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Background Laboratory bioassays using treated leaf disks of peach were conducted to determine the efficacy of nine insecticides against the green peach aphid (GPA), Myzus persicae (Sulzer). The effects of these insecticides on aphid feeding behaviors and rates of transmission of Turnip mosaic virus (TuMV) to potted rutabaga plants were also determined. Results Median lethal concentration (LC50 ) values after 48 h feeding varied considerably, ranging from lows of 1.5 and 4.6 μg a.i./L for sulfoxaflor and λ-cyhalothrin, respectively, to 97.2 and 167.9 μg a.i./L for flonicamid and spirotetramat. LC50 values were lowest and roughly equivalent for λ-cyhalothrin (1.2) acetamiprid (2.1), sulfoxaflor (0.23) and flupyradifurone (2.3) after 72 h feeding. Electrical penetration graph (EPG) recordings showed modest effects on feeding behaviors for certain insecticides, with sulfoxaflor, spirotetramat, and acetamiprid non-significant reduction in feeding duration and number of pathway and potential drop phases occurring during the first 5 min compared with the control. However, greenhouse experiments carried out to investigate the effect of these insecticides on rates of transmission of TuMV, which is transmitted non-persistently by GPA, resulted in only modest non-significant reductions in infection rates for acetamiprid, pymetrozine, λ-cyhalothrin, and flonicamid of 27%, 23%, 20%, and 17%, respectively. Conclusion All test materials were efficacious to GPA at differing levels, and some such as sulfoxaflor and acetamiprid non-significantly reduced the duration and number of pathways and potential drop phases of feeding within the first 5 min. None, however, resulted in significant reductions in rates of transmission of TuMV. © 2020 Her Majesty the Queen in Right of Canada. Pest Management Science © 2020 Society of Chemical Industry.

A single step Direct real-time quantitative reverse transcription quantitative polymerase chain reaction (DRT-qPCR) was applied and optimized for the detection of Grapevine rupestris stem-pitting associated virus (GRSPaV) in grapevine. A probe and primer set were designed from conserved genomic regions from eight GRSPaV accessions from GenBank and used with the Direct Plant Extraction Buffer (DiPEB) system in place of total nucleic acid extraction using commercial kits to identify 14 grapevine accessions and field grapevine isolates for the presence of GRSPaV. Optimal detection of GRSPaV was obtained using foliar tissue macerated first in ELISA extraction buffer that was further diluted in DiPEB buffer (1:9 v/v). Middle-aged grape leaves on young canes gave acceptably low Cq (quantification cycle) values, with no significant differences detected between bisected half leaves, refrigerated and frozen leaves, and frozen leaf macerates made in ELISA buffer. Cq values of macerates derived from cambial tissue were comparable to those of leaf tissue macerates. The DRT-qPCR was shown to be a reproducible, specific test capable of detection of as little as 4.59 pg of viral RNA. This is the first study to use this simplified method for the detection of viruses that infect grapevine. Because this system does not require the use of a commercial nucleic acid extraction kit, it provides savings in cost and time allowing higher throughput testing.

In 2000, the Dideron (D) strain of Plum pox virus (PPV) was detected in commercial peach and nectarine orchards in the Niagara region of Ontario where most of Canada’s stone fruit crops are produce...

Turnip yellow mosaic virus (TYMV) has been reported throughout Europe, New Zealand, and Australia. In 1994, this virus was identified in two field plantings of Bok Choi and one planting of Pak Choi (Brassica campestris Chinensis group var. communis) in Durham and Haldimand-Norfolk counties, respectively. In early October, approximately 25% of the plants were infected at each site. Both the striped flea beetle (Phyllotreta striolata (F.)) and the crucifer flea beetle (P. Cruciferae(Goeze)), reported vectors of the virus (1), were present at each site. Infected plants exhibited bright yellow to yellow-green mosaic mottling and often showed chlorotic lesions on the lower leaves. Vein clearing was also seen on several plants. Plants were often coinfected with turnip mosaic virus. Four symptomatic plants were taken from each field site for testing. Spherical virus particles (28 nm) were identified as TYMV by electron microscopy following post-antibody decoration and enzyme-linked immunosorbent assay with the TYMV Agdia test kit. Symptoms were reproduced on both Bok and Pak Choi by mechanical inoculation into healthy plants. Extended host range susceptibility tests with 14 differential hosts were consistent with those reported in the VIDE database (1). This virus, in the presence of the flea beetle vectors, may pose a threat to susceptible traditional cruciferous vegetables grown extensively in this area. Reference: (1) A. A Brunt et al., eds. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 16th January 1997.

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at –20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada. References: (1) E. Boudon-Padieu. Bull. O I V, 79:299, 2003. (2) M. Rott et al. Plant Dis. 91:1682, 2007. (3) E. Tanne et al. Phytopathology 91:741, 2001.

The residual activity of horticultural mineral oil (HMO) on the ability of green peach aphids, Myzus persicae (Sulzer), (GPA) to transmit Plum pox virus (PPV) to peach was measured by infection rates of detached leaves from plants sprayed with either HMO or water as a control that were inoculated using transfer of 25 viruliferous aphids per leaf at 0, 2, 4, 7, 9, 11 and 14 days after treatment (DAT). Persistent effects of HMO residue on the probing and feeding behaviours of GPA were also monitored with the electrical penetration graph (EPG) system. For glasshouse-grown peach seedlings, the residual activity of HMO reduced PPV infection rates by more than 58% for up to 4 DAT following an initial reduction of approximately 81%. EPG recordings of GPA feeding behaviour showed that HMO significantly delayed first feeding probes and first intracellular punctures by more than 50 min without changing the ensuing stylet penetration behaviour. Applying HMO reduced virus infection rates for up to a week depending on the environmental conditions. EPG monitoring of aphid probing showed that HMO reduced the mean duration and mean number of potential drop (PD) phase feeding occurrences, compared with the water control. A reduction in the PD that has been shown to be related to the transmission of non-persistently transmitted viruses may partly explain the reduction in PPV infection rates.

A method was developed to evaluate susceptibility of peach leaves to Plum pox virus (PPV) infection by aphids. We examined whether virus multiplication could be detected in aphid-inoculated detached leaves and if transmission efficiency of PPV by green peach aphids to detached leaves was comparable with that of peach seedlings. Results demonstrated that transmission efficiencies of viruliferous aphids transferred to detached peach leaves subsequently maintained on an agar layer for 3 weeks was not significantly different from that for intact seedlings. Overlaying infected PPV plum or peach leaf segments on the healthy peach leaves with subsequent application of aphids to the infected leaf pieces provided a comparable transmission efficiency. Reduced handling of the aphids using this method minimized the possibility of damaging the aphids and facilitated higher throughput testing. Comparable infection rates were obtained for detached leaves using either 50 or 25 viruliferous aphids per leaf. Residu...

Early viroid detection in chrysanthemum cultivars depends on both an efficient RNA extraction method as well as a sensitive detection assay. In this study, we evaluated RNA purification methods and optimized a one-step RT-qPCR assay for the detection of Chrysanthemum stunt viroid (CSVd). Three commercially available RNA extraction technologies (silica and silicon carbide spin column as well as the phenol/chloroform extraction) were compared for efficacy in CSVd detection sensitivity. We found a 10- to 50-fold improvement in CSVd detection sensitivity when using silicon carbide (SiC) spin columns to purify RNA compared with the silica-based column or the traditional phenol/chloroform extraction method. To optimize detection sensitivity and reduce detection time, a commercial CSVd end-point PCR primer set was adapted to a SYBR Green based one-step RT-qPCR assay. Standard curve analysis estimated that the limit of detection was about 54 CSVd copies and the specificity test against six related viroids...

Greenhouse trials demonstrated the ability of bumblebees (Bombus impatiens Cresson) to transmit Pepino mosaic virus (PepMV) from infected tomato plants to perennial climbing nightshade (Solanum dulcamara L) in 2 of 3 trials (5.1% and 5.6% frequency, respectively). The efficiency of transmission was lower than that between tomato plants in previous studies (80%). Low rates of transmission were also seen in bee transmission from nightshade plants back to tomato (6.3%, 3.7% and 2.8%), and between nightshade plants (8.3% and 2.8%). Nightshade was easily infected by mechanical inoculation in controls. Bumblebees used by growers to pollinate tomatoes can move freely in and out of the production houses, and bees carrying virus inoculum from infected greenhouse tomatoes could establish and spread PepMV in nearby climbing nightshade populations. This overwintering reservoir could allow for ongoing virus introduction from the field through pollinating bees back into tomato production houses seasonally. The ...

Leafhoppers (Hemiptera: Cicadellidae) are pests of many temperate crops, including grapevines ( Vitis species). Uncontrolled populations can induce direct and indirect damage to crops due to feeding that results in significant yield losses and increased mortality in infected vineyards due to virus, bacteria, or phytoplasmas vectored by leafhoppers. The main objective of this work was to determine the diversity of leafhoppers found in vineyards of the three main Canadian production provinces, i.e., in British Columbia, Ontario, and Quebec. Approximately 18,000 specimens were collected in 80 commercial vineyards from 2006 to 2008. We identified 54 genera and at least 110 different species associated with vineyards, among which 22 were predominant and represented more than 91% of all the leafhoppers. Species richness and diversity were estimated by both Shannon’s and Pielou’s indices. For each province, results indicated a temporal variation in species composition. Color photographs provide a tool to quickly identify 72 leafhoppers commonly associated with vineyards.

Surveys of native weed species growing in and within 30 m of tomato greenhouses infected with Pepino mosaic virus (PepMV) identified Solanum dulcamara (climbing nightshade), Solanum nigrum (black nightshade), and Solanum ptycanthum (eastern black nightshade) as susceptible host plants. All plants exhibited mild mosaic symptoms and virus was back-inoculated and confirmed in tomato (Lycopersicon esculentum). In transmission tests using both mechanical methods and bumblebees (Bombus impatiens), S. ptycanthum, Solanum sarrachoides (hairy nightshade), and Datura stramonium (jimsonweed) were infected. Weak positive assays by ELISA or DRT-PCR for Amaranthus blitoides (prostrate pigweed), Chenopodium album (lamb’s-quarters), and Plantago lanceolata (narrow-leaved plaintain) were possibly a result of either a hypersensitive localized response to infection or low level virus contamination by bees since the virus could not be recovered by back-inoculation into tomato. Bumblebees exposed to PepMV infected D. stramoni...

A new procedure is described for the detection of Plum pox virus (PPV) in samples from large-scale field tests. The new direct real-time polymerase chain reaction (drtPCR) procedure was based on crude supernatants collected from peach (Prunus persica) leaves macerated in a buffer that was specially developed for this purpose and named “direct pathogen extract buffer.” Specific TaqMan primers and probes were designed for PPV detection: two sets specific to PPV D and M strains, respectively, and one for universal detection of PPV strains D, M, C, EA, and W. These primer and probe sets can be used singly or for duplex differentiation of D and M strains. Using Prunus spp. tissue infected with 30 known fruit tree viruses, the universal primer and probe set correctly identified PPV-infected and PPV-free samples, with no nonspecific cross-reactions. Based on endpoint analysis of the drtPCR reaction, a threshold cyle value of 36 was suggested as the maximum threshold to declare a sample positive for PPV....

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

Plum pox virus (PPV), one of the potyviruses originally from Europe and recently found in Canada, is the causative agent of sharka in Prunoideae. The virus infects many types of ornamental as well as stone-fruit Prunus spp., such as peach, nectarine, plum, cherry, and apricot, resulting in extensive economic losses. Immediately after it was found in Canada, a PPV eradication, grower compensation, and research program funded by the Canadian federal and provincial governments was implemented. This review summarizes general information about PPV, including the physical and molecular features of the viral pathogen, symptoms of the disease, geographical distribution, and current control strategy, and highlights research progress and future prospects for control of the disease in Canada.

Water spinach, a specialty vegetable and a member of the sweet potato family, is cultivated for Asian markets in California (United States) and Ontario (Canada). Foliar diseases of this vegetable observed in commercial greenhouses of Ontario in 1993 and 1994, as well as in greenhouses of California in 1998, were attributed respectively to Phyllosticta ipomoeae, Cercospora ipomoeae, and Pseudomonas syringae pv. syringae in the present study. In Ontario, C. ipomoeae infected less than 1% of plants, with less than 25% of the foliage affected, while Phyllosticta ipomoeae occurred on 100% of plants in some greenhouses, with leaf spotting affecting up to 25% of the foliage. In California, Pseudomonas syringae pv. syringae occurred on less than 25% of plants with up to 25% of the foliage affected. In Ontario, no virus was detected in eight suspect foliage samples while damage from vectors of plant viruses such as the green peach aphid (Myzus persicae) was minor and from Western flower thrips (Frankliniella occid...

Plum pox virus (PPV), genus Potyvirus, is a serious threat to the fruit and ornamental horticulture industry in Canada. Genomic resources of its natural hosts, Prunus spp., currently available to researchers studying PPV are very poor. Systematic sequencing of expressed sequence tags (ESTs) can be used as a discovery base for genes associated with PPV infection, replication, and symptom development. Here we describe the construction of two directional, size-selected cDNA libraries derived from PPV-infected and PPV-free peach leaves, and the generation of 3839 ESTs. Based on their partial sequences and putative functions, the ESTs are classified into 611 clusters and 1153 singletons. About 65.36% of the genes (2509 ESTs) show homology to genes from other organisms, and 34.64% of cDNAs (1330 ESTs) have no matches in the GenBank database. Comparison analyses suggest that genes involved in defence, cellular transport, development, protein synthesis, proteins with binding function in the PPV-infected peach lea...

Extensive surveys of native weed populations in peach orchards heavily infected with Plum pox virus strain D (PPV-D) in the Niagara Region quarantine area, Ontario, Canada, failed to identify natural infection in any of the species examined. Surveys of rural and urban residential properties within areas of high PPV incidence did not detect widespread infection of susceptible hosts, with infected Prunus glandulosa (dwarf flowering almond) being found only at one site. The prominent color-breaking observed in blossoms of PPV-infected P. glandulosa would make this an excellent sentinel species for early detection of virus in Prunus orchards. Surveys of susceptible ornamental Prunus spp. in Niagara nurseries failed to demonstrate PPV infection in any of the nursery field plantings.

(1990). Susceptibility of Ontario weed species to turnip mosaic virus. Canadian Journal of Plant Pathology: Vol. 12, No. 3, pp. 255-262.

In this paper, plans are given for the construction of an inexpensive enzyme-linked immunosorbent assay plate washer from readily available materials. The wash unit uses an intermittent wash cycle based on a wash manifold cycling over the microdilution plates for a predetermined time. Laboratory tests showed that the unit provided reliable, rapid washing of plates with tap water, with no detectable contamination between wells. Substrate absorbance values for test samples from machine-washed plates were equal to or greater than absorbance values for corresponding samples from plates washed manually by an accepted protocol, by using either enzyme-linked immunosorbent assay wash buffer or tap water.

Onion yellow dwarf virus (OYDV) (1) was identified in a commercial planting of garlic (Allium sativum L.) near Delhi, Ontario, in 1998. Infected plants exhibited mild mosaic symptoms that became less noticeable by mid-July. Many plants were co-infected with garlic latent virus (GLV) (4), which was mechanically transmitted to leek (A. porrum L.). Since leek is not susceptible to OYDV (3), it is an appropriate host for the differentiation of GLV and OYDV. OYDV was transmitted nonpersistently by Myzus persicae (Schultz) from garlic to garlic and onion (A. cepa L.). Infected onion exhibited yellow striping, leaf curling, and pronounced stunting. Necrotic lesions were not present on inoculated leaves of Chenopodium amaranticolor Coste & Reyn. and C. quinoa Willd., which are often associated with co-infection with leek yellow dwarf virus (2). The isolate reacted strongly in enzyme-linked immunosorbent assay with OYDV antisera (from M. Fukami, Chiba Prefectural Agric. Exp. Stn., Chiba 266, Japan; D. Z. Maat, DLO Research Instit., Plant Prot., Wageningen, The Netherlands). Electron microscopic observations of negatively stained preparations of infected leaf tissues revealed virus particles averaging 765 ± 45 nm with typical pinwheel inclusions. References: (1) L. Bos. CMI/AAB Descrip. Plant Viruses no. 158, 1976. (2) K. Graichen. Nachrichtenbl. Pflanzenschutz DDR 32: 245, 1978. (3) K. Graichen and H. U. Leistner. Arch. Phytopathol. Pflanzenschutz 23:165, 1987. (4) L. W. Stobbs et al. Plant Dis. 80:343, 1996.

Fifty-four tobacco cultivars, seven species, and four somatic hybrids were examined for resistance to a Canadian isolate of potato virus Y necrotic strain (PVY N ). Four cultivars were resistant to infection: Virgin A Mutante (VAM), NC744, TN86, and PBD6. Virus was not detected in inoculated plants by enzyme-linked immunosorbent assay, electron microscopy, or back-inoculation to susceptible tobacco plants. Resistant plants were similar in appearance to the healthy uninoculated controls. A single recessive gene from VAM is believed to be common to the four resistant varieties. Havana 307, Wanda, and Wisana were tolerant to PVY N infection, exhibiting mild mosaic symptoms with or without veinal necrosis, and similar in overall growth to the healthy controls. All the registered Canadian cultivars tested were susceptible to PVY N infection. Of the species tested, Nicotiana kawakami exhibited mild symptoms and reduced virus titer and N. rustica 1. var. NRT exhibited tolerance when inoculated with PVY N . No resistance or tolerance to PVY N infection was found in any of the somatic hybrids tested. These results demonstrate that genetic resistance to PVY N is present both within the tabacum genome and among its wild relatives. The utility of the sources of resistance is variable, however

Rows of rutabaga plants, grown in a greenhouse groundbed, were inoculated with turnip mosaic virus (TuMV) at 2-wk intervals during root development. Plants inoculated within 12 wk after seeding produced unmarketable roots that were small and elongated with severe goosenecking of the upper root and stem. Dry matter content of healthy roots infected at different stages in their development did not differ significantly. Infected roots contained low levels of virus, independent of the stage of root development at which they were inoculated. The presence of TuMV in roots and decay of the roots in storage were not correlated (...)

This study examined the influence of turnip mosaic virus (TuMV) on rutabaga (Brassica napusssp. rapifera (Metzg.) Sinsk) root yield, mineral composition and storability from plants infected during the late stages of first year growth. TuMV‐infected plants from four commercial fields in Ontario, Canada were sampled when one‐sixth to one‐fourth of the plant foliage displayed TuMV symptoms. TuMV was detected in the roots of infected plants but at. low levels. Root yield, root dry matter percentage and the elemental composition of roots were similar from TuMV non‐infected and infected plants in the same field. Nitrogen, ?, ?n and Zn contents in the root tissues were observed to vary among the fields. There was no obvious differences in storability between TuMV non‐infected and infected roots after 5 months at 1°C and 87% humidity

The effect of shoot thinning on the production of wood suitable for grafting was evaluated on four commercial rootstocks. Sonona and SO4 yielded the largest quantity of graftable wood when thinned to seven shoots, although only a slight increase was apparent over unthinned Sonona. Lesser volumes of wood were obtained from Couderc 3309 and 4453 Maleque with unthinned vines producing marginally higher quantities of wood per vine.Key words: Rootstock, wood production

Rutabaga (Brassica napus L. Group Napobrassica) cultivars were screened for resistance to turnip mosaic virus (TuMV), and the inheritance of resistance was studied. Immunity and resistance were found in four of 49 rutabaga cultivars evaluated. The cultivar ‘Sensation’ was immune to the Ontario strain of TuMV. In crosses between susceptible ‘Laurentian’ and resistant Line 165 and susceptible triazine-resistant ‘Laurentian’ and Line 165, TuMV resistance was determined by a single dominant gene.

Evaluation de l'efficacite de l'ajout d'un conjugue aux cupules enrobees d'anticorps specifique de l'immobilisation du conjugue par sechage a l'air ou par lyophilisation ou du matricage sur gelatine semi-solide et conservation des plaques