The Strawberry FaWRKY1 Transcription Factor Negatively Regulates Resistance to Colletotrichum acutatum in Fruit Upon Infection.

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.

Viromes in Xylariaceae fungi infecting avocado in Spain.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

A Novel Hypovirus Species From Xylariaceae Fungi Infecting Avocado.

The white rot root disease caused by Rosellinia necatrix is a major concern for avocado cultivation in Spain. Healthy escapes of avocado trees surrounded by diseased trees prompted us to hypothesize the presence of hypovirulent R. necatrix due to mycovirus infections. Recently, we reported the presence of another fungal species, Entoleuca sp., belonging to the Xylariaceae, that was also found in healthy avocado trees and frequently co-infecting the same roots than R. necatrix. We investigated the presence of mycoviruses that might explain the hypovirulence. For that, we performed deep sequencing of dsRNAs from two isolates of Entoleuca sp. that revealed the simultaneous infection of several mycoviruses, not described previously. In this work, we report a new member of the Hypoviridae, tentatively named Entoleuca hypovirus 1 (EnHV1). The complete genome sequence was obtained for two EnHV1 strains, which lengths resulted to be 14,958 and 14,984 nt, respectively, excluding the poly(A) tails. The genome shows two ORFs separated by a 32-nt inter-ORF, and both 5'- and 3'-UTRs longer than any other hypovirus reported to date. The analysis of virus-derived siRNA populations obtained from Entoleuca sp. demonstrated antiviral silencing activity in this fungus. We screened a collection of Entoleuca sp. and R. necatrix isolates and found that EnHV1 was present in both fungal species. A genetic population analysis of EnHV1 strains revealed the presence of two main clades, each of them including members from both Entoleuca sp. and R. necatrix, which suggests intra- and interspecific virus transmission in the field. Several attempts failed to cure Entoleuca sp. from EnHV1. However, all Entoleuca sp. isolates collected from avocado, whether harboring the virus or not, showed hypovirulence. Conversely, all R. necatrix isolates were pathogenic to that crop, regardless of being infected by EnHV1.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive.

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.

Novel, diverse RNA viruses from Mediterranean isolates of the phytopathogenic fungus, Rosellinia necatrix: insights into evolutionary biology of fungal viruses.

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.