The Virome Of Fusarium Isolates Collected From Three Wheat Planting Regions Of China And Characterization Of Hypovirulence-associated Viruses In Fusarium Asiaticum

Fusarium Head Blight(FHB)caused by wheat Fusarium species is an important fungal disease worldwide,resulting in serious losses in the yield and quality as well as severe mycotoxin pollution of wheat,which posed a significant threat to food safety as well as human and animal health.The primary pathogens of FHB were F.graminearum and F.asiaticum.Mycoviruses are viruses that replicate and proliferate in fungi.Hypovirulence-associated mycoviruses have great potential and application prospects for controlling crop disease.Exploring and investigating the diversity of mycoviruses in the wheat fusarium population as well as in individual isolates and screening candidate virocontrol agents not only helps to discover and identify new viruses,provide alternative resources for green prevention and control of FHB,provide important materials for virus classification and the evolution research of the virosphere,but also provide tools to exploring the characteristics of their fusarium host.This thesis analyzed the diversity of mycoviruses in Fusarium species from Sichuan Basin(SC),the middle and lower reaches of the Changjiang/Yangtze River(CJML)and the HuangHuai-Hai region(HHH)in China,and explored the biological characteristics of the three hypovirulent strains as well as the molecular characteristics of the corespongding hypovirulent-related viruses,and the specific research content is as follows:The species and distribution of mycoviruses in wheat fusarium isolates were analyzed.A total of 1607 isolates of wheat fusarium isolates were isolated from diseased wheat head from 44 counties or cities in wheat planting areas of SC,CJML and HHH.Molecular identification showed that F.graminearum was predominant in the HHH wheat region,while F.asiaticum was predominant in the SC and CJML region,accounting for 96%,80% and 88% of corresponding Fusarium isolate groups,respectively.The virus diversity of wheat fusarium groups from three wheat planting regions(with 50 strains from each region)in China was analyzed for the first time,19 different viruses including 10 new viruses with partial or complete genome sequences were obtained.The positive single-stranded RNA(+ss RNA)virus sequences accounted for 30.43%,88.89% and 81.82% of each wheat planting region sample virome,respectively;the negative single-stranded RNA(-ss RNA)virus sequences accounted for 39.13%,11.11% and 18.18% of each wheat planting region sample virome,respectively;the double-stranded RNA(ds RNA)virus sequences with complete ORF were only detected in the SC sample,accounting for 30.43% of SC sample virome.In all,the majority of the putative viruses were predicted with +ss RNA genomes accounting for 68.75% of the total viruses;then-ss RNA genomes accounting for 18.75%of the total viruses and ds RNA viruses accounting for 12.5% of the whole viruses.The+ss RNA viruses detected belong to Mitoviridae,Narnaviridae,Botourmiaviridae,Martellivirales,Hypoviridae,Picornavirales,and Orfanplasmovirus(with uncertain taxonomic status).The detected-ss RNA viruses belong to Bunyavirales and Mymonaviridae,the bunyavirus was only found in SC samples,while mymonaviruses were distributed in all three wheat planting region samples.The double-stranded RNA viruses were only detected in SC samples,and mainly belonged to Chrysoviridae and Polymycoviridae.Of the 19 virus species,there are 10 new viruses,belonging to the Martellivirales,Picornavirales,Narnaviridae,Mitoviridae,Botourmiaviridae,Polymycoviridae,Orfanplasmovirus,Bunyavirales and Mymonaviridae,respectively.In wheat fusarium species,viruses belonging to Martellivirales,Picornavirales,Narnaviridae,Polymycoviridae and Orfanplasmovirus were reported for the first time in this thesis.Viruses belonging to Martellivirales,Bunyavirales,Chrysoviridae,and Polymycoviridae were only detected in SC samples;Orfanplasmovirus was only detected in CJML samples;The narnaviruses were only detected in the samples from SC and CJML;Botourmiaviridae viruses were only detected in CJML and HHH samples;hypovirus was only detected in HHH samples,Mitoviridae,Picornavirales and Mymonaviridae viruses were detected in all three wheat region samples.This discovery enriches the viral resource pool and increases our understanding of the diversity,ecology and evolution of the virosphere.F.asiaticum strain BZ6 was a hypovirulent strain co-infected by two mycoviruses.With normal or mild abnormal clone morphology,the sexual and asexual reproduction,as well as the pathogenicity of strain BZ6,was under restriction,and the mycelial branches were abnormal.Metavirome-sequencing and RT-PCR results confirmed that strain BZ6 was only infected by two different positive single-stranded RNA viruses:Fusarium asiaticum virga-like virus 1(Fa VLV1)and Fusarium asiaticum mitovirus 1(Fa MV1).Fa VLV1 is a new member of Martellivirales in Kitrinoviricota.The genome of Fa VLV1 is 9641 nt in length(poly(A)-like region at the 3’ end not included)and consists of three segments(RNA1-3)with two methyltransferase(Mtr)conserved domains,the virus particles of Fa VLV1 were symmetrical spiral rod or filamentous,with diameter between 10.00-15.93 nm and length between 149.02-1289.48 nm.The all three genome nucleic acid fragments of Fa VLV1 have a 78 nt conserved sequence rich in "CAAA(A)" at the 5 ’end,and a poly(A)-like region at the 3’ end.Phylogenetic analysises of conserved domains of Rd Rp,Mtr,and Hel of Fa VLV1 indicate that Fa VLV1 and some other virga-like viruses isolated from arthropod,plant,fungi and/or oomycetes hosts are new members of Martellivirales,the two Mtr of Fa VLV1 had different origins,and a new family should be established to accommodate these virgalike viruses.Fa VLV1 could not be transmitted vertically by ascospores,but could be transmitted by conidia with 81% efficiency.The effort of transmission Fa VLV1 from BZ6 to XG-7-1(virulent strain without virus)was failed(0/50),but BA2(ascospores derived strain of BZ6)was succeed in getting Fa VLV1 from BZ6 by hypha anastomosis with an efficiency of 41.67%.Biological experiments showed that the infection of Fa VLV1 increased the growth rate of the host,decreased the yield of conidia,and reduced the pathogenicity on the wheat heads in vitro.Fa MV1 is a fusarium-infected strain of Plasmopara viticola lesion associated mitovirus 13(Pv LAMV13)as they share94.47% sequence similarity of Rd Rp.Result from phylogenetic analysis place Fa MV1 in a clade with mitoviruses described in other fungal or oomycete genus which different with the rest of mitoviruses described in genus of Fusarium.Fa MV1 could be transmitted vertically by asexual and sexual reproduction with an efficiency of 100%,and could be transmitted to virulent F.asiaticum strain XG-7-1 by mycelium anastomosing with an efficiency of 21.62%,resulting in decreased mycelial growth rate,conidia yield and germination rate as well as pathogenicity of the host,indicating that the Fa MV1 could impact host biological phenotype and lead to the decline of host pathogenicity.In addition,FAMV1 has a titer effect on host phenotype,and the accumulation of FAMV1 could lead to host degeneration.The biological characteristics and infected fungal viruses of two hypovirulent strains LJX70 and YT54-2 were also studied.With relatively normal colony morphology,growth rate and asexual sporulation,LJX70 was not a typical hypovirulent strain.The results of RT-PCR and Viral Metagenome sequencing indicate that LJX70 was co-infected by a new mycovirus Fusarium asiaticum Bunyavirus 1(Fa BV1/LJX70)and a reported virus Fusarium graminearum negative-stranded RNA virus1(Fg NRV1).We can infer confidently that it is Fa BV1/LJX70 that caused the hypovirulence of strain LJX70 as previous research reports indicated that Fg NRV1 does not affect the virulence of its F.graminearum host.Phylogenetic analysis of Rd Rp indicated that Fa BV1/LJX70 clustered with several unclassified Bunyavirales viruses in a branch on the periphery of the Bunyavirales viruses,indicating that Fa BV1/LJX70 represents a new group of Bunyavirales viruses,it was suggestted that a new genus or even a new family of Bunyavirales should be established to accommodate such viruses.Strain YT54-2 is a hypovirulent strain with the obvious abnormal colony morphology,slow growth rate,and poor asexual and sexual sporulation.Metavirome sequencing and RT-PCR results indicate that strain YT54-2 is co-infected by a new virus discovered in this study(Fa VLV1)and two reported viruses: Betachrysovirus fugramineari(trival name:Fusarium graminearum chrysovirus)and Fg NRV1.Blast X results showed that the protein coded by RNA5(P5)share the lowest similarity in 5 segments among three Fusarium graminearum chrysovirus strains and C-terminus of P5 of Fusarium chrysovirus YT54-2(FCV-YT54-2,the chrysovirus strain infecting YT54-2)contains13 C2H2 zinc fingers.According to our study combined with the reported literature,it was speculated confidently that Fa VLV1 and FCV-YT54-2 were the hypovirulent factors of strain YT54-2.In this study,we analyzed the mycoviral diversity of three groups of Fusarium species isolated from SC,CJML,and the HHH region in China,as well as three single hypovirulent strains from SC.Ten noval mycoiruses were discovered,a novel Martellivirales virus Fa VLV1 from strain BZ6 was first discovered in wheat fusarium species,two hypovirulence associated factors: Fa VLV1 and Fa MV1,for strain BZ6 were corroborated,the accumulation of Fa MV1 lead to fungal host degeneration was discovered,and two candidate hypovirulence associated factor: Bunyavirales virus Fa BV1/LJX70 and Betachrysovirus FCV-YT54-2 were identified from relatively normal strain LJX70 and obviously abnormal strain YT54-2,respectively.This study not only helps expand the diversity of mycoviruses and potential virocontrol agent resources such as Fa VLV1,Fa MV1,Fa BV1/LJX70,and FCV-YT54-2,but also provides new materials to investigate the mechanism involved in multiple infection and hypovirulence of mycoviruses.