This is the first report that describes the existence of a wild-type B. cinerea strain simultaneously infected with two different viral particles types that are possibly related to the hypovirulence of the host fungus. One of the mycoviruses would be a putative partitivirus because of its morphology and particle size, the molecular mass of its capsid protein, its possession of a dsRNA bipartite genome in the molecular size range of members of the Partitiviridae family and its high capsid protein sequence similarity with Fusarium poae dsRNA mycovirus 1. In this context, the initial observations were supported by the fact that the CsCl gradient fractions 1 and 2 contained only particles of 32 nm in diameter associated with dsRNAs of 2.2 kbp. These results confirm that these viral particles contain two dsRNA molecules of approximately 2.2 kbp with identical electrophoretic migration in agarose gel, minimal separation in polyacrylamide gel, and most likely different nucleotide sequences, with one of them encoding for the 70 kDa polypeptide (capsid protein) and the another one for the viral RNA-dependent RNA polymerase (RdRp) .
In fractions 3–8, two types of mycoviruses (32 and 23 nm in diameter) were observed to be associated with five dsRNA molecules. The larger particles (32 nm) were considerably more abundant than the smaller particles (Figure 2C, panel 5 and Figure 3A), which correlated perfectly with the amount of dsRNAs detected in these fractions. In fact, the densitometric analysis of the nucleic acid bands in fractions 4–6 showed that the larger dsRNA band was present in an amount at least five-times greater than that of the sum of all the smaller dsRNAs. At the same time, the 70 kDa polypeptide band was also found in a quantity at least five-times greater than that of the 48 kDa polypeptide. How the virus-host interaction regulates the stoichiometry of both viral particle types is unknown. We do not have a clear explanation for the appearance of the polypeptide band of approximately 65 kDa in fractions 7–10. This band may be a proteolytic fragment originating from the degradation of the 70 kDa polypeptide.
By directly visualizing ultrathin mycelium sections of the fungus with electron microscopy to determine the subcellular location of mycoviruses, we were able to detect both types of viral particles in the same fungal cell (Figure 3B), which is very solid evidence for the co-infection of B. cinerea CCg378 by two different mycoviruses.
Because the intracellular quantity of 32 nm viral particles was considerably greater than the quantity of 23 nm particles in the CCg378 strain, we expected to obtain monosporic clones infected only with the 32 nm virus and containing only the 2.2 kbp dsRNAs when a virus-free fungal strain was transfected with purified viral particles obtained from fraction 5 of the CsCl gradient. Our results with the above mentioned fungal clones demonstrate that it is possible to separate the 32-nm mycovirus and that its stable intracellular maintenance does not depend on the presence of the 23 nm particles or the smaller dsRNA molecules (Figure 3C and D).
The physiological parameters associated with the virulence, laccase activity and sporulation rate of the CCg378 strain were the lowest compared to those of B. cinerea CKg54, a virus-free strain, and B. cinerea CKg54vi378 (a clone infected only with the 32-nm mycovirus). Suppressed laccase activity is a common phenotypical change associated with hypovirus infection in C. parasitica [36, 37] and Diaphorte ambigua . It has been suggested that laccase secreted by B. cinerea may act as a detoxifying enzyme to protect the fungus from the toxic metabolites formed during pathogenesis and to reduce the host’s lignification activity . In fact, B. cinerea laccase is able to degrade pterostilbene and resveratrol, two phytoalexins from grapes . In addition, it has been shown that the repression of laccase production transforms B. cinerea into a weak pathogen , confirming the importance of this enzyme in the virulence of this fungus . Both the laccase activity and sporulation rate are physiological parameters that have been used as virulence parameters in other dsRNA-containing phytopathogenic fungi [34, 41, 42].
Our results show that B. cinerea CCg378 is infected by two different dsRNA mycoviruses. Experimental evidence suggests that the genome of the 32-nm mycovirus consist of the two dsRNAs of about 2.2 kbp. Furthermore, it is known that the monocistronic dsRNAs of the partitiviruses are encapsidated separately in identical capsids, one of them containing the dsRNA encoding the capsid protein and the other the dsRNA encoding the RdRp . We presented confirmatory evidence that the 32 nm mycovirus is a new member of the partitiviruses, since the sequence analysis of the cDNA corresponding to the gene of the capsid protein showed high similarity with members of the Partitiviridae family. On the other hand, the genome of the 23-nm mycovirus would correspond to one, two or the three smaller dsRNA molecules; however, due to the small size of the virion, they should be separately encapsidated. Another possibility is that some of the smaller dsRNAs correspond to a satellite dsRNA or to defective-interfering dsRNA (DI-dsRNAs), a common feature of mycovirus infections . Atkinsonella hypoxylon, Discula destructiva, Gremmeniella abietina and Rosellinia necatrix [43, 44] are some fungal species infected with partitiviruses in which satellite dsRNAs or DI-dsRNAs have been detected. Therefore, it is clear that the genome composition of the 23-nm mycovirus remains to be elucidated.
Finally, our results suggest that the low virulence of the CCg378 strain may be partially caused by the presence and/or gene expression of the putative 32-nm partitivirus because the infected clone CKg54vi378 presents an intermediate virulence between that of the CKg54 and CCg378 strains. Therefore, the complete hypovirulence phenotype would be conferred by the presence of both mycoviruses and/or by the expression of both mycoviral genomes with the consistent generation of gene products that regulate the attenuation of host fungus virulence at the molecular level.