Blue-White Colony Selection of Virus-Infected Isogenic Recipients Based on a Chrysovirus Isolated from Penicillium italicum

Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum(P. italicum). In this study, we isolated and characterized a new double-stranded RNA(ds RNA) virus, designated Penicillium italicum chrysovirus 1(Pi CV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that Pi CV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1 Dpks P defective in the polyketide synthase gene(pks P), which is involved in pigment biosynthesis, and these mutants formed albino(white) colonies. Then we applied hyphal anastomosis method to horizontally transmit Pi CV1 from the white virus-donors(i.e., HSPi-YN1 mutants) to wild-type recipients(i.e., P.italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable Pi CV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic ds RNA profile analysis. This bluewhite colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized Pi CV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen Pi CV1-infected isogenic recipients.