To reveal mycovirus diversity, we conducted a search of as-yet-unexplored Mediterranean isolates of the phytopathogenic ascomycete Rosellinia necatrix for virus infections. Of seventy-nine, eleven fungal isolates tested RNA virus-positive, with many showing coinfections, indicating a virus incidence of 14%, which is slightly lower than that (approximately 20%) previously reported for extensive surveys of over 1000 Japanese R. necatrix isolates. All viral sequences were fully or partially characterized by Sanger and next-generation sequencing. These sequences appear to represent isolates of various new species spanning at least 6 established or previously proposed families such as Partiti-, Hypo-, Megabirna-, Yado-kari-, Fusagra- and Fusarividae, as well as a newly proposed family, Megatotiviridae. This observation greatly expands the diversity of R. necatrix viruses, because no hypo-, fusagra- or megatotiviruses were previously reported from R. necatrix. The sequence analyses showed a rare horizontal gene transfer event of the 2A-like protease domain between a dsRNA (phlegivirus) and a positive-sense, single-stranded RNA virus (hypovirus). Moreover, many of the newly detected viruses showed the closest relation to viruses reported from fungi other than R. necatrix, such as Fusarium spp., which are sympatric to R. necatrix. These combined results imply horizontal virus transfer between these soil-inhabitant fungi.

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Strawberry ( Fragaria × ananassa ) is a major food crop worldwide, due to the flavor, aroma and health benefits of the fruit, but its productivity and quality are seriously limited by a large variety of phytopathogens, including Colletotrichum spp. So far, key factors regulating strawberry immune response remain unknown. The FaWRKY1 gene has been previously proposed as an important element mediating defense responses in strawberry to Colletotrichum acutatum . To get further insight into the functional role that FaWRKY1 plays in the defense mechanism, Agrobacterium -mediated transient transformation was used both to silence and overexpress the FaWRKY1 gene in strawberry fruits ( Fragaria × ananassa cv. Primoris), which were later analyzed upon C. acutatum inoculation. Susceptibility tests were performed after pathogen infection comparing the severity of disease between the two agroinfiltrated opposite halves of the same fruit, one half bearing a construct either for FaWRKY1 overexpression or RNAi-mediated silencing and the other half bearing the empty vector, as control. The severity of tissue damage was monitored and found to be visibly reduced at five days after pathogen inoculation in the fruit half where FaWRKY1 was transiently silenced compared to that of the opposite control half and statistical analysis corroborated a significant reduction in disease susceptibility. Contrarily, a similar level of susceptibility was found when FaWRKY1 overexpression and control fruit samples, was compared. These results unravel a negative regulatory role of FaWRKY1 in resistance to the phytopathogenic fungus C. acutatum in strawberry fruit and contrast with the previous role described for this gene in Arabidopsis as positive regulator of resistance against the bacteria Pseudomonas syringae . Based on previous results, a tentative working model for WRKY75 like genes after pathogen infection is proposed and the expression pattern of potential downstream FaWRKY1 target genes was also analyzed in strawberry fruit upon C. acutatum infection. Our results highlight that FaWRKY1 might display different function according to species, plant tissue and/or type of pathogen and underline the intricate FaWRKY1 responsive defense regulatory mechanism taking place in strawberry against this important crop pathogen.

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. ‘Picual’ were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae.