| Cherry is one of the fastest developing fruit trees in China in the past 20 years.Because of its early flowering and maturity character,it enjoys the reputation of“the firstly ripening fruit in spring”.However,cherry diseases have restricted the further development of the cherry industry.Cherry black spot disease seriously endangers cherry cultivation and production.It causes early leaf fall and tree vigor decline,which directly affects the flower bud differentiation and yield of the next year.Different cherry cultivars exhibit different resistance to black spot disease,however,the black spot disease resistant mechanism is still unclear.In this study,the pathogen of cherry black spot disease was isolated and identified,and the resistance level of different cherry cultivars to black spot disease was comprehensively evaluated.After inoculation with pathogen,the resistant and susceptible cherry cultivars at different infectious stages were used for transcriptome sequencing and metabolome analysis.Then,the candidate genes of cherry disease resistance were screened,those genes function was verified,and the disease resistance mechanism was explored,which provided theoretical basis and scientific guidance for cherry disease resistance breeding.The main results are as follows:1.Samples of cherry leaves and fruits with black spot disease were collected,and the pathogen was isolated and purified by tissue separation technology,and the pathogenicity was determined by Koch’s rule.The ITS and protein-coding genes（ATPase,calmodulin and Alt a1）specific primers were used for PCR amplification,and the phylogenetic tree was constructed through sequence comparison analysis.Finally,the species of the pathogens were determined based on their morphology and molecular features.The main pathogen causing cherry black spot in Shaanxi Province was identified as Alternaria alternata.2.The resistance of 34 cherry cultivars to black spot disease was evaluated.According to the lesion diameter and relative disease resistance index,the disease resistance of cherry was divided into six grades:immune,highly resistant,resistant,medium resistant,susceptible and highly susceptible.Field investigation and evaluation showed that five Prunus cerasus cultivars‘Aode’,‘Meilei’,‘Meili’,‘Aojie’and‘ZY-1’,and one P.avium cultivar‘13-33’were highly resistant,while P.avium‘Qinying I’,‘Kordia’,‘Valeriy’,‘Ranier’and‘Rita’were highly susceptible,and no immune cultivars were found.The resistance evaluation was carried out by artificial inoculation of A.alternata on detached leaves,and it was found that the detached-leaf inoculation evaluation was positively correlated with the field evaluation results.Among them,the P.cerasus showed highly resistant,and the P.avium‘Qinying I’,‘Kordia’,‘Valeriy’,‘Ranier’and‘Rita’still showed highly susceptible,and the plant inoculation again verified the reliability of the results.In addition,in the comparison of resistance between different species,it was found that the resistance of P.cerasus was greater than that of P.avium,P.tomentosa,P.pseudocerasus and P.mahaleb.3.The dynamic changes of defense-related enzyme activities were determined after the selected resistant cultivar（RC）P.cerasus‘Aode’and susceptible cultivar（SC）P.avium‘Rita’were inoculated with A.alternata.It was found that the infection of A.alternata activated the outbreak of reactive oxygen species（ROS）in cherry leaves.In the early stage of inoculation,H2O2 content,the enzyme activities of peroxidase（POD）,catalase（CAT）,superoxide dismutase（SOD）and polyphenol oxidase（PPO）in RC were significantly higher than those in SC,and the reaction of pathogenesis-related proteins chitinase（CHI）andβ-1,3 glucanase（GLU）in RC was also earlier than that in SC.In addition,the malondialdehyde（MDA）content in RC did not change much during the whole inoculation process,while the MDA content in SC continued to increase.Field emission scanning electron microscopy was used to observe the leaf surface of RC and SC inoculated with A.alternata.On the leaf surface of SC,it was found that a large number of conidiophores directly invaded the host epidermal cells,and the conidiophores were about to differentiate into more conidia.On the leaf surface of RC,only scattered hyphae and single conidia were observed.4.According to the data of physiological changes of cherry leaves after inoculation with A.alternata,combined with the development of leaf disease,the plant tissues at the junction of disease and health were collected for transcriptome and metabolomics analysis at 0,1 and5 days post inoculation（dpi）.Transcriptome sequencing results showed that there were 10645differentially expressed genes（DEGs）in RC and SC during the defense response of cherries to A.alternata.DEGs were enriched in multiple metabolic pathways,including phenylpropanoid biosynthesis,α-linolenic acid metabolism,plant-pathogen interaction,flavonoid biosynthesis,brassinolide biosynthesis,sesquiterpene and triterpene biosynthesis.A total of 1088 metabolites were detected in the metabolome detection.Orthogonal partial least squares discriminant analysis showed that the metabolites petunidin-3-O-（6’’-O-p-Coumaroyl）glucoside,isorhamnetin-3-O-rutinoside,L-Arginine,quercetin-5-O-β-D-glucoside,isohyperoside,and coumarin were likely to be important variables leading to the separation of resistant and susceptible cherry cultivars.Differentially accumulated metabolites（DAMs）were mapped to KEGG database,and the flavonoids biosynthesis,ABC transporters,cyanoamino acid metabolism,tropine,piperidine and pyridine alkaloids biosynthesis and phenylpropanoid biosynthesis made positive responses in cherry defense reaction.5.Through the combined analysis of transcriptome and metabolome,it was found that the key genes involved in jasmonic acid synthesis in RC were significantly up-regulated at 0and 1 dpi,which promoted the accumulation of jasmonic acid,indicating that the resistance of cherry to A.alternata was related to the early signal of jasmonic acid.In the phenylpropanoid metabolic pathway,the key genes involved in lignin synthesis were also significantly up-regulated in RC,promoting the accumulation of lignin,and the metabolites phenylalanine,tyrosine and coumarin were also significantly accumulated.In vivo and in vitro inhibition tests showed that coumarin could effectively inhibit the growth of A.alternata.In addition,there were 943 transcription factors（TFs）in response to A.alternata infection,and some TFs,such as MYB,NAC,WRKY,ERF and b HLH,may participate in A.alternata-mediated defense response by activating disease resistance signals and downstream defense pathways.6.A key transcription factor PcMYB63 regulating cherry resistance to A.alternata was screened from differentially expressed MYB TFs in phenylpropanoid pathway,which was specifically up-regulated in resistant cherry cultivars.PcMYB63 gene was cloned from P.cerasus cherry‘Aode’by homologous cloning method.It was found that PcMYB63 was located on chromosome 4 of cherry.The nucleotide sequence of the gene coding region was1098 bp in length,encoding 365 amino acids.Subcellular localization experiment result showed that PcMYB63 protein was mainly located in cell nucleus to perform a transcriptional regulatory function.The PcMYB63 fusion overexpression vector was constructed,and the transient overexpression of PcMYB63 in cherry leaves could improve the resistance to A.alternata.Furthermore,the disease resistance function of PcMYB63 was verified by heterologous transformation of Arabidopsis thaliana and Populus tomentosa,it was found that overexpression of PcMYB63 induced up-regulation of lignin synthesis-related genes and promoted lignin accumulation.Yeast one-hybrid assay and dual luciferase reporter gene assay demonstrated that PcMYB63 could directly bind to the promoter of the downstream target gene PcCCoAOMT in the lignin synthesis pathway.In order to verify whether the downstream target gene PcCCoAOM also has disease resistance function,transient overexpression of PcCCoAOMT in cherry leaves and stable transformation of PcCCoAOMT gene in Arabidopsis showed that transgenic plants also had higher resistance to A.alternata.In summary,this study isolated and identified that A.alternata is one of the important pathogens leading to the cherry black spot disease.and The resistant cultivars of cherry were screened out.Combined with transcriptome and metabolome analysis,the functions of black spot disease resistant key metabolites and genes were verified.The findings of this study have important implications for cherry black spot resistance breeding and genetic improvement. |
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