Preliminary Study On The Function Of CcCas And CcSCD1 Of Corynespora Cassiicola In Strawberry

Corynespora cassiicola is an important plant pathogen worldwide,and caused the leaf spot of strawberry for the first time in 2018 in Zhongmu,Henan Province of China,which subsequently occurred in Shandong Province and Florida of the United States.In view of its characteristics of high variability,wide transmission route and strong infection and destruction,this pathogen poses a potential threat to the healthy development of strawberry industry.At present,research on C.cassiicola mainly focuses on the disease symptoms,biological characteristics,control methods and genetic differentiation,while the study on its molecular pathogenic mechanism is still in the primary stage.Phytotoxins are important pathogenic factors in many necrotrophic plant pathogenic fungi,including secondary metabolite phytotoxins and phytotoxic peptides.Many studies have reported that C.cassiicola can produce a small molecular weight proteinaceous toxin,cassiicolin,which acts as an important virulence factor.In order to further understand the role of the protein toxin in the interaction between strawberry and C.cassiicola,the deletion mutant of CcCas gene and the complementary strain were obtained by using the reverse genetics research approach and the homologous recombination principle.The phenotype was analyzed and the main results are as follows:PCR amplification of C.cassiicola were carried out by using the known specific primers of Cas toxin subtype.The subtype of the protein toxin contained in C.cassiicola isolated from strawberry leaf spot was identified as Cas2 by sequencing and multiple alignment.Four △ CcCas deletion mutants were obtained by hygromycin resistance screening and detection with four pairs of primers.Phenotype analysis of △CcCas deletion mutants showed that the deletion of CcCas gene did not affect the morphology,vegetative growth,response to various stresses,conidia production and pathogenicity at all.These results suggest that the CcCas gene may have lost its pathogenic function in the C.cassiicola isolated from strawberry.In plant pathogenic fungi,DHN melanin can not only improve the survivability but closely influence the infection ability in some cases.Deletion of any of the genes involved in the melanin synthesis pathway will hinder the biosynthesis of melanin.Scytalone dehydratase(SCD)is one of the key enzymes of the melanin biosynthesis pathway,and plays an important role in the pathogenic process of plant pathogenic fungi.In this study,four △CcSCD1 knockout mutants of C.cassiicola were obtained by homologous recombination,which were also verified by complement and RT-PCR.The main results are as follows:Phenotype analysis of △CcSCD1 deletion mutants showed that there was no difference in colony growth rate between wild-type and CcSCD1 deletion mutants,but the colony color of the latter changed significantly.The content of melanin decreased significantly after deletion of the CcSCD1 gene,and the other related genes in melanin biosynthesis pathway were differentially expressed,suggesting that CcSCD1 gene plays an important role in the melanin biosynthesis pathway.In the determination of different stresses,the △CcSCD1 deletion mutant was more sensitive to oxidative stress,but under the hyperosmotic stress caused by Sorbitol,the growth rate of the mutant was higher than that of the wild-type and the complementary strain,indicating that the CcSCD1 gene may be involved in different stress signaling pathways.The spore production of the mutant decreased significantly,and the colorless conidia were smaller.qRT-PCR analysis of sporulation related genes also showed that the expression of homologous genes CcStuA,CcBrlA and CcWetA related to conidial production and morphological development were significantly down-regulated,whereas the expression of another gene CcMedA related to the development of conidiophore was significantly up-regulated.These results suggest that CcSCD1 gene may be involved in the regulation of conidial formation and development.The pathogenicity of the mutant strain was significantly lower than that of the wild-type and the complementary strain,indicating that CcSCD1 gene is involved in the regulation of the pathogenicity of C.cassiicola.This study confirmed that CcCas gene may have lost its pathogenic function in C.cassiicola,and clarified the importance of CcSCD1 gene in melanin biosynthesis and pathogenic process,which laid a theoretical foundation for further understanding the pathogenic mechanism of C.cassiicola.