Identification And Characterization Of Two Novel Dsrna Viruses From Penicillium Digitatum

Citrus green mood caused by Penicillium digitatum is one of the major destructive pathogen of citrus fruits, triggering fruit decay during post-harvest packaging, storing and transportation, thus causing extremely economic losses. Drug-resistant strains appeared due to excessive use of chemical fungicides,e.g., triazoles fungicides,which urges the exploitation of more effective and sustainable biocontrol method. Mycovirus is a kind of virus which can infect and replicate in fungi, some of them can cause hypovirulence of their host fungi and have great potential for controlling the plant pathogenic fungi. While there is no mycovirus isolated from P. digitatum being reported until now, thus isolating and analyzing mycovirus from P. digitatum is likely to provide new proposal for controlling citrus green mold. Meanwhile, this experiment may give reference data for the evolution and taxonomy of mycovirus as well as mechanism of fungi-mycovirus interaction system.In this paper, two mycoviruses were isolated and identified from P. digitatum. Their biological effect on host fungi has also been analyzed to explore their potential for biological control. All major results are listed as following.1 A dsRNA about 5kb long was detected in P. digitatum strain HS-RH1. The full-length cDNA of it was made and sequenced, which revealed that the whole sequence of the dsRNA is 5211 bp and was submitted to GenBank with accession number KU933932. The dsRNA is predicted to contain two open reading frame (ORF) encoding Coat Protein (CP) and RNA-dependent RNA polymerase (RdRp) respectively according to NCBI BLAST. Multiple alignments and phylogenetic analysis using the amino acid sequences of CP and RdRp indicated that the dsRNA is the genome of mycovirus belonging to the Victorivirus genus in Totiviridae. A H-type pseudoknot structure is predicted to located at 38nt directly upstream of an AUGA motif containing CP stop codon and RdRp start codon. This genomic characteristic is coincide with members of Victorivirus genus. Viral particles observation using TEM showed that it has spherical particles about 35 nm in diameter within the range reported for Totiviridae members.This mycovirus was named P. digitatum virus 1 (PdV1).2 In comparison, HS-RH1 generates a much smaller sized colony on PDA than a standard P. digitatum strain HS-F6, the mycelial growth of HS-RH1 on the citrus fruit's surface is lower than that of HS-F6 as well. A virus-cured strain HS-RH1F was obtained using protoplast regeneration and ribavirin treatment, and a transfected strain HS-F6V was obtained by infecting HS-F6 with PdV1 virion. Under the same growth condition(25?, cultivated 4d), the colony size on PDA and the lesion diameter on citrus fruit of HS-RH1 are 9.04cm2 and 0.78 cm. they are much smaller than that of HS-RH1F(17.35cm2 and 4.89cm). Meanwhile, the colony area of HS-F6V was 14.29 cm2, which was almost 30% smaller than that of HS-F6 (20.53 cm2). Besides, the lesion diameter of HS-F6 decreased after infected with PdV1 from 5.76cm to 3.95cm.3 Five dsRNAs (dsRNA 1-5) were detected from strain HS-RH2 and subjected to sequencing and BLAST analysis, the result showed that dsRNA 1-3 and dsRNA 5 are the genomic components of a novel member of the family Polymycoviridae (tentatively designated P. digitatum polymycovirus 1 (PdPmV1)); while dsRNA4 is the genomic component of a novel member of the family Narnaviridae (tentatively designated P.digitatum Narna-like virus 1 (PdNLV1)). This paper studied PdPmV1 in depth and the full-length of its four dsRNAs are 2382bp, 2336bp, 2016bp and 1292bp respectively.Each of them contains an ORF, encoding an RdRP (766aa, 83.8kDa), a hypothetical protein (695aa, 75.4kDa),a methyltransferase (612aa, 66.5kDa) and a P-A-S-rich protein(261aa,27.3kDa).4 The infection of PdPmV1 and PdNLV1 has no significant impact on the growth rate and virulence of P. digitatum. The susceptibility to prochloraz of strains infected with PdPmV1 and PdNLV1 was measured. The prochloraz EC50 of PdPmV1 and PdNLV1 infected strains HS-F61 and HS-E91 are 3.809mg/L and 0.639mg/L respectively,which is significant lower than that of their isogenome strains HS-F6 and HS-E9(7.896mg/L, 0.939mg/L). Moreover, the virulence of HS-F61 and HS-E91 is weaker than that of HS-F6 and HS-E9 under the same concentration of prochloraz.