Diversity Of Diaporthe Species Associated With Pear Shoot Canker In China And Interactions Between Diaporthe Fusicola And Pyrus Bretschneideri

China is a major producer and consumer of pears,and its cultivated area and yield rank first in the world.In recent years,pear shoot canker is serious in major pear-cultivation areas in China,causing death of the pear branches,brown canker tissue around buds on the shoots,twigs or large branches,and always killing the infected shoots or branches and the attached blossom and leaf buds.It has caused serious losses to the economic benefits of pear orchards in the major pear-cultivation areas and greatly affected the development of pear industry in our country.In this study,to clarify the species and distribution of pathogens of pear shoot canker in different pear-cultivation areas in China,as well as the differences in morphological characteristics,pathogenicity and gene sequences of the genus Diaporthe from different regions and different pear cultivars,we conducted extensive surveys,sample collection,pathogen isolation and species diversity analysis of pear shoot canker in the major pear-cultivation areas in China.In addition,to elaborate the pathogenic mechanism of Diaporthe in different infection stages and the corresponding defense strategy of the host pear from the transcriptome level,the simultaneous transcriptome analysis of an hypervirulent isolate from the Diaporthe species and inoculated pear branch tissue by this isolate were systematically carried out.This study provides information for further understanding pathogen diversity of Diaporthe and the interaction mechanism between Diaporthe and host plants.The main research results were as follows:1.Identification and diversity of Diaporthe species associated with pear shoot canker in ChinaFrom 2014 to 2017,a total of 286 pear samples(shoots,branches,and twigs)affected by pear shoot canker collected from 12 provinces(including Chongqing,Fujian,Guizhou,Hebei,Henan,Hubei,Jiangsu,Jiangxi,Liaoning,Shandong,Yunnan and Zhejiang)were subjected to fungal isolation,resulting in a total of 453 Diaporthe isolates identified based on morphology and ITS sequence data.A total of 113 representative isolates were chosen for further phylogenetic and taxonomic analyses.Multi-locus phylogenetic and morphological analyses revealed 19 species belonging to Diaporthe,among them,there are 13 known species,including D.caryae,D.cercidis,D.citrichinensis,D.eres,D.fusicola,D.ganjae,D.hongkongensis,D.padina,D.pescicola,D.sojae,D.taoicola,D.unshiuensis and D.velutina.Of these,six species are newly described here,namely D.acuta,D.chongqingensis,D.fulvicolor,D.parvae,D.spinosa and D.zaobaisu.Representative strains of 19 species of Diaporthe inoculated on live P.pyriforia cv.Cuiguan seedlings produced the same symptoms as in the field,and the results confirmed that these species were the pathogens of pear shoot canker.And Diaporthe species are closely linked to the sampling area,with a higher diversity in the south of the Yangtze River than that in the north Analysis of the abundance of Diaporthe species on pear species revealed P.pyrifolia was the most sensitive to the Diaporthe.The results showed that D.eres was the most prevalent species in China.To our knowledge,this is the first report that these species infecting pear are responsible for pear shoot canker besides D.eres.The pathogens of pear shoot canker in China showed high diversity.The classification results showed that 19 species of Diaporthe belonged to three Diaporthe species complexes,D.eres complex D.sojae complex and D.arecae complex.The observation of morphology showed that there were significant difference in colony pigment and alpha conidia sizes among the three Diaporthe species complexes.The results of virulence and host range tests showed that there were significant differences in virulence between different species of Diaporthe,of which,D.fusicola and D.chongqingensis had the strongest virulence.The host P.pyrifolia was the most susceptible to the Diaporthe species,while P.communis and P.ussuriensis were more resistant.In addition to pears,13 of 19 species of Diaporthe could infect Malus pumila,Prunus persica,Actinidia chinensis and Citrus reticulata.The results of mating type test showed that most of the species were heterothallic,only D.sojae was homothallic,while the remaining species(D.eres,D.unshiuensis,D.hongkongensis,D.cercidis)contained potentially homothallic as well as heterothallic isolates.2.Simultaneous transcriptome analysis of D.fusicola and P.bretschneideriIn this study,RNA-Seq analysis was performed on strain DK371 of D.fusicola and its infected P.bretschneideri branches tissue at different infection stages(pre-onset and post-onset).Transcriptome data after quality control showed that the data amount,Q20 and Q30,and CG values all met the requirements of high quality.2103 and 9788 differentially expressed g enes(DEGs)were screened from the D.fusicola transcriptome and the P.bretschneideri transcriptome respectively.The GO annotation classification and KEGG enrichment analysis were performed on these DEGs.The results showed that the metabolic process of D.fusicola was more vigorous at pre-onset stage than post-onset stage.In contrast,the host P.bretschneideri began to activate multiple signaling pathways to resist the further invasion of pathogens D.fusicola.Using the transcriptome data to further explored the host P.bretschneideri in response to D.fusicola infection,and it was clear that the plant-pathogen interaction pathway,plant hormone signal transduction pathway,phenypropanoid pathway,and a variety of transcription factors plays a key role in the immune response of P.bretschneideri in response to D.fusicola infection.During the interaction between P.bretschneideri and D.fusicola,P.bretschneideri mainly depended on PTI reaction at pre-onset stage,and ETI reaction was gradually activated at post-onset stage.At the same time,the response of P.bretschneideri also depended on the combined action of plant hormone signaling pathways such as SA,ABA and ET,while the signal pathways such as JA and IAA are not activated by antagonistic action.In the pre-onset stage of P.bretschneideri in response to D.fusicola,the phenypropanoid pathway was activated,in which the genes related to lignin and anthocyanin synthesis were up-regulated.In this study,we analyzed the adaptability of D.fusicola to the P.bretschneideri by annotating the carbohydrate active enzyme in the process of infection.The results showed that the amount of carbohydrate active enzymes and plant cell wall degrading enzymes in D.fusicola were close to the hemibiotrophic fungi,and the infection of D.fusicola to P.bretschneideri was mainly completed by degrading lignin,hemicellulose and pectin.In addition,we predicted the candidate effectors of strain DK371 of D.fusicola.Finally,a total of 14 candidate effectors were identified,among them,4 genes were significantly up-regulated in pre-onset and post-onset stages.Two candidate effectors(Dfcp1 and Dfhp1)were selected for full-length cloning and sequence analysis.The results showed that the cDNA sequencing results were completely consistent with the RNA-seq data,and the sequences were more conserved within the genus Diaporthe.Quantitative real-time PCR showed that Dfcpl and Dfhp1 unigenes were significantly up-regulated during D.fusicola infection,indicating that they may be involved in the process of D.fusicola infection.