Characterization of the RNA Mycovirome Associated with Grapevine Fungal Pathogens: Analysis of Mycovirus Distribution and Their Genetic Variability within a Collection of Botryosphaeriaceae Isolates.

Botryosphaeriaceae are fungi involved in the decay of various woody species, including the grapevine, leading to significant production losses. This fungal family is largely ubiquitous, and seven species of Botryosphaeriaceae have been identified in French vineyards, with variable levels of aggressiveness, both in vitro and in planta. Mycoviruses can impact the life traits of their fungal hosts, including aggressiveness, and are one of the factors influencing fungal pathogenicity. In this study, the RNA mycovirome of fifteen Botryosphaeriaceae isolates was characterized through the high-throughput sequencing of double-stranded RNA preparations from the respective samples. Eight mycoviruses were detected, including three potential novel species in the Narnaviridae family, as well as in the proposed Mycobunyaviridae and Fusagraviridae families. A large collection of Botryosphaeriaceae isolates was screened using RT-PCR assays specific for 20 Botryosphaeriaceae-infecting mycoviruses. Among the mycoviruses detected, some appeared to be specialists within a single host species, while others infected isolates belonging to multiple Botryosphaeriaceae species. This screening allowed us to conclude that one-third of the Botryosphaeriaceae isolates were infected by at least one mycovirus, and a significant proportion of isolates (43.5%) were found to be coinfected by several viruses, with very complex RNA mycoviromes for some N. parvum isolates.

Characterization of the Mycovirome of the Phytopathogenic Fungus, Neofusicoccum parvum.

Neofusicoccum parvum is a fungal plant-pathogen belonging to the family Botryosphaeriaceae, and is considered one of the most aggressive causal agents of the grapevine trunk disease (GTD) Botryosphaeria dieback. In this study, the mycovirome of a single strain of N. parvum (COLB) was characterized by high throughput sequencing analysis of total RNA and subsequent bioinformatic analyses. Contig annotations, genome completions, and phylogenetic analyses allowed us to describe six novel mycoviruses belonging to four different viral families. The virome is composed of two victoriviruses in the family Totiviridae, one alphaendornavirus in the family Endornaviridae, two mitoviruses in the family Mitoviridae, and one narnavirus belonging to the family Narnaviridae. The presence of the co-infecting viruses was confirmed by sequencing the RT-PCR products generated from total nucleic acids extracted from COLB. This study shows that the mycovirome of a single N. parvum strain is highly diverse and distinct from that previously described in N. parvum strains isolated from grapevines.

Molecular characterization of a novel fusarivirus infecting the plant-pathogenic fungus Neofusicoccum luteum.

Double-stranded RNAs from an isolate of Neofusicoccum luteum collected from grapevines were analyzed by high-throughput sequencing. Contig annotations revealed the presence of a potential novel virus belonging to the newly proposed family Fusariviridae. Completion of the viral genome sequence was performed. The genome is 6,244 nucleotide long, excluding the poly(A) tail and contains two putative open reading frames (ORFs). The first one encodes a large polypeptide of 1,552 amino acids (aa) with conserved RNA-dependent RNA polymerase and helicase domains typical of viral replicases. The second ORF encodes a putative 475-aa-long polypeptide showing weak homology to the corresponding ORF of Macrophomina phaseolina single-stranded RNA virus 1, for which no function is known so far. Phylogenetic analyses indicated that this virus should be considered a novel mycovirus belonging to the proposed family Fusariviridae, for which the name "Neofusicoccum luteum fusarivirus 1" (NlFV1) is proposed.

Determination of the complete genomic sequence of Neofusicoccum luteum mitovirus 1 (NLMV1), a novel mitovirus associated with a phytopathogenic Botryosphaeriaceae.

Neofusicoccum luteum species belongs to the Botryosphaeriaceae family and is involved in grapevine wood decay diseases. The present study reports the discovery and the molecular characterization of a novel mitovirus infecting this fungus. Double-stranded RNAs were purified from cultivated N. luteum and analysed by next generation sequencing. Using contigs showing BlastX homology with the RNA-dependent RNA polymerase (RdRp) gene of various members of the family Narnaviridae, a single contig of approximately 1.2 kb was constructed. The genomic sequence was completed and phylogenetic analyses indicated that this virus represents a new member of the genus Mitovirus, for which the name of "Neofusicoccum luteum mitovirus 1" is proposed. The genome is 2,389 nucleotides long and, based on the fungal mitochondrial genetic code, it encodes a putative protein of 710 amino acids, homologous to the RdRps of members of the Narnaviridae family. The neofusicoccum luteus mitovirus 1 (NLMV1) RdRp contains the six conserved motifs previously reported for mitoviral RdRps. Our findings represent the first evidence that a mycovirus can infect N. luteum, an important pathogenic fungus of grapevine.

A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds.

Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate their incidence levels. In the present study, we designed two sets of primers that target the ß-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across ß-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. lata during the dormant season. Experimental grapevines were located in two counties of northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation method, respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata-complex in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.

Phylogenetic and experimental evidence for host-specialized cryptic species in a biotrophic oomycete.

Assortative mating resulting from host plant specialization has been proposed to facilitate rapid ecological divergence in biotrophic plant pathogens. Downy mildews, a major group of biotrophic oomycetes, are prime candidates for testing speciation by host plant specialization. Here, we combined a phylogenetic and morphological approach with cross-pathogenicity tests to investigate host plant specialization and host range expansion in grapevine downy mildew. This destructive disease is caused by Plasmopara viticola, an oomycete endemic to North America on wild species and cultivated grapevines. Multiple genealogies and sporangia morphology provide evidence that P. viticola is a complex of four cryptic species, each associated with different host plants. Cross-inoculation experiments showed complete host plant specialization on Parthenocissus quinquefolia and on Vitis riparia, whereas cryptic species found on V. aestivalis, V. labrusca and V. vinifera were revealed to be less specific. We reconstructed the recent host range expansion of P. viticola from wild to cultivated grapevines, and showed that it was accompanied by an increase in aggressiveness of the pathogen. This case study on grapevine downy mildew illustrates how biotrophic plant pathogens can diversify by host plant specialization and emerge in agrosystems by shifting to cultivated hosts. These results might have important implications for viticulture, including breeding for resistance and disease management.