Integration of Microbial Pesticides in Pome-Fruit Production in the Pacific Northwest

Objectives 1-3

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.