Evaluation of gray spot resistance and mining of resistance genes in Eriobotrya japonica leaves

【Objective】Gray spot disease is one of the most serious diseases affecting the yield and quality of loquat in the production. This study investigates the molecular mechanisms underlying loquat resistance to Pestalotiopsis infection and identifies the responsive genes in loquat leaves during pathogen invasion, aiming to lay the genetic foundation for resistance breeding. It is generally believed that the causative agent of loquat gray spot disease belongs to the deuteromycete, melanospora, and the pestalotiopsis, which can infect several organs of loquat, and when it infects the leaves, they become rigid and fall easily; when it infects the flowers, fruits and root necks, it causes them to rot, which has a great impact on the growth and development of the tree and leads to a serious decrease in the yield and quality of loquat. Currently, loquat gray spot is mainly defended and controlled using chemical agents, but it tends to lead to increased resistance of the pathogen and pollution of the environment. Therefore, improving the disease resistance of loquat germplasm is the most economical and effective way to solve this problem, and a good assessment of the disease resistance of different loquat germplasm is a prerequisite for breeding disease-resistant varieties, which is also important for the study on the mechanism of loquat response to infestation by the gray spot pathogen and breeding for disease resistance. At present, due to the lack of disease-resistant germplasm and its genetic information, there are few studies on the mechanism of interactions between loquat and pestalotiopsis, especially on the pathogenicity of pathogenic bacteria and the mechanism of plant disease resistance, and the breeding of disease-resistant germplasm is progressing slowly. In this study, loquat gray spot disease was taken as the research object to explore the molecular mechanism of loquat resistance to the infection of gray spot pathogen, and to screen the relevant response genes when gray spot pathogen damaged loquat leaves, so as to provide the genetic basis for resistance breeding.【Methods】The resistance of 52 loquat germplasm resources collected and preserved to Cercospora cinerea was evaluated by using software and visual methods, combined with the method of artificial in vitro leaf inoculation. In addition, transcriptome sequencing technology was used to analyze the changes of gene expression levels in leaves of highly susceptible and highly resistant germplasm resources after inoculation, and to screen the main disease-resistance-related genes, so as to find out the disease resistance factors of loquat germplasm, in order to reveal the disease resistance mechanism of loquat gray spot disease, and provide some guidance and theoretical basis for the breeding of loquat gray spot disease resistance.【Results】Among the 52 loquat germplasm resources, eleven were consistent with the results of field investigation and artificial inoculation in vitro, including three high-resistance germplasms (Baili, Eriobotrya fragrans and E. serrate); Four disease-resistant germplasms (E. fulvicoma Chun & Liao, E. deflexa f. koshunensis, Mogi, Shiromogi); Two susceptible germplasms (Dahongpao and E. kwangsiensis); And two highly susceptible germplasms (Guangrongben and E. cavaleriei.). By analyzing the transcriptome of the leaves of the inoculation treatments and the control of the high resistance germplasm 'Baili' and the high sensitivity germplasm Guangrongben with loquat leaf gray spot disease, a total of 8664 DEGs were detected in the comparison of BL-33h and BL-33ck of Baili cultivar, of which 4049 were up-regulated and 4615 were down regulated. In the comparison of subsequent time points, both the number of up-regulated and down regulated DEGs showed a downward trend, while Guangrongben variety was just the opposite. In the comparison of GRB-48h and GRB-48ck, there were a total of 7483 DEGs, 3098 of which were up- regulated and 4385 were down regulated. It is speculated that the difference in the number of DEGs between the two varieties at different time points may be related to their different resistance to gray spot disease. The analysis found that a total of 684 (585 up- regulated and 99 down regulated) DEGs responded in Baili and Guangrongben, and a total of 25 (12 up- regulated and 13 down regulated) DEGs responded only in Baili. GO and KEGG analysis were performed on these co- responsive genes and special responsive genes, and their transcription factors and disease resistance genes were identified. 64 transcription factors and 59 disease resistance genes were identified in the co-responsive genes, and one transcription factor and three disease resistance genes were identified in the special responsive genes.【Conclusion】In this study, 52 loquat germplasm resources were evaluated for resistance to Cercospora cinerea more accurately, and some key genes in response to Cercospora cinerea infection in loquat were analyzed at the transcriptome level. AP2/ERF transcription factors and three disease resistance genes were found in the differentially- expressed genes of high resistance germplasm. The results could help to enrich the regulation mechanism of resistance to Cercospora cinerea in loquat. The differences in resistance related pathways between resistant and susceptible germplasm resources were helpful to reveal the reasons for the differences in resistance. The key metabolic pathways and resistance-related genes in response to Cercospora cinerea infection in Loquat germplasm with high resistance were identified. The molecular mechanism of pathogen interaction provides a reference for the theory.