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.

Résumé

Des essais en serre ont démontré la capacité du bourdon (Bombus impatiens Cresson) à transmettre le virus de la mosaïque du pépino (VMPép) de tomates infectées à la morelle douce-amère (Solanum dulcamara L.), et ce, au cours de deux essais sur trois (fréquence de 5.1 et 5.6%, respectivement). L’efficacité de la transmission était plus faible que lors d’études précédentes faites avec la tomate seulement (80%). Les faibles taux de transmission ont également été observés chez les bourdons allant de la morelle douce-amère à la tomate (6.3, 3.7 et 2.8%) et de plants de morelles à d’autres plants de morelles (8.3 et 2.8%). Chez les plantes témoins, la morelle était facilement infectée par inoculation mécanique. Les bourdons utilisés par les producteurs pour polliniser les tomates peuvent entrer et sortir librement des serres et ceux qui y ont contracté le VMPép peuvent l’établir et le disséminer dans les populations de morelle douce-amère poussant à proximité. Ce réservoir d’hivernage pourrait contribuer à introduire le virus en continu grâce aux bourdons revenant des champs pour une nouvelle saison de pollinisation dans les serres. De même, les bourdons pourraient répandre le virus chez les tomates poussant en champ après avoir pollinisé des morelles douces-amères infectées.

Keywords:

• bumblebees
• climbing nightshade
• Pepino mosaic virus
• tomato
• virus transmission

Mots clés:

• bourdons
• morelle douce-amère
• tomate
• transmission
• virus de la mosaïque du pépino

Introduction

Pepino mosaic virus (PepMV) was first reported in tomato greenhouses in Ontario in 2001 (French et al. 2001) and has been responsible for moderate to severe losses (5–15%) in production within Canada (Jones 2005). Reductions in fruit production by up to 2 kg m⁻² of production area have been reported (Ontario Greenhouse Vegetable Growers, Leamington, ON – personal communication). Phylogenetic analysis demonstrated that the Ontario isolate of PepMV formed a separate subclade that might indicate introduction of the virus into the region with subsequent molecular evolution (French et al. 2005).

Bumblebees (Bombus impatiens Cresson) have been shown to be an efficient vector in spreading PepMV between tomato plants in greenhouses (Lacassa et al. 2003; Shipp et al. 2008). In commercial greenhouses in Ontario where PepMV was present, 92–100% of the plants and 88–100% of the bumblebees tested positive for PepMV (Shipp et al. 2008). Bee movement between greenhouses and the field commonly occurs, and susceptible weed species both in and around greenhouses could become reservoirs of virus inoculum. A number of studies have examined weed hosts for this virus; many of the systemically susceptible hosts are annual species and therefore do not present significant sources of over-wintering virus carryover (Jorda et al. 2001; Roggero et al. 2001; Salamone & Roggero 2002; Cordoba et al. 2004; Kazinczi et al. 2005; Stobbs et al. 2009; Hanssen & Thomma 2010).

Three species of nightshade (Solanum dulcamara L. (climbing nightshade, perennial), S. nigrum L. (black nightshade, annual) and S. ptycanthum Dunal (eastern black nightshade, annual)) were shown to be susceptible to PepMV by mechanical inoculation in previous assays (Stobbs et al. 2009). The annual species S. sarrachoides Sendtn (black nightshade) and S. ptycanthum were susceptible to natural infection by bumblebees and served as reservoirs for seasonal transmission back to tomato (Stobbs et al. 2009). Annual carryover of PepMV in nightshade spp. is negligible since seed transmission of PepMV could not be detected (Kazinczi et al. 2006). Low levels of seedborne transmission have been demonstrated in tomato (Córdoba-Sellés et al. 2007; Hanssen et al. 2010) with the virus found only on the seed coat and not in the embryo or endosperm (Krinkels 2001; Ling 2008), implying that seed transmission likely occurs as a result of contact between the germinating seedling and the virus-contaminated seed coat (Sabaratnam 2008; Hanssen et al. 2010). Thorough disinfection of the seed coat of tomato seed from infected fruits has been shown to significantly reduce or eliminate infection of the seedlings (Ling 2010).

The aim of this study was to examine bumblebee transmission to and from perennial climbing nightshade plants which was not tested in the previous study. This species could represent an overwintering reservoir for the virus in the field, and represent a significant source of inoculum for reintroduction by bees back into tomato production houses.

Materials and methods

Source of PepMV

The Ontario isolate of PepMV, isolated and characterized previously by French et al. (2001) was used in this study. Infective plant sap was prepared by grinding young symptomatic leaves from infected tomato plants in ELISA extraction buffer (1:7; tissue:buffer) and used to inoculate carborundum-dusted leaves of either tomato or nightshade plants included in the study as positive source material in the vector trials.

Vector trials

Due to variances in research facilities available over the three years required to complete the study, different bee containment enclosures were used in three separate trials. In trial 1, a wood frame enclosure (3.7 m long × 3.7 m wide × 2.1 m high) (Fig. 1) covered with nylon mesh was constructed in a greenhouse and sealed to the concrete floor to prevent bumblebee egress. Two metal greenhouse benches (3.1 m long × m 1.2 m wide) were placed side to side in the enclosure. To examine bee transmission from PepMV-infected tomato to nightshade, eight tomato plants (Solanum lycopersicum L. ‘Glamour’) were mechanically inoculated with PepMV and grown in the greenhouse to flowering. Small climbing nightshade plants (S. dulcamara L.), collected from the field in the spring, were potted into 20 cm diameter plastic pots and similarly maintained in the greenhouse until flowering.

Fig. 1 Left: Bee containment cage constructed in a greenhouse used in one of the transmission trials. Right: Bumblebee feeding on nectar from tomato in PepMV transmission trials.

To check for absence of PepMV infection, all nightshade plants were tested by DAS-ELISA as previously described (Shipp et al. 2008) using a PepMV polyclonal antiserum obtained from Plant Research International (Wageningen, the Netherlands). In all ELISA tests, plants were considered positive if their substrate absorbance values (405 nm) exceeded two times those of the healthy controls for each plant species. PepMV infection was confirmed by ELISA for each of the infected tomato plants that were placed at the ends of the chamber. Nightshade plants were located in the centre with no less than 0.5 m distance from infected tomato plants. A drip irrigation system was installed to minimize physical contact between the plants. A hive containing 10–20 bumblebees (Bombus impatiens, Plant Products, Leamington, ON) was suspended over the plants for 14 days. PepMV-free pollen (Canadian Hydrogardens, Ancaster, ON) was confirmed by ELISA and added to each bee cage for bee feeding. Plants were then removed and incubated in a separate greenhouse for 4 weeks.

To examine transmission from infected nightshade plants to tomato, the enclosure was disinfected and eight PepMV-infected nightshade plants were placed at the ends of the benches, and 16 healthy tomato plants were located in the centre area of the benches. Plants were maintained as described for Trial 1. All plants were tested by ELISA for PepMV infection.

In trial 2, two screened tents (3.7 m long × 3.7 m wide × 2.1 m high) were set up in a greenhouse and sealed to the concrete floor. The greenhouse vents were screened to prevent ingress of any bees from the outside. To examine transmission from PepMV infected tomato plants to nightshade, 39 small nightshade plants were collected from the field in the spring, potted in 8 L pots and once established, compositely tested by ELISA for PepMV. Nine tomato plants (S. lycopersicum ‘Glamour’) grown in the greenhouse in 8 L pots were infected with PepMV. Both tomato and nightshade plants were used when they were approximately 0.5 m high and had begun flowering. Nightshade plants were located around the inside perimeter of the first screen tent while the tomato plants were placed in the centre.

To examine transmission from infected nightshade plants to tomato, 27 healthy tomato plants were placed around the inside perimeter of the second screen tent, and 9 PepMV-positive nightshade plants were placed in the centre. Watering and bumblebee release were as described previously. After 14 days, plants were removed from each tent and grown in the greenhouse for an additional 4 weeks and tested by ELISA for PepMV. Any infected nightshade plants resulting from bee transmission from tomato were mechanically inoculated back into tomato and tested as previously described.

In the third trial, three sealed greenhouse compartments (15 m long × 9 m wide × 10 m high) were used to examine bee transmission of PepMV between nightshade and nightshade and further replications on tomato to nightshade and nightshade to tomato. The experimental procedure was similar to trial 2 except 12 infected donor plants and 36 healthy test plants were used in each of these experiments. In this trial, nightshade plants were grown from seed (provided by P. Cavers, Dept. of Biology, University of Western Ontario) rather than using field collected plants. In all trials, nine test plants of the inoculation host plant were also tested by mechanical inoculation with PepMV.

Overwintering of PepMV in infected S. dulcamera plants

To determine whether PepMV-infected S. dulcamera plants overwinter and new year growth is infected, 10 PepMV-infected climbing nightshade plants were planted in a field plot at the Jordan research farm and maintained over winter, 2010. In June 2011, the plants were tested by ELISA for the presence of PepMV.

Results and discussion

In all trials, tomato and climbing nightshade could be easily mechanically infected (100%) with PepMV by rub-inoculation from either infected climbing nightshade or tomato. Bumblebees were shown to transmit PepMV from tomato to climbing nightshade at a low incidence in the second and third trials (5.1% and 5.6%, respectively, Table 1). This transmission efficiency is lower than that previously reported between tomato plants (80%) (Shipp et al. 2008) and between annual nightshade species including eastern black nightshade (S. ptycanthum) (94%) and hairy nightshade (S. sarrachoides) (81.3%) (Stobbs et al. 2009). In the first trial, where fewer plants were used, no transmission from tomato to nightshade was found. Heavy blossom damage in the nightshade plants did occur early in this trial, likely as a result of physical damage to the blossoms from higher density feeding activity of the bees on the smaller number of nightshade plants in the trial. In trials 2 and 3, back inoculation of infected climbing nightshade to tomato reproduced infection in the tomato plants.

Table 1. Transmission of PepMV by bumble bees between tomato and climbing nightshade.

Transmission direction	No. of samples	No. infected
Trial 1
PepMV-tomato to nightshade	16	0
PepMV-nightshade to tomato	16	1
Trial 2
PepMV-tomato to nightshade	39	2
PepMV-nightshade to tomato	27	1
Trial 3
PepMV-nightshade to nightshade
Replication 1	36	3
Replication 2	36	1
PepMV-tomato to nightshade	36	2
PepMV nightshade to tomato	36	1

Transmission of PepMV by bumblebees from infected climbing nightshade to tomato was demonstrated in all three trials at a frequency of 6.3%, 3.7%, and 2.8%, respectively (Table 1). Mechanical back-inoculation from infected tomato into nightshade reproduced symptoms in nightshade. Low transmission efficiency from climbing nightshade to tomato may reflect different physiological factors with each host. Tomato, with large glandular pilose trichomes (Bailey & Bailey 1976) terminating in sticky sap exudate laden with virus (L. Stobbs, unpublished results) provides a greater source of virus acquisition on mechanical contact with the bumblebees. This is likely a principal factor responsible for the higher rate of bee transmission between tomato plants as shown in a previous study (Shipp et al. 2008). The non-glandular stellate pubescent trichomes in climbing nightshade would provide less opportunity for virus acquisition by the bumblebees. Low rates of bee transmission were also observed between infected to non-infected nightshade (8.3% and 2.8% in two trials, Table 1). Mechanical injury and necrosis of the flowers by bee activity may limit systemic spread of any virus transferred to the flowers. However, bumblebee presence was commonly noted on other parts of the plants, suggesting that other factors may also be involved.

The ability of bumblebees to vector PepMV to a perennial nightshade species introduces a new weed reservoir for potential seasonal carryover of this virus. Infected plants were shown to overwinter and all 10 plants tested positive by ELISA the following year (data not shown). Build up of PepMV inoculum in climbing nightshade in the field represents an increasing risk of bumblebees introducing virus into tomato greenhouses where it would be further spread by pollinating bees. Similarly, field tomatoes could be adversely affected by virus acquired by pollinating insects from climbing nightshade. A survey completed in 2008 did not find PepMV in susceptible weed populations outside greenhouses with a past history of PepMV infection (Stobbs et al. 2009); however, with increasing inoculum pressure from greenhouses, this could change. The primary source of virus infection in commercial greenhouses is believed to be the introduction of young infected plants grown from contaminated seed in nurseries (Córdoba-Sellés et al. 2007). Virus is subsequently spread from infected plants through mechanical contact by workers or pollinating bees in the greenhouses. It is important to minimize the incidence of PepMV in greenhouses, possibly by more diligent treatment of seed for virus elimination (Ferguson 2001; Ling 2008, 2010; Hanssen et al. 2010), before the virus becomes established in weed populations and is much more difficult to control in both field and greenhouse crops.

Acknowledgements

The authors are grateful to Michelle Behar, Ashley De Foa, Vishesh Duggal, Carly Dumoulin, Zain Khan, Anna Krzywdzinski, Rebecca Matthews and Darlene Nesbitt for conducting the ELISA and infectivity assays and assisting with the bee transmission studies. Special appreciation is given to the greenhouse and facility management staff for construction or modification to existing greenhouses to accommodate the study. The research was funded in part by a grant from the Ontario Greenhouse Vegetable Growers and Agriculture and AgriFood Canada Matching Investment Initiative.