| Mulberry has been cultivated in China for more than 5000 years.Mulberry leaves can be used not only for sericulture,but also for vegetables,tea,feed,medicine,and so on.Mulberry anthracnose is a serious fungal disease,which can cause withered spots or yellow.The diseaseincidence can reach about 40% in autumn,and even the incidence can reach close to100%,which causes serious harm to the mulberry industry.Cultivating and planting resistant mulberry varieties is the most economical and effective method to controlthe disease.To date,the research on mechanism of mulberry resistance to anthracnose is weak.in view of this situation,the etiology of anthracnose,the evaluation of resistance,the process of mulberry responding to the infection of pathogen,and the changes of transcriptome and metabolome were analyzedto reveal the molecular mechanism of mulberry resistance to anthracnose and accelerate the mining of mulberry resistance genes and the cultivation of mulberry resistant varieties.The main results are as follows:1.Fourteen strains were isolated and purified from the leaves of diseased mulberry trees collected from various area of Hubei Province in 2018.Through pathogenicity test,morphological and molecular biology identification,it was found that seven strains were Colletotrichum aenigma,four strains were C.fructicola,and three strains were C.gloeosporioides.C.aenigma strain C1 had the strongest pathogenicity,followed by C.gloeosporioides,and C.fructicola did not cause disease.According to the classification of the severity of mulberry anthracnose,and the determination results expressed by the incidence rate and disease index,11 mulberry varieties were evaluated for disease resistance.A high-resistant cultivar Zheza 1,six moderate-resistant cultivars,including 69 × 851,Fengchi mulberry,Sangteyou 2,Shandong 2020,Tang10,Yuesang 51,three medium-resistant cultivars,including Guisang 5,Guisang 6,Guisangyou 12,and one highsensitive cultivar,Guisangyou 62 were selected.2.Light microscope and scanning electron microscope were used to observe the process of C.aenigma infecting the leaves of high-resistant cultivar Zheza 1 and highly susceptible cultivar Guisangyou 62.The results showed that there was no significant difference in the infection process of resistant and susceptible mulberry cultivars.After 6 h of inoculation,the conidia began to germinate and bud tubes formed at the top or side of the spores;24 h after inoculation,the tip of the germ tube expanded to form oval or irregular appressorium,the appressorium gradually blackened and the cytoplasm became sticky;48 h and 72 h after inoculation,the hyphae showed crisscross arrangement on the leaf surface,followed by branches and gradually reticulated arrangement,and the tips of some hyphae formed secondary conidia.According to the observation of scanning electron microscope,the C.aenigma could invade the host from the leaf surface by the germination of spores or the hyphae formed from elongation of the germ tubes,and also can invadethe leaves through the penetration peg produced by appressorium,there are two ways to invade the host.3.Transcriptome sequencing results showed that 25495 expression genes were detected in four stages(0 h,24 h,48 h,96 h)of inoculation of highly resistant and highly susceptible mulberry varieties.The response period of mulberry to pathogens was mainly 24 h after inoculation,and the number of up-regulated genes was significantly more than the number of down-regulated genes among comparison groups after inoculation,indicating that mulberry leaves responded to pathogen infection more through positive regulation of genes.The main signaling pathways involved in the differential expression genes of resistant materials are "plant pathogen interaction","flavonoid biosynthesis","semi terpene and triterpenoid biosynthesis","MAPK signaling pathway","plant hormone signaling",and "phenylpropane biosynthesis".In this experiment,10 differentially expressed genes including PR1,WRKY33,ZAT11,kiwellin,CNGC,JRL3,CHS,PAL,CYP73 A and unidentified proteins were selected for q RT PCR analysis.The results showed that the trend was consistent with the transcriptome sequencing results,indicating that the transcriptome data sequencing expression was credible.The results of metabolomic detection of high resistance and high sensitivity varieties at the above four times showed that 865 metabolites were obtained,and the differential metabolic pathways mainly involved "biosynthesis of phenylpropanoid compounds","ABC transport vehicles","phenylalanine metabolism","phenylpropanoid biosynthesis",and "biosynthesis of flavonoids".Through the joint analysis of transcriptome data and metabolome data,it was found that "phenylpropane biosynthesis" and "flavonoid biosynthesis" metabolic pathways played an important role in the process of mulberry resistance to anthracnose.Among them,metabolites phenylalanine,pcoumarinylquinic acid,and colorless anthocyanins may be important metabolites in the infection response of mulberry trees to hidden anthracnose bacteria.PAL,COMT,4CL,CYP73 A,CHS,FLS,DFR,LAR may be the key genes playing a crucial role. |
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