The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi.

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing of near-isogenic lines of B. cinerea was used to map a vic locus to a 60-kb region of the genome. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus.

The Potential of Molecular Indicators of Plant Virus Infection: Are Plants Able to Tell Us They Are Infected?

To our knowledge, there are no reports that demonstrate the use of host molecular markers for the purpose of detecting generic plant virus infection. Two approaches involving molecular indicators of virus infection in the model plant Arabidopsis thaliana were examined: the accumulation of small RNAs (sRNAs) using a microfluidics-based method (Bioanalyzer); and the transcript accumulation of virus-response related host plant genes, suppressor of gene silencing 3 (AtSGS3) and calcium-dependent protein kinase 3 (AtCPK3) by reverse transcriptase-quantitative PCR (RT-qPCR). The microfluidics approach using sRNA chips has previously demonstrated good linearity and good reproducibility, both within and between chips. Good limits of detection have been demonstrated from two-fold 10-point serial dilution regression to 0.1 ng of RNA. The ratio of small RNA (sRNA) to ribosomal RNA (rRNA), as a proportion of averaged mock-inoculation, correlated with known virus infection to a high degree of certainty. AtSGS3 transcript decreased between 14- and 28-days post inoculation (dpi) for all viruses investigated, while AtCPK3 transcript increased between 14 and 28 dpi for all viruses. A combination of these two molecular approaches may be useful for assessment of virus-infection of samples without the need for diagnosis of specific virus infection.

A novel chrysovirus from a clinical isolate of Aspergillus thermomutatus affects sporulation.

A clinical isolate of Aspergillus thermomutatus (Teleomorph: Neosartorya pseudofischeri) was found to contain ~35 nm isometric virus-like particles associated with four double-stranded (ds) RNA segments, each of which coded for a single open reading frame. The longest dsRNA element (3589 nt) encodes a putative RNA-dependent RNA polymerase (1114 aa), the second longest dsRNA element (2772 nt) encodes a coat protein (825 aa), and the other two dsRNAs (2676 nt, 2514 nt) encode hypothetical proteins of 768 aa and 711 aa, respectively. Phylogenetic analysis of the amino acid sequences showed 41-60% similarity to the proteins coded by the dsRNAs of the most closely related virus, Penicillium janczewskii chrysovirus 2, indicating that it is a new species based on the International Committee on Taxonomy of Viruses criteria for the genus Chrysovirus. This is the first virus reported from A. thermomutatus and was tentatively named Aspergillus thermomutatus chrysovirus 1. A virus free line of the fungal isolate, cured by cycloheximide treatment, produced large numbers of conidia but no ascospores at both 20°C and 37°C, whereas the virus infected line produced ten-fold fewer conidia at 20°C and a large number of ascospores at both temperatures. The effects of the virus on fungal sporulation have interesting implications for the spread of the fungus and possible use of the virus as a biological control agent.

The Effect of Aspergillus Thermomutatus Chrysovirus 1 on the Biology of Three Aspergillus Species.

This study determined the effects of Aspergillus thermomutatus chrysovirus 1 (AthCV1), isolated from Aspergillus thermomutatus, on A. fumigatus, A. nidulans and A. niger. Protoplasts of virus-free isolates of A. fumigatus, A. nidulans and A. niger were transfected with purified AthCV1 particles and the phenotype, growth and sporulation of the isogenic AthCV1-free and AthCV1-infected lines assessed at 20 °C and 37 °C and gene expression data collected at 37 °C. AthCV1-free and AthCV1-infected A. fumigatus produced only conidia at both temperatures but more than ten-fold reduced compared to the AthCV1-infected line. Conidiation was also significantly reduced in infected lines of A. nidulans and A. niger at 37 °C. AthCV1-infected lines of A. thermomutatus and A. nidulans produced large numbers of ascospores at both temperatures, whereas the AthCV1-free line of the former did not produce ascospores. AthCV1-infected lines of all species developed sectoring phenotypes with sclerotia produced in aconidial sectors of A. niger at 37 °C. AthCV1 was detected in 18% of sclerotia produced by AthCV1-infected A. niger and 31% of ascospores from AthCV1-infected A. nidulans. Transcriptome analysis of the naturally AthCV1-infected A. thermomutatus and the three AthCV1-transfected Aspergillus species showed altered gene expression as a result of AthCV1-infection. The results demonstrate that AthCV1 can infect a range of Aspergillus species resulting in reduced sporulation, a potentially useful attribute for a biological control agent.

Taxonomy of the order Mononegavirales: update 2017.

In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

First complete genome sequence of vanilla mosaic strain of Dasheen mosaic virus isolated from the Cook Islands.

We present the first complete genome of vanilla mosaic virus (VanMV). The VanMV genomic structure is consistent with that of a potyvirus, containing a single open reading frame (ORF) encoding a polyprotein of 3139 amino acids. Motif analyses indicate the polyprotein can be cleaved into the expected ten individual proteins; other recognised potyvirus motifs are also present. As expected, the VanMV genome shows high sequence similarity to the published Dasheen mosaic virus (DsMV) genome sequences; comparisons with DsMV continue to support VanMV as a vanilla infecting strain of DsMV. Phylogenetic analyses indicate that VanMV and DsMV share a common ancestor, with VanMV having the closest relationship with DsMV strains from the South Pacific.

Differential distribution and titre of selected grapevine leafroll-associated virus 3 genetic variants within grapevine rootstocks.

In this study of three grapevine leafroll-associated virus 3 (GLRaV-3) genetic variants in two grapevine rootstock hosts, GLRaV-3 detection was shown to be affected by the virus distribution, titre, and the genetic variant. Group VI and NZ2 GLRaV-3 variants had reduced detectability compared with the group I variant. Differences in the genomic and subgenomic RNA (sgRNA) expression levels, and differences in the level of expression between the genetic variants were also observed. The observed differences in virus titre and sgRNA expression levels suggest differences in plant-virus interactions by the various GLRaV-3 genetic variants.

Molecular characterisation of a novel recombinant Ribgrass mosaic virus strain FSHS.

BACKGROUND: The genus Tobamovirus (Virgaviridae) comprises 33 accepted species with the recent addition of eight new viruses and is divided in to three subgroups based on the origin of assembly of the virion and host range. Within the subgroup 1 tobamoviruses the orchid-associated tobamovirus was hypothesized to be a chimeric derivative of recombinations between genome fragments from subgroup 3 and 1. Recombination events involving RdRp, movement and coat protein genes are recorded within subgroup 1 and 2. However natural recombinations have not previously been reported between subgroup 3 tobamoviruses. FINDINGS: The organization and phylogenetic analyses of the complete genome and the different ORFs placed the new isolate within the Ribgrass mosaic virus clade of subgroup 3 tobamoviruses. Recombination detection analyses indicated that the isolate was a chimeric genome with fragments of high similarity to Ribgrass mosaic virus (RMV) strains NZ-439 (HQ667978) and Actinidia-AC (GQ401365.1) infecting herbaceous Plantago sp. and woody Actinidia spp., respectively. The recombinant differed across the whole genome by 3-8 % from other published RMV genomes. CONCLUSION: In this investigation we report an intra-specific recombination between RMV strains NZ-439 (HQ667978) and Actinidia-AC (GQ401365.1), in the replicase component between viral-methyltransferase and viral-helicase regions, resulting in a novel RMV strain FSHS (JQ319720.1) that represents the first described natural recombinant within the RMV cluster of subgroup 3 tobamoviruses.

Comparison of Illumina de novo assembled and Sanger sequenced viral genomes: A case study for RNA viruses recovered from the plant pathogenic fungus Sclerotinia sclerotiorum.

The advent of 'next generation sequencing' (NGS) technologies has led to the discovery of many novel mycoviruses, the majority of which are sufficiently different from previously sequenced viruses that there is no appropriate reference sequence on which to base the sequence assembly. Although many new genome sequences are generated by NGS, confirmation of the sequence by Sanger sequencing is still essential for formal classification by the International Committee for the Taxonomy of Viruses (ICTV), although this is currently under review. To empirically test the validity of de novo assembled mycovirus genomes from dsRNA extracts, we compared the results from Illumina sequencing with those from random cloning plus targeted PCR coupled with Sanger sequencing for viruses from five Sclerotinia sclerotiorum isolates. Through Sanger sequencing we detected nine viral genomes while through Illumina sequencing we detected the same nine viruses plus one additional virus from the same samples. Critically, the Illumina derived sequences share >99.3 % identity to those obtained by cloning and Sanger sequencing. Although, there is scope for errors in de novo assembled viral genomes, our results demonstrate that by maximising the proportion of viral sequence in the data and using sufficiently rigorous quality controls, it is possible to generate de novo genome sequences of comparable accuracy from Illumina sequencing to those obtained by Sanger sequencing.

Complete genome sequence of Colocasia bobone disease-associated virus, a putative cytorhabdovirus infecting taro.

We report the first genome sequence of a Colocasia bobone disease-associated virus (CBDaV) derived from bobone-affected taro [Colocasia esculenta L. Schott] from Solomon Islands. The negative-strand RNA genome is 12,193 nt long, with six major open reading frames (ORFs) with the arrangement 3'-N-P-P3-M-G-L-5'. Typical of all rhabdoviruses, the 3' leader and 5' trailer sequences show complementarity to each other. Phylogenetic analysis indicated that CBDaV is a member of the genus Cytorhabdovirus, supporting previous reports of virus particles within the cytoplasm of bobone-infected taro cells. The availability of the CBDaV genome sequence now makes it possible to assess the role of this virus in bobone, and possibly alomae disease of taro and confirm that this sequence is that of Colocasia bobone disease virus (CBDV).

Characterisation of a novel hypovirus from Sclerotinia sclerotiorum potentially representing a new genus within the Hypoviridae.

A novel mycovirus tentatively assigned the name Sclerotinia sclerotiorum hypovirus 2 (SsHV2/5472) was detected in the phytopathogenic fungus Sclerotinia sclerotiorum. The genome is 14581 nucleotides (nts) long, excluding the poly (A) tail. A papain-like cysteine protease (Pro), an RNA-dependent RNA polymerase (RdRp) and a helicase (Hel) domain were detected in the polyprotein. Phylogenetic analysis based on multiple alignments of the aa sequence of the polyprotein placed it in a distinct clade from Alphahypovirus and Betahypovirus. The distinct aa sequence plus the fact that SsHV2/5472 possesses the longest reported genome for a hypovirus, suggests that SsHV2/5472 may represent a new genus in the family Hypoviridae. Eliminating SsHV2/5472 from S. sclerotiorum significantly increased the virulence of the protoplast virus-free derivative 5472-P5, although SsHV/5472-containing isolates showed significant variation in their virulence. In addition, membrane-bound vesicles (25-50 nm) were observed in ultrathin mycelial sections of SsHV2/5472 containing isolates but not in SsHV2/5472-free isolate.

Molecular characterisation of novel mitoviruses associated with Sclerotinia sclerotiorum.

Seven putative mitoviral genomes, representing four species from three Sclerotinia sclerotiorum isolates, were fully sequenced. The genome lengths ranged from 2438 to 2815 nucleotides. The RNA-dependent RNA polymerase (RdRp) of one genome shared high amino acid (aa) sequence identity (98.5 %) with the previously described Sclerotinia sclerotiorum mitovirus 2 (SsMV2/NZ1) and was provisionally assigned the name SsMV2/14563. The RdRps of three of the genomes with closest aa sequence identity of 78.8-79.3 % to Sclerotinia sclerotiorum mitovirus 1 (SsMV1/KL1) were provisionally considered to represent a new species, and the corresponding virus was named Sclerotinia sclerotiorum mitovirus 5 (SsMV5/11691, SsMV5/14563 and SsMV5/Lu471). The remaining two novel genomes, for which the viruses were provisionally named Sclerotinia sclerotiorum mitovirus 6 (SsMV6/14563 and SsMV6/Lu471) and Sclerotinia sclerotiorum mitovirus 7 (SsMV7/Lu471), showed closest aa sequence identities to Sclerotinia sclerotiorum mitovirus 3 (SsMV3/NZ1; 57.5-57.8 %) and Cryphonectria cubensis mitovirus 1a (CcMV1a; 32 %), respectively. The RdRp proteins of all seven genomes contained the conserved aa sequence motifs (I-IV) previously reported for mitoviruses, and their 5' and 3' untranslated regions (UTRs) have the potential to fold into stem-loop secondary structures.

Molecular characterisation of an endornavirus infecting the phytopathogen Sclerotinia sclerotiorum.

The complete sequence and genome organisation of an endornavirus from the phytopathogenic fungus Sclerotinia sclerotiorum isolate 11691 was described and the name Sclerotinia sclerotiorum endornavirus 1 (SsEV1/11691) proposed. The genome is 10,513 nucleotides (nts) long with a single open reading frame (ORF) that codes for a single polyprotein of 3459 amino acid (aa) residues. The polyprotein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), phytoreo_S7 (S7) and RNA-dependent RNA polymerase (RdRp) domains. The polyprotein and the conserved domains are phylogenetically related to endornaviruses. However, the coding strand of SsEV1/11691 does not contain a site-specific nick characteristic of most previously described endornaviruses. The elimination of SsEV1/11691 did not result in any significant changes in the host phenotype and virulence.

Molecular characterization of a novel victorivirus from the entomopathogenic fungus Beauveria bassiana.

New Zealand isolates of the entomopathogenic fungus Beauveria were examined for the presence of dsRNAs and virus-like particles. Seven out of nine isolates contained one or more high-molecular-weight dsRNAs and all seven contained isometric virus particles ranging in size from 30 to 50 nm. B. bassiana isolate ICMP#6887 contained a single dsRNA band of ~6 kb and isometric virus-like particles of ~50 nm in diameter. Sequencing revealed that the virus from ICMP#6887 had a genome of 5,327 nt with two overlapping ORFs coding for a putative coat protein (CP) and an RNA-dependent RNA-polymerase (RdRp). The sequence showed a highest CP identity of 58.3 % to Tolypocladium cylindrosporum virus 1 (TcV1) and a highest RdRp identity of 48.8 % to Sphaeropsis sapinea RNA virus 1 (SsRV1). Since both TcV1 and SsRV1 belong to the genus Victorivirus, the new virus from B. bassiana ICMP#6887 was tentatively assigned the name Beauveria bassiana victorivirus 1 (BbVV1-6887).

Molecular characterization of three mitoviruses co-infecting a hypovirulent isolate of Sclerotinia sclerotiorum fungus.

Three double-stranded RNAs (dsRNAs) of 2438 nts (A), 2588 nts (B), and 2744 nts (C), from a single isolate of Sclerotinia sclerotiorum were sequenced. All three sequences showed similarity to known mitoviruses, consisting of a single open reading frame (ORF) with the characteristic conserved motifs of RNA-dependent RNA polymerase (RdRp). Mitochondrial malformations and reduced virulence and growth were associated with the presence of the dsRNAs. The terminal sequences of the (+) strand of the three dsRNAs could be folded into stem-loop structures and the inverted terminal complimentary sequences of dsRNA-A potentially form a panhandle structure. Sequence A showed 91.6% aa similarity to the previously described Sclerotinia sclerotiorum mitovirus 2 and was tentatively assigned the acronym SsMV2/NZ1. Sequences B and C showed only 16.4% similarity to each other and 15-48% aa similarity to the previously described mitoviruses and consequently appear to be new mitoviruses.

Characterization of the complete genome of a novel citrivirus infecting Actinidia chinensis.

A ssRNA virus from kiwifruit (Actinidia spp.) was identified as a member of the family Betaflexiviridae. It was mechanically transmitted to the herbaceous indicators Nicotiana benthamiana, N. clevelandii, N. glutinosa and N. occidentalis. The complete genome was comprised of three ORFs and a 3'poly (A) tail. Phylogenetic analysis of the entire genome indicated it was a novel member of the genus Citrivirus (family Betaflexiviridae). The complete nucleotide sequence differed from that of citrus leaf blotch virus (CLBV) by ~ 26 %. The movement protein (ORF2) and coat protein (ORF3) shared 95-96 % and 90-92 % amino acid sequence identity, respectively, with CLBV. The replicase polyprotein (ORF1) was distinctly different from published CLBV sequences, with 78-79 % amino acid sequence identity, while the 5' UTR and 3' UTR differed from CLBV by 28 % and 29 %, respectively. The sequence differences indicate that the citrivirus from Actinidia is either a divergent strain of CLBV or a member of a new citrivirus species.

Viruses of botrytis.

Botrytis cinerea (gray mold) is one of the most widespread and destructive fungal diseases of horticultural crops. Propagation and dispersal is usually by asexual conidia but the sexual stage (Botryotinia fuckeliana (de Bary) Whetzel) also occurs in nature. DsRNAs, indicative of virus infection, are common in B. cinerea, but only four viruses (Botrytis virus F (BVF), Botrytis virus X (BVX), Botrytis cinerea mitovirus 1 (BcMV1), and Botrytis porri RNA virus) have been sequenced. BVF and BVX are unusual mycoviruses being ssRNA flexous rods and have been designated the type species of the genera Mycoflexivirus and Botrexvirus (family Betaflexivirdae), respectively. The reported effects of viruses on Botrytis range from negligible to severe, with Botrytis cinerea mitovirus 1 causing hypovirulence. Little is currently known about the effects of viruses on Botrytis metabolism but recent complete sequencing of the B. cinerea genome now provides an opportunity to investigate the host-pathogen interactions at the molecular level. There is interest in the possible use of mycoviruses as biological controls for Botrytis because of the common problem of fungicide resistance. Unfortunately, hyphal anastomosis is the only known mechanism of horizontal virus transmission and the large number of vegetative incompatibility groups in Botrytis is a potential constraint on the spread of an introduced virus. Although some Botrytis viruses, such as BVF and BVX, are known to have international distribution, there is a distinct lack of epidemiological data and the means of spread are unknown.

Molecular characterisation of two divergent variants of grapevine leafroll-associated virus 3 in New Zealand.

Partial genomic sequences of two divergent grapevine leafroll-associated virus 3 (GLRaV-3) variants, NZ1-B and NZ2, from New Zealand were determined and analysed (11,827 nt and 7,612 nt, respectively). At the nucleotide level, both variants are more than 20 % different from the previously published GLRaV-3 sequences, from phylogenetic groups 1 to 5. Phylogenetic analysis indicated that NZ1-B is a variant of the previously identified divergent NZ-1, while NZ2 is a novel sequence with only 76 % nucleotide sequence identity to GLRaV-3 variants NZ-1, GH11, and GH30. Therefore, NZ2 is a new variant of GLRaV-3. Amino acid sequence analysis of the NZ1-B and NZ2 coat proteins indicated significant substitutions that are predicted to alter the coat protein structure, which potentially leads to the observed reduced immunological reactivity of both variants to the Bioreba anti-GLRaV-3 conjugated monoclonal antibody.

Generic and sequence-variant specific molecular assays for the detection of the highly variable Grapevine leafroll-associated virus 3.

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus, which is found in all grapevine growing regions worldwide. Its accurate detection in nursery and field samples is of high importance for certification schemes and disease management programmes. To reduce false negatives that can be caused by sequence variability, a new universal primer pair was designed against a divergent sequence data set, targeting the open reading frame 4 (heat shock protein 70 homologue gene), and optimised for conventional one-step RT-PCR and one-step SYBR Green real-time RT-PCR assays. In addition, primer pairs for the simultaneous detection of specific GLRaV-3 variants from groups 1, 2, 6 (specifically NZ-1) and the outlier NZ2 variant, and the generic detection of variants from groups 1 to 5 were designed and optimised as a conventional one-step multiplex RT-PCR assay using the plant nad5 gene as an internal control (i.e. one-step hexaplex RT-PCR). Results showed that the generic and variant specific assays detected in vitro RNA transcripts from a range of 1×10(1)-1×10(8) copies of amplicon per µl diluted in healthy total RNA from Vitis vinifera cv. Cabernet Sauvignon. Furthermore, the assays were employed effectively to screen 157 germplasm and 159 commercial field samples. Thus results demonstrate that the GLRaV-3 generic and variant-specific assays are prospective tools that will be beneficial for certification schemes and disease management programmes, as well as biological and epidemiological studies of the divergent GLRaV-3 populations.