Final Report for GW12-022
Project Information
In the Pacific Northwest, an emerging complex of viruses in raspberry has caused symptoms of crumbly fruit, resulting in lowered fruit quality, and shortened life of the field. One of the important viruses is Raspberry leaf mottle virus (RLMV), transmitted by the large raspberry aphid, Amphorophora agathonica. Infection rates of 100% are commonly seen in fields only four years old. Control of RLMV depends on effective management of the aphid by targeting life stages that are responsible for virus spread to new fields, such as the winged morph of the aphid, or exposed life stages, such as the overwintering egg. Additionally, late-season raspberry aphid populations can be lowered by naturally-occurring parasitoids, as many mummified aphids (indicating parasitism) are observed in the late summer period. Given the potential overlap of viruliferous aphids and parasitoids in the field, the interactions between the two can affect control.
Introduction
In the Pacific Northwest region, an emerging complex of virus diseases in raspberry has caused symptoms of crumbly fruit, resulting in lowered fruit quality, crop loss, and shortened life of the field. As a result, control of these viruses has consistently been one of the top priorities of the Washington Red Raspberry Commission and the Northwest Center for Small Fruit Research. One of the viruses implicated in these symptoms is Raspberry leaf mottle virus (RLMV), a closterovirus that is transmitted by the large raspberry aphid, Amphorophora agathonica. RLMV is widespread in the top raspberry producing counties of Washington, which produce 95% of the processed red raspberries in the U.S. Infection rates of 100% are commonly seen in fields only four years of age and contribute to the loss of the raspberry crop and short life of the field. Control of RLMV depends on effective management of the aphid vector by targeting life stages that are most responsible for virus spread to new areas, such as the winged morph of the aphid, or broadly exposed life stages, such as the overwintering egg.
To improve current management of this aphid vector, this study: 1) determined if control of the fall flight period of A. agathonica is required for control of virus spread, 2) evaluated organic fungicides and oils for their efficacy in suppressing egg hatch, and 3) compared the development of parasitoids on aphids feeding on healthy or virus-infected plants.
Amphorophora agathonica has two flight periods throughout the season in northern Washington: in mid-June and September. Transmission of virus diseases into newly planted fields and uninfected fields occurs during these flight times, as aphids carrying the virus migrate into these fields. It is uncertain how important the September flight period is for new infections of raspberry plants. This is because the leaves on which the aphid is feeding may senesce and drop off before the virus has enough time to replicate and move systemically through the plant and root system. The first objective of this study exposed raspberries in the field to viruliferous (virus carrying) aphids to determine if there is a time after which new infections are unlikely. Defining this time eliminates the need to control aphids after this point and reduces insecticide inputs into the system without compromising plant health. Raspberry plants were systematically exposed to viruliferous aphids by caging the aphids to randomly selected plants weekly from September through November. After the plants overwintered, they were tested for presence of the virus via RT-PCR to determine if there was a point after which plants are unlikely to acquire the virus.
One of the challenges of controlling A. agathonica throughout the growing season is that it can be difficult to get effective insecticide coverage because aphids are protected on the undersides of leaves. During my PhD research, I found that the large raspberry aphid overwinters as an egg on the bare raspberry canes, primarily near the ground, where they are exposed. These eggs on bare canes may be a more vulnerable stage effectively targeted with an insecticide spray. Reduction of the overwintering population would result in lowered spring emergence and decreased aphid pressure at the beginning of the growing season. Current management practices, such as application of lime sulfur for control of fungal diseases before bud break, may affect aphid egg hatch. Additionally, horticultural oils and neem oils are organic products registered for use in raspberry, but their efficacy against aphid eggs is unknown. The second objective of the study was to measure aphid emergence rates in the lab when exposed to lime sulfur or oils. Aphid eggs were obtained and stored outside over the winter. Prior to egg hatch, the eggs were taken into the lab and exposed to lime sulfur, lime sulfur with dormant oil, neem oil, or a water control. Emergence rates were evaluated to determine which applications may be most useful in the field for decreasing overwintering success of A. agathonica.
Biological control of A. agathonica may play a significant role in aphid population suppression, as aphid mummies are frequently observed under field conditions. The most common parasitoid found is an Aphidius spp. (Braconidae). Because of the intimate relationship between plant viruses and their insect vectors, as well as the close relationship between aphids and parasitoids, the development of parasitoids may be impacted by the virus status of the aphids’ host plant. With the presence of both plant viruses and parasitoids in the field, interactions between the two may affect biological control of aphids. The last objective of this study examined whether the virus infection status of the host plant affected the development time and size of Aphidius spp. developing in A. agathonica. Aphid nymphs feeding on either healthy plants, plants infected with RLMV, or plants infected with Raspberry latent virus (RpLV, another aphid transmitted virus commonly found in raspberry cropping systems) were exposed to mated female parasitoids. The amount of time for the parasitoids to develop and the wing length of the newly emerged parasitoid (a measure of parasitoid size) were compared. Differences between treatments indicated that biological control is being impacted by the presence of raspberry viruses in the field.
Objective 1: Determine the late-season interval when control of aphids is important for reducing raspberry leaf mottle virus (RLMV) in raspberry, and if there is a date after which aphid control is unnecessary.
Objective 2: Evaluate the effectiveness of lime sulfur, lime sulfur with dormant oil, and neem oil for suppressing aphid egg hatch.
Objective 3: Compare the development time and offspring size of the parasitoid wasp Aphidius spp. (Braconidae) that develop on aphids feeding on infected versus healthy host plants.
Cooperators
Research
Objective 1: Determine the late-season interval when control of aphids is important for reducing raspberry leaf mottle virus (RLMV) in raspberry, and if there is a date after which aphid control is unnecessary.
This field study was conducted at the Lewis-Brown Experimental Farms in Corvallis, Oregon. An experimental field was used for this study because of the risk of introducing RLMV into a grower’s field. Additionally, virus and aphid pressure is low in this area, reducing the likelihood of contamination of the field experiment from surrounding fields.
In spring 2011, 100 virus-free raspberry plants (cultivar ‘Meeker’) were planted at Lewis-Brown farms. During the first week of September 2011, ten randomly selected raspberry plants were exposed to RLMV by five aphids each (adults and late-instar nymphs) carrying RLMV. The aphids were caged to the leaves to ensure that they did not move to other plants in the study. After four days, the aphids and any offspring were removed from the plants. The protocol was repeated with a new set of ten randomly selected plants on a weekly basis for ten weeks, until mid-November. The raspberries overwintered in the field and, in spring 2012, leaf tissue was collected from each plant and tested for the presence or absence of RLMV with RT-PCR using previously developed primers.
Because of difficulties achieving inoculation of RLMV during any week in 2011, infection of raspberry with viruliferous aphids was repeated in September 2012 with two year old Meeker raspberry plants., The protocol was as above, with the addition of a greenhouse control. Ten randomly selected plants in the field and ten young ‘Meeker’ raspberry propagated in the greenhouse were exposed to RLMV by five aphids each. The aphids were caged to the plants for four days. Plants in the field overwintered and, in spring 2013, leaf tissue was collected from each plant and tested for presence of RLMV using RT-PCR. Greenhouse control plants were treated with insecticides throughout the winter, then also tested for RLMV.
To further study the transmission biology of RLMV, inoculation experiments with varying densities of aphids were conducted in spring 2014. A colony of aphids was maintained on plants confirmed positive for RLMV. Clean recipient plants were then inoculated with 7, 20, or 50 aphids. After one week, aphids were counted and removed, and plants were treated with a systemic insecticide to prevent further feeding from insects. Plants were maintained in the greenhouse for one to two months to allow the virus to replicate and then checked for presence of RLMV with PCR tests.
Objective 2. Evaluate the effectiveness of various compounds for suppressing aphid egg hatch.
To determine which chemical treatments have the greatest impact on aphid egg survival, this study was conducted in the laboratory using a Potter Precision Spray Tower. The recommended field rates were evaluated for lime sulfur, lime sulfur with a dormant oil, and neem oil (Neemix), as well as a water control.
Raspberry aphid eggs were obtained by caging the sexual (egg laying) morphs of aphids with potted raspberry plants when these sexual aphids appeared in fall 2011. The plants were located outside to provide autumn temperatures and photoperiod. In January, the caged potted plants were dissected and the eggs gently removed from leaves and stems using a paintbrush. Eggs were surface sterilized for two minutes using a 10% bleach solution, rinsed with distilled water, then divided among 50mm Petri dishes lined with filter paper, and returned outside for the remainder of the diapause. In mid-February, each Petri dish of eggs was sprayed in a Potter spray tower with either lime sulfur, stylet oil, lime sulfur with stylet oil, neem oil, or water. After application, dishes remained open until the lime sulfur volatilized, then were closed and returned outside.
Aphid eggs were monitored daily to count the number of aphids that emerged each day in each treatment. The percentage of eggs that hatched in each treatment were compared using an ANOVA to determine which treatment was the most effective at reducing aphid emergence. Analyses were conducted in SAS 9.2.
Objective 3: Compare the development time and offspring size of the parasitoid wasp Aphidius spp. developing on aphids on RLMV infected, RpLV infected, or healthy host plants.
Parasitoids were reared on aphid nymphs feeding on plants with different infection statuses. Aphid nymphs were parasitized through exposure to mated Aphidius spp. in a Petri dish, then moved onto a host plant that was either healthy, infected with RLMV, or infected with Raspberry latent virus, RpLV (another aphid transmitted raspberry virus). Offspring parasitoid development was measured by both the number of days required for the parasitoid to pupate (aphid turns into a mummy), and the number of days required for pupation (days spent as a mummy before adult emergence). The size of the emerging parasitoids was estimated using the wing length, which is correlated with adult body size in parasitoid wasps. The study was replicated three times. Data were analyzed using an ANOVA to test for differences in the number of days for parasitoid development and for differences in wing length between treatments.
Objective 1:
Over two years, September – November 2011 and August – November 2012, 200 raspberry canes (‘Meeker’) were inoculated with RLMV by the aphid vector Amphorophora agathonica. Additionally, a set of 10 control plants were inoculated in the greenhouse each week with viruliferous aphids in 2012 (100 plants total). None of the plants experimentally inoculated in the fall tested positive for the virus in the spring. These results indicate either a) late season is a poor time for successful inoculation of the virus, or b) that the aphids were unsuccessful at inoculating with RLMV for some other reason. The fact that the control plants also did not become infected with virus suggests that fairly high aphid densities, or differences in the inoculation period, are needed to inoculate plants with virus. These results warrant further study on the transmission biology of the virus.
To examine the effect of aphid density on RLMV transmission, plants were infested with either 7, 20, or 50 viruliferous aphids. Only one of the 21 plants tested acquired the virus, and it had received seven aphids. This suggests that virus inoculation is not always consistent with aphid density. Additionally, these results indicate that are other factors involved in transmission of RLMV that are not understood at this time. Because in some cases high aphid densities did not result in infection, alternative control options such as biological control or use of resistance varieties which reduce but do not eliminate aphid populations may be suitable for pest control and virus management. However, further studies on virus transmission needs to be done to fully understand RLMV transmission biology.
Objective 2:
Amphorophora agathonica eggs were obtained during fall and winter 2011 by caging adult aphids with potted ‘Meeker’ raspberry plants. Eggs were treated in February 2012 with lime sulfur, stylet oil, lime sulfur + stylet oil, Neem oil, or water control in a Potter spray tower. Eggs sprayed with lime sulfur and lime sulfur + stylet oil had approximately 99% egg hatch suppression over eggs that were sprayed with water only (Figure 1). These results suggest that lime sulfur typically used for disease suppression as a current management practice may also have a beneficial impact at reducing egg survival of raspberry aphid. By reducing egg hatch in the field, the population increase of the pest may be delayed. Stylet oil alone was not successful at reducing egg hatch as has been observed in other systems and may be because of the late-winter timing of this study.
Objective 3:
Development time and size (wing length) of emerging Aphidius spp. adults was recorded after parasitized aphid nymphs were reared on healthy plants or hosts infected with RLMV or RpLV. Plant infection status did not affect the number of days required for developing parasitoids to pupate (turn into a mummy) or emerge from the mummy as an adult. However, plant infection status did significantly affect the size of the adult parasitoids (Figure 2). Parasitoids developing in aphid hosts feeding on healthy plants were larger than parasitoids developing in aphids feeding on plants infected with RpLV. Parasitoids developing in aphids on RLMV were intermediate in size between the healthy and RpLV treatments. Oftentimes, size of adults affects their longevity and/or fecundity, with larger adults able to survive longer and produce more offspring. Therefore, it is possible that the presence of virus in fields can negatively impact biological control by decreasing the overall number of aphids parasitized.
Research Outcomes
Education and Outreach
Participation Summary:
Extension
Lightle, D. and J. Lee. 2013. Large Raspberry Aphid Amphorophora agathonica. A Pacific Northwest Extension Publication. PNW 648, August 2013. http://ir.library.oregonstate.edu/xmlui/bitstream/handle/1957/41195/pnw648.pdf
Lightle, D. and Quito, D. Update on raspberry viruses and raspberry aphids. WSU Mount Vernon Northwestern Washington Research & Extension Center Field day, July 2012.
Lightle, D., Quito, D., and J. Lee. Seasonal phenology of Amphorophora agathonica and aphid transmitted diseases in Northern Washington. Washington Small Fruit Workshop, December 2012.
Publications
Lightle, D.M., D. Quito-Avila, R.R. Martin, J.C. Lee. 2014. Seasonal phenology Amphorpohora agathonica (Hemiptera: Aphididae) and spread of viruses in red raspberry in Washington. Env. Ent. 43:467-473.
Lightle, D. and J.C. Lee. 2013. Raspberry viruses affect the behaviour and performance of Amphorophora agathonica in single and mixed infections. Entomologia Exp. Appl. 151: 57-64.
Presentations
Quito, D., Lightle, D., Lee, J., Finn, C., Zasada, I., Johnson, D.T., Burrack, H., Fernandez, G., Clark, J.R., Sabanadzovic, S., Wintermantel, W.M., Tzanetakis, I., and R.R. Martin. Three Viruses Contribute to the Raspberry Crumbly Fruit Phenotype. Am. Society for Horticultural Science, August 2012.
Lightle, D. and J. Lee. Seasonal activity and biological control of large raspberry aphid (Amphorophora agathonica) in northern Washington. Ent. Soc. of Am., National Meeting, December 2010.