Evidence of Grapevine enamovirus 1 Infecting Grapevine (Vitis vinifera L.) in Canada.

Grapevine enamovirus 1 (GEV1) belongs to the genus Enamovirus, in the family Solemoviridae. It has been reported from several countries infecting grapevines including Brazil (Silva et al. 2017), China (Ren et al. 2021) and France (Hily et al. 2022). To assess the prevalence and diversity of economically important grapevine viruses in nine Canadian vineyards, total RNA and double-stranded RNA (dsRNA) (Fall et al. 2020) were extracted from 30 and 100 composite samples respectively, with each consisting of five vines of the same cultivars. The cultivars included in this study are Frontenac noir (n=34), Vidal (n=32), Marquette (n=33), Riesling (n=31), and Pinot noir (n=31). The total RNA and dsRNA samples were subsequently multiplexed and diagnosed by high-throughput sequencing (HTS) on NovaSeq (600 S4 PE100) and MiSeq (2 × 250 cycle PE) respectively. From NovaSeq and MiSeq sequencing, an average of 410,000 to 1.3 million reads/sample were obtained, respectively, with mapped viral reads representing 10.92% to 12.48% of the total reads. After sequence quality was verified using Trimmomatic v.0.40 (Bolger et al. 2014), the clean sequences were screened against all possible viruses in the databases using the Virtool (Rott et al. 2017) and VirFind virus detection pipelines (Ho and Tzanetakis 2014). GEV1 was detected in clean sequences from two, three, and two leaf samples of cultivars 'Marquette' 'Riesling' and 'Frontenac noir' respectively. Six of the seven HTS-assembled GEV1 genomes were partial, ranging from 4,523 to 6,000 nucleotide (nt) with genome coverage varying from 71% to 89%. Only one 6,314 nt long assembled contig (Accession No. OR021829), represented a nearly complete genome, being only 53 and 3 nt shorter than Sd-CG (MT536978) at 5' and 3' untranslated regions (UTR), respectively. Isolate 3- Riesling-CAN (OR021829) shares 90.56 to 94.19% nt identities with several GEV1isolates at 96-99% of query coverage. Phylogenetically, OR021829 is closer to GEV1 isolates from France and China (Figure S1). To validate the HTS results, the developed primer pair SetF and Set1R (Silva et al., 2017) was used for RT-PCR detection. The amplicons from all seven HTS-positive samples were sequenced using Sanger sequencing, confirming the presence of GEV-1 in three studied grape cultivars in Canadian vineyards. Symptoms associated with the specific GEV1-infected vines could not be explained as composite samples were used. Each of the combined samples HTS library also tested positive for at least one of the known grape virus/viroids, namely grapevine leafroll associated-virus -3, grapevine pinot gris virus, grapevine rupestris stem pitting-associated virus, Marafivirus syrahense grapevine Syrah virus-1 and hop stunt viroid. To our knowledge, this is the first report of GEV1 being detected in grapevines in Canada, or in any North American vineyard. GEV1 is a relatively new virus, and its biology remains largely unknown. Based on this sequence new GEV1 primers can be developed to know the genetic variability among GEV-1 and improve the detection of this virus in vineyards.

NanoViromics: long-read sequencing of dsRNA for plant virus and viroid rapid detection.

There is a global need for identifying viral pathogens, as well as for providing certified clean plant materials, in order to limit the spread of viral diseases. A key component of management programs for viral-like diseases is having a diagnostic tool that is quick, reliable, inexpensive, and easy to use. We have developed and validated a dsRNA-based nanopore sequencing protocol as a reliable method for detecting viruses and viroids in grapevines. We compared our method, which we term direct-cDNA sequencing from dsRNA (dsRNAcD), to direct RNA sequencing from rRNA-depleted total RNA (rdTotalRNA), and found that it provided more viral reads from infected samples. Indeed, dsRNAcD was able to detect all of the viruses and viroids detected using Illumina MiSeq sequencing (dsRNA-MiSeq). Furthermore, dsRNAcD sequencing was also able to detect low-abundance viruses that rdTotalRNA sequencing failed to detect. Additionally, rdTotalRNA sequencing resulted in a false-positive viroid identification due to the misannotation of a host-driven read. Two taxonomic classification workflows, DIAMOND & MEGAN (DIA & MEG) and Centrifuge & Recentrifuge (Cent & Rec), were also evaluated for quick and accurate read classification. Although the results from both workflows were similar, we identified pros and cons for both workflows. Our study shows that dsRNAcD sequencing and the proposed data analysis workflows are suitable for consistent detection of viruses and viroids, particularly in grapevines where mixed viral infections are common.

First report of Alstroemeria necrotic streak virus infecting greenhouse bell pepper (Capsicum annuum) in Canada.

Alstroemeria necrotic streak virus (ANSV) is an Orthotospovirus that has been isolated from symptomatic Alstroemeria plant in 2010 (Hassani-Mehraban et al. 2010). It has been shown to infect crops of bell pepper (Capsicum annuum) and tomato (Solanum lycopersicum) (Olaya et al. 2017) which are two of the three biggest greenhouse crops in Canada in terms of production volume and value (Statistic Canada. 2020). In July of 2022, the entire production of bell pepper (all plants) from a greenhouse in Québec was presenting necrotic rings and discoloration in fruit and seemingly healthy leaves. Samples from these infected bell pepper were found to be negative for twenty two common viruses infecting bell pepper by ELISA immunoassay by the Laboratoire d'expertise et de diagnostic en phytoprotection (LEDP) (Québec, Canada). To identify the causal agent, double-stranded RNA was extracted from leaf and fruit of one plant to form two separate samples (leaf and fruit) and used for cDNA library preparations with Nextera XT DNA Sample Prep kit (Illumina, USA). The libraries were sequenced using Illumina Miseq (Fall et al. 2020). The same dsRNA were also sequenced with MinION nanopore sequencing method as described previously (Javaran et al. 2021; Javaran et al. 2023). The obtained raw FASTQ data were processed following the methodology described in Fall et al. 2020 and Javaran et al. 2023. The Miseq sequencing yielded over 2 million reads per sample with a percentage of mapped viral reads ranging from 26.92 to 47.29% of the total number of reads. The leaf samples were positive to Bell pepper endornavirus (BPEV) with the full genome covered 16713 times and Alstroemeria necrotic streak virus (ANSV) with 98% of the genome covered 4929 times. The MinION sequencing yielded 1,028,460 reads and the same viruses were detected with 1288 long reads (mean length of 745bp) assigned to ANSV genome. Both viruses were detected in the leaf and fruit samples. The complete ANSV genome comprising three segments (L, M, and S) was assembled and deposited in GenBank: (OQ261731-OQ261733). These L, M and S segments shown 99% nt identity with an isolate from the Columbia (GenBank: MF469036, MF469037, MF469038). It is interesting that read coverage at near the 2000th position of the S segment, was very low. This phenomenon may suggest a cleavage site nearby by a viral or host factor. ANSV was mainly found in leaf samples and very low numbers of reads in fruit samples. The presence of ANSV was confirmed by RT-PCR using the primers specific to the ANSV nucleocapsid gene Tospo_S_F (5'- CAG AAT CAG GCT GCA TTT AAT TTC C-3') and Tospo_S_R (5'-CAA CGC TTC CTT TAG CAT TAG G-3') (Gallo et al. 2019). The sequences of ∼600 bp amplicons were determined using Sanger sequencing and showed 100% nt identity with Miseq-derived sequences of ANSV. The virus has previously been detected in Colombia (Hassani-Mehraban et al. 2010) and then in California in 2018 (Tian et al. 2020). This is to our knowledge the first detection of ANSV in Canada. Bell pepper is one of the most important crops in Canada and the ANSV vector, the western flower thrips (Frankliniella occidentalis), known to spread the tomato spotted wilt virus (TSWV) is established in Canada (Allen et al. 1986). The detection of ANSV in Canada is line with the hypothesis of an international spread of this virus (Tian et al. 2020) as is it not known to spread through seeds.

Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics.

Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus-plant interactions, as well as the characterization of viral RNA modifications.

First report of Grapevine yellow speckle viroid 1 infecting grapevine (Vitis vinifera L.) in Canada.

Quebec is the third largest wine grape producer in Canada in acreage, tonnage, and wine grape sales (Carisse et al. 2017; Ben Moussa et al. 2019). To evaluate the diversity of viruses infecting grapevine in Quebec, a total of 77 leaf tissue samples (cv. Vidal) were collected from July to October in 2020 in three different vineyards located in Frelighsburg, Hemmingford and Saint-Jacques-le-Mineur in Quebec, Canada. Double-stranded RNA was extracted from each sample and used for cDNA library preparation with the Nextera XT DNA Library Preparation Kit (Illumina) as described previously (Kesanakurti et al. 2016). High-throughput sequencing (HTS, 2x300 bp) was conducted on dual-indexed libraries in a v3 flow cell using the Illumina MiSeq platform (Adkar-Purushothama et al. 2020). The obtained raw FASTQ data was de-multiplexed into 154 separate sequence files, and the adapters and barcode sequences were trimmed. The quality of the sequences was verified using Trimmomatic V.0.32 and the "clean" sequences were analyzed using Virtool and VirFind virus detection pipelines described elsewhere (Ho and Tzanetakis 2014; Rott et al. 2017) to screen for all possible viruses in the databases. Over 100,000 reads per sample were obtained with a percentage of mapped viral reads ranging from 1.47 to 19.43% of total number of reads. Out of 77 samples, 16 revealed the sequence of grapevine yellow speckle viroid 1 (GYSVd-1), for which the length coverage ranged from 98.5 to 99.1%; the depth ranged from 2X to 856X. The GYSVd-1 positive sequence files were subjected to whole genome assembly on CLC genomics Workbench v20.0.4 with the isolate SY-BR from Brazil (KU880715) used as reference. Seven complete genomes of GYSVd-1 of 366-368 nucleotides (nt) in size were deposited (GenBank Acc. MW732682 to MW732688). BLASTN analysis of the sequences showed 98-100% nt identities with isolate SY-BR. Other viruses and viroids such as Grapevine fleck virus, Grapevine rupestris stem pitting-associated virus, Grapevine rupestris vein feathering virus and Hop stunt viroid were also detected. To confirm GYSVd-1 presence in Quebec vineyards, seven of the 16 HTS-positive grapevine leaf tissue samples were subjected to total RNA extraction, followed by RT-PCR assay as before (Adkar-Purushothama et al. 2015; Sahana et al. 2013); all were positive by RT-PCR. The PCR products were directly Sanger-sequenced, and they showed 100% nt identity to the HTS derived sequences. Three of the seven GYSVd-1 positive grapevines exhibited yellow leaf spots and flecks and tiny yellow leaves, but their mixed infection status makes definitive symptoms association difficult to determine. Previously, Hop stunt viroid was reported from grapevines in Canada (Xiao et al. 2019; Fall et al. 2020) but to the best of our knowledge, this is the first report of GYSVd-1 infecting grapevines in Canada, specifically in the province of Quebec. Further research is required to assess the GYSVd-1 related yield loss. Monitoring and testing for GYSVd-1 infection is necessary to prevent propagation of infected materials, spread, and potential negative impact for the Canadian grapevine industry.

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLRaV) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLRaV-3 and 2. Our data show that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLRaV-3 and GLRaV-2, in gradient order. However, co-occurrence analysis revealed that the presence of GLRaV species was randomly associated with the development of virus-like symptoms. These findings and their implications for grapevine leafroll disease management are discussed.

Management of Blueberry Maggot With High Temperatures.

High temperatures were investigated to manage blueberry maggot (Rhagoletis mendax Curran; Diptera: Tephritidae) in field and postharvest situations. To estimate lethal combinations of high temperatures/duration of exposure, blueberry maggot pupae were immersed in water at various temperatures during either 1 or 30 s in the laboratory. Treatments such as 70°C (1 s) or 55°C (30 s) caused 100% mortality of blueberry maggot pupae. In a lowbush blueberry field, soil temperatures at 0, 1, 2, 3, 4, and 5 cm depth were measured with thermocouples following the passage of a flamethrower attached to a tractor in the fall. While the temperatures reached up to ca. 80°C for ca. 1 s at the soil surface, they were <10°C at depths of 3, 4, and 5 cm. In field situations, the energy required to kill all pupae would be expensive to deliver with a flamethrower and this would also cause environmental concerns. An example of application concerning the use of high temperatures in a postharvest situation is discussed, notably immersion of reusable containers in hot water in compliance with Canadian Food Inspection Agency Directive D-02-04 (2015) to prevent dissemination of R. mendax in uninfested areas.

Controlled-release of Bacillus thurigiensis formulations encapsulated in light-resistant colloidosomal microcapsules for the management of lepidopteran pests of Brassica crops.

Bacillus thuringiensis (B. t.) based formulations have been widely used to control lepidopteran pests in agriculture and forestry. One of their weaknesses is their short residual activity when sprayed in the field. Using Pickering emulsions, mixtures of spores and crystals from three B. t. serovars were successfully encapsulated in colloïdosomal microparticles (50 µm) using innocuous chemicals (acrylic particles, sunflower oil, iron oxide nanoparticles, ethanol and water). A pH trigger mechanism was incorporated within the particles so that B. t. release occurred only at pH > 8.5 which corresponds to the midgut pH of the target pests. Laboratory assays performed on Trichoplusia ni (T. ni) larvae demonstrated that the microencapsulation process did not impair B. t. bioactivity. The best formulations were field-tested on three key lepidopteran pests that attack Brassica crops, i.e., the imported cabbageworm, the cabbage looper and the diamondback moth. After 12 days, the mean number of larvae was significantly lower in microencapsulated formulations than in a commercial B. t. formulation, and the effect of microencapsulated formulations was comparable to a chemical pesticide (lambda-cyhalothrin). Therefore, colloïdosomal microcapsule formulations successfully extend the bioactivity of B. t. for the management of lepidopteran pests of Brassica crops.

Extreme cold temperature to kill blueberry maggot (Diptera: Tephritidae) in reusable containers.

Pupae of the blueberry maggot, Rhagoletis mendax Curran (Diptera: Tephritidae), were subjected to -20 degrees C for 2, 4, 7, 10, and 15 d (control = 0 d at 4 degrees C) in fall 2011 and 2012 and were allowed to overwinter for at least 181 d at 4 degrees C. Mean adult emergence in the control was 64% in 2012 and 39% in 2013. No adults emerged in May 2012 from 500 pupae treated for > 2 d at -20 degrees C in fall 2011. Two adults emerged in May 2013 from 100 pupae treated for 2 d at -20 degrees C in fall 2012. No adults emerged in May 2013 from 400 pupae subjected to -20 degrees C for durations of 4, 7, 10, and 15 d. No adults emerged from larvae concealed in berries subjected to -20 degrees C for > 2 d. Exposure of larvae and pupae to cold temperatures (i.e., -20 degrees C) for > 2 d can be a valuable nonpesticidal method for killing R. mendax larvae and pupae associated with reusable containers.

Kaolin affects blueberry maggot behavior on fruit.

This study assessed the effects of Surround (kaolin) on several behavioral parameters of female blueberry maggot, Rhagoletis mendax Curran (Diptera: Tephritidae). First fruit visited, walking, cleaning, and oviposition behavior were quantified in two-choice and no-choice assays where females encountered Surround-treated and untreated fruit of highbush blueberries (Vaccinium spp.). In two-choice assays, females had a propensity (68%) to first visit untreated blueberries. In two-choice and no-choice tests, number of walking bouts and duration of walking bouts were significantly shorter on the Surround-treated than on untreated fruit. Few oviposition attempts on fruit were observed, irrespective of treatments and assays. Chromameter measurements showed significant modification of the blueberry skin color parameters lightness, hue, and saturation between untreated fruit compared with fruit dipped once or twice in a suspension of Surround. Oviposition trials with field-treated fruit showed that blueberries treated with Surround had fewer oviposition scars than the control, and this was more pronounced with weekly applications of Surround. Uneven coating of the fruit by Surround in field applications may have resulted in higher acceptance rates by flies than in blueberries individually dipped and used in laboratory trials.