Pathogen Identification And Whole Genome Cloning And Structural Analysis Of Mycovirus In Pathogen Of Areca Catechu Diaporthe Leaf Spot

The Areca catechu L.,belonging to the Palmaceae family,is one of the most important economic crops in Hainan.A.catechu is also a precious tropical medicinal plant,that make it becomes the most important of the four famous medicines in south China.With the continuous expansion of planting area,leaf spot disease has become one of the important factors restricting the sustainable development of areca industry due to the reduction of land resources in traditional planting areas,and the years of cultivation and improper cultivation and management.This article first carried out preliminary studies on areca leaf spot,including pathogen identification,biological characteristics,the full-length nucleotide sequence cloning and identification of mycovirus and the effect on the host.The results are as follows:Diaporthe sp.,the causal agent of areca leaf spot,mainly damaged the leaves of areca.Early stage of the pathogen infection,note the small yellow spots.Middle infection stage,the lesions turned dark grey overtime with distinct yellow halos.Lesions were long oval,sometimes irregular with black to brown,small black spots and distinct yellow halos.Late infection stage,the necrotic spots were enlarged,gradually gray,and then combined to form larger necrotic areas.Isolation and purification of pathogenic fungi,and then tested for pathogenicity,morphological characteristics and multi-gene(ITS、TUB、CAL、TEF and HIS)phylogeny analysis,all the results indicated that the pathogenic fungi belongs to the species of Diaporthe pseudophoenicicola,D.limonicola and D.ueckerae,causing areca leaf spot disease.The biological characteristics of Diaporthe sp.were further studied.PDA culture media was the most suitable medium for the growth of the pathogens HNWC01,HNQH01,and HNQH02.The most suitable temperature for the growth of mycelia was 28℃.The most suitable p H was 7,and the light had no effect on the growth of mycelia.Double stranded RNA(ds RNA)was extracted from the collected mycelia of 14 strains of Diaporthe sp.Interestingly,the mycelia of HNWC02 was detected to contain a ds RNA mycovirus which has 6 ds RNA fragments.The full-length c DNAs of ds RNA1-6were 3335 bp,3039 bp,3030 bp,2980 bp,963 bp,and 780 bp,respectively.Sequence analysis indicated the presence of nine open reading frames(ORFs)in strain HNWC02.Among them,ORF1 in ds RNA-1 putatively encoded the RNA-dependent RNA polymerase(Rd Rp)and ORF3 in ds RNA-2 encoded a capsid protein(CP),respectively.The seven remaining ORFs,ORF2 in ds RNA-2,ORF4,5 in ds RNA-3,ORF6,7 in ds RNA-4,ORF8 in ds RNA-5,and ORF9 in ds RNA-6,encoded proteins with unknown functions.Phylogenetic analysis based on the Rd Rp and CP sequences revealed that Dp CV1 is closely related to members of the cluster I group within the family Chrysoviridae but formed a separate branch.The virus particles were spherical virions that approximately 40 nm in diameter.Importantly,all the six segments of Dp CV1 were cured successfully through single spore isolation to obtain the isogenic virus-free strains.Dp CV1 exerts minor influence on the growth of Diaporthe pseudophoenicicola but can confer hypovirulence to the fungal host,enhance ability of pigment production and decrease resistance to prochloraz.Compared with the virus-free strain,the result of the mycelia of Dp CV1 infected strain stained with calcofluor-white showed that the cell wall was loose and enlarged.The results provide a potential view for the biocontrol of areca leaf spot caused by Diaporthe sp.strains by using mycoviruses.The research results above not only provide relevant theoretical basis for the prevention and treatment of areca disease,but also has great significance to the sustainable development of areca industry in Hainan Province.