-regulated and 13,624 down-regulated genes.Among them,the transcriptomes in the roots of the susceptible genotype M.26 were reflected by overrepresented DEGs.Based on a Map Man analysis,an enormous number of DEGs were involved in the response of apple plants to Fpmd MR5 stress.The important functional groups identified via GO and KEGG enrichment were those responsible for fundamental biological regulation,secondary metabolism,plant-pathogen identification,and phytohormone signaling（ethylene and jasmonate）.5.An endophytic Bacillus licheniformis XNRB-3 was isolated from the root tissue of healthy apple trees,and the addition of strain XNRB-3 in Fusarium infested soils significantly reduced the number of pathogens in the soil,thus resulting in a lower disease incidence,and the relative control effect on F.oxysporum and Fpmd MR5 reached the highest of 66.11% and64.66%.The fermentation broth can also protect the roots of the plants from Fpmd MR5 infection.Among the antimicrobial substances secreted by strain XNRB-3,α-bisabolol and2,4-di-tert-butylphenol had significant inhibitory effects on various planted pathogenic fungi.Butanedioic acid,monomethyl ester and dibutyl phthalate promote root development in Arabidopsis plants.Strain XNRB-3 has multifarious plant growth promoting traits and antagonistic potential.The optimal liquid and solid carrier fermentation components and conditions of strain XNRB-3 were obtained.In pot and field experiments,the addition of strain XNRB-3 significantly promoted the growth of plants,and the activity of enzymes related to disease resistance was also significantly enhanced.It also reduced the abundance of four species of Fusarium and the content of phlorizin,cinnamic acid（CA）,ferulic acid（FA）,benzoic acid（BA）,and p-hydroxybenzoic acid（PHBA）in the rhizosphere soil,and increased soil microbial diversity and activity,as well as soil enzyme activity. |
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