L. W. Stobbs

Plum pox virus in Canada : progress in research and future prospects for disease control

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.

Distribution of Plum pox virus in Residential Sites, Commercial Nurseries, and Native Plant Species in the Niagara Region, Ontario, Canada

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.

Direct real-time PCR detection of Plum pox virus in field surveys in Ontario

Abstract 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. A comparative analysis comparing drtPCR and ELISA using 12 200 field samples revealed that drtPCR was approximately 100- to 1000-fold more sensitive than ELISA and was able to detect PPV at an earlier stage of infection than ELISA. The drtPCR is a valuable tool for PPV diagnosis and may also be applicable to other studies, including pathogen population dynamics and vector transmission efficiency.

A comparative survey, by expressed sequence tag analysis, of genes expressed in peach leaves infected with Plum pox virus (PPV) and free from PPV

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 leaf tissue are more active than those in PPV-free leaves. Moreover, a number of gene transcripts, including β-1,3- glucanase, cytochrome-P450-like protein, cytochrome P450 monooxygenase, heat-shock protein 70, thioredoxin, alcohol dehydrogenase, catalase, cysteine protease inhibitor, translation factor EF-1α, and pathogenesis-related protein (PR1) are highly prevalent only in the PPV-infected peach leaf. Consistent with results in several recent publications from other laboratories, our finding supports the view that positive-stranded viruses may elicit common responses in susceptible plants. The results of this study help better understand the molecular mechanisms associated with susceptibility to PPV disease in Prunus persica. and provide a studying point for the development of novel strategies to control the virus.

Diversity and Abundance of Leafhoppers in Canadian Vineyards

Abstract 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.

Evaluation of viroid extraction methods and application of a one-step reverse transcription real-time polymerase chain reaction assay (RT-qPCR) for the rapid detection of Chrysanthemum stunt viroid (CSVd) infection

Abstract 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 showed no cross-reactivity. As a part of the validation, the one-step RT-qPCR detection system was compared with RT-PCR to detect CSVd in both randomly selected greenhouse plants and mechanically inoculated chrysanthemum varieties. Cultivars selected from a commercial greenhouse with >50% infection levels were tested for CSVd presence. Cultivars ‘Pelee’, ‘Puma’ and ‘Shamrock’ tested positive by both the one step RT-qPCR and RT-PCR but CSVd was not detected in ‘Icecap’ and ‘Snowball’. In mechanical inoculation studies, CSVd was detected in 5 of 6 chrysanthemum cultivars – ‘Chesapeake’, ‘Durango’, ‘Juneau’, ‘Pelee’ and ‘Viron’ but not in ‘Pueblo’. No differences in detection were evident between the RT-qPCR and RT-PCR methods.

Aphid Transmission of the Ontario Isolate of Plum Pox Virus

ABSTRACT Utilization of timed virus acquisition access probes in studies of plum pox virus (PPV) transmission by aphids demonstrated that endemic species transmitted the virus readily from plum, Prunus domestica (L.) Batsch; peach, P. persica (L.); or dwarf flowering almond, P. glandulosa Thunberg., to peach seedlings. The green peach aphid, Myzus persicae (Sulzer), was shown to be the most efficient vector. Acquisition of virus by green peach aphids from infected peach leaves resulted in 18–28% infected peach seedlings, while aphids previously fed on infected leaves of plum transferred virus to 36% of peach seedlings. Although the spirea aphid, Aphis spiraecola (Patch), was a less efficient vector than M. persicae it is perhaps more important for the spread of PPV due to its greater abundance and occurrence earlier in the season when peach trees are thought to be more susceptible to infection. Virus transmission rates varied depending on the virus source and healthy test plant species. In contrast to many previous studies, aphid inoculation of the experimental host Nicotiana benthamiana Domin occurred at a low rate, never exceeding 4%. Acquisition of PPV by M. persicae from infected peach fruit was greatly reduced compared with acquisition from leaves. The results of this research indicate that the Ontario isolate of PPV-D is readily transmissible by aphids to peach and natural spread of the virus needs to be considered in future management or eradication programs.

Development of a detached leaf procedure to evaluate susceptibility to Plum pox virus infection by the green peach aphid (Myzus persicae (Sulzer)) in peach

Abstract 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. Residual PPV was not detected by direct quantitative reverse transcriptase polymerase chain reaction assay (DqRT-PCR) on non-host plants probed by viruliferous aphids. The effect of short-term storage temperatures pre- or post-inoculation did not significantly alter the susceptibility of peach leaves to PPV infection or the transmission rate. Application of the leaf overlay method to evaluate seasonal changes in susceptibility of peach leaves in the field is the subject of an ongoing study.

Horticultural mineral oil influences Plum pox virus transmission by Myzus persicae

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.

First report of bumblebee (Bombus impatiens Cresson) transmission of Pepino mosaic virus between tomato (Solanum lycopersicum L.) and perennial climbing nightshade (Solanum dulcamara L.)

Abstract 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 virus could also spread from infected climbing nightshade into tomato field plantings through similar bee activity.