Survey for major viruses in commercial Vitis vinifera wine grapes in Ontario

Viruses and the diseases they cause present a major roadblock to the sustainable production of quality grapes and wines [1]. Infections with individual viruses, and mixed infections with multiple viruses as is commonly seen in grapes, are responsible for considerable and perpetual economic losses to grape and wine industries worldwide [2‐6]. Grapevines are known to be infected with the largest number of viruses of all cultivated plant crops. At present, over 80 distinct virus species from 17 virus families and 27 genera have been identified in grapevines [7]. The most damaging and widespread viruses are those involved in the four major disease complexes known as (1) leafroll disease complex [Grapevine leafroll-associated virus (GLRaV)-1, − 2, − 3, − 4, − 7 and − 13]; (2) rugose woody complex [grapevine virus A (GVA), grapevine virus B (GVB), grapevine rupestris stem pitting-associate virus (GRSPaV)]; (3) infectious degeneration and decline [grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), arabis mosaic virus (ArMV)]; and (4) fleck complex [grapevine fleck virus (GFkV), grapevine asteroid mosaic-associated virus (GAMaV), grapevine rupestris vein feathering virus (GRVFV), grapevine redglobe virus (GRGV)] [7]. Recently, a new disease, grapevine red blotch, caused by the DNA virus ‘grapevine red blotch virus’ (GRBV) was discovered in the USA [8, 9] and Canada [10, 11]. Another new virus, grapevine Pinot gris virus (GPGV) was identified in grapevine plants showing symptoms of chlorotic mottling and leaf deformations [12] and was soon reported worldwide from many countries in Europe, Asia, South and North America [13]. In Canada, GPGV has been reported in Ontario and British Columbia [14, 15].

The grapevine and wine industry in Canada is young, first established in the 1970s. Ontario is the largest producer of grape and wine in Canada, followed by British Columbia, Nova Scotia and Quebec [16]. The grape and wine industries constitute an important cornerstone for the economy of Ontario. Grapes rank as the second largest fruit crop in Ontario, with a farm gate value of $100 million annually. Ontario wines have garnered prestigious recognition in the international market, and produce an economic impact estimated at $4.4 billion in 2017. However, starting in 2013, the industry has experienced major outbreaks of viral diseases across the province. Infected vines showed severe symptoms suggesting infections with two disease complexes: leafroll and decline as well as the newly identified red blotch disease. Infected vineyards exhibited poor vigor, fewer and smaller berry clusters, reduced yield and sugar content, and delay and inconsistency in fruit maturity. Furthermore, viral diseases reduce winter hardiness, leading to increased susceptibility to damage or even death resulting from freezing temperatures over severe winters. Some growers had to endure total crop losses, even abandoned their vineyards. As shown in the 2016 annual report of Grape Growers of Ontario, the sales of Vitis vinifera wine grapes had decreased by 40% in 2014 and by 44% in 2015 as compared with that of 2013 due to the colder than normal winters in 2014 and 2015 [17]. The additional pressure of viruses and viral diseases has therefore become major concern to Ontario grape/wine community and threatens the sustainability of the grape and wine industry.

Understanding the type of viruses, their prevalence and the severity of viral diseases is the first step in winning the battle against viruses and viral diseases. However, the situation of grapevine viruses and their economic impact to grape and wine production in Canada is very limited. An earlier nation-wide survey for the distribution of four major viruses was conducted in the 1990s by Centre for Plant Health, Agriculture and Agri-Food Canada [16]. This survey targeted only four viruses, two involved in the infectious degeneration (arabis mosaic virus, ArMV and grapevine fanleaf virus, GFLV) and two involved in the grapevine leafroll,grapevine leafroll associated virus 1 (GLRaV-1) and GLRaV-3. Results of this survey showed low incidence of ArMV (0.53%) and GFLV (0.25%) but higher prevalence for GLRaV-3 (10.8%), followed by GLRaV-1 (1.67%) [16]. Though over 85% the samples included in this survey were collected from Ontario, only a small percentage of these samples (20.9%) were from V. vinifera cultivars. Two recent surveys were conducted in British Columbia on the distribution and genetic diversity of GRBV [11] and four viruses associated with leafroll disease [18]. GLRaV-3 was shown to be the most prevalent, being detected in 16.7% of composite samples, whereas GLRaV-1, − 2 and − 4 were detected in a small percentage of samples (< 4%). Interestingly, GRBV was shown to have a low incidence (1.6%).

Ontario is the predominant grape and wine producer in Canada. However, very little is known about the distribution and prevalence of viruses in the province. In the past two decades, there has been major shifts in the grape and wine industry in Ontario, with increased acreage of V. vinifera cultivars in replacement of non-vinifera grapes, such as those of the V. labrusca type and French-American hybrids. However, there have been no further studies on the distribution of grapevine viruses in Ontario. As a response to the disease outbreaks since 2013, a small-scale survey was conducted by McFadden-Smith and others, which revealed the presence of three grapevine leafroll-associated viruses (GLRaV-1, 2, 3), and to a lesser extent, grapevine red blotch virus in numerous vineyards (McFadden-Smith, personal communication).

This work represents the most comprehensive and up to date survey for all major viruses in V. vinifera wine grapes in Ontario, the major grape and wine producer in Canada. In recent years, there have been outbreaks of viral diseases in commercial vineyards in Ontario, resulting in severe damage and even total crop losses. To investigate the viruses that may be involved in the disease outbreaks, a province-wide survey for commonly targeted viral pathogens in commercial V. vinifera wine grape vineyards in Ontario were carried out in 2015 and 2016. Using a multiplex RT-PCR format we recently developed, we have tested for 17 viruses from 657 composite samples representing 3285 vines collected from the three major grape-growing regions in the province. All but three of these viruses were detected. These viruses have varying degrees of prevalence, ranging from 0.2 to 84% (Table 3). Importantly, the most destructive viruses involved in leafroll, red blotch, and rugose wood were all widely detected. Mixed infections are very common. The most widely distributed viruses include GLRaV-3 (47.9%), GRBV (18.3%), GPGV (21.6%), GFkV (21.8%) and GRSPaV (84%), which are present in large proportions of vineyard blocks we have surveyed. We also showed that all major wine cultivars are heavily infected with multiple viruses.

It is important to note that much higher prevalence of both GLRaV-3 and GRBV was detected in Ontario compared to British Columbia. For instance, only 1.6% of the 2000 samples tested positive for GRBV in British Columbia [11] while GLRaV-3 was detected in < 17% of V. vinifera wine grape samples [18]. Possible reasons for these discrepancies may include differences in the infection status of propagating materials used to establish vineyards, difference in the duration and efficiency of transmission by insect vectors, and efficiencies of nucleic acid isolation systems and test methods. For example, we replied entirely on PCR-based tests for both viruses whereas ELISA was used for the survey of GLRaV-3 in British Columbia.

What factors were responsible for the heavy infection of commercial wine grapes with multiple viruses in Ontario vineyards? Undoubtedly, one of the main factors would be the use of uncertified, virus-infected planting material as illustrated by the high percentage of blocks with 100% infection rate in this survey (Table 6). Another potentially important factor would be the presence of arthropod vectors in Ontario, which have been confirmed in other countries to transmit some of these viruses. It has been reported that GLRaV-3 can be transmitted by multiple species of mealybugs (family Pseudococcidae) and soft scales (family Coccidae) [19‐24]. GRBV was reportedly transmitted under greenhouse conditions by the three-cornered alfalfa treehopper, Spissistilus festinus (Say) [25]. However, it remains unknown if it is the only vector or one of the vectors that transmit GRBV in nature. In a recent study, Poojari et al. [18] demonstrated the presence of grape mealybug (Pseudococcus maritimus), the soft scale insect Parthenolecanium corni and likely other species in British Columbia. The grape mealybug and cottony maple scale (Neopulvinaria innumerabilis) were also detected in Ontario (McFadden-Smith, personal communication). The higher incidence of GLRaV-3 in older vineyards compared to younger vineyards points to the possibility of GLRaV-3 spread by vectors in Ontario, resulting in higher infection rate over time. Interestingly, the prevalence of GLRaV-3 in Ontario as surveyed in the mid 1990s was 3.42% among samples collected from V. vinifera grape cultivars [16]. A similar trend is also observed for GVA and GVB. Interestingly, both GVA and GVB are known also to be transmitted by mealybugs. Much further work is required in order to identify the vectors that can transmit these important viruses and the role they play in the spread of these viruses and their diseases in Ontario.

The disease Grapevine leaf mottling and deformation (GLMD) was first reported on Pinot Gris and Pinot Blanc in Northern Italy in 2003. Symptoms of GLMD include short internodes, mottling and deformation of leaf blades, smaller clusters and reduced yield [13]. In 2012, the genome of its putative causal agent, GPGV, was determined through next generation sequencing [12]. Within a short period of time following its discovery, the distribution of GPGV has reached a global scale as it has been reported from 16 countries so far [13], including Canada [14, 15]. Based on the data from this survey, GPGV was one of the most prevalent viruses, which was detected in different V. vinifera wine grapes grown in Ontario, with a prevalence of 21.6% (Table 3). It was recently reported that, in controlled conditions, GPGV was transmissible by the eriophyid mite Colomerus vitis in Italy [26]. It remains unknown if similar vectors exist in Ontario or other viticulture regions of Canada.

Our survey results suggest that GRBV and GPGV were more recently introduced into Ontario. This is based on the observation that in vineyards that were established between 1974 and 1990, both viruses were present in a small percentage of samples. In contrast, both viruses were detected in much higher percentage of grapes that were planted since 1991. For example, only 2.2% of grapes planted during 1974–1990 tested positive for GRBV. In sharp contrast, GRBV was detected in 17.9% of samples from vineyards established during 1991–2005. Similarly, the incidence of GPGV increased from 13.3% in grapes planted between 1974 and 1990 to 21.8% in grapes planted in the period of 1991–2005, to 25.7% in vines planted since 2006. These results may suggest increased introduction of both viruses along with infected planting materials in the past two decades. Both GRBV and GPGV were new viruses that were recently identified [8, 9, 12]. It is most likely that both viruses might have already existed in other grape growing regions for quite some time prior to their identification. As these two viruses were not included in the early list of viruses that were regulated or targeted in the clean stock and certification programs, the propagation material used may have been infected with either virus and became a source of further infection in newly established vineyards. The high percentage of vineyard blocks in which 100% of the composite samples were positive for GRBV (Table 5) lends support to this possibility. It is therefore of critical importance for the nurseries to survey their propagation material to ensure freedom of GRBV and GPGV, as well as other major viruses.

GRSPaV is the most widespread virus with a worldwide distribution [27]. It is not surprising that GRSPaV was detected in 84% of the samples tested. Though GRSPaV has been associated with RSP [28‐30] and vein necrosis [31], definitive evidence that it is the causal agent of these diseases is still lacking. It was reported that GRSPaV infection has no major impact on growth and yields [32] and that it may be even beneficial to the grapevine host by enhancing tolerance against drought [33, 34]. It is important to note that GRSPaV comprises a family of strains that differ vastly in genome sequence and possibly also in pathogenicity. In line with this, strains SY and PN were detected in declining vines of cultivars Syrah and Pinot Noir vines [35‐37]. Furthermore, GRSPaV infection was correlated to decreased defense responses (Gambino et al. [33]), which may render the vine infected with GRSPaV more susceptible to infection by other viruses. As revealed in this survey, a vast majority of the samples were infected GRSPaV in combination with one of more other viruses. The real economic impact of GRSPaV, either alone or in combination with other viruses with multiple viruses, remains to be determined.

Several viruses of the family Tymoviridae that are associated with the Fleck complex [38] were also detected in this survey. GFkV was detected in 21.8% of the samples with the other three viruses being detected much more rarely. GFkV was also commonly detected in British Columbia, with 29.7% of the samples tested positive for this virus [39]. The real economic impact of GFkV, and the related viruses, awaits to be seen.

Several important questions remain to be addressed. Non-vinifera grapes (table grapes, juice grapes, and hybrid wine grapes) constitute an important part of Ontario’s grape/wine industry. These locally important, non-vinifera grapes had been widely planted in the province, though large acreages of these grapes have been replaced by vinifera grapes in recent decades. As a result, both types of grapes are often grown in close vicinity to each other. As such, they could serve as reservoirs of viruses for infecting commercial wine grape vineyards through vector transmission. While this survey as well as the two surveys conducted in British Columbia [11, 18] provided compelling evidence for the wide distribution of major viruses in vinifera wine grapes, virtually no information is available pertaining to the distribution of viruses among non-vinifera grapes. We are currently conducting surveys for major viruses in these non-vinifera grapes in order to obtain a holistic assessment on the scope and magnitude of viral diseases in Ontario. This is important as all types of grapes, regardless of their genetic background or uses, would serve as host to many of the viruses that are destructive to the grape and wine industry, and consequently, would serve as natural reservoirs for the spread of viruses.

The ultimate solution to viruses and viral diseases is the generation and broad implementation of propagating materials that are free of all major viruses. This has been practiced in many grape-growing regions in the world, including the European Union, California and other states of the USA, and Australia. These survey results demonstrate the wide distribution of major viruses and the severity of viral diseases, urging the establishment of a nationwide clean stock certification program in Canada. In the meantime, it would be prudent to screen planting materials for certain most pathogenic viruses, producing the so called “best available materials’ for use in the establishment of new vineyards or replantation in existing vineyards before the full implementation of clean stock certification program. It is also crucial to identify biological vectors present in Ontario that are responsible for the transmission of highly detrimental viruses, which would allow the establishment and implementation of integrative strategies for the effective control of viral vectors.