Clone And Characterization Of A Mycovirus From Bothyosphseria Dothidea Strain XA-3

Botryosphaeria dothida(Moug:Fr) Ces﹠de Not is an important pathogenic fungus causing fruit rot, leaf and stem ring spots and die-back, stem canker, stem death or stool mortality and decline of apple trees. In the present research, a strain XA-3 of B. dothidea was obtained from apple showing stem ring spot symptoms, and showed normal growth rate and colony morphologies but attenuated virulence.Its genome was composed of five segments of dsRNA(double-stranded RNA), with sizes of 2399,2188,1965,1131 and 1059 bp, respectively. With each dsRNA containing a single ORF(Open Reading Frame Finder) in one of the strands, i.e., ORF1 to ORF5. Five segments of dsRNA was 99-100% sequence similarities with Botryosphaeria dothidea RNA virus 1(BdRV1), showing that they should be the same virus.The ORF to ORF5 putatively encoded proteins of 758, 694, 613, 275 and 279 amino acids(aa) with cal-culated molecular masses of 82, 75, 66, 28, and 30 kDa, respectively. The ORF1, 3 and 4 was predicted to encode RdRp( RNA-dependent RNA polymerase),Methyltransferase(MET) and coat protein, respectively. However, the functions of the putative protein encoded by ORF2 and 5 were unknown. The 5’untranslated regions(5’ UTRs) of the coding strands of dsRNA1 to dsRNA5 were 53-, 70-, 57-, 89-, and 98-nt long, respectively, and shared 51 to 83% identity with each other and contained conserved sequences(CGATTAAAA). While the corresponding 3’ UTRs were 69-, 33-, 66-, 214- and 121-nt long,respectively, and shared 54 to 66% identity with each other and contained sequences conserved in the five dsRNAs, contained conserved sequences(GGGG). Moreover, phylogenetic reconstruction of the complete sequence of the RdRp encoded by ORF1 together with Aspergillus fumigatus tetramycovirus-1,was clustered separately from other dsRNA viruses in an intermediate position between dsRNA and(+) ssRNA viruses. It suggests that the virus appears in the early stages of viral evolution from(+) ssRNA to dsRNA virus.Several approaches were applied to with try to eliminate mycoviruses from the isolate XA-3, including single conidial isolation, hyphae-tipping, hyphae-tipping and chemotherapy, protoplast isolation and regeneration. Regardless of the treatments applied, the dsRNA elements were detected,suggesting that BdRV1 is stably associated with B. dothidea. The results that the virus could be detected in conidia showed that the virus could be vertically transmitted though conidia. Confront culture suggested that the virus could be transmitted into strain MAO-2 from XA-3, but not into another strain MAO-1 genetically closer to MAO-2(one nucleotide mutation between their ITS sequences). Instead, the virus could be transmitted into strain MAO-1 from XA-3 mediated by MAO-2. The results showed that the virus was horizontally transmitted depending on the strains. Comparative biological characteristics of the strains infected and uninfected by the virus showed that the virus did not change the host growth rate and colony morphologies, and had no significant impact on the virulence. The study on BdRV-1 viral provides new insights for further studies on mycovirus diversity and evolution.