Diversity Analysis Of Amphobotrys Ricini

Gray mold disease is a worldwide disease caused by Botrytis and Botrytis-like fungi,causing serious losses.Amphobotrys is a Botrytis-like genus and only contains one species,Amphobotrys ricini?Buchw.?Hennebert.A.ricini mainly infects Euphorbiaceae plants,including castor bean,copperleaf,poinsettia,Euphorbia milii and Jatropha podagrica.However,the etiology study of this pathogen is relatively weak from its history and current situation.In this study,reclassification of the status of Amphobotrys and determination of species within Amphobotrys,genetic diversity,mating type detection,MAT 1-1 and MAT 1-2 gene structure analysis and mycovirus diversity were studied.The main results obtained were as follows:?1?It was clarified that Amphobotrys belongs to the Sclerotinaceae and that the 200 strains studied are the same species of A.ricini.The genome of the strain WHA-1 was sequenced and the genome size was 34.99Mbp.A phylogenetic tree containing 15 species of fungi was constructed based on amino acid sequences translated by 1813 core genes in the genome.The results showed that the strain WHA-1 was clustered with the other 7 species of Sclerotinaceae.The result showed strain WHA-1 belongs to Sclerotinaceae.The colony morphology,mycelial growth rate,conidia and conidiophore morphology of the tested strains were consistent with Amphobotrys ricini?Buchw.?Hennebert.The sequences of ITS,G₃PDH,HSP₆₀ and RPB₂ of 200 strains were alignmented and phylogenetic trees were constructed based on G₃PDH,HSP₆₀ and RPB₂.The results showed that all the strains were clustered together.The results of morphological and phylogenetic tree indicated that 200 strains belonged to the same species of A.ricini.?2?RAPD analysis showed A.ricini strains existed a high degree of genetic diversity.Eight primers with strong polymorphism amplification and stability were selected from 50 RAPD primers and genetic diversity of 82 A.ricini strains were analyzed.The results showed that eight primers produced 63 DNA bands,of which 49 were polymorphic bands,and the percentage of polymorphic rate was up to 77.8%.The polymorphism information content?PIC?values of these markers varied from 0.47 to0.84 with an average of 0.72.The average value of the observed number of alleles?Na?,effective number of alleles?Ne?,Nei's gene diversity?H?and Shannon's Information index?I?were 2.0000,1.3577,0.2275 and 0.3631.Cluster analysis showed that range of genetic similarity?GS?was 0.4082 to 1.0000 and strains tested could be divided into6 groups at the GS of 0.74.The RAPD groups had a certain correlation with the geographical origin and the host of the strains.?3?Mating type distribution and MAT 1-1 and MAT 1-2 gene structure were clarified.Mating types of 200 strains were detected.The result showed that 170 of the 200strains were MAT l-1 accounting for 85%and 21 were MAT 1-2 type accounting for10.5%and 9 were MAT l-1/MAT 1-2 type accounting for 4.5%.MAT l-1 is a dominant mating type among the tested strains.MAT 1-1 gene locus is 8226 bp in length and contains four gene loci:APN2?DNA lyase?,SLA2?cytoskeleton assembly control?,MAT 1-1-1 and MAT 1-1-5.MAT 1-1-1 encodes a conserved Alpha box domain.MAT1-2 gene locus is 11992 bp in length and contains five gene loci:APN2?DNA lyase?,SLA2?cytoskeleton assembly control?,MAT 1-2-1,MAT 1-2-4 and part sequence of the MAT 1-1-1 gene?truncated MAT 1-1-1?.MAT 1-2-1 encodes a conserved high mobility protein box domain?HMG domain?including 197 amino acids.This result also showed that sexual reproduction of A.ricini is mainly heterothallism.?4?Mycovirus diversity of 200 A.ricini strains was analyzed.The total RNA of 200 strains was sequenced using high-throughput sequencing.The contigs obtained were subjected to NCBI using Blastx.A total of 948 virus sequences were obtained with an average length of 1247 bp.Based on the type of nucleic acid,these virus sequences were divided into positive single-stranded RNA viruses,negative single-stranded RNA viruses and double-stranded RNA viruses,in which positive single-stranded RNA viruses accounted for 88.1%,negative single-stranded RNA viruses accounted for 0.9%and dsRNA viruses accounted for 11%.The RdRp phylogenetic analysis revealed that the mycoviruses in A.ricini population contained 12 families and 18 genera and several new mycoviruses.The result showed that A.ricini was infected by a diverse mycoviruses.Two specific primers were designed to confirm existence of putative mycoviruses in 40 groups?5 strains as one group?by RT-PCR.The result showed that 87%of 40 groups could amplify virus fragments indicating that the two virus sequences are widely distributed.The above findings help to understand the population structure and biological characteristics of A.ricini.