State-of-the-Art Plant Virus Research in Australasia.

The Special Issue 'State-of-the-Art Plant Virus Research in Australasia' in Viruses provided a fascinating snapshot of plant and fungus virus research being undertaken in Australasia during the final year of the official COVID-19 pandemic [...].

A Chronological Study on Grapevine Leafroll-Associated Virus 2 in Australia.

Grapevine leafroll disease affects the health status of grapevines worldwide. Most studies in Australia have focused on grapevine leafroll-associated viruses 1 and 3, while little attention has been given to other leafroll virus types, in particular, grapevine leafroll-associated virus 2 (GLRaV-2). A chronological record of the temporal occurrence of GLRaV-2 in Australia since 2001 is reported. From a total of 11,257 samples, 313 tested positive, with an overall incidence of 2.7%. This virus has been detected in 18 grapevine varieties and Vitis rootstocks in different regions of Australia. Most varieties were symptomless on their own roots, while Chardonnay showed a decline in virus-sensitive rootstocks. An isolate of GLRaV-2, on own-rooted Vitis vinifera cv. Grenache, clone SA137, was associated with severe leafroll symptoms after veraison with abnormal leaf necrosis. The metagenomic sequencing results of the virus in two plants of this variety confirmed the presence of GLRaV-2, as well as two inert viruses, grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine rupestris vein feathering virus (GRVFV). No other leafroll-associated viruses were detected. Among the viroids, hop stunt viroid and grapevine yellow speckle viroid 1 were detected. Of the six phylogenetic groups identified in GLRaV-2, we report the presence of four groups in Australia. Three of these groups were detected in two plants of cv. Grenache, without finding any recombination event. The hypersensitive reaction of certain American hybrid rootstocks to GLRaV-2 is discussed. Due to the association of GLRaV-2 with graft incompatibility and vine decline, the risk from this virus in regions where hybrid Vitis rootstocks are used cannot be overlooked.

The ABI5-dependent down-regulation of mitochondrial ATP synthase OSCP subunit facilitates apple necrotic mosaic virus infection.

Apple necrotic mosaic virus (ApNMV) is associated with apple mosaic disease in China. However, the mechanisms of ApNMV infection, as well as host defence against the virus, are still poorly understood. Mitochondrial ATP synthase plays a fundamental role in the regulation of plant growth and development. However, mitochondrial ATP synthase function in response to virus infection remains to be defined. In the present study, a yeast two-hybrid (Y2H) screening revealed that the apple mitochondrial ATP synthase oligomycin sensitivity-conferring protein (OSCP) subunit (MdATPO) interacts with ApNMV coat protein (CP). It was further verified that overexpression of MdATPO in Nicotiana benthamiana inhibited viral accumulation. In contrast, silencing of NbATPO facilitated viral accumulation, indicating that ATPO plays a defensive role during ApNMV infection. Further investigation demonstrated that ApNMV infection accelerated abscisic acid (ABA) accumulation, and ABA negatively regulated ATPO transcription, which was related to the ability of ABA insensitive 5 (ABI5) to bind to the ABA-responsive elements (ABREs) of the ATPO promoter. Taken together, our results indicated that transcription factor ABI5 negatively regulated ATPO transcription by directly binding to its promoter, leading to the susceptibility of apple and N. benthamiana to ApNMV infection. The current study facilitates a comprehensive understanding of the intricate responses of the host to ApNMV infection.

A Metagenomic Investigation of the Viruses Associated with Shiraz Disease in Australia.

Shiraz disease (SD) is an economically important virus-associated disease that can significantly reduce yield in sensitive grapevine varieties and has so far only been reported in South Africa and Australia. In this study, RT-PCR and metagenomic high-throughput sequencing was used to study the virome of symptomatic and asymptomatic grapevines within vineyards affected by SD and located in South Australia. Results showed that grapevine virus A (GVA) phylogroup II variants were strongly associated with SD symptoms in Shiraz grapevines that also had mixed infections of viruses including combinations of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine leafroll-associated virus 4 strains 5, 6 and 9 (GLRaV-4/5, GLRaV-4/6, GLRaV-4/9). GVA phylogroup III variants, on the other hand, were present in both symptomatic and asymptomatic grapevines, suggesting no or decreased virulence of these strains. Similarly, only GVA phylogroup I variants were found in heritage Shiraz grapevines affected by mild leafroll disease, along with GLRaV-1, suggesting this phylogroup may not be associated with SD.

Genetic Diversity of Grapevine Virus A in Three Australian Vineyards Using Amplicon High Throughput Sequencing (Amplicon-HTS).

Shiraz disease (SD) is one of the most destructive viral diseases of grapevines in Australia and is known to cause significant economic loss to local growers. Grapevine virus A (GVA) was reported to be the key pathogen associated with this disease. This study aimed to better understand the diversity of GVA variants both within and between individual SD and grapevine leafroll disease (LRD) affected grapevines located at vineyards in South Australia. Amplicon high throughput sequencing (Amplicon-HTS) combined with median-joining networks (MJNs) was used to analyze the variability in specific gene regions of GVA variants. Several GVAII variant groups contain samples from both vineyards studied, suggesting that these GVAII variants were from a common origin. Variant groups analyzed by MJNs using the overall data set denote that there may be a possible relationship between variant groups of GVA and the geographical location of the grapevines.

Identification and molecular characterization of a novel carlavirus infecting rose plants (Rosa chinensis Jacq.).

In the present study, the genome sequence of a potential novel virus, tentatively named "rose virus C" (RVC), was mined from publically available transcriptomic data from a Rosa chinensis plant. The complete genome sequence of RVC consists of 8,386 nt, excluding a 3' poly(A) tail, and contains five ORFs. Phylogenetic analysis showed that RVC clustered with members of the genus Carlavirus, family Betaflexiviridae. The replicase gene had 48.8-52.1% nt sequence identity to those of other carlaviruses, while the CP gene had 40.4-45.9% nt sequence identity, which is far below the species demarcation cutoff of 72%. The incidence of RVC in rose plants was low (5.4%). Overall, our data suggest that RVC is a novel atypical virus of the genus Carlavirus.

Molecular characterization of rose spring dwarf-associated virus isolated from China rose (Rosa chinensis Jacq.) in China.

China rose (Rosa chinensis Jacq.) is an important ornamental plant grown widely in China. In May 2019, we sampled and analyzed a China rose plant by high-throughput sequencing using small RNAs. A luteovirus, rose spring dwarf-associated virus (RSDaV), was detected in this plant, and its complete nucleotide sequence of 5816 nucleotides was determined. The China rose isolate of RSDaV contains five major open reading frames (ORFs) and three putative small ORFs, typical of members of the genus Luteovirus. It shares 94.4% nt sequence identity with the Californian (USA) isolate of the virus. Genomic analysis revealed a deletion of a single U at nt position 5295, which introduced a frameshift mutation, and an insertion of nine nucleotides (AUAAAUGAU) at position 5706-5714, which did not change the reading frame. The aa sequence in that portion of the protein was 90.5% identical to that of the Californian isolate. This is the first report on the occurrence of RSDaV infecting rose plants in China.

First report of grapevine rupestris vein feathering virus in grapevine in Australia.

Grapevine rupestris vein feathering virus (GRVFV; tentative genus Marafivirus; family Tymoviridae ) was first detected from a Greek grapevine (Vitis vinifera), with asteroid mosaic-like symptoms (El Beaino et al. 2001; Ghanem-Sabanadzovic et al. 2003) and was also infected with grapevine fleck virus. GRVFV has been detected in the United States, South Africa, Canada, Spain, China, New Zealand, Brazil, Germany, Korea, Slovakia, Hungary and Pakistan (Cho et al. 2018; Mahmood et al. 2019).Transmission vectors are currently unknown. In 2018, nine grapevine samples were collected between May to July in South Australia (SA) and Western Australia (WA) (Table S1), were analysed by high-throughput sequencing (HTS) to characterise grapevine viruses in Australian vineyards. Total RNA or double stranded RNA was extracted from grapevine canes using RNeasy 96 QIAcube HT kit (Qiagen) with MacKenzie buffer (MacKenzie et al. 1997) or using CF-11 (Balijja et al. 2008). Libraries were prepared using the NEBNext® Ultra II RNA library Prep Kit (NEB) or TruSeq® Stranded mRNA Prep kit (Illumina) with Ribo-Zero®gold plant kit for ribosomal depletion (Illumina, San Diego, CA). Libraries were sequenced using Illumina Miseq (SA) or Hiseq (WA) technology with 2x300 (SA) or 2x100 (WA) paired end reads which were trimmed using Trim Galore! (0.4.0) or BBmap (38.20), respectively. De novo assembly, using the SPAdes (version 3.12.0) genome assembler with default settings, resulted in twelve near full length GRVFV genomes (6713-6737nt), eight sequences from the WA samples and four from the SA samples. WA samples 171 and 178 and SA sample BV each had two distinct GRVFV molecular variants. Variants 171-1 and 171-2 (GenBank accessions MT084811, MT084812) from sample 171 shared 83.39% nucleotide (nt) identity. Variants 178-1 and 178-2 (MT084813, MT084814) from sample 178 shared 83.54% nt identity. Variants BV6799 and BV8822 (MN974274, MN974275) from sample BV shared 82.85% nt identity. Only one GRVFV sequence was obtained from all other samples. The genome of SA isolate LC1 (MN974273) was confirmed by RT-PCR amplification and Sanger sequencing of overlapping genome regions. Tissue from the infected LC1 isolate has been deposited into the Victorian plant pathogen reference collection (VPRI accession No. 43698). When the genomes of all Australian isolates were compared, they had 78.94% to 94.37 % nt identity with each other. The SA isolates LC1, BV8822, BV6799, and SEL-L (MN974276), and the WA isolates 172 (MT084807), 179 (MT084808), 180 (MT084809), and 182 (MT084810) were most closely related to the Swiss isolate CHASS (KY513702; 82.87% to 85.46% nt identity). The WA isolates 171-1, 171-2, 178-1 and 178-2 were most closely related to the New Zealand isolate Ch8021 (MF000325; 83.21% to 93.87%). Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-3, GLRaV-4 (strain 6 and 9), grapevine virus A, grapevine rupestris stem pitting associated virus, grapevine yellow speckle viroid 1 and hop stunt viroid were also identified in the sequencing data. This is the first report of GRVFV in Australia. All WA samples were collected during dormancy and symptoms were not observed. Sample LC1 from SA had Shiraz disease, the other SA samples were asymptomatic, and none had asteroid mosaic-like symptoms. Further research is required to determine its distribution and association with disease in Australia.

Molecular characterization and pathogenicity analysis of prunus necrotic ringspot virus isolates from China rose (Rosa chinensis Jacq.).

Prunus necrotic ringspot virus (PNRSV) is a viral pathogen with worldwide distribution, infecting many commercial fruit trees and ornamental plants. So far, the correlation between PNRSV infection and China rose mosaic disease has not been studied. Rose mosaic disease is characterized by severe symptoms, including mosaic, line pattern, and ringspot. Six viruses that were potentially associated with mosaic disease, including PNRSV, were tested in China roses. Only PNRSV was detected in China roses showing mosaic disease, and asymptomatic samples tested negative for this virus. This result was confirmed by small RNA sequencing, but rose leaf rosette-associated virus and rose spring dwarf-associated virus were also identified in both samples with mosaic disease and asymptomatic samples. This implied that PNRSV might be associated with China rose mosaic disease. Full genome sequences of two PNRSV isolates were determined, and the RNA1, 2 and 3 segments were found to be 3,332, 2,594 and 1,951 nucleotides (nt) in length, respectively. The three RNA segments shared 88.7-89.1% nt sequence identity in the 3'UTR, while RNA2 and RNA3 shared 98.2-99.4% identity. The higher variability in RNA1 suggests that it might have been under greater selection pressure. Phylogenetic analysis showed that the two PNRSV isolates clustered in group PV-32. Full-length infectious cDNA clones of PNRSV from China rose were constructed and used to agroinfiltrate cucumber seedlings. The inoculated cucumber leaves showed yellowing, chlorotic spots, necrosis, dwarfing, and decline at 23 to 39 days post-inoculation, demonstrating the virulence of the PNRSV isolate from China rose. These data lay a foundation for determining the molecular mechanism of rose mosaic disease caused by PNRSV.

Virus Pathogens in Australian Vineyards with an Emphasis on Shiraz Disease.

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on "Shiraz Disease" (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Complete nucleotide sequence of a new virus, peach chlorotic leaf spot virus, isolated from flat peach in China.

Contigs with sequence homologies to apple chlorotic leaf spot virus (ACLSV) were identified by high-throughput sequencing analysis in three peach samples. Complete genomic sequences of RP19-1 and RP19-2 of the virus consisted of 7,466 and 7,465 nucleotides (nts), respectively, excluding the poly (A) tails. They shared the highest identity with Ta Tao 5, but lower than 70% of sequence similarity with other ACLSV isolates. Furthermore, phylogenetic analysis revealed that these two isolates clustered with Ta Tao 5, which is distinct from other ACLSV isolates. According to the criteria for species demarcation within the genus Trichovirus, these two isolates as well as Ta Tao 5 should be recognized as a new virus species, tentatively named "Peach chlorotic leaf spot virus".

Improved detection of grapevine latent viroid by RT-qPCR, its bioassay analysis, and its rare occurrence worldwide.

Three of the five well-known viroids infecting grapevine belong to the genus Apscaviroid. Grapevine latent viroid (GLVd) is a novel grapevine viroid. Although GLVd has the typical sequence motifs of the genus Apscaviroid, it is still an unassigned viroid. In this study, a sensitive, convenient, and rapid one-step RT-qPCR method using hydrolysis probes for the detection of GLVd was developed. Survey and bioassays were also performed for this viroid. Using this method in the survey of GLVd, a low infection rate of 2/226 in a grapevine germplasm resource nursery and a demonstration vineyard in China was determined. Bioassays using agroinfiltration showed that GLVd can infect 'Kyoho' grapevine but not any of the tested herbaceous plants. Furthermore, sequence variability of GLVd was analyzed in six GLVd-infected grapevines. Sequencing revealed a predominant variant with only a few nucleotide changes compared with the reference variant of GLVd. Therefore, the developed RT-qPCR method should be helpful for determining GLVd in other vineyards of the world. The low infection rate, host range, and sequence variability of GLVd have important implications to further improve our knowledge on this novel grapevine viroid.

The recent importation of Grapevine Pinot gris virus into Australia.

A total of 575 commercial grapevine (Vitis vinifera) samples from Australia have been tested for a newly emerging virus, Grapevine Pinot gris virus (Trichovirus, Betaflexiviridae) during 2015-2017. Nine samples from two states tested positive. Six of these were from New South Wales and 3 from South Australia in a total of eight varieties. All these varieties were imported from Europe within the last 4-19 years. A fragment of 431 nucleotides on the coat protein gene of Grapevine Pinot gris virus was targeted for virus identification by RT-PCR. The virus specificity of each positive sample was confirmed by sequencing followed by the BLASTn analysis which showed an identity of up to 99.3% to the virus sequences in the NCBI database. The phylogenetic tree as well as pairwise sequence identity showed that, although the virus sequence in each variety was unique, they all grouped with the isolates from Europe and well away from the South Korean and several Chinese isolates. This is the first report of the occurrence of Grapevine Pinot gris virus in Australia.

Effect of temperature on symptom expression and sequence polymorphism of grapevine yellow speckle viroid 1 in grapevine.

Symptom expression of yellow speckle disease was studied in a row of 32 individual Vitis vinifera cv. Chardonnay vines in the warmer summer of 2009/2010 as compared with the cooler summer of 2011/2012 in South Australia. RT-PCR analysis showed that all these vines were positive for hop stunt viroid, grapevine yellow speckle viroid 1 (GYSVd-1) and grapevine rupestris stem pitting-associated virus. Four vines named Vines 1, 8, 11 and 15 were selected for further analyses. Vines 1 and 8 had never shown yellow speckle (YS) symptoms, Vine 11 had always been symptomatic, and Vine 15 showed YS symptoms only in the summer of 2009/2010, but not in 2011/2012. Analysis of partial nucleotide (nt) sequence of GYSVd-1 from these vines showed two major sequence polymorphisms in the pathogenicity domain coinciding with the YS symptoms and the prevailing temperature in each season. One group designated UA group had a uridine (U) at position 309 and an adenine (A) at position 311, while another group designated AU group had the other way around: an "A" at position 309 and a "U" at position 311. The AU group had never been reported before. The AU group was a minor variant in the GYSVd-1 population and not present in symptomatic Vine 11. In contrast, the UA group was dominant and present in all the vine samples. Surprisingly, all the asymptomatic vines, but not symptomatic vines, had the signature of the AU group. Whether the AU group is associated with the YS symptom expression is interesting. Our result provides a new insight into the sequence variability of viroid-inducing symptoms during two significantly different growing seasons.

New Iranian and Australian peach latent mosaic viroid variants and evidence for rapid sequence evolution.

Peach latent mosaic viroid isolates from peach and plum in Iran have been compared with an Australian isolate from nectarine. Thirteen sequence variants 336-338 nt in size were obtained. All variants clustered phylogenetically with variants reported from several hosts and countries. A total nucleic acid extract, a slightly longer than full-length RT-PCR amplicon, and a recombinant plasmid clone from the Australian isolate were all infectious to, and symptomatic in, mechanically inoculated peach seedlings. The infectious clone generated two progeny viroid molecules, which each showed 10 different mutations compared with the parent clone inoculated 30 days previously.

Extensive variation of sequence within isolates of Grapevine virus B+.

Four regions covering 1247 nucleotides of the RNA genome of 20 isolates of a Vitivirus, Grapevine virus B (GVB), from three countries were analyzed. All the regions in these isolates varied in sequence as compared to the published GVB sequence. Of these, the intergenic region varied the most, with 73.2% nucleotide sequence homology, while ORF4 encoding coat protein varied the least when compared both at nucleotide sequence (80.3% homology) and at amino acid sequence levels (90.6% homology). The variations were scattered along each region length and were higher at the nucleotide level than at the amino acid level, but none resulted in a frame shift or stop codon. These results indicate that GVB may exist as a heterogeneous population, possibly resulting from mixing different strains by grafting practices or by RNA-RNA recombination in the grapevine, the only known natural host of this virus. Although it has been reported that GVB is associated with corky bark disease, no corky bark symptoms were observed in any of the GVB positive grapevine sample collected from Australia.

Temporal and Spatial Analysis of Grapevine Leafroll-Associated Virus 3 in Pinot Noir Grapevines in Australia.

An epidemic of grapevine leafroll disease (GLD), caused by grapevine leafroll-associated virus 3 (GLRaV-3), was monitored over an 11-year period in Nuriootpa, South Australia. Inoculum originated from infected budwood, and initial GLD incidence at the time of transplanting in 1986 was 23.1%. Infected vines were planted in a random spatial pattern. Change in disease incidence was not observed until 8 years after planting, when disease incidence increased to 27.9%. Disease incidence increased to 51.9% by 1996. Disease progress and rate curves (dy/dt versus time) indicated that the logistic (R2 = 96.2) and Gompertz (R2 = 96.3) growth models would best describe disease progress. However, the logistic model, which has a simpler data transformation with fewer model assumptions, was chosen for the purpose of comparing this epidemic (South Australia) with a GLRaV-3 epidemic in Cabernet Sauvignon grapevines in New Zealand. The logistic rate of GLD spread with respect to time was 0.35 logit/year in South Australia and was nearly three times faster (1.19 logits/year) for GLRaV-3 spread in New Zealand. Ordinary runs analyses indicated that the arrangement of infected vines within rows in South Australia was random up to 8 years after transplanting but subsequently became highly aggregated. Thus, GLD-infected plants are contributing to new infections (i.e., there is evidence for plant-to-plant spread), and a biotic vector with a steep dispersal gradient from each point source is likely to be involved.