| Corynespora leaf fall disease caused by Corynespora cassiicola is an important leaf disease on Hevea brasiliensis,and is also an internationally regarded as a destructive disease like the South American leaf disease.The current research on this disease mainly focuses on pathogen biology,disease epidemiology,prevention technology,and pathogenic mechanism of toxin protein.The pathogen was divided into seven different types of strains according to the type of pathogenic toxin protein.It is reported that there are mainly two types of strains of Cas2 and Cas5 in domestic Corynespora cassiicola on rubber tree.However,in recent years,it has been found that the damage of Corynespora leaf fall disease in Hainan rubber trees in nurseries and young trees has increased,indicating that there may be new strains of toxin protein.Therefore,this study carried out the collection,isolation and identification of pathogen in major rubber producing areas such as Wuzhishan City,Danzhou City,Baisha Li Autonomous County,Ledong Li Autonomous County and Baoting Li Miao Autonomous County in Hainan Province from May to December 2017.Subsequently,the identification and expression analysis of toxin and genomic analysis of the main population were performed to clarify the toxin protein types and genomic information of the dominant population of Corynespora leaf fall disease in Hainan rubber tree.This study can provide basis and information for the prevention and control of this disease and further function gene research.The specific results are as follows.A total of 35 strains causing Corynespora leaf fall disease were obtained by separation and purification.These strains had black and gray colonies,gray and white edges,slow hyphae growth rate,and few conidiospore.Some strains are reddish brown concentric wheel pattern on the back.According to Koch postulates,morphology observation,combined with ITS,elongation factor TEF-1,β-tubulin gene analysis,35 strains were identified as Corynespora cassiicola,and these strains are no genetic differentiation.35 strains of pathogen were inoculated into 5 different resistant rubber strains.The results showed that all the strains could develop disease on the leaves of susceptible rubber strain RRIM600,and strains DZ-27,DZ-123,DZ-131,BS-35,BS-37 and BS-43 can cause disease in 5 different resistant rubber strains,while strains DZ-33,BS-36,WZS-65,WZS-72,BT-110 and LD-114 can only cause disease in the susceptible rubber strain RRIM600,indicating that the pathogen may have a pathogenicity variation.Through the detection of toxin protein subtypes,all 35 strains of pathogen only contain Cas5 toxin protein gene(Cas5),indicating that Corynespora cassiicola in rubber tree is mainly Cas5 subtypes,and no other toxin protein subtypes were detected.Through cloning and structural analysis of the Cas5 toxin protein gene,We found that the Cas5 toxin protein gene includes 3 exons and 2 introns,encoding 57 amino acids.Quantitative PCR technology was used to analyze the expression level of Cas5 toxin protein gene.The results showed that the expression level of Cas5 toxin protein gene was the highest when the mycelium was cultured for 6 days,and the expression level was lower in other growth periods.When interacting with the rubber strains RRIM600 and Wenchang 11 for 3 days,the expression of Cas5 toxin protein gene was the highest,and the expression of other interaction time points was lower.Moreover,the expression of Cas5 toxin protein gene of susceptible rubber strain RRIM600 was higher than that of the disease-resistant rubber strain Wenchang 11.We speculated that the expression of Cas5 toxin protein gene may be regulated by certain specific factors.The whole genome of the DZ-27 strain was sequenced and analyzed.The results showed that 162 scaffolds were obtained from the genome sequencing,with a total length of 46.2 Mb.Among them,the maximum length of scaffold is 4.6 Mb,and the minimum length of scaffold is 0.4 Mb.Meanwhile,the assembled scaffold N50 has a length of 2.5 Mb,a gap of zero and an average content of(G + C)% of 51.07%.In the whole genome,14,953 genes,1,526,476 tandem repeats,41,976 exons,and 27,023 introns were predicted,of which 90.24% of the genes were functionally annotated.The above whole genomic information will provide basic information for the follow-up study of the gene function of this pathogen in Hainan Province. |
