vaporariorum (Hemiptera: Aleyrodidae) as vectors
of strawberry viruses in Quebec, Canada
Résumé
Dans le cadre du dépérissement des fraisières cultivées (Fragaria x ananassa Duchesne) au Québec, qui est principalement d’origine virale, notre objectif était de faire le suivi d’abondance et d’activité des deux insectes vecteurs: le puceron du fraisier, Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae), et l’aleurode des serres, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae). En comparant l’efficacité de deux outils de dépistage, les résultats ont démontrés que les pièges-collants jaunes sont plus efficaces que les pièges-bols jaunes pour détecter les envolées des vecteurs. Nous avons déterminé les périodes de vol des deux vecteurs à l’échelle du Québec : le pic d’abondance des pucerons du fraisier se situe durant les deux premières semaines d’août, alors que celui de l’aleurode des serres se situe durant les deux dernières semaines de septembre. Enfin, nous avons inventorié 55 espèces différentes de pucerons ailés et une nouvelle mention pour une espèce d’aleurode présentes dans les fraises.
Abstract
Strawberry decline disease, predominantly viral in origin, poses a serious threat to Quebec’s strawberry fields (Fragaria x ananassa Duchesne). Our aim was to monitor the abundance and activity of two insect vectors: the strawberry aphid, Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae) and the greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae). By comparing the effectiveness of two monitoring techniques, the results showed that the yellow sticky trap is more effective than the yellow pan-trap in capturing alates in flight. We determined the peak flight period for each of the two vectors in several locations within the province of Quebec: the peak abundance of winged strawberry aphids is during the first two weeks of August, while the peak abundance of the greenhouse whitefly is in the last two weeks of September. Overall, trap captures in strawberries field also found 55 different species of winged aphids and one new record of whitefly species.
Introduction
The Province of Quebec is the largest producer of strawberries (Fragaria x ananassa Duchesne) in Canada, with 52% of the country’s production, and ranks third in North America, after California and Florida (Agriculture and Agri-Food Canada, 2015). Most commercial strawberry growers in Quebec manage their crops using the conventional matted row system, under which production lasts 2 or 3 years (Hancock et al. 1997). Every summer, plants are mowed immediately after harvest in order to eliminate the old leaves infested with diseases, stimulate the proliferation of runners (stolons) and fruit buds, and promote a bountiful yield the following year (Thireau & Lefebvre, 2004).
Strawberry decline disease has become a severe issue for growers worldwide (Tzanetakis & Martin, 2012). Serious damage has been sustained by Quebec’s matted row strawberry fields since 2012 (Lambert et al. 2014). Symptoms include a reduction of runners produced by the plant, a weakened root system and failure to thrive after planting. One outcome is plant death, which represents an economic loss to the grower. The predominant causal factor of the disease is infection by two or more strawberry viruses, including both persistent (SMYEV and SCV) and semi-persistent types (SMoV, SVBV and SPaV) (Martin & Tzanetakis, 2013).
The only known vector of Strawberry pallidosis virus (SPaV) is the greenhouse whitefly Trialeurodes vaporariorum Westwood (Tzanetakis et al. 2014). Only a few whitefly species have been documented as colonizers of commercial strawberry plants in the United States and Canada: the iris whitefly, Aleyrodes spiraeoides Quaintance; the strawberry whitefly, Trialeurodes packardi Morrill, Trialeurodes fernaldi Morrill and Trialeurodes ruborum Cockerell (Evans, 2007). Other sources have reported the presence of the banded-wing whitefly, Trialeurodes abutiloneus Haldeman; the silverleaf whitefly, Bemisia tabaci Gennadius (Tzanetakis et al. 2006); and the cabbage whitefly Aleyrodes proletella Linné (Brisson, 2015a) in cultivated strawberry. Surprisingly, the most recent strawberry crop profile does not identify the greenhouse whitefly as an insect pest in commercial strawberry in Canada (Agriculture and Agri-Food Canada, 2015), similar to local lists in Quebec (Lambert et al. 2007). Although, many studies have demonstrated that whitefly pose a risk to strawberry (e.g. Martin & Tzanetakis, 2013; Tzanetakis & Martin, 2013; Martin & Tzanetakis, 2006; Tzanetakis et al. 2006). There seems to be limited published information about whiteflies in Quebec, given that the greenhouse whitefly is the only species listed for the province, based on the only known database for
Hemiptera (and Homoptera) insects in Canada, which currently contains more than 3900 species (Maw et al. 2000).
The SPaV, or Strawberry pallidosis, virus is propagated and introduced into other strawberry fields by the greenhouse whitefly during the winged stage. The vector’s flight peak thus corresponds to the maximum virus transmission. There is no available information about the flight period of the greenhouse whitefly in Quebec. However, it is mentioned that this vector has been observed exiting greenhouse structures at the end of summer (Lambert et al. 2014).
The main vector of Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV), Strawberry crinkle virus (SCV) and Strawberry vein banding virus (SVBV) is the strawberry aphid Chaetosiphon fragaefolii Cockerell (Martin & Tzanetakis, 2006). However, many aphid species can be present in strawberry fields, either as colonizers or as errants (Stultz, 1968). Among them, the yellow rose aphid Rhodobuim porosum (Sanderson), the green peach aphid Myzus persicae (Sulzer), the Foxglove aphid Aulacorthum solani (Kaltenbach), the large blackberry aphid Amphorophora rubi
(
Kaltenbach), the melon aphid Aphis gossypii Glover, and Chaetosiphon spp. may transmit strawberry viruses as well (Craig & Stultz, 1964; Tzanetakis & Martin, 2013).Among the viruses responsible for the strawberry decline disease, there are two different transmission modes: semi-persistent and persistent (circulative and/or propagative) (Sylvester, 1989). The semi-persistent viruses such as SMoV, SVBV and SPaV are acquired quickly by the insects and located in their stylet for a few hours (Watson & Plumb, 1972). Since the semi-persistent virus is only present in the stylet of the vector, the vector-virus specificity is thereby moderate, which indicates that other aphid species could transmit these viruses as well (Pelletier et al, 2012;. Andret-Link & Fuchs, 2005). However, the circulative persistent viruses such as SMYEV are acquired during feeding, circulating into the body for several days without replicating inside the insect. The circulative propagative persistent viruses such as SCV are also acquired during feeding, circulating in the body and replicating inside the insect for a few weeks or the lifetime of the aphid (Gray & Banerjee 1999). Since sustained feeding is necessary for the acquisition of persistent virus, the number of vector species is very limited. Since its virus-vector specificity is high, the strawberry aphid C. fragaefolii is the only known vector of SMYEV and SCV (Andret-Link & Fuchs, 2005; Sylvester, 1980).
Over the complex life cycle of aphids, the alate forms occur depending on the crowding conditions, the nutritional quality of the host plant (Wadley, 1923), temperature and photoperiod or when conditions for growth are optimal (Kawada, 1987). This can also be induced by the presence of natural enemies and pathogens (Irwin et al. 2007). Throughout the strawberry growing season, most winged aphids emerge, including the strawberry aphid. The winged morph is adapted for dispersion over great distances, being carried by the wind to other strawberry fields (Dixon & Kindlmann, 1999). Moreover, the probing behaviour of alate aphids during host selection involves intensive probing activity with several plants at once (Boquel, 2011; Klingauf, 1987). These features play a key role in the spread of viruses and the effectiveness of winged aphids as virus vectors. The presence of strawberry aphids in strawberry fields often coincides with high virus incidence in these same fields (Lavandero et al. 2012). The flight peak of the greenhouse whitefly also corresponds to the virus transmission peak of SPaV. There is currently no available information on the flight period of the strawberry aphid in Quebec. However, a report published by Lewis (2013) indicates that the flight period of the strawberry aphid in Western and Valley regions of Nova Scotia is set from beginning of June until the end of July.
The most common and relatively inexpensive screening techniques to monitor the flight activity of insects are the combined interception and attraction traps: pan-traps and sticky traps (Mukhopadhyay, 2011). As part of efforts to understand the strawberry decline problem, yellow sticky traps were used in Nova Scotia to record flight periods of winged strawberry aphids (Lewis, 2013). Pelletier et al. (2012) used yellow pan-traps filled with propylene glycol (40-60%) to conserve the RNA of the potato virus Y (PVY) from the captured aphids. The success of these two types of screening techniques comes from the yellow color, which is very attractive to many insects, including aphids and whiteflies (Eastop 1955; Berlinger, 1980). In order to measure the seasonal variation in the abundance of winged virus vectors, yellow pan-traps and yellow sticky traps seem to be appropriate methods.
As mentioned before, there is limited information about aphid populations and the flight activity of vectors of strawberry viruses in strawberry fields in Quebec. In order to help Quebec’s agronomists and strawberry producers, the objectives of this study were to 1) compare the effectiveness of sticky traps and pan-traps to monitor strawberry aphid and greenhouse whitefly in strawberry fields; 2) determine the flight peak of these two vectors;
3) examine aphid and whitefly populations; and 4) document aphid and whitefly species diversity in commercial strawberry fields.
Materials and Methods
Trapping Technique Comparison
Monitoring techniques to capture aphids and whiteflies as vectors of strawberry viruses were evaluated: yellow sticky traps and yellow pan traps. The yellow sticky traps were recycled PVC and sticky on both faces (Bug-Scan Dry, Biobest, Ilse Velden 18 2260 Westerlo BE – Belgium) (Figure 3). The glue was non-toxic, waterproof and did not dry out (Biobest, 2016). The trap measured 25 cm X 10 cm, for a total surface of 250 cm2. The sticky traps were hooked on two metal stakes to remain stable in the fields. Yellow pan- traps were setup following the collecting protocol used to monitor aphid flight in Nova Scotia (D. Moreau, personal communication). Stainless steel bowls were painted matte yellow, using Rustoleum Painter’s Touch 2X Ultra Spray Paint No 253712. Bowls were 19,7 cm in diameter and 6 cm deep for a surface of 305 cm2 (Figure 4). Pan-traps were filled twice a week with propylene glycol solution (55%-70% propylene glycol mixed with water) using protocol described in Pelletier et al. 2012. Propylene glycol concentration was higher because of evaporation in the fields. An individual bowl was hooked on to a modified tomato cage to remain stable in the fields.
Experimental Setting
Strawberry fields were selected 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) and Bellechasse (46°74’12.7’’N 70°92’04.1’’W), for a total of six fields. In both years (2014 and 2015), each field measured 1 ha and was in the first year of production using a conventional matted row system. Ten sticky traps and ten pan- traps were placed in a single corner in each field, with a 1-meter distance between traps. The corner was chosen according to the prevailing wind direction. From June 23to August 25, 2014 and June 22 to August 24, 2015, sticky traps were collected once a week and pan-traps were collected twice a week. When collected, sticky traps were wrapped in plastic wrap. For pan-traps, a strainer was used to empty the specimens in Whirl-pack ® Nasco bags and 95% ethanol was added for preservation. Samples were carried to the laboratory on ice packs and then placed in a refrigerator until processing. Abundance
counts and identification of C. fragaefolii aphid and whitefly specimens were performed using a stereomicroscope and based on morphological characteristics described in Foottit & Richards (1993) and Brisson (2015b). For the purpose of this objective, alate aphids were sorted as ‘strawberry aphid C. fragaefolii’ or ‘other species’, and all the whiteflies species were combined.
Statistical Analysis
Counts of winged aphids across time were analysed using a Generalized Randomized Block Anova model with repeated measures. In order to deal with the over- dispersed count outcome variables, the negative binomial distribution was chosen with a log link function. The experimental unit was a group of 4 to 10 traps, depending on the number of traps recovered. There were 12 experimental units for each type of traps, as there were 6 strawberry fields for 2 years. Since the number of traps was not constant, this variable was used as an offset in the model in the log scale. The fixed effects were the type of traps (pan-traps vs. sticky traps) and the dates, while the random effects were the sites. The total catch of winged aphids across the season was also analysed using a similar approach, but this time without repeated measurements. Analyses were done using the Glimmix procedure of SAS (release 9.4, SAS inc. NC). Following significant effects in the ANOVA table, protected LSD multiple comparisons were done to evaluate the differences between the average counts.
Vectors’ Flight Activity Across the Province
We measured the vectors’ flight activity, for both strawberry aphid and whiteflies using the yellow sticky traps as described above.
Experimental Setting
Seventeen sites throughout the province of Quebec were selected for the vectors’ flight activity measurements. For each region mentioned, one strawberry field was selected: 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). For 2014 and 2015, each field had a surface area of 1 ha, and was in the first or second year of production using a conventional matted row system. Five sticky traps were placed in one corner of each field, with a distance of 1 meter between traps. The corner was chosen according to the prevailing wind direction. From May 26thto October 27th (in 2014) and May 25th to October 26th (in 2015), sticky traps were collected once a week and wrapped in plastic wrap. Traps were promptly shipped to Dr. Fournier’s laboratory at Laval University and placed in a refrigerator upon arrival. Abundance counts and identification of C. fragaefolii aphid and whitefly specimens were performed using a stereomicroscope and based on the morphological characteristics described in Foottit & Richards (1993) and Brisson (2015b). Again, for the purpose of this part of the study, alate aphids were sorted as either ‘strawberry aphid C. fragaefolii’ or ‘other species’ and all whitefly species were combined.
Abundance and Diversity of Aphids and Whiteflies in Selected Strawberry Fields
Experimental Setting for whitefly diversity
Data on the abundance and diversity of whiteflies was obtained by counting the specimens caught from May 25th to October 26th, 2015 on the yellow sticky traps used at the 17 sites described previously. Specimens were identified under stereomicroscope based on morphological characteristics (Brisson, 2015b) and identification was validated by taxonomist J. D. Brisson.
Experimental Setting for aphid diversity
The abundance of aphid species and genera reported in this paper was measured by collecting the aphids caught in yellow pan-traps as described above. Three sites, located in St-Nicolas (46°68’88.1’’N 71°45’49.9’’W), Portneuf (46°77’54.1’’N 71°64’21.3’’W) and Île d’Orléans (46°86’27.7’’N 71°04’92.3’’W) were selected for the survey of aphid species and genera. We selected these fields due to the reduced number of overall pesticide applications that prevailed in them. Trap setup and collection methods
previously outlined under Experimental Setting for Trapping Technique Comparison. Once collected from the field, aphids were sorted and then mounted on microscope slides in Canada balsam, according to an established protocol (Maw, 1999). Mounted specimens were identified under stereomicroscope based on morphological characteristics (Foottit & Richards, 1993; Gualtieri & McLeod, 1994; Quednau, 1985; MacGillivray, 1979). Identifications were validated by taxonomist Eric Maw (Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa).
Results
Trapping Technique Comparison
The effectiveness of pan-traps versus sticky traps in capturing alate aphids, excluding the strawberry aphid, is shown in Figure 5A. The mean of the total catch, all sites combined, was 24.0 (±5.8) aphids per pan-trap, while it was 133.2 (± 32.4) aphids per sticky trap. This difference was significant (Type of traps (F=173.29; df=1, 11; p <0.0001)).
The ability of pan-traps versus sticky traps to capture strawberry aphids is shown in Figure 5B. The mean of the total catch, all sites combined, was 0.9 (±0.2) strawberry aphids per pan-trap, while it was 1.8 (±0.4) strawberry aphids per sticky trap. This difference is also significant (Type of traps (F=21.04; df=1, 11; p=0.0008)), even though the numbers are very low.
No whitefly was captured in pan-traps in 2014 or 2015. Therefore, no comparison was made between the two types of traps for this taxon.
Vectors’ Flight Activity Across the Province
Figure 6A shows the mean number of whiteflies caught per yellow sticky trap throughout the season. In 2014, the highest count per trap was 4 whiteflies, captured in mid-September. There was a peak of 56 whiteflies per trap at the end of September in 2015. Based on this, highest abundance of whiteflies was observed near the end of the growing season, although, whiteflies were usually present throughout the entire season, from the end of May until the end of October in both years. Mean numbers of whiteflies/trap were higher in 2015 vs. 2014.
Figure 6B represents the mean number of strawberry aphids captured per trap over the strawberry season. There was a peak of 0.5 strawberry aphids per trap in mid-August 2014, and 0.7 strawberry aphids per trap around the same time in 2015. The mean numbers of strawberry aphids/trap for each year were quite similar, though there were slightly more aphids in 2015 (N=3,1) than 2014 (N=2,9). They are mostly present from early June to the end of September, with a recurrence in mid-October in both years.
Abundance and Diversity of Aphids and Whiteflies in Selected Strawberry Fields
Table 7 shows the relative abundance of whiteflies captured in 2015, from the 17 sites throughout the province of Quebec using yellow sticky traps. Two different species of whiteflly were identified: the greenhouse whitefly, Trialeurodes vaporariorum (West.), and the iris whitefly, Aleyrodes spiraeoides (Quaintance). The highest number of greenhouse whiteflies was found in the region Lanaudière, with 1398 individuals. The iris whitefly was most abundant in St-Nicolas, with 88 individuals. The greenhouse whitefly represents 96% of all the catches, the iris whitefly, 4%.
Table 8 presents the list of all aphid species, or genera, that were captured using yellow pan-traps in selected three strawberry fields (St-Nicolas, Portneuf and Île d’Orléans) in 2014. A total of 1595 specimens were captured, representing 35 different genera and 55 species. The ten most abundant species were: Aphis spp. (764); chenopodium aphid, Hayhurstia atriplicis Linnaeus (239); pea aphid, Acyrthosiphon pisum Harris (123); soybean aphid, Aphis glycines Matsumura (98); green peach aphid, Myzus persicae Sulzer (54); mustard aphid, Lipaphis pseudobrassicae Davis (43); yellow clover aphid, Therioaphis trifolii Monell (39); strawberry aphid, Chaetosiphon fragaefolii Cockerell (24); Macrosiphum sp. (21) and large raspberry aphid Amphorophora agathonica Hottes (20).
Discussion
In the context of the strawberry decline disease, managing viruses starts with managing the winged form of the virus-carrying insects. Our aim was therefore to gather new and crucial information on the flight activity of the strawberry aphid and the greenhouse whitefly throughout the province of Quebec. We compared the effectiveness of two monitoring techniques: 1) yellow sticky traps, as used in Nova Scotia to monitor the flight activity of the strawberry aphid (Lewis, 2013); and 2) yellow pan-traps, as used in
New Brunswick to monitor aphid vectors of PVY in potato fields (Pelletier et al. 2012). Our results showed higher effectiveness of the yellow sticky trap technique over the pan-traps. Our study also unveils a new record of whitefly species in the province of Quebec.
Trapping Technique Comparison
The ANOVA results demonstrate that the yellow sticky traps were by far the most efficient screening technique for all species of aphids found in strawberry fields combined, as they collected the highest number of insects (Figure 5). This further confirms previous findings, where sticky traps were very effective for measuring the relative abundance of aphids (Harrington et al. 2007; A’Brook, 1973). Sticky traps are also very easy to use, as they require less equipment and manipulation than pan-traps. One of the disadvantages of using sticky traps is that identification of the aphid species can be difficult because the specimens are often in poor condition and key taxonomic characteristics are obscured.
Regarding C. fragaefolii, statistical analysis indicated a significant difference between the trap type, with the yellow sticky traps being the most effective. However, although statistically significant, the actual difference between capture numbers of C. fragaefolii was very low, 0.9. The overall low captures of C. frageafolii likely impacted the