Exploration And Function Research Of Mycovirus From Trichoderma Spp.

Mycovirus or Fungal virus is a type of virus that infects filamentous fungi,mushrooms,yeast and replicates in them.Trichoderma spp.are important biocontrol fungi.Trichoderma mycoviruses remain poorly researched in general.To explore mycovirus resources of Trichoderma spp.,analysing their genomic structures and functions,and researching their diversities,especially the effect of mycoviruses on the biocontrol and biological properties of Trichoderma spp.have the important theoretical and practical value.In this experiment,there were four strains of Trichoderma containing mycovirus screened from the Trichoderma resources isolated from soil from Xinjiang and Inner Mongolia.Two mycoviruses had been completed genome sequencing from the Trichoderma harzianum strain 525（T525）and T.harzianum strain 137（T137）.And their genomic structures and taxonomic status were also analyzed.In addition,the biological characteristics of mycoviruses,mode of transmission and the effects on the biocontrol functions of Trichoderma spp.were all evaluated.This study enriched the resources of endophytic viruses of Trichoderma spp.and provided insights and resources for the classification of mycoviruses.The main results were as the followings.1.The screening of Trichoderma strains possibly possessing mycoviruses.Through the analysis of the differences on the morphology and the antagonism of Trichoderma spp.from the soil collected from Xinjiang and Inner Mongolia in 2014-2016,163 strains of Trichoderma spp.were selected out to be extracted for dsRNA,and from which the four strains（T525,T137,T444 and T111）were possibly possessing the mycoviruses.T525,T137,T444 belonged to Trichoderma harzianum,while T111 belonged to Trichoderma rossicum.2.The genome sequences of two explored mycoviruses and the analysis of the classification statuses.Through the Metatranscriptomic sequencing,most of the genome fragments of two mycoviruses from T525 and T137 respectively were obtained,and the 5’and 3’terminals of the two mycovirus were obtained by RACE technology,and the complete genome of the viruses were obtained by assembling the sequences,and then the genomic structures of the two mycoviruses were analyzed.The mycovirus from T525 has double-stranded RNA（dsRNA）with a complete genome sequence of 3160 base pairs and two open reading frames（ORFs）on the negative strand.ORF A encodes an RdRP,ORF B encodes a putative protein or coat protein.Phylogenetic analysis indicated the mycovirus was a new virus that belonged to an unclassified family of dsRNA mycoviruses,named as Trichoderma harzianum mycovirus 1（ThMV1）.The mycovirus of T137 contained two genomic segments,one segment was 2088 bp and another segment was 1634 bp.Each segment contained a single open reading frame.ORF1 was predicted to encode the RdRP.The C-terminal domain and catalytic palm subdomain were detected in the regions encompassing amino acids 300-448 and 326-454 in ORF1.ORF2 was predicted to encode a hypothetical protein.The 5’UTRs of dsRNA1 and dsRNA2 had the same conserved element（CUGAGUUAACAAGCCAC UGUUUUACUCUCGU）necessary for virus replication.Through phylogenetic analysis,we suggested it as a novel unclassified dsRNA mycovirus species and named it as Trichoderma harzianum bipartite mycovirus 1（ThBMV1）.3.The transmission characteristics of the mycoviruses between Trichoderma strains with nutritional incompatibility.Through the dual culture assay,T.harzianum strain 989（T989）was screened out with the characteristics of nutritional incompatibility with T525 and T137 respectively.By the protoplast transformation,T.harzianum strain 989^hyg containing hygromycin resistance gene(T989^hyg)was obtained.The experiment of dual-culture of T989^hyg with T525 and T137 respectively showed that neither of the two viruses could overcome the nutritional incompatibility to transmit to T989^hyg.4.The effect of mycovirues on the biocontrol capability of Trichoderma.Research platform of mycovirus elimination was constructed by the combination methods of protoplast transformation and Ribavirin,and T525 virus-free strain（T525-F）was gotten.The differences between T525 and T525-F mycelia had been shown on the growth rate,colony morphology and biomasses,especially the significant differences were focus on the mycelial growth rate and biomass.The antagonisms between T525 and T525-F to three pathogens fungi（F.oxysporum f.sp.vasinfectum,B.cinerea and Fusari-um oxysporum f.sp.cucumerinum）were no obvious differences.The effect of mycovirus on control effectivity of T525 to Fusarium Wilt of cucumber seedlings caused by Fusarium oxysporum f.sp.cucumerinum was reduced but promoted the growth of cucumber seedlings,which was the first focus on the effect of mycovirus to the biocontrol function of fungus in the world.