Study On The Diversity Of Pathogenic Fungi And Mycovirues In The Botryosphaeriaceae From Fruit Tree Branches

Botryosphaeriaceae fungi can be found all over the world with a wide range of hosts,which can harm many important fruit trees,including peach,apple and so on.It has caused serious losses to the development of fruit tree industry in regions of the middle and lower reaches of the Yellow River in China.In this study,the diseases of different fruit trees in regions of the middle and lower reaches of the Yellow River were investigated extensively,the species identification and diversity analysis of pathogenic fungi were carried out,in order to clarify the pathogen species and distribution of fruit trees in regions of the middle and lower reaches of the Yellow River,and the morphological characteristics,pathogenicity and gene diversity of the pathogenic fungi were analyzed,which provided useful information for in-depth understanding of the characteristics of the fungi of the Botryosphaeriaceae.In addition,the species and diversity of mycoviruses in the Botryosphaeriaceae were analyzed,and fungal viruses with weakened virulence were identified and biocontrol potential was studied.The main content is as follows:1.Identification and diversity of Botryosphaeriaceae species.From 2019 to 2022,348 samples of branch diseases from five provinces of the middle and lower reaches of the Yellow River(Henan,Shandong,Shaanxi,Shanxi and Hebei Province)and fruit growing areas of Beijing were isolated and purified.A total of 434 strains of Botryosphaeriaceae were obtained.According to their morphological characteristics and Internal Transcribed Spacers(ITS)sequences,97 representative strains were selected for multi-gene phylogenetic analysis(ITS,tef1,tub2 and rpb2)and morphological identification.The results showed that 15 Botryosphaeriaceae species were inditified,including four new species,which named Botryosphaeria eriobotryae,Lasiodiplodia regiae,L.xinyangensis and L.ziziphin,six new host species(B.kuwatsukai,Dothiorella acericola,Do.plurivora,L.chiangraiensis,L.citricola and L.huangyanensis),and five known species(B.dothidea,Diplodia mutila,D.seriata,L.pseudotheobromae and L.theobromae).In addition,according to morphological characteristics and nucleotide sequence,the taxonomic status of some species was revised,including L.paraphysoides and L.nanpingensis are reduced to synonymy with L.citricola,L.fujianensis is reduced to synonymy with L.iraniensis and L.henanica is reduced to synonymy with L.huangyanensis.Four novel species and six new host species in this study were selected for inoculations on the original host to prove Koch’s postulates in vitro and in vivo.The results indicated that these species are pathogenic.The host range of the 21 representative isolates from 15 Botryosphaeriaceae species was assessed by inoculating mycelial discs(one to three isolates per species)onto detached shoots of four fruit trees,including apple,pear,peach and walnut.The significant differences in virulence were observed among Botryosphaeriaceae species.L.theobromae and L.pseudotheobromae were strongly virulence for walnut,peach and apple trees,while Do.plurivora,D.mutila and D.seriata were weakly virulence for four fruit trees.Diversity of Botryosphaeriaceae species were observed from fruit tree branch diseases in regions of the middle and lower reaches of the Yellow River in China.A total of 15 Botryosphaeriaceae species belonging to four genera(including Botryosphaeria,Diplodia,Dothiorella and Lasiodiplodia)were identified.B.dothidea could infect 12 different fruit trees,distributed 5 provinces and Beijing.It is the dominant pathogen of fruit tree branch disease in regions of the middle and lower reaches of the Yellow River in China.2.Diversity of mycoviruses in Botryosphaeriaceae.Morphological characteristics and pathogenicity were conducted of Botryosphaeriaceae isolates from 12 different fruit trees in regions of the middle and lower reaches of the Yellow River in China.The study found that some isolates had abnormal colony morphology and hypovirulence.Double-stranded RNA(ds RNA)viruses were detected from these isolates with molecular weights from 1 to 10 kb,indicating the diversity of mycoviruses in Botryosphaeriaceae isolates.230 Botryosphaeriaceae isolates were selected to explore the diversity of mycoviruses using RNA＿Seq analysis,and 61 mycoviruses were obtained.There are 40 +ss RNA viruses were predicted;18 ds RNA viruses;one-ss RNA virus.Moreover,two viruses with unclear classification status were also found.Further analysis showed that 61 viruses were classified into 13 viral families,including +ss RNA viral families:Botourmiaviridae,Tombusviridae,Hypoviridae,Narnaviridae,Mitoviridae and Fusariviridae;ds RNA viral families: Polymycoviridae,Partitiviridae,Totiviridae,Bipartitiviridae,Chrysoviridae,Endornaviridae and unclassified virus;-ss RNA viral:Bunyavirales.The results revealed that there are not only abundant viral resources in Botryosphaeriaceae,but increased our understanding of viral structure and evolution,and enriched the existing viral resources.3.Molecular and biological characteristics of LzPV1 and BdPV2 in PartitiviridaeA novel partitivirus was identified from Lasiodiplodia ziziphi,named Lasiodiplodia ziziphi partitivirus 1(LzPV1).The LzPV1 contains two ds RNAs with the lengths of1905 bp and 1773 bp,respectively.Ds RNA1 encodes an RNA-dependent RNA polymerase(Rd Rp),ds RNA2 encodes an Coat Protein(CP).Blastp search showed that the amino acid sequence of Rd Rp and CP were similar to those of Lichen partiti-like RNA virus 2(63.99% and 52.60% identity).Phylogenetic analysis using Rd Rp and CP amino acid sequences revealed that LzPV1 was closely clustered with the genus“Alphapartitivirus” of the Partitiviridae.To the best of our knowledge,LzPV1 was a novel partitivirus in the family Partitiviridae.The virion of LzPV1 was spherical with a diameter of ～32 nm.The growth rate of CDZM004(LzPV1-infected)and CDZM004-T(LzPV1-free)were no significant difference,but strain CDZM004-T increased aerial hyphae and exhibited virulence.In addition,LzPV1 could be transimitted by conidia,and horizontally cotransmitted through hyphal contact at the intraspecific.A novel partitivirus was described in Botryosphaeria dothidea strain from walnut,named as Botryosphaeria dothidea partitivirus 2(BdPV2).The genome contains three ds RNAs.The lengths of ds RNA1、ds RNA2 and ds RNA3 were 1751 bp,1568 bp and1198 bp,respectively,encoding an Rd Rp,CP and unknown protein.Sequence comparisons indicated that this virus is similar to those of Penicillium stoloniferum virus S(Ps V-S).Phylogenetic analysis based on Rd Rp and CP sequences revealed that BdPV2 was closely clustered with the genus “Gammapartitivirus” of the Partitiviridae.In summary,this study systematically investigated and analyzed the diversity of fungal species of fruit tree branch diseases in the regions of middle and lower reaches of the Yellow River,identified B.dothidea as the dominant pathogen of fruit tree branch diseases.We identified four new species and six new host species.This result will provide first-hand data for the identification and prevention of fruit tree branch diseases in the middle and lower reaches of the Yellow River.Meanwhile,we initially identified the diversity of mycoviruses in Botryosphaeriaceae.The viruses LzPV1 and BdPV2 can be used as potential biological control resources,which is of great significance for the prevention and control of fruit tree branch diseases in the middle and lower reaches of the Yellow River.