Résumé
Le dépérissement des fraisières cultivées (Fragaria x ananassa Duchesne) au Québec est causé en grande partie par des virus (SMYEV, SCV, SMoV, SVBV et SPaV) transmis par des insectes vecteurs. L’objectif était d’examiner les sources de contamination virale dans les fraisières du Québec: 1) déterminer la prévalence de SMYEV et SCV dans le puceron du fraisier Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae); 2) déterminer la prévalence des virus dans le fraisier des champs (Fragaria virginiana Miller); et 3) évaluer la contamination des plants de fraisiers avant et après leur plantation en fraisières commerciales. Les résultats indiquent que 38% des pucerons analysés (N=205) et que 67% des fraisiers sauvages analysés (N=12) étaient infectés par un virus ou plus. Nos résultats démontrent que 5% des plants provenant de pépiniéristes (N=56) étaient contaminés à l’implantation, alors que 29% des plants sains exposés en champ (N=96) sont devenus infectés suite à la présence des vecteurs.
Abstract
The decline of cultivated strawberry (Fragaria x ananassa Duchesne) in Quebec is mostly caused by viruses (SMYEV, SCV, SMoV, SVBV and SPaV) transmitted by insect vectors. The objective of this study was to determine the diverse sources of viral contamination in strawberry fields in Quebec: 1) determine the prevalence of SMYEV and SCV in winged strawberry aphids Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae); 2) examine the prevalence of viruses in wild strawberries (Fragaria virginiana Miller) near commercial plantings; and 3) evaluate the contamination of strawberry plants before and after planting in commercial strawberry fields. The results indicated that 38% of the aphids (N=205) and 67% of the wild strawberry patches (N=12) were infected. Finally, our results showed that 5% of the plants from nurseries (N=56) were infected before planting, whereas 29% of the healthy exposed plants in the fields (N=96) became infected within a year of having been planted.
Introduction
Worldwide strawberry production is constantly growing every year, exceeding 4 million tonnes since 2007 (Wu et al. 2012). The largest producer is the United States, with 1.3 million tonnes (Martin & Tzanetakis, 2013). Canada contributes 18, 500 tonnes of strawberry produced in 2014 (MAPAQ, 2016). Hybrid species of strawberry (Fragaria x ananassa Duchesne) are grown in every region of the world, although most of the production is situated in the northern hemisphere (Hancock, 1999).
With more than 500 farms growing strawberries, the province of Quebec represents 52% of Canadian production (MAPAQ, 2016). The crop production system that is the most widely adopted is the conventional matted row system. This system needs a first year of new plants without fruit production and then two to three years of sustained production of strawberries (Hancock et al. 1997). However, continuous production over several consecutive years can result in an increased risk of accumulation of viruses and vectors (Black et al. 2002). Some 75% of Quebec producers buy strawberry transplants produced in Quebec’s nurseries or in vitro culture laboratories located in the province. The other 25% of strawberry transplants come from the US and Nova Scotia (S. Tellier, Conference Laurentides 2015). Although there are a lot of plant movements throughout North American, there are currently no certification programs (Martin & Tzanetakis, 2013).
In recent years, Eastern Canadian strawberries in matted row plantations have faced symptoms of decline. Infected strawberry plants often struggle when newly planted, as the root system is weakened and there is significant reduction of the number of stolons, causing reduced yields and important losses for Canadian producers (Martin & Tzanetakis, 2006; Lambert et al. 2014). New data show that the recent strawberry decline disorder is caused mainly by viral complexes of at least two viruses transmitted by insects (Tzanetakis & Martin, 2013).
In the province of Quebec, six viruses were detected in declining cultivated strawberries in 2013: persistent viruses Strawberry mild yellow edge virus (SMYEV) and Strawberry crinkle virus (SCV), and semi-persistent viruses such as Strawberry mottle virus (SMoV), Strawberry vein banding virus (SVBV) and Strawberry pallidosis virus (SPaV) (Tzanetakis & Martin, 2013; R. Hogue, Conference Lanaudière 2015; G. Gilbert, Conference RAP 2013). A sixth virus, named Strawberry Polerovirus 1 (SPV1) and newly
detected in Canada (Moreau, 2015; Xiang et al. 2015), was not included in the present study.
In addition to the cultivated hybrid species of strawberry, closely related plant species such as Virginian wild strawberry Fragaria virginiana Miller, are known hosts of SMYEV, SMoV, SCV and SVBV (Tzanetakis & Martin, 2013; Martin & Tzanetakis, 2006; Thompson et al. 2003). The wild strawberry is one of the most common host strawberry species in North America as it occurs in open woods, old fields, roadsides and meadows (Angevine, 1983). The virus SPaV shows a broader host range, infecting other plants than Fragaria spp., that includes Rosacea family member Sibbaldia procumbens L., and few Solanaceae family members Nicotiana benthamiana Domin, Nicotiana clevelandii A. Gray, Physalis wrightii A. Gray, and Malvaceae family member Malva parviflora L. (Tzanetakis et al. 2006).
The main insect vector of SMYEV, SCV, SMoV and SVBV is the Strawberry aphid Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae), while the SPaV is only known to be transmitted by the greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) (Martin & Tzanetakis, 2006; Tzanetakis et al. 2006). The SPaV alone has no impact on the decline of strawberry plants but in combination with two or more viruses transmitted by aphids, it can causes decline symptoms in plants (Tzanetakis & Martin, 2013). For both insect vectors, it is the winged form that can introduce viruses to adjacent fields, under the influence of the prevailing winds, though aphids can transmit viruses at all stages of development (Lavandaro et al. 2012; Krczal, 1980; Tzanetakis et al. 2014).
Potential host plants of strawberry aphid include various varieties of cultivated strawberry Fragaria x ananassa, Potentilla spp, wild strawberries Fragaria spp, and possibly Rosa spp., all part of the Rosacea family (Moreau, 2015; Blackman et al. 1987; Blackman & Eastop, 2006; Martin & Tzanetakis, 2015; Dicker, 1952). Meanwhile, the greenhouse whitefly has many host plants, including cultivated strawberry plant Fragaria x ananassa and wild strawberry F. virginiana plant (Tzanetakis & Martin 2013).
Among the mentioned viruses, there are two modes of transmission by vectors: 1) semi-persistent stylet-borne viruses (SMoV, SVBV and SPaV) are quickly acquired and reside in the stylets of aphids and whiteflies for a few hours (Waston & Plumb, 1972; Verbeek et al. 2014). Because the vector-virus specificity is moderate, other species of
aphids and whiteflies could possibly transmit semi-persistent strawberry viruses (Pelletier et al. 2012; Andret-Link & Fuchs, 2005; Tzanetakis et al. 2006); 2) persistent viruses are called «circulative», as they circulate into the insect body cavity (Gray & Banerjee, 1999). The non-propagative persistent virus (SMYEV) is acquired through feeding but does not replicate inside the insect. The propagative persistent virus (SCV) is also acquired during feeding and replicates inside the vector. Because feeding is needed for acquisition of persistent viruses, the specificity vector-virus is more specific than that of semi-persistent viruses (Andret-Link & Fuchs, 2005). Species from the aphid genus Chaetosiphon are the only known vectors of SMYEV and SCV (Tzanetakis & Martin, 2013).
The general aim of this study was to examine the importance of potential sources of virus contamination in Quebec strawberry fields. The specific objectives were to 1) determine the prevalence of persistent viruses (SMYEV and SCV) in winged strawberry aphids captured in strawberry fields; 2) examine the prevalence of persistent and semi- persistent viruses in Virginian wild strawberries near commercial plantings; and 3) determine to what extent healthy strawberry plants accumulate viruses, whether nursery stock is infected, or whether they get infected after planting in commercial strawberry fields, during the growing season and when exposed to the main virus-carrying insects. This knowledge will contribute to the establishment of provincial certification programs as well as identifying efficient strategies to reduce the prevalence of viruses in Quebec’ strawberry fields.
Materials and Methods
Prevalence of SMYEV and SCV in the Strawberry Aphid C. fragaefolii
Winged C. fragaefolii aphid specimens were collected using: yellow pan-traps installed in strawberry fields and strawberry leaf samples.
Yellow pan-traps were installed in 2014 and 2015 in six strawberry fields: Portneuf (46°77’54.1’’N 71°64’21.3’’W), Côte-de-Beaupré (46°91’50.2’’N 71°10’60.0’’W), Île d’Orléans (46°86’27.7’’N 71°04’92.3’’W), St-Nicolas (46°68’88.1’’N 71°45’49.9’’W), Beauce (46°41’92.5’’N 70°97’20.9’’W) and Bellechasse (46°74’12.7’’N 70°92’04.1’’W). Yellow pan- traps were setup following the collecting protocol used to monitor aphid flight in Nova Scotia (D. Moreau, personal communication). We painted stainless steel bowls with the Rustoleum Painter’s Touch 2X Ultra Spray Paint matte yellow. Bowls were 19.7 cm in
diameter and 6 cm deep for a surface of 305 cm2. Pan-traps were filled twice a week with propylene glycol solution (in 2014: 55% propylene glycol, 45% water; in 2015: 70% propylene glycol, 30% water) using protocol described in Pelletier et al. 2012. Propylene glycol concentration was higher in 2015 because of evaporation in the fields. The bowls were hooked on modified tomato cage to remain stable in the fields, about 40 cm from the ground. In both years, fields were 1 ha, first year production using conventional matted row system with cv Jewel, Cavendish, Mira, Sunset Valley, Wendy, Cabot, Sable and Clery. Ten pan-traps were placed in one corner of each field, 1-meter distance in between traps and the corner was chosen facing the prevailing wind direction. Pan-traps were collected twice a week for the following periods: in 2014, from May 26thto August 25th at 4 sites and until October 27th, 2014 in St-Nicolas and Île d’Orléans; in 2015, from May 25th to August 24th, at 4 sites and until October 26th in St-Nicolas and Île d’Orléans. A strainer was used to transfer the pan traps and collect specimens in Whirl-pack bags with 95% ethanol. Samples were transported to the laboratory using a cooler and were later placed in a refrigerator waiting for sorting and identification. Identification of aphid specimens was based on morphological characteristics, using a compound microscope (Foottit & Richards, 1993).
Leaf samples were collected from seventeen strawberry fields: Gaspésie (48°04'39.0"N 65°35'38.0"W), Bas-Saint-Laurent (47°54'52.1"N 69°26'06.7"W), Lac Saint- Jean (48°29'23.3"N 72°18'44.5"W), Portneuf (46°77’54.1’’N 71°64’21.3’’W), Côte-de- Beaupré (46°91’50.2’’N 71°10’60.0’’W), Île d’Orléans (46°86’27.7’’N 71°04’92.3’’W), St- Nicolas (46°68’88.1’’N 71°45’49.9’’W), Beauce (46°41’92.5’’N 70°97’20.9’’W), Bellechasse (46°74’12.7’’N 70°92’04.1’’W), Laurentides (45°35'55.6"N 73°55'35.1"W), Lanaudière (45°54'53.3"N 73°21'24.6"W), Mauricie (46°33'19.4"N 72°13'08.9"W), Outaouais (45°34'19.0"N 75°27'41.2"W), Montérégie Est (45°42'06.8"N 72°57'52.2"W), Montérégie Ouest (45°15'34.7"N 73°47'49.3"W), Estrie (45°26'49.5"N 72°00'15.4"W) and Centre-du- Québec (46°05'15.2"N 72°50'19.9"W). In both years (2014 and 2015), each field was 1 ha, first year or second year production using conventional matted row system with cv Jewel, Cavendish, Mira, Sunset Valley, Wendy, Cabot, Sable and Clery. Thirty young, newly unfolding leaf were collected from as many randomly selected plants, once a week, from May 26 to August 25, 2014 or until October 27 2014 in St-Nicolas, Île d’Orléans, Lanaudière and Centre-du-Québec as well as from May 25 to August 24, 2015 and until October 26, 2015 in St-Nicolas, Île d’Orléans, Lanaudière and Centre-du-Québec. The leaf
samples were kept in a sealed plastic bag and brought to the laboratory, then examined for winged aphids. Specimens were placed in vials with ethanol 95%. Strawberry aphid identification was done as previously described for pan-trap samples.
RT-PCR detection of SMYEV and SCV
Dr. Richard Hogue’s team from IRDA’s Laboratoire d’analyse biologique (LAB), in Quebec City, Canada, performed the RT-PCR detection of SMYEV and SCV for C. fragaefolii specimens. A cytochrome c oxidase mRNA from C. fragaefolii was used as an internal control using He et al. (2006)’s protocol. SMYEV and SCV were detected by one- step reverse transcription polymerase chain reaction (RT-PCR) using Zhang et al. 2013 protocol with QIAGEN OneStep RT-PCR Kit to extract viral RNA, using whole aphids.
Prevalence of five Viruses in Wild Strawberry Plants F. virginiana
We tested foliage sampled from Virginian wild strawberries patches nearby commercial strawberry fields. RT-PCR tests were done for the detection of the following five strawberry viruses: SMYEV, SCV, SMoV, SVBV and SPaV. Wild strawberry leaf samples were collected adjacent to twelve strawberry fields in the following localities: Portneuf (46°77’54.1’’N 71°64’21.3’’W), Côte-de-Beaupré (46°91’50.2’’N 71°10’60.0’’W), Île d’Orléans (46°86’27.7’’N 71°04’92.3’’W), St-Nicolas (46°68’88.1’’N 71°45’49.9’’W), Beauce (46°41’92.5’’N 70°97’20.9’’W), Bellechasse (46°74’12.7’’N 70°92’04.1’’W), Lanaudière (45°54'53.3"N 73°21'24.6"W), Mauricie (46°33'19.4"N 72°13'08.9"W), Montérégie Ouest (45°15'34.7"N 73°47'49.3"W)(N=2) and Estrie (45°26'49.5"N 72°00'15.4"W)(N=2) for a total of 12 samples. All the commercial strawberry fields adjacent to the collection sites were 1 ha, first year or second year production under conventional matted row system. For each site, we collected 2 leaves at each of 4 different patches of wild strawberry plants between May 28 and June 15, 2015. Once collected, the leaf samples were sent to IRDA’s LAB for RT-PCR analyses.
RT-PCR detection of five strawberry viruses
Detection of the five strawberry viruses for the wild strawberry leaves samples was done using RT-PCR. A plant mRNA specific internal control, Cox (Cox-F / Cox R), was used as a control as in Thompson et al. (2003) protocol. The band position of this control (79b) does not obstruct the interpretation of virus results (Qu et al. 2008). SMYEV, SCV,
SMoV and SVBV were detected by one-step reverse transcription polymerase chain reaction (RT-PCR) using Constable et al. (2010) protocol with QIAGEN OneStep RT-PCR Kit to extract viral RNA, using 0.3 g of homogenized wild strawberry leaves. The RNA samples were diluted 1/5 to eliminate any RT-PCR inhibitory effects.
Monitoring the Accumulation of Viruses in Strawberry Fields
We used «sentinel» strawberry plants for the monitoring of virus accumulation in 2015. These sentinels, consisting of young plants (cv Jewel) provided by various nurseries (new plantings) and by Phytoclone Inc (established fields), were tested for viruses using RT-PCR as described in the previous section, «Prevalence of viruses in wild strawberry plants» prior to in-field planting. Only sentinels that tested negative for all viruses (SMYEV, SMoV, SCV, SVBV and SPaV) were kept for the study. A total of 14 strawberry fields 1-ha matted-row system cv Jewel, Cavendish, Mira, Sunset Valley, Wendy, Cabot, Sable and Clery were used.
Experimental Setting
Eight of the 14 fields were new plantings in 2015 while the six other fields were established fields from the previous year (2014). In each field, a total of seven sentinel plants were selected to be planted between May 20 and June 19, 2015 at randomly chosen locations within the field and identified with a flag and ribbon. These seven sentinel plants were to be exposed to the presence of insect-carrying viruses throughout the growing season. Overall, a total of 98 sentinel plants were selected for planting.
The 14 strawberry fields were: Laurentides (45°35'55.6"N 73°55'35.1"W), Lanaudière (45°54'53.3"N 73°21'24.6"W), Estrie (45°26'49.5"N 72°00'15.4"W), Centre-du- Québec (46°05'15.2"N 72°50'19.9"W), Mauricie (46°33'19.4"N 72°13'08.9"W), Portneuf (46°77’54.1’’N 71°64’21.3’’W), Côte-de-Beaupré (46°91’50.2’’N 71°10’60.0’’W), Île d’Orléans (46°86’27.7’’N 71°04’92.3’’W), St-Nicolas (46°68’88.1’’N 71°45’49.9’’W), Beauce (46°41’92.5’’N 70°97’20.9’’W), and Bellechasse (46°74’12.7’’N 70°92’04.1’’W).
At each site, aphid and whitefly densities were monitored using yellow sticky traps, made of recycled PVC and sticky on both faces (Bug-Scan Dry, Biobest, Ilse Velden 18 2260 Westerlo BE – Belgium). The traps measured 25 cm X 10 cm, for a surface of 250 cm2 and were hooked on two metal stakes to remain stable in the fields. Ten traps were
placed in one corner of each field, 1-meter distance in between traps. The corner was chosen according to the prevailing wind direction. From May 25 to October 26, 2015, sticky traps were collected once a week and then wrapped in plastic wrap for preservation. Specimens captured on sticky traps were examined under the stereomicroscope to identify C. fragaefolii and T. vaporariorum according to morphological traits (Foottit & Richards, 1993; Brisson, 2015).
At the end of the growing season, from September 30 to October 28, 2015, 3 leaves were sampled from each sentinel strawberry plants and tested using RT-PCR for the presence of five viruses, as described in the previous section.
Statistical Analysis
A binomial regression model with overdispersion correction was used to describe the infection rate of sentinel strawberry plants in response to the number of strawberry aphids captured. Analyses were done using the LOGISTIC procedure of SAS (release 9.4, SAS inc. NC).
Results
Prevalence of SMYEV and SCV in the Strawberry Aphid C. fragaefolii
A total of 205 winged specimens of C. fragaefolii were sampled from yellow pan- traps and strawberry foliage during the 2014 (N=74) and 2015 (N=131) growing seasons. Figure 7 shows the prevalence of persistent viruses detected in samples collected from both pan-traps and leaves, combined for each year. In 2014, 43% of the winged strawberry aphids were infected: 31% with SMYEV only, 5% with SCV only while 7% of the aphids were infected with both viruses (SMYEV and SCV). In 2015, 35% of the winged strawberry aphids were infected: 15% with SMYEV only, 12% with SCV only while 8% of the aphids were infected with both viruses (SMYEV and SCV). Overall, 38% of winged strawberry aphids captured were infected with one or two persistent strawberry viruses when both years are combined (N=205).
Prevalence of Viruses in Wild Strawberry Plants F. virginiana
Figure 8 shows the prevalence of the five strawberry viruses detected in wild strawberries patches found nearby commercial plantings (N=12). One wild strawberry
patch was infected with SMYEV only, 1 patch with SVBV only, 2 patches were infected with SMoV, 1 patch were infected with SMoV + SMYEV, 1 patch were infected with SMoV + SCV and 1 patch were infected with SMoV + SPaV. In total, eight samples of the wild strawberry patches out of 12 were infected by one or two viruses.
Monitoring the Accumulation of Viruses in Strawberry Fields
A total of 98 «sentinel» strawberry plants were selected, but only 54 were planted in fields that were planted in 2015 (in a total of eight new plantings) and 42 sentinels planted in fields that were planted in 2014 (in a total of six established fields). Prior to in- field implantation, three sentinel strawberry plants from nurseries tested positive for virus: in Lanaudière region, two plants (tested positive for SMoV and SMYEV) were removed from the experiment, and in Mauricie region, one plant (tested positive for SMoV) was removed and replaced, for a total of 96 sentinel plants planted and monitored over the season (N=96).
Figures 9A and 9B show the viral infection rate (%) of sentinel strawberry plants comparison between new plantings and established fields at the end of the growing season. In new plantings, ten sentinel plants were infected (N=54) (Figure 9A). In established fields, 18 sentinel plants were infected (N=42) (Figure 9B).
Figure 10 shows the prevalence of strawberry viruses at the end of the growing season in infected sentinel plants, according to new plantings and established fields. In new plantings (N=54), ten sentinel plants were infected with SMoV only. In established fields (N= 42), a total of 18 sentinel plants were infected: ten plants were infected with SMoV only, one plant with SMYEV only, one plant with SCV only, four plants with SMoV and SMYEV combined and there were two plants infected with three viruses combined (SMoV, SMYEV and SCV).
Figure 11 shows the prediction of viral infection rate of sentinel strawberry plants at the end of the growing season, in response to the total number of winged strawberry aphids found on sticky traps during the season. The regression line demonstrates that the higher the population of winged strawberry aphids, the higher the viral accumulation among strawberry plants through the season occurs. The discrimination power of the model is judged fair by Hosmer and Lemeshow (2013) since the AUROC statistic take the value 0.771. The best cut-off point of the predicted probability of infection was determined
in order to have the highest sensitivity and specificity. The value of this cut-off point is equal to 0.30, which indicates that we can predict a plant infection when the estimated probability of infection derived from the model is greater than 0.30. By doing so, the sensitivity and specificity are equal to 0.70 and 0.75 respectively. Because no sentinel plants were infected with SPaV, which is only transmitted by greenhouse whitefly, the latter was not counted in a prediction graph.
Figures 12A and 12B show the sources of viral contamination in new plantings and established fields. Because nurseries provided sentinel plants for new plantings only, Figure 12A shows the nurseries contamination in new plantings. The RT-PCR tests indicate that three sentinel plants out of 56 total were infected (two with SMoV and one with SMYEV) prior to in-field implantation, which represents 5.4% of infection rate. Figure 12B shows the insects vectors contamination in new plantings and established fields combined. The RT-PCR tests indicate that 28 sentinel plants out of 96 total, as mentioned above, were infected throughout the season, after being exposed to strawberry viruses’ vectors, which represents 29.2% of infection rate. Figures 12A and 12B thus compare the two main sources of viral contamination, such as nurseries versus exposition to virus- carrying insects following implanting.
Discussion
This is the first study to test for the presence of viruses in specimens of the Strawberry aphid C. fragaefolii. We are also the first ones to compare the role played by two sources of viral contamination in commercial strawberry fields: 1) contaminated plants from nurseries and 2) contaminated plants from the feeding of insect carrying viruses. Our findings will contribute to a better understanding of viral contamination in Quebec’s strawberry fields.
Prevalence of SMYEV and SCV in the Strawberry Aphid C. fragaefolii
Boonham et al. (2002) studied the prevalence of Tomato spotted wilt virus (TSWV) in single thrips caught in sticky traps and universal bottles, using RT-PCR based on