| The areas around the Tarim Basin are an important apple-producing region in Xinjiang,China.In recent years,apple Valsa canker has caused increasingly damages to the apple industry in these areas,and specifically resulted in weakened tree vigor and decreased yields,and even the death of entire apple trees in severe cases.In this study,we collected samples from apple Valsa canker in apple-producing areas around Tarim Basin,and conducted isolation and identification of the pathogens.We also studied the population composition and genetic structure of Cytospora,the causative agent of apple Valsa canker.Additionally,we screened and identified potential endophytes for biocontrol,and conducted genomic analysis,biocontrol potential evaluation,and antibacterial mechanism research on Bacillus velezensis SY01,which was a promising candidate.These research findings provide a theoretical basis for the green prevention and control of apple Valsa canker in the Tarim Basin,and offer insights for the development of safe and efficient biocontrol agents.The main research findings are as follows:1.Analysis of the composition of pathogens causing apple Valsa canker in the Tarim Basin.A total of 310 isolates of apple Valsa canker pathogens were obtained from 32 apple orchards in 16 counties(cities)in 4 regions around the Tarim Basin.Through morphological characteristics,ITS sequence analysis,and pathogenicity determination,33 representative strains were screened out.Phylogenetic analysis and morphological identification of multi-gene locus sequences(including ITS,ACT,TEF,and TUB)revealed that these strains belonged to 9 species of the Cytospora species,namely Cytospora pyri,C.mali,C.chrysosperma,C.nivea,C.leucostoma,Cytospora parasitica,C.melnikii,C.tritici,and C.euonymina.Among them,C.pyri was found to be the dominant species causing apple Valsa canker in the Tarim Basin,and C.melnikii,C.tritici,and C.euonymina were newly recorded species causing apple Valsa canker in China.Based on the results of host range determination,the pathogenicity of these 9 Valsa fungal species varied among 7 common economic and protective forests.Among them,C.pyri and C.mali were capable of infecting all tested plants and exhibited strong pathogenicity.Moreover,eight strains of Cytospora(including C.pyri,C.nivea,and C.chrysosperma)isolated from 7 economic and protective forests could infect apple twigs.Notably,C.pyri isolated from Pyrus sinkiangensis Yü showed the strongest pathogenicity.2.Genetic structure analysis of Cytospora genus populations causing apple Valsa canker.Based on transcriptome data analysis,18 pairs of EST-SSR primers were developed.Among them,13 pairs of primers could be used for analyzing the genetic diversity and taxonomic identification of C.pyri related species(including C.mali,C.nivea,C.chrysosperma,and C.parasitica),and 10 pairs of primers could be used for analyzing the genetic diversity of C.pyri geographical and host populations.The study found significant genetic differentiation and gene flow restriction among different pathogenic fungi of C.pyri related species.The genetic differentiation of C.pyri populations was found to be less related to their host associations,while geographic isolation was identified as a crucial factor leading to the genetic differentiation of this species’ population.Different geographic populations displayed varying degrees of gene flow,and the level of genetic diversity gradually decreased from Alar to the east and west.Furthermore,the genetic diversity of populations located north of the Tarim Basin was significantly higher than that of populations south of it.The study also revealed that C.pyri was a heterothallic fungus,and its distribution in the field was relatively balanced.3.Screening and identification of endophytes for biocontrol of apple Valsa canker.Eighty-two strains of endophytic bacteria were isolated from healthy apple tree bark tissues in the Tarim Basin.Through plate confrontation assays,a strain named SY01 with strong antagonistic activity against the C.pyri was screened,with initial and re-screening inhibition rates of 83.7% and 82.6%,respectively.The fermentation filtrate of SY01 at different concentrations showed inhibitory effects on C.pyri,with an inhibition rate of 82.4% at a concentration of 10%.SY01 exhibited protective and therapeutic effects against apple Valsa canker,with relative protection efficiencies of 72.3% and 55.8%,respectively.SY01 showed good inhibitory effects against 13 tested strains,with the best inhibition rate of 88.9% against C.mali,a pathogen causing apple black rot.Based on morphological observations,physiological and biochemical tests,as well as 16 S r DNA sequence analysis,SY01 was identified as B.velezensis.4.Genomic analysis of B.velezensis strain SY01 and evaluation of its biocontrol potential..The genome of strain SY01 was 3,904,915 bp in length with a GC content of 46.47%.It contains 3,745 coding genes.Among them,128 genes were annotated in the CAZy database,encoding enzymes involved in the degradation of plant pathogenic fungal cell walls.Additionally,12 secondary metabolite gene clusters were predicted in SY01,which were capable of synthesizing various secondary metabolites,such as surfactin,bacillaene,and fengycin,known to inhibit plant pathogenic fungi.Furthermore,SY01 harbors genes were found to be related to promoting plant growth,motility,and biofilm synthesis.Observation of hyphal morphology and ultrastructure revealed that SY01 could cause distortion,breakage,or swelling of C.pyri hyphae,leading to degradation of spore cell walls,nuclear condensation,and disruption of the original microstructure.The fermentation broth of SY01 significantly induced the activity of defense enzymes in apple twigs,including SOD,CAT,POD,PPO,and PAL.Colonization assays demonstrated the ability of SY01 to colonize,migrate,and reproduce within apple plants,highlighting its potential for sustained biological control within the plant.5.The expression regulation of pathogenic genes of C.pyri.in response to SY01 stress.Comparative transcriptomics was performed to investigate the transcriptome differences between C.pyri grown on PDA agar,C.pyri infecting apple twigs for 24 h and 48 h,and C.pyri co-cultured with SY01 on twigs.The results revealed significant changes in the expression levels of 2,280 and 2,821 genes in C.pyri infecting apple twigs for 24 h and 48 h,respectively,compared to C.pyri grown on PDA agar.Moreover,when C.pyri was treated with SY01,the expression levels of 5,043 genes were altered compared to C.pyri infecting twigs for 24 h,indicating that SY01 had a significant impact on the gene expression of C.pyri.Furthermore,compared to C.pyri grown on PDA agar,835 genes were consistently up-regulated in C.pyri infecting apple twigs for 24 h and 48 h.However,after exposure to SY01,approximately 80% of these consistently up-regulated genes showed down-regulation in their expression levels compared to C.pyri infecting twigs for 24 h,including 164 genes encoding cell wall-degrading enzymes such as glycosyl hydrolases,and 13 genes encoding peroxidases such as catalase.Meanwhile,several genes related to amino acid biosynthesis,energy supply,and antioxidant defense were up-regulated in C.pyri in response to SY01 stress. |
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