Final Report for SW08-049
A new isolate of the codling moth granulovirus, CpGV, was found to be equally effective as the commercial Mexican isolate, showing it to be a viable alternative to the Mexican isolate in cases of resistance development. A wood-based, anti-desiccant foam improved the efficacy of entomogenous nematode treatments on tree trunks for control of codling moth. Fumigation of adult and larval codling moth with volatile compounds produced by the fungus Muscador alba resulted in high mortality of all stages. Pandemis granulovirus killed all Pandemis pyrusana larvae prior to pupation and reduced feeding damage. Ongoing studies show the potential for auto-dissemination of the Pandemis virus by male moths. Seven grower presentations were made focusing on the use of codling moth granulovirus, and three technical articles were produced on some of these topics. Organic growers showed high satisfaction with the efficacy of CydX for codling moth control, except for requirements for repeated applications, largely due to solar inactivation.
1. Monitor the effectiveness of operational spray programs, including microbials (chiefly CM virus) in apple and pear orchards. (This monitoring was terminated after 2009 and superseded by New Objective 5)
2. Conduct research components as part of Objective 1:
a) obtain baseline data of efficacy of commercial formulations of CpGV to facilitate resistance monitoring among regional pest populations;
b) evaluate new CpGV virus strain in laboratory and field trials;
c) test new formulations (lignin- and particle-film based adjuvants) to improve the stability (UV tolerance) and effectiveness of CpGV under field conditions.
3. Determine the effects of volatile organic compounds emitted by the fungus Muscador albus on codling moth adults, neonate larvae, larvae in infested apples and cocooned larvae in diapause in simulated storage conditions.
4. Evaluate the effects of anti-desiccant agents in mulch and on tree trunks for improvement of larvicidal activity of Steinernema carpocapsae and S. feltiae for CM control.
5. Evaluate the efficacy of the Pandemis granulovirus on the Pandemis leafroller (Objective added to replace 1 and 2a, above).
6. Disseminate relevant information such as optimal application strategies or issues of concern regarding secondary pests through specified outreach activities.
Historically, the tree-fruit industry of the Pacific Northwest has depended on broad-spectrum pesticides, such as Guthion, to control damage from codling moth and leafrollers. EPA will phase out this pesticide by 2012, leaving conventional producers looking to the practices of organic farms to maintain profitability. Organic producers have been targeting codling moth with microbial pesticides, a sustainable pest management approach. This project studied the effectiveness of microbial pesticides to help producers through this transition. Efforts to develop improved formulations of the microbial pesticides continued, and research addressed the pesticide’s ability to kill secondary pests. Improving formulations and discovering the appropriate application strategies for microbial pesticides will encourage their usage in this large region of tree-fruit production.
For evaluation of CpGV efficacy, the Cyd-X product was used in field and laboratory studies in 2009, and Cyd-X was compared to the new Andemart CpGV isolate (Plus). Trees were sprayed with five treatment levels: Control, Cyd-X at 2 oz, Cyd-X at 4 oz, Plus 1 oz and Plus 2 oz. Ten undamaged fruit were picked from each of four trees zero, one, three and seven days after application. There were four reps per treatment; 40 pieced of fruit per treatment per day collected. The four reps were divided between the Red and Golden blocks. Treated apples were individually suspended from the lid of the 16-20 oz plastic ventilated containers with a binder clip attached to the apple stem. Five neonate larvae were added to the cheek of each apple, and the containers incubated for seven days, incubated at 25°C. After seven days the apples were evaluated. The number of stings for each apple was recorded, and then each sting was dissected out to determine if it was shallow <3 mm or deep >3 mm. The number of live, dead and missing larvae was recorded along with their larval instar. All of the above, including field sprays, sampling of fruit with fresh to aged residues and laboratory evaluations were replicated three times. Similar methods were applied to test UV protectant formulations, but treated fruit were exposed to a controlled UV exposure using a solar simulator. See Arthurs et al. (2008) for experimental details of using the solar simulator.
In field studies (2008), the effect of CpGV that had been stored at different temperatures was assessed by spraying trees and subsequently sampling fruit from 30 trees per site per treatment using a random-stratified method, and 50 fruit per tree were examined to determine percent fruit damage. Infested fruit (CM) were dissected in the laboratory to determine percentage mortality due to CpGV virus infection. The time of death and cadaver structure was used to estimate mortality due to CpGV and to determine fruit damage categories (shallow cosmetic damage and deep stings resulting in culling of the fruit) and ultimately to estimate population suppression.
(Objective 4) Studies of the bio-fumigant producing fungus Muscador albus were completed during 2008 and methods employed are extremely detailed and reported in Lacey et al., (2009).
(Objective 5) Studies of antidessicant formulations to enhance activity of entomogeneous nematodes were completed in 2009 and are reported in detail in Lacey et al(2010 EPNs for all experiments were produced in greater wax moth larvae, Galleria mellonella (L.), using procedures described in Lacey et al. 2010). Briefly, G. mellonella larvae were infected with S. carpocapsae S. feltiae or Heterorhabditis bacteriophora. Three days afterwards, the cadavers were dipped in a 1% starch suspension and then rolled in clay, until adhesion of the powder was complete. Viability of emerging EPNs from this formulation was estimated at 97%, based on an average of at least three counts for S. carpocapsae and S. feltiae.
Two antidessicant treatments were tested: a wood-flour based foam and a fire retardant gel, Barricade† II, were compared. Complete details for the formulation of these antidessicant sprays are provided in Lacey et al. (2010). To assess value of cover sprays of nematodes with either the wood-foam or gel was based on using sentinel codling moth larvae cocooned in cardboard strips (8x1.9 cm; double-faced, B flute). Perforated cardboard bands (7.6 cm wide and long enough to wrap around the trunk of each tree) were used for field testing the effects of gel or foam on the efficacy of S. carpocapsae and S. feltiae. Twenty diapausing CM larvae were used to infest each strip, and 75 larvae were used to infest each band.
To test the efficacy of the formulated G. mellonella larvae infected with of S. carpocapsae, S. feltiae or H. bacteriophora, the following Petri dish assay was used. Five Petri dishes (25mm deep and 150mm diameter) were used for each nematode species and untreated control for each type of sentinel larvae in cardboard strips in relation to formulated cadavers. The dishes were first filled 1 cm deep with potting soil (75 g and 20% moisture), and lightly patted down. One cadaver and one cardboard strip infested with 20 diapausing cocooned CM larvae were placed on top of the soil, covered with a layer of fine wood mulch at three different distances from the source cadavers. Five control dishes were set up in an identical manner to those of the nematode treatments. Because CM larvae are repelled by dampness, the lids of the dishes were secured with two rubber bands to prevent escape of larvae. The mulch was lightly misted daily to maintain moisture. The dishes were then incubated at 25° C for seven days and then mortality was determined. A portion of the cadavers were examined microscopically to confirm infection with nematodes. The experiment was replicated on three separate dates for the 1-cm position and twice for the 2- and 3-cm position experiments.
The efficacy of S. carpocapsae and S. feltiae in G. mellonella larval cadavers in mulch with and without wood flour foam and 120 mL of water was determined at the USDA experimental orchard near Moxee, WA. Thirty-one one-square meter plots in a Golden Delicious apple block were used for the research. Six treatments consisted of control plots treated with water (0.05% Silwet) or foam, and plots treated with cadavers infected with S. feltiae or S. carpocapsae with and without foam. Each treatment was replicated five times. Two sentinel strips were placed on the soil in the center of each control plot and two were placed 2-3 cm on each side of the cadaver in EPN-treated plots, and then covered with approximately 2 cm of mulch. The orchard was irrigated the evening before the tests. After adding sentinel larvae and infected larvae, the plots were lightly wetted with 120 mL of water per plot. Half of the plots were then sprayed with approximately 1 cm depth of foam.(See Lacey et al. 2010, for application details). After seven days, the sentinel strips were collected and taken to the laboratory to assess mortality. Temperatures in this and all other field trials were recorded with a Hobo H8 Pro data logger mounted in the scaffold branches of one of the treated trees.
Field tests on the effect of adjuvants on the efficacy of EPNs on tree trunks were also conducted at the YARL experimental orchard. Cardboard bands infested with cocooned CM larvae, as described above, were stapled onto the trunks of 35 trees in the block of Golden Delicious apples. Prior to application of treatments, trees were lightly misted with water until moist. Treatments consisted of water or foam controls each applied to six and five trees, respectively, and 24 trees sprayed with 300 mL of IJ suspensions (1000 IJs/mL) (3_105 IJs/tree); 12 with S. carpocapsae and 12 with S. feltiae. After application, six trees for each treatment immediately received a light misting of water or approximately 1.25 L of foam for each EPN species as described above. The amount of foam applied was determined by what was needed to evenly cover the bands and approximately 10-12 cm of tree trunk above and below the bands. Two days after applications, the bands were collected and incubated in the laboratory for five days at 25C and mortality was assessed.
Two additional field trials were conducted in May and September 2009. Infested bands were stapled onto the trunks of 45 trees in a block of Red Delicious apples. Treatments consisted of water, gel or foam controls each applied to five trees; 30 trees were sprayed with 300 mL suspensions of 1000 IJs/mL; 15 with S. carpocapsae and 15 with S. feltiae. After application of IJs, five trees for each treatment were misted with water, 1.25 L of foam, or 135 mL of fire retardant gel for each EPN species. The foam was applied as described above, and the gGel was applied to bands and tree trunks with an eductor nozzle designed to deliver a gel/water mixture at a ratio of 1:100 from the 3.8-L application container in which the product is sold. Three days after applications, the bands were removed and taken to the laboratory and were incubated seven days at 25° C prior to assessment of mortality. Treatment effects in all experiments were detected through ANOVA as described in Lacey et al. (2010).
(Objective 6) More complete methods are provided in Unruh et al., submitted. The Pandemis leafroller granulovirus, PapyGV, used in our research was originally isolated in Wenatchee, WA and propagated in 4th instar P. pyrusana taken from a clean colony of P. pyrusana maintained at the USDA-YARL facility on a bean-based diet. Recently killed or moribund, PapyGV-infected larvae were stored at -80° C until used. Fresh virus suspensions were prepared from the infected larvae by grinding them in distilled water (1 ml of water/larva). The number of PapyGV OBs/larva, approximately 2-4 x 109, was estimated microscopically.
Bioassays of PapyGV against P. pyrusana consisted of covering the surface of bean diet with a prescribed concentration of virus suspension described in Unruh et al. (submitted). Concentration-response bioassays were performed with five to six concentrations per test, which ranged between 20 and 10000 OBs/mm2 spread on the diet surface. Larvae were incubated at 25C and mortality of each larva was evaluated and recorded at 7, 10, and 14 days after treatment.
Measurements of leaf damage were based on the area consumed by fourth instar larvae of P. pyrusana of diet treated with 0, 20, 200 or 2000 OBs mm-2. After two days of feeding on the diet, 30 larvae for each concentration were transferred individually to 9 cm dia. plastic Petri dishes with a clean unsprayed apple leaf (var. Red Delicious). Larval status was assessed every two days until 22 days had elapsed or larvae stopped feeding for two consecutive interval, had pupated, or had died. A new leaf was provided every two days. Leaf area consumed by each larva was estimated with a leaf area meter as the difference in area between leaves before they were fed upon and again after the two day feeding interval in cm2. The influence of concentration of the granulovirus and time to cessation of feeding due to death or pupation (treated as a covariate) on cumulative leaf area consumed at the end of the feeding trial was evaluated with one way ANOVA . Time to feeding cessation was also considered as a response variable in a separate analysis.
A greenhouse study was conducted to measure the effects of PapyGV on neonate larvae colonizing trees containing the virus. Apple seedlings, var. Red Delicious, were sprayed with a solution of ca. 3 x 106 OBs/ml of PapyGV in 115 ml of water on each side of the seedling. Other seedlings were sprayed with a water control. After the spray had dried, an egg mass of P. pyrusana adhering to wax paper was stapled to each seedling. Fourteen days after virus application, living and dead larvae were enumerated on each seedling and living larvae were transferred to clean diet. Larval survival, pupation and apparent disease states were assessed every three-seven days until tests were terminated. The experiment was repeated on five dates conducted sequentially in a single greenhouse, allowed to vary in temperature between 12-27° C.
Tolerance of PapyGV to exposure to solar radiation was estimated using a solar simulator to irradiate virus on greenhouse-produced apple seedlings (var. Red Delicious). Seedlings were sprayed as in the green house study above but at ca. 107 OBs/ml. After drying, seedlings were exposed in a solar simulator as described in Lacey and Arthurs et al. (2008). Exposures were 765 W/m2 (=2.754 x 106 joule/m2) over one hour or 27,021 W/m2 over 6 h. Immediately after irradiation treatments, each of seven plants were infested with 20 and 30 larvae in the three consecutive replicates. Mortality was assessed at 7, 10 and 14 days after infestation of the plants. Treatment combinations tested represented a complete factorial: no virus and no irradiation, no virus with irradiation, virus with no irradiation and virus with irradiation. Each combination was replicated with three plants. Mortalities measured on three dates were analyzed using a repeated measure factorial ANOVA with virus and irradiation treatments as crossed factors.
Monitoring was conducted at two orchards in 2009, one organic and one treated with mixed chemicals and biopesticides. The organic orchard showed less effective control of codling moth using the granulovirus than it had in 2004-2008 as determined by pheromone trapping and apple damage. (This objective was terminated for lack of suitable study orchards as superseded by New Objective 5).
Field studies to estimate baseline susceptibility of Cyd-X as a representative commercial formulation of CpGV showed efficacy similar to that of previous years. In late 2008, we concluded research on storage and formulation of the codling moth granulovirus. CpGV retained 100% of the original larvicidal activity for over two years when stored at 2C. Comprehensive reviews on CpGV and use of microbial control in integrated pest management of orchard pests were published in 2008 and widely distributed to scientists, organic growers and crop consultants. Concentrated formulations) were compared with the original Certis formulation (Cyd-X). No substantial difference in efficacy was noted.
A new CpGV strain from from Andermatt Labs (the main supplier of Certis USA) was reported to overcome codling moth resistance to the Mexican strain of CpGV used in all commercial products. Our bioassays and field-testing indicated that the new strain is efficacious as the original Cyd-X formulation and both strains performed well. They are compared in Figure 1. Research on dry formulations of codling moth granulovirus (CpGV) produced by BioTepp, Inc. was concluded in 2009. Mortality produced by the dry formulation was inferior to liquid formulations currently on the market. Testing of adjuvant formulations (lignin- and particle-film based adjuvants) to improved CpGV longevity in the field showed a lignin formulation provided significant protection from UV radiation using a solar simulator in the laboratory. The protection of the virus was considerably less in the field.
The endophytic fungus, Muscador albus, produces volatile compounds (VOCs) that are biocidal for several pest organisms including plant pathogens and insect pests. The objectives of our research were to determine the effects of M. albus VOCs on codling moth adults, neonate larvae, larvae in infested apples and diapausing cocooned larvae in simulated storage conditions. Fumigation of adult codling moth with VOCs produced by M. albus for 3 d and incubating in fresh air for 24 h at 25C resulted in 81% corrected mortality. Four- and five-day exposures resulted in higher mortality (84 and 100%, respectively), but control mortality was also high due to the short life span of the moths. Exposure of neonate larvae to VOCs for 3 d on apples and incubating for 7 d resulted in 86% corrected mortality. Treated larvae were predominantly 1st instars, while 85% of control larvae developed to 2nd and 3rd instars. Exposure of apples that had been infested for 5 d, fumigated with M. albus VOCs for 3 d and incubated as above resulted in 71% corrected larval mortality.
Exposure of diapausing cocooned codling moth larvae to VOCs for 7 or 14 d resulted in 31 and 100% mortality, respectively, with negligible control mortality. Our data on treatment of several stages of codling moth with M. albus VOCs indicate that the fungus could provide an alternative to broad spectrum chemical fumigants for codling moth control in storage and contribute to the systems approach to achieve quarantine security of exported apples.
Evaluation of three new formulations of entomopathogenic nematodes (EPNs) was conducted in the fall 2008, spring 2009 and fall 2009. A foam formulation consisting of wood flour and adjuvants was applied immediately after application of EPNs for control of the overwintering stage of the moth (cocooned larvae). The foam maintained moisture enabling survival of the infective stages (IJs) of Steinernema carpocapsae and S. feltiae, permitting greater possibility of encountering cocooned larvae under bark and in leaf litter. Larval mortality was increased significantly with the foam formulation compared to aqueous suspensions of IJs. Cooler fall temperatures enabled greater survival of IJs and increased mortality of codling moth. A gel formulation (a biodegradable flame retardant) was also evaluated and, significantly, survival of the EPNs and increased mortality in codling moth larvae in 2009 spring and fall tests. A formulation consisting of infected wax moth larvae provided effective control in laboratory tests but failed to produce significant mortality in field tests against cocooned larvae under mulch. Evaluation of the effects of anti-desiccant agents in mulch and on tree trunks for improvement of larvicidal activity of Steinernema carpocapsae and S. feltiae for CM control. Figure 2.
The Pandemis leafroller granulovirus (PapyGV) caused 100% mortality before pupation of exposed larvae that showed symptoms of the disease. The onset of mortality was both a function of larval age and dosage. Even moderately low dosages (5 and 50 virus occlusion bodies (OBs)/mm2 of feeding substrate) resulted in high mortality when the observation period was extended to 20 days. One-hundred % mortality of neonates exposed to 100 and 1000 OBs/mm2 was observed at 20 and 7 days, respectively, following initial exposure. Whereas, mortality in fourth instar larvae was 95% after 14 days of exposure to 1000 OBs/mm2. Fourth instar Pandemis treated with 20-2000 OBs/mm2 consumed 20-80% less area of apple leaves. In a greenhouse study, neonates that feed on apple seedlings treated with 0.3 x 1013 OBs/hectare rates (dosage used with codling moth granulovirus) showed no capacity to complete. Exposure of plants sprayed with virus and exposed to 765 Watts/m2 for one hour in a solar simulator showed virtually complete loss of virus activity. This virus shows promise for population regulation but not for rapid cessation of feeding, despite reduced feeding rate.
Our outreach program has significantly increased in large part due to the objectives of the Western SARE grant. The overall goal is to disseminate information generated from our research to orchardists, farm managers and crop consultants. The presentations and publications listed below for late 2008-2009 have resulted in substantial transfer of microbial control technology to these groups. Another objective is to recruit and train young scientists, especially from under-represented minorities (Native Americans, Hispanics and women). Training this year has included the hiring of a recently graduated woman and familiarizing her with the production, quantification, bioassay and field assessment of insect pathogens that can be used for control of orchard pest insects. She is currently enrolled in a teaching credential program at Central Washington University and has spoken of the potential of transferring her skills learned in our lab to students in the near future. She will work with us part-time until obtaining her teaching credential. We also employed an under-graduate Hispanic male for our summer intern program. We have also trained a college educated Hispanic male in the laboratory and field technology. He also functions as our link with Hispanic workers and orchardists during presentation of workshops and field demonstrations. Part of our outreach is through the TechReach project for early (middle school) students at risk. I have attended two meetings of the American Indian Society of Engineering and Science (AISES) as a representative of the USDA Agricultural Research Service to encourage recruitment and diversity within the USDA.
Contribution: The new CpGV virus is not yet commercially available, but our research results will enable its more rapid registration when it is produced on a commercial scale.
Contribution: The foam formulation of EPNs provides a potential means of maintaining moisture, allowing longer survival and greater infectivity of EPNs.
Outcome: Some orchardists are interested but have not tried EPNs in their orchards because this formulation is not commercially available in a ready-to-use form. The increase in efficacy caused both by the formulation and the movement to Stienernema feltiae will provide incentive to use EPNs as part of integrated management of codling moth.
Contribution: The demonstration of complete prevention of maturation of Pandemis pyrusana larvae following feeding on PapyGV laced diet or leaves demonstrates the remarkable potential of this virus for population suppression.
Outcome: PapyGV would be inexpensive to produce, but revision of rules controlling the production and sale of such a virus must be revised before it would be available to growers.
Education and Outreach
Arthurs, S., Lacey, L.A., Behle, R.W. 2008. Evaluation of Lignins and Particle Films as Solar Protectants for the Granulovirus of the Codling Moth, Cydia pomonella. Biocontrol Science and Technology. 18:829-839.
Lacey, L.A., D.I. Shapiro-Ilan and G.M. Glenn. 2010. Post-application of anti-desiccant agents improves efficacy of entomopathogenic nematodes in formulated host cadavers or aqueous suspension against diapausing codling moth larvae (Lepidoptera: Tortricidae). Biocontrol Science and Technology, 20, 909-921.
Lacey, L.A., Headrick, H.L., Arthurs, S.P. 2008. The Effect of Temperature on the Long Term Storage of Codling Moth Granulovirus Formulations. Journal of Economic Entomology 101(2):288-294.
Lacey, L.A., Horton, D.R., Jones, D.C., Headrick, H.L., Neven, L.G. 2009. Efficacy of the Biofumigant Fungus Muscodor albus (Ascomycota: Xylariales) for Control of Codling Moth (Lepidoptera: Tortricidae) in Simulated Storage Conditions. Journal of Economic Entomology 102:43-49.
Lacey, L.A., Shapiro Ilan, D.I. 2010. Formulation to Enhance the Insecticidal Activity of Entomopathogenic Nematodes for Control of Insect Pests of Orchards. International Colloquium on Invertebrate Pathology and Microbial Control Proceedings. July 11-14, 2010, Trabzon, Turkey. pp. 20-27.
Unruh, T.R., Lacey, L.A., Headrick, H.L. and Pfannenstiel, R. The Effect of a Granulovirus on Larval Mortality and Feeding Behavior of Pandemis pyrusana (Lepidoptera: Tortricidae), For Biocontrol, Science and Technology (submitted).
Outreach presentations. Seven presentations were made in late 2008 -2010 on various topics in microbial control and integrated pest management of orchard pests at five grower and scientific society meetings:
Entomological Society of America. November 16-20, 2008, Reno, NV
Western Orchard Pest and Disease Management Conference January, 2009, Portland OR
Blue Mountain Horticultural Society. February 3, 2009, Milton-Freewater, OR
Washington State University sponsored field day July 11, 2009, Wapato, WA
Society for Invertebrate Pathology, August 16-20, 2009, Park City, UT
Entomological Society of America, December 13-17, 2009, Indianapolis, IN
Society of Nematologists was held from July 11- 14, 2010 in Boise, Idaho
Education and Outreach Outcomes
Areas needing additional study
- Research to discover improved adjuvants for the codling moth granulovirus that protect the virus from solar radiation and enhance feeding on the virus by codling moth are still needed.
A wood flour foam provided significantly better control than a gel or water. A paper was published on the results of this research with a SARE acknowledgement. Further research with industry needs to be conducted to make the foam formulation simpler to use and available from nematode producers.
We concluded research on factors that govern larvicidal activity of a virus of Pandemis pyrusana and its effect on larval feeding. A SARE acknowledgement will be added to the paper now in review. Ongoing research indicates that the Pandemis virus can be auto-disseminated if a pheromone trap/inoculator can be developed. Lophilized virus in the traps would be mixed with a carrier. Evidence for the potential of auto-dissemination is provided by the results of our research thus far: directly contaminated females contaminated their egg masses, but more importantly contaminated males can contaminate females during mating and they in turn contaminate their eggs. So far an impressive about of transmission takes place via sexual transmission and larvae die at a young age! We have considered this as a model system that could be used for CM auto-dissemination of virus. Further studies on the trans-generational epidemiology of the Pandemis granulovirus are needed.