The New Zealand perspective of an ecosystem biology response to grapevine leafroll disease.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevines worldwide resulting in grapevine leafroll disease (GLD), reduced fruit yield, berry quality and vineyard profitability. Being graft transmissible, GLRaV-3 is also transmitted between grapevines by multiple hemipteran insects (mealybugs and soft scale insects). Over the past 20 years, New Zealand has developed and utilized integrated pest management (IPM) solutions that have slowly transitioned to an ecosystem-based biological response to GLD. These IPM solutions and combinations are based on a wealth of research within the temperate climates of New Zealand's nation-wide grape production. To provide context, the grapevine viruses present in the national vineyard estate and how these have been identified are described; the most pathogenic and destructive of these is GLRaV-3. We provide an overview of research on GLRaV-3 genotypes and biology within grapevines and describe the progressive development of GLRaV-3/GLD diagnostics based on molecular, serological, visual, and sensor-based technologies. Research on the ecology and control of the mealybugs Pseudococcus calceolariae and P. longispinus, the main insect vectors of GLRaV-3 in New Zealand, is described together with the implications of mealybug biological control agents and prospects to enhance their abundance and/or fitness in the vineyard. Virus transmission by mealybugs is described, with emphasis on understanding the interactions between GLRaV-3, vectors, and plants (grapevines, alternative hosts, or non-hosts of the virus). Disease management through grapevine removal and the economic influence of different removal strategies is detailed. Overall, the review summarizes research by an interdisciplinary team working in close association with the national industry body, New Zealand Winegrowers. Teamwork and communication across the whole industry has enabled implementation of research for the management of GLD.

Molecular Characterization of Two Totiviruses from the Commensal Yeast Geotrichum candidum.

Mycoviruses can infect many of the major taxa of fungi including yeasts. Mycoviruses in the yeast fungus Geotrichum candidum are not well studied with only three G. candidum-associated viral species characterized to date, all of which belong to the Totiviridae genus Totivirus. In this study, we report the molecular characteristics of another two totiviruses co-infecting isolate Gc6 of G. candidum. The two totiviruses were tentatively named Geotrichum candidum totivirus 2 isolate Gc6 (GcTV2-Gc6) and Geotrichum candidum totivirus 4 isolate Gc6 (GcTV4-Gc6). Both viruses have the typical genome organization of totiviruses comprising two ORFs encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) at the N and C termini, respectively. The genomes of GcTV2-Gc6 and GcTV4-Gc6 are 4592 and 4530 bp long, respectively. Both viruses contain the-frameshifting elements and their proteins could be expressed as a single fusion protein. GcTV2-Gc6 is closely related to a totivirus isolated from the same host whereas GcTV4-Gc6 is related to insect-associated totiviruses. The phylogenetic analysis indicated that GcTV2-Gc6 and GcTV4-Gc6 belong to two different sister clades, I-A and I-B, respectively. It is interesting that all viruses identified from G. candidum belong to the genus Totivirus; however, this might be due to the lack of research reporting the characterization of mycoviruses from this fungal host. It is possible that the RNA interference (RNAi) mechanism cannot actively suppress totivirus accumulation in G. candidum Gc6.

A National Catalogue of Viruses Associated with Indigenous Species Reveals High-Throughput Sequencing as a Driver of Indigenous Virus Discovery.

Viruses are important constituents of ecosystems, with the capacity to alter host phenotype and performance. However, virus discovery cued by disease symptoms overlooks latent or beneficial viruses, which are best detected using targeted virus detection or discovered by non-targeted methods, e.g., high-throughput sequencing (HTS). To date, in 64 publications, 701 viruses have been described associated with indigenous species of Aotearoa New Zealand. Viruses were identified in indigenous birds (189 viruses), bats (13 viruses), starfish (4 viruses), insects (280 viruses), and plants (126 viruses). HTS gave rise to a 21.9-fold increase in virus discovery rate over the targeted methods, and 72.7-fold over symptom-based methods. The average number of viruses reported per publication has also increased proportionally over time. The use of HTS has driven the described national virome recently by 549 new-to-science viruses; all are indigenous. This report represents the first catalogue of viruses associated with indigenous species of a country. We provide evidence that the application of HTS to samples of Aotearoa New Zealand's unique fauna and flora has driven indigenous virus discovery, a key step in the process to understand the role of viruses in the biological diversity and ecology of the land, sea, and air environments of a country.

Grapevine Leafroll-Associated Virus 3 Genotype Influences Foliar Symptom Development in New Zealand Vineyards.

Grapevine leafroll disease (GLD) constrains wine production worldwide. In New Zealand, the main causal agent of GLD is grapevine leafroll-associated virus 3 (GLRaV-3). To control GLD, an integrated management program is used and includes removing (roguing) GLRaV-3-infected vines from the vineyard. The classical foliar symptoms from virus-infected red-berry cultivars are leaves with dark red intervein, green veins, and downward rolling of margins. Growers use these phenotypic cues to undertake visual symptom identification (VSI) for GLD. However, the influence of the known large genetic variation among GLRaV-3 isolates on the foliar symptoms from different grapevine cultivars remains undescribed, especially in cool-climate growing environments, such as New Zealand. Over three vintages (2015, 2016, and 2017), VSI for GLD was undertaken at three field sites in New Zealand (Auckland, Hawke's Bay, and Marlborough), each including four cultivars (Merlot, Pinot noir, Sauvignon blanc, and Pinot gris) infected with three GLRaV-3 genotypes (Groups I, VI, and X) or GLRaV-3-uninfected control plants. Throughout this study, no visual symptoms were observed on white-berry cultivars infected with GLRaV-3. For red-berry cultivars, the greatest variability in observed foliar symptoms among regional study sites, cultivars, and GLRaV-3 genotypes was observed early in the growing season. In particular, Group X had significantly delayed symptom expression across all three sites compared with Groups I and VI. As the newly infected, young vines matured in years 2 and 3, the GLRaV-3 genotype, cultivar, region, and environmental conditions had minimal influence on the accuracy of VSI, with consistently high (>95%) within-vintage identification by the end of each vintage. The results from this study strongly support the use of VSI for the GLD management of red-berry cultivar grapevines, Merlot and Pinot noir, as a reliable and cost-effective tool against GLD.

Characterization of a Novel Double-Stranded RNA Virus from Phytophthora pluvialis in New Zealand.

A new dsRNA virus from the oomycete Phytophthora pluvialis has been characterized and designated as Phytophthora pluvialis RNA virus 1 (PplRV1). The genome of the PplRV1 reference genome is 6742 bp that encodes two predicted open reading frames (ORFs). ORF1 and ORF2 overlap by a 47 nt "slippery" frameshift sequence. ORF1 encodes a putative protein of unknown function. ORF2 shows high similarity to the RNA-dependent RNA polymerase (RdRp) of other dsRNA viruses. Phylogenetic analysis of the putative PplRV1 RdRp and its most closely related viruses showed PplRV1 is distinct from other known viruses (below 33% amino acid similarity), which indicates this virus may belong to a new virus family. Analyses of the geographical distribution of PplRV1 in relation to two genetically distinct classes of its host revealed two corresponding genotypes of the PplRV1 (termed a and b), which share 92.3% nt identity. The reference genome for the second genotype is 6760 bp long and a prediction of its genetic organization shows three ORFs, with ORF2 being split into two ORFs, ORF2a and ORF2b, that is conserved in seven of eleven genotype b isolates. Additionally, a quick and simple diagnostic method using qPCR has been developed, which is suitable for large scale screens to identify PplRV1 in Phytophthora.

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.

Mycovirus-encoded suppressors of RNA silencing: Possible allies or enemies in the use of RNAi to control fungal disease in crops.

Plants, fungi, and many other eukaryotes have evolved an RNA interference (RNAi) mechanism that is key for regulating gene expression and the control of pathogens. RNAi inhibits gene expression, in a sequence-specific manner, by recognizing and deploying cognate double-stranded RNA (dsRNA) either from endogenous sources (e.g. pre-micro RNAs) or exogenous origin (e.g. viruses, dsRNA, or small interfering RNAs, siRNAs). Recent studies have demonstrated that fungal pathogens can transfer siRNAs into plant cells to suppress host immunity and aid infection, in a mechanism termed cross-kingdom RNAi. New technologies, based on RNAi are being developed for crop protection against insect pests, viruses, and more recently against fungal pathogens. One example, is host-induced gene silencing (HIGS), which is a mechanism whereby transgenic plants are modified to produce siRNAs or dsRNAs targeting key transcripts of plants, or their pathogens or pests. An alternative gene regulation strategy that also co-opts the silencing machinery is spray-induced gene silencing (SIGS), in which dsRNAs or single-stranded RNAs (ssRNAs) are applied to target genes within a pathogen or pest. Fungi also use their RNA silencing machinery against mycoviruses (fungal viruses) and mycoviruses can deploy virus-encoded suppressors of RNAi (myco-VSRs) as a counter-defence. We propose that myco-VSRs may impact new dsRNA-based management methods, resulting in unintended outcomes, including suppression of management by HIGS or SIGS. Despite a large diversity of mycoviruses being discovered using high throughput sequencing, their biology is poorly understood. In particular, the prevalence of mycoviruses and the cellular effect of their encoded VSRs are under-appreciated when considering the deployment of HIGS and SIGS strategies. This review focuses on mycoviruses, their VSR activities in fungi, and the implications for control of pathogenic fungi using RNAi.

A Mechanically Transmitted DNA Mycovirus Is Targeted by the Defence Machinery of Its Host, Botrytis cinerea.

Eukaryotic circular single-stranded DNA (ssDNA) viruses were known only to infect plants and vertebrates until the discovery of the isolated DNA mycovirus from the fungus Sclerotinia sclerotiorum. Similar viral sequences were reported from several other sources and classified in ten genera within the Genomoviridae family. The current study reports two circular ssDNA mycoviruses isolated from the phytopathogen Botrytis cinerea, and their assignment to a newly created genus tentatively named Gemydayirivirus. The mycoviruses, tentatively named botrytis gemydayirivirus 1 (BGDaV1) and BGDaV2, are 1701 and 1693 nt long and encode three and two open reading frames (ORFs), respectively. Of the predicted ORFs, only ORF I, which codes for a replication initiation protein (Rep), shared identity with other proteins in GenBank. BGDaV1 is infective as cell-free purified particles and confers hypovirulence on its natural host. Investigation revealed that BGDaV1 is a target for RNA silencing and genomic DNA methylation, keeping the virus at very low titre. The discovery of BGDaV1 expands our knowledge of the diversity of genomoviruses and their interaction with fungal hosts.

Characterisation and Distribution of Karaka Okahu Purepure Virus-A Novel Emaravirus Likely to Be Endemic to New Zealand.

We report the first emaravirus on an endemic plant of Aotearoa New Zealand that is, to the best of our knowledge, the country's first endemic virus characterised associated with an indigenous plant. The new-to-science virus was identified in the endemic karaka tree (Corynocarpus laevigatus), and is associated with chlorotic leaf spots, and possible feeding sites of the monophagous endemic karaka gall mite. Of the five negative-sense RNA genomic segments that were fully sequenced, four (RNA 1-4) had similarity to other emaraviruses while RNA 5 had no similarity with other viral proteins. A detection assay developed to amplify any of the five RNAs in a single assay was used to determine the distribution of the virus. The virus is widespread in the Auckland area, particularly in mature trees at Okahu Bay, with only occasional reports elsewhere in the North Island. Phylogenetic analysis revealed that its closest relatives are pear chlorotic leaf spot-associated virus and chrysanthemum mosaic-associated virus, which form a unique clade within the genus Emaravirus. Based on the genome structure, we propose this virus to be part of the family Emaravirus, but with less than 50% amino acid similarity to the closest relatives in the most conserved RNA 1, it clearly is a novel species. In consultation with mana whenua (indigenous Maori authority over a territory and its associated treasures), we propose the name Karaka Okahu purepure virus in te reo Maori (the Maori language) to reflect the tree from which it was isolated (karaka), a place where the virus is prevalent (Okahu), and the spotted symptom (purepure, pronounced pooray pooray) that this endemic virus appears to cause.

Retention and Transmission of Grapevine Leafroll-Associated Virus 3 by Pseudococcus calceolariae.

Grapevine leafroll-associated virus 3 (GLRaV-3), an economically significant pathogen of grapevines, is transmitted by Pseudococcus calceolariae, a mealybug commonly found in New Zealand vineyards. To help inform alternative GLRaV-3 control strategies, this study evaluated the three-way interaction between the mealybug, its plant host and the virus. The retention and transmission of GLRaV-3 by P. calceolariae after access to non-Vitis host plants (and a non-GLRaV-3 host) White clover (Trifolium repens L. cv. "Grasslands Huia white clover"), Crimson clover (T. incarnatum), and Nicotiana benthamiana (an alternative GLRaV-3 host) was investigated. For all experiments, P. calceolariae first instars with a 4 or 6 days acquisition access period on GLRaV-3-positive grapevine leaves were used. GLRaV-3 was detected in mealybugs up to 16 days on non-Vitis plant hosts but not after 20 days. GLRaV-3 was retained by second instars (n = 8/45) and exuviae (molted skin, n = 6/6) following a 4 days acquisition period on infected grapevines leaves and an 11 days feeding on non-Vitis plant hosts. Furthermore, GLRaV-3 was transmitted to grapevine (40-60%) by P. calceolariae second instars after access to white clover for up to 11 days; 90% transmission to grapevine was achieved when no alternative host feeding was provided. The 16 days retention period is the longest observed in mealybug vectoring of GLRaV-3. The results suggest that an alternative strategy of using ground-cover plants as a disrupter of virus transmission may be effective if mealybugs settle and continue to feed on them for 20 or more days.

Investigating the Pea Virome in Germany-Old Friends and New Players in the Field(s).

Peas are an important legume for human and animal consumption and are also being used as green manure or intermediate crops to sustain and improve soil condition. Pea production faces constraints from fungal, bacterial, and viral diseases. We investigated the virome of German pea crops over the course of three successive seasons in different regions of pea production to gain an overview of the existing viruses. Pools from 540 plants, randomly selected from symptomatic and asymptomatic peas, and non-crop plants surrounding the pea fields were used for ribosomal RNA-depleted total RNA extraction followed by high-throughput sequencing (HTS) and RT-PCR confirmation. Thirty-five different viruses were detected in addition to nine associated nucleic acids. From these viruses, 25 are classified as either new viruses, novel strains or viruses that have not been reported previously from Germany. Pea enation mosaic virus 1 and 2 were the most prevalent viruses detected in the pea crops, followed by pea necrotic yellow dwarf virus (PNYDV) and turnip yellows virus which was also found also in the surrounding non-legume weeds. Moreover, a new emaravirus was detected in symptomatic peas in one region for two successive seasons. Most of the identified viruses are known to be aphid transmissible. The results revealed a high virodiversity in the German pea fields that poses new challenges to diagnosticians, researchers, risk assessors and policy makers, as the impact of the new findings are currently unknown.

Tenth Scientific Biennial Meeting of the Australasian Virology Society-AVS10 2019.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

Comparison of two different host plant genera responding to grapevine leafroll-associated virus 3 infection.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.

A Novel Totivirus Naturally Occurring in Two Different Fungal Genera.

Mycoviruses are widely distributed across different phyla of the fungal kingdom. Viruses that share significant sequence similarities have been reported in different fungi, suggesting descent from a common ancestor. In this study, two fungal genera isolated from the same sample, Trichoderma koningiopsis isolate Mg10 and Clonostachys rosea isolate Mg06, were reported to have identical double-stranded RNA (dsRNA) profiles that consist of two virus-like, dsRNA elements (dsRNA-L and dsRNA-S). The complete sequence and genome organization of dsRNA-L from isolate Mg10 was determined. It is 4712 nucleotides (nt) long and contains two non-overlapping open reading frames (ORFs) that code for proteins with similarities to totiviruses. Consequently the virus was given the proposed name Trichoderma koningiopsis totivirus 1 (TkTV1/Mg10). The TkTV1/Mg10 genome structure resembles that of yeast totiviruses in which the region preceding the stop codon of ORF1 contains the elements required for -1 ribosomal frameshifting which may induce the expression of an ORF1-ORF2 (CP-RdRp) fusion protein. Sequence analyses of viral dsRNA-L from C. rosea isolate Mg06 revealed that it is nearly identical with that of TkTV1/Mg10. This relatedness was confirmed by northern blot hybridization and indicates very recent natural horizontal transmission of this virus between unrelated fungi. TkTV1 purified isometric virions were ∼38-40 nm in diameter and were able to transfect T. koningiopsis and C. rosea protoplasts. This is another report of a mycovirus present naturally in two taxonomically distinct fungi.

A New Era for Mild Strain Cross-Protection.

Societal and environmental pressures demand high-quality and resilient cropping plants and plant-based foods grown with the use of low or no synthetic chemical inputs. Mild strain cross-protection (MSCP), the pre-immunization of a plant using a mild strain of a virus to protect against subsequent infection by a severe strain of the virus, fits with future-proofing of production systems. New examples of MSCP use have occurred recently. New technologies are converging to support the discovery and mechanism(s) of action of MSCP strains thereby accelerating the popularity of their use.

Development of sensitive molecular assays for the detection of grapevine leafroll-associated virus 3 in an insect vector.

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically significant virus of grapevines, with secondary spread mediated by several species of mealybug and soft scale insects. To better understand virus-vector interactions, sensitive virus detection in these insects is a key tool. In this research, two new hydrolysis-probe-based real-time assays for GLRaV-3 detection were developed and compared to three existing assays. Of the five assays compared, the one-step RT-qPCR probe-based assay was the most sensitive and reliable, with as few as 10 virus RNA copies detected. This is the first description of a real-time molecular assay for virus detection in mealybugs with such sensitivity.

Common Variants of the Plant microRNA-168a Exhibit Differing Silencing Efficacy for Human Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1).

BACKGROUND: The discovery that a plant microRNA (miRNAs) from rice (Oryza sativa miR168a) can modify post-transcriptional expression of the mammalian. Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1) gene highlights the potential for cross-kingdom miRNAmRNA interactions. OBJECTIVE: To investigate whether common variants of the conserved miR168a family have the capability for similar cross-kingdom regulatory functions, we selected sequences from three dietary plant sources: rice (Oryza sativa), tomato (Solanum lycopersicum), apple (Malus domestica) and compared their ability to regulate human LDLRAP1 expression. METHODS: Target prediction software intaRNA and RNAhybrid were used to analyze and calculate the energy and alignment score between the miR168a variants and human LDLRAP1 mRNA. An in vitro cell-based Dual-Luciferase® Reporter Assay (pmirGLO, Promega), was then used to validate the miRNA-mRNA interaction experimentally. RESULTS: Computational analyses revealed that a single nucleotide difference at position 14 (from the 5' end of the miRNA) creates a G:U wobble in the miRNA-mRNA duplex formed by tomato and apple miR168a variants. This G:U wobble had only a small effect on the free energy score (-33.8-34.7 kcal/mol). However, despite reasonable hybridization energy scores (<-20 kcal/mol) for all miR168a variants, only the rice miR168a variant lacking a G:U wobble significantly reduced LDLRAP1 transcript expression by 25.8 + 7.3% (p<0.05), as measured by relative luciferase activity. CONCLUSION: In summary, single nucleotide differences at key positions can have a marked influence on regulatory function despite similar predicted energy scores and miRNA-mRNA duplex structures.

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.

Grapevine virus I, a putative new vitivirus detected in co-infection with grapevine virus G in New Zealand.

A novel virus, with characteristics of viruses classified within the genus Vitivirus, was identified from a sample of Vitis vinifera cv. Chardonnay in New Zealand. The virus was detected with high throughput sequencing (small RNA and total RNA) and its sequence was confirmed by Sanger sequencing. Its genome is 7507 nt long (excluding the polyA tail) with an organisation similar to that described for other classifiable members of the genus Vitivirus. The closest relative of the virus is grapevine virus E (GVE) with 65% aa identity in ORF1 (65% nt identity) and 63% aa identity in the coat protein (66% nt identity). The relationship with GVE was confirmed with phylogenetic analysis, showing the new virus branching with GVE, Agave tequilina leaf virus and grapevine virus G (GVG). A limited survey revealed the presence of this virus in multiple plants from the same location where the newly described GVG was discovered, and in most cases both viruses were detected as co-infections. The genetic characteristics of this virus suggest it represents an isolate of a new species within the genus Vitivirus and following the current nomenclature, we propose the name "Grapevine virus I".