Project Title: Exploring control of foliar cranberry pests: Fireworm, Tipworm, Dearness scale with a new natural pesticide (Neem formulation)

Project Title: Exploring control of foliar cranberry pests: Fireworm, Tipworm, Dearness scale with a new natural pesticide (Neem formulation) Date: October 25, 14 Report to: BC Cranberry Marketing Commission Attn: Dianne Driessen From: Institute for Sustainable Horticulture Kwantlen Polytechnic University 12666 72th Ave Surrey BC, V3W 2M8 Trial Site: Institute for Sustainable Horticulture Research Laboratory 91 Langley Bypass, Langley BC, V3 8G9 Director: Deborah Henderson 64-599-346, Cell 64-992-466 deborah.henderson@kpu.ca Research Trial Participants: Dr. Rita Seffrin, Institute for Sustainable Horticulture Jason Lussier, E.S. Cropconsult Ltd. Commission Research Priorities High: Tipworm reduced risk pesticide Fireworm reduced risk pesticide Medium: Dearness Scale reduced risk pesticide Long term: Organic production Objective Conduct Tier 1 (direct contact) and Tier 2 efficacy trials (on cranberry foliage) with 4 new neem oil formulations (Terramera Inc.) for control of cranberry foliar pests; Fireworm, Tipworm, Dearness scale Institute for Sustainable Horticulture October 14 Page 1

INTRODUCTION There is currently a need for more insecticide options for organic and conventional cranberry producers in the Fraser Valley. In response to this need, the Kwantlen Institute of Sustainable Horticulture has conducted preliminary experiments with two newly formulated neem-based botanical insecticides which are in development by Terramera Inc.; named in this report as TER397 and TER 447. It was intended to screen two additional, unformulated products, however the company requested that we test their formulations destined for registration. More replicates were completed since fewer products were screened. Neem essential oil contains compounds which have been shown to have insecticidal properties. The products used in our experiment were new neem formulations created by Terramera which contained additional ingredients to facilitate the application and effectiveness of these products. These new formulations are in their infancy; therefore our research was conducted in hopes to determine the effectiveness, in addition to identifying potential improvements. The products were tested on three common cranberry pests collected from cranberry fields in the Fraser Valley. The pests belonged to three different orders (Lepidoptera, Homoptera and Diptera) and are all problematic for organic and conventional growers with current insecticide options. This report is grouped into sections for each insect and includes several trials conducted to determine the efficacy of the neem products. Each section will have a pest overview, followed by methodology, results and discussion. A final project discussion is also given to interpret the overall potential of these neem products and to outline areas for future research. BLACK-HEADED FIREWORM Figure 1. 2 nd instar fireworm Larva on cranberry leaf. Scientific Name: Rhopobota naevana Order: Lepidoptera Family: Tortricidae The black-headed fireworm is one of the most challenging pests in the production of cranberries. Larvae may cause significant damage to crops if populations are not controlled. In conventional practice several insecticides are registered for this pests (including: Diazinon, Altacor, Intrepid, Delegate, Confirm), but options for organic producers are limited. The primary product available to organic growers in Canada is Entrust (Spinosad), and a maximum of three applications may be applied during a growing season. In fields with large and staggered populations, three Entrust applications may not suffice. Dipel (Bacillus thuringienses) may also be used to lower Institute for Sustainable Horticulture October 14 Page 2

fireworm populations, but experience has been that efficacy can be variable. It is this limitation on available products to control the black-headed fireworm larvae which is often most challenging for the pest management of organic crops. Current and new organic cranberry growers within the region may benefit greatly by the addition of new organic insecticide for this pest. LIFE CYCLE The black-headed fireworm overwinters in the egg stage on cranberry foliage. Eggs which are about.65mm in diameter, flattened, circular and pale to dark yellow, hatch in the spring when cranberry plants break dormancy. Fireworm larvae are caterpillar-like, with a distinct head (Fig. 1) and several pairs of distinct legs. When fully grown, the larvae range from cream, gray, green coloration; the head and shield (just behind the head) are dark brown to shiny black. First generation larvae generally occur between mid-may and mid-june. First instar larvae enter leaves to feed as leaf miners. They emerge from the leaf as second instar larvae and move to the upright tips to feed on new growth. Once mature, these larvae will form yellowish brown e which develop into adults after a two to three week period (depending on temperature). First generation adult moths are greyish-brown and lay eggs from late-may to late-june. The second generation eggs are laid towards the tips of the uprights, and the larvae occur from mid-june to mid-july. A second generation of adults flies from mid-july through mid- August. This generation primarily lays overwintering or diapausing eggs which tend to be lower in the canopy than summer eggs (but still mostly on the current year's growth). Although there are two generations per year, there is a partial third generation in BC which can develop into adults in a warm fall and continue to lay eggs (mostly diapausing) while weather is favorable; thus augmenting the population for the following spring. BIOASSAYS A.1- Indirect Contact and residual toxicity of TER 397and TER 447 on (small 2 nd to 3 rd instar) cranberry black-headed fireworm (Rhopobota naevana) larvae METHOD Cranberry shoots were placed into small plastic cups. Larvae (2 nd - 3 rd instar) were placed on the cranberry shoots to feed on the growing tips. For treatment, shoots with larvae in tents were removed from the cups and placed on paper towel then sprayed with the formulations using a small spray bottle. Afterwards, all shoots and larvae were placed back into the cups; with one larva and one shoot in each cup. Holes were made on the lids to avoid potential fumigation effect. Each treatment contained ten replicates (Fig. 2). The control was sprayed with tap water. Mortality was determined under a dissecting microscope 24 hours, 6 hours, and 8 hours after treatment (Fig. 3). Institute for Sustainable Horticulture October 14 Page 3

Percent mortality Percent mortality Figure 2. Cranberry tips with single small fireworm larvae in individual cups after treatment Figure 3. Cranberry tips with small fireworm larvae, at mortality assessment RESULTS No mortality was observed after 24 hours. After 6 hours, greatest mortality was observed in the TER447 treatment at 67%. In comparison, the mortality with the TER397 treatment was 5% and in the control 17% (Graph 1). The assessment after 84 hours showed a mortality increase in treatment TER397 to 67%, while theter 447 and control treatment remained the same (Graph 2). 8 6 4 397 447 Control Graph 1. Percent of black-headed fireworm (2 nd -3 rd instar) mortality; 6 hours after treatment. 8 6 4 397 447 Control Graph 2. Percent of black-headed fireworm (2 nd 3 rd instar) mortality: 84 hours after treatment Institute for Sustainable Horticulture October 14 Page 4

Percent mortality A. 2- Direct contact toxicity of TER 397 and TER 447 on cranberry (2 nd 3 rd instar) black-headed fireworm (Rhopobota naevana) larvae METHOD Larvae were placed on a paper towel and treated with the formulations using a fine brush to spread the product directly on the larva. Following this treatment larvae were then placed on cranberry uprights within small plastic cups. Each cup contained one larvae and one shoot, with ten replicates for each treatment. The Control was treated with tap water. Mortality was determined under a dissecting microscope 65 hours after treatment. RESULTS With direct contact, % of mortality of larvae occurred with the TER447 formulations. Mortality levels were observed to be at 6% with the TER397 treatment and at 1% in the control treatment (Graph 3). 9 8 7 6 5 4 3 1 397 447 Control Graph 3. Percent of black-headed fireworm mortality (2 nd 3 rd ) with direct contact; 6 hours after treatment. DISCUSSION In both of the above experiments both TER397 and TER447 treatments appeared to induce higher mortality than the control. It was also noted that some larval death occurred within the control treatment in both trials; ranging from 1-17%. The presence of dead larvae in the control indicates a flaw within the experimental design where other variables may be contributing to the mortality of larvae, possibly due to lack of humidity in the cups, which was noted at 48 hours. To address this possible source of mortality, a new methodology was used to create a better microclimate and prevent dehydration. Institute for Sustainable Horticulture October 14 Page 5

B. 1-Indirect Contact and residual toxicity of TER 397and TER 447 on cranberry (2 nd instar) blackheaded fireworm (Rhopobota naevana) larvae. METHOD Cranberry shoots were placed into plastic drink cups. Very small larvae (2 nd instar) from the lab colony (established with field collected larvae in 14) were placed on the cranberry shoots to allow feeding on new growing tips for a few minutes. For treatment, shoots with larvae were removed from the cups and placed on paper towel and then sprayed with the formulations; using a small spray bottle. Afterwards all shoots and larvae were placed back into the cups; one larva and one shoot in each cup. The cups were filled ¾ with tap water and crystal soil to prevent foliage dehydration. A paper sheet was placed above the crystal soil in order to avoid direct contact with the upright. A second cup was inverted over the first, and sealed with parafilm, to retain humidity. Small holes were made on the top cup to avoid potential fumigation effects (Figure 4 and 5). Five replicates were prepared for each treatment. Control was sprayed with tap water. Mortality was determined under a dissecting microscope 24 hours and 6 hours and eight days after treatment. Figure 4. Cranberry tip with small fireworm larva in cup Figure 5. Cranberry tips with small fireworm larvae at mortality assessment RESULTS No mortality was observed within 24 hours. At 6 hours after treatment, the highest mortality was observed in the TER447 formulation (4% mortality). In both the control and TER397 treatments, 1% of larvae were observed to be dead (Graph 4). Larvae assessment after 8 days showed % mortality in the TER447 group, with 3% mortality in the TER397. Mortality levels did not change in the control group (Graph 5). Institute for Sustainable Horticulture October 14 Page 6

Percentage of mortality Percent mortality 8 6 4 397 447 Control Graph 4. Percent of black-headed fireworm mortality (2 nd instar), 6 hours after treatment 8 6 4 397 447 Control Graph 5. Percent of black-headed fireworm mortality (2 nd instar), 8 days after treatment It was also observed during this experiment that larvae in the control and formulation 397 group showed similar development and size. In contrast, the only larva remaining in the 447 group after 8 days appeared to have a stunted growth (Figure 6). Figure 6. Comparison of larval size 8 days after indirect treatment Institute for Sustainable Horticulture October 14 Page 7

Percent mortality B. 2 Indirect contact and residual toxicity of TER 397and TER 447 on cranberry (3-5 th instar) blackheaded fireworm (Rhopobota naevana) larvae. METHOD Cranberry shoots were placed into small plastic drink cups. Larvae of 3 rd - 5 th instar were placed on the cranberry shoots to feed on the newly growing tips. For treatment, the cranberry shoots with larvae in tents were removed from the cups and placed on paper towel then sprayed with the formulations using a small spray bottle. Afterwards all shoots and larvae were placed back into the cups; one larva and one shoot in each cup. The cups were filled ¾ with tap water and crystal soil to prevent foliage dehydration. A paper sheet was placed above the crystal soil in order to avoid direct contact with the upright. A second cup was inverted over the first, and sealed with parafilm to maintain humidity for the plants. Small holes were made on the lids to avoid fumigation. Each treatment contained ten replicates. Control was sprayed with tap water. Mortality was determined under a dissecting microscope 24 and 6 hours after treatment. RESULTS No mortality was observed after 24 hours. Mortality was observed only in treatment TER447 (3% of larvae). All live larvae after 6 hours were healthy and survived the treatment (Graph 6). 8 6 4 397 447 Control Graph 6. Percent of black-headed fireworm mortality (3 rd -5 th instar); 6 hours after treatment. DISCUSSION The new experimental design used for these trials appeared to create a better environment for cranberry and black-headed fireworm larvae, as mortality in the control was reduced in the second set of trials. In all trials, the TER447 formulation appeared to be the most toxic to fireworm larvae. Institute for Sustainable Horticulture October 14 Page 8

Both the TER477 and TER397 products appeared to be more effective on smaller larvae (2 nd instar). Upon initial treatment many larvae appeared sick as movement was slower and upright feeding damage was reduced. With treatment on smaller larvae, many sick fireworm did not recover and were found dead after a prolonged period of 8 days (Graph 5). When treatments were applied to larger larvae (Trial B2), some fireworm also appeared sick but the recovery rate was observed to be higher. After 6 hours all larvae which had survived the treatment appeared healthy and recovered fully (Graph 6). For all sizes the mode of action of these products on fireworm larvae appears to be slow acting as limited larval mortality was observed in the 24 hour period after application. These products should be further investigated in the field to determine effectiveness of these products in reducing crop damage. Further research should also be done to investigate the lethal dose required to kill 5% of larvae (LD5 value), in order to determine adequate rates in the field. It may be important to note that in the 2 nd instar larvae trial (Trial B1), the only surviving fireworm in the TER447 treatment was much smaller than larvae in the TER397 treatment or control (Fig. 6). This suggests either a feeding deterrent or growth inhibition effect of this formulation. Of course, many factors may have contributed to this reduced size and further testing is required to confirm the observation and determine the reason. DEARNESS SCALE Common Name: Dearness scale Scientific Name: Rhizaspidiotus dearnessi Order: Homoptera Family: Diaspididae Figure 7. Dearness scale adults with thick shells The dearness scale is a localized pest which is found in specific regions of the lower Fraser Valley. This pest is relatively harmless in low levels but populations may grow quickly; causing extensive damage to vines. Diazinon is currently the only product used for control in the Fraser Valley. This limitation on registered insecticides is problematic as the optimal application-time for dearness scale is typically during bee pollinations from early June to July. As Diazinon is toxic to bees, some growers may choose to not spray for this pest. In addition to this dilemma, Diazinon is projected to be un-registered for cranberries in the upcoming years. This emphasizes the need to find a viable control options for this increasing cranberry pest. The 5 ES Cropconsult Ltd. study on Dearness Scale in the field Institute for Sustainable Horticulture October 14 Page 9

demonstrated that a neem oil product reduced crawlers significantly over other treatments at 1 and 4 weeks post treatment. For this reason, Dearness Scale was included in these trials. LIFE CYCLE Egg Stage The abdomen of the female contains a granular viscous yellow fluid that oozes out when poked with a needle. This fluid contains eggs in the early developmental stage. In late April and into May, the fluid becomes thicker and granules appear larger. By mid- May, the eggs hatch into crawlers which remain inside the shell of the female until emerging on stems in the first week of June. The formation of eggs and therefore crawlers, is staggered. Crawler Stage The crawler stage is observed on the vines from the first week of June. They are found both on new growth and older vines. Dearness Scale crawlers are minute (less than 1mm.), highly mobile, yellow ovals with three pairs of legs. The crawlers have needle-like mouthparts which they inject into the live vine to feed continuously. Crawlers escape from the females shell (Fig. 7) through a small split between the shells upper and lower portions at the tapered flattened end. The crawlers move easily to new growth and settle there. Once settled, they stopped moving and very quickly developed a small thin shell. BIOASSAYS The objective of these bioassays was to determine if the provided formulations would affect the crawler s ability to develop a new thin shell and crawlers survival. The first trial (C1) treated the adult females and cranberry stem just before the crawlers emerged; thus crawlers would contact the treatment only after leaving the female shell. The second (C2) set of trials was designed to provide more direct contact for the crawlers by partially lifting the female shell before the treatments. This was to mimic more closely a field application when crawlers were emerging and moving on vines C1- Residual toxicity of TER 397 and TER 447 on dearness scale crawlers METHOD A small cranberry shoot infested with one adult female scale was treated with the formulation then placed into a plastic drink cup. Two young cranberry shoots were sprayed with the formulation at the same time, using a spray bottle, and were placed in the cups as food supplies for crawlers. The cups were filled ¾ with tap water and crystal soil to prevent foliage dehydration. Another cup was inverted over the first one, and sealed with parafilm, to maintain humidity. The bottoms of the inverted cups were replaced with fine mesh to avoid fumigation (Fig. 8). Each treatment was replicate ten times. Controls were treated with tap water. In order to assess the efficacy of treatments, the cranberry shoots Institute for Sustainable Horticulture October 14 Page 1

were removed from the cups and observed under a dissecting microscope days after treatment (Figure 9).. Figure 8. Dearness scale on cranberry uprights treated with neem formulations Figure 9. Dearness Scale on cranberry stems treated with Neem formulations after days RESULTS Most crawlers were able to form new scales in the control treatment (9%) by days after treatment. In contrast, new scale formation was reduced in both neem treatments. The TER447 formulation saw the most reduction as only 5% of uprights contained new scales. Treatment TER397 saw a slightly lower reduction as 7% of uprights containing new scales (Graph 7). Institute for Sustainable Horticulture October 14 Page 11

(%) of uprights with new scale shells 9 8 7 6 5 4 3 1 397 447 Control Graph 7. Percent of uprights with new scale formation days after treatment C2-Contact toxicity of TER 397 and TER 447 on dearness scale crawlers METHOD Stems with single stationary female scale were painted with the formulation using a paint brush. The shell was opened slightly prior to treatment to exposure the crawlers to the formulation. After treatment uprights were placed in Petri dishes and covered with a small clip cage containing a net on the top to avoid fumigation. The stems were analyzed under a dissecting microscope 24 hours after treatment and the number of stems with a dead crawlers was determined. Ten replicates were prepared for each formulation, and controls were water treated. The bioassay was repeated three times in different days. RESULTS Very consistent results were observed in both the TER447 and control treatment in all three experiments. No stems with dead crawlers was observed in any control group but % of stems with a dead crawlers occurred in all TER447 groups. The number of stems treated with TER 397 with dead crawlers had more variation between trials, from 3% to 8%. Institute for Sustainable Horticulture October 14 Page 12

% of stems with a dead staionary scale % of stems with dead stationary scale % of stems with dead stationary scale 8 6 4 397 447 Control Graph 8 Percent of stems with dead stationary scale (Trial C2a) 24 hours after treatment 8 6 4 397 447 Control Graph 9. Percent of stems with dead stationary scale (Trial C2b) 24 hours after treatment 8 6 4 397 447 Control Graph 1. Percent of stems with dead stationary scale (Trial C2c) 24 hours after treatment DISCUSSION Scale trials were somewhat limited by difficulties in collecting and maintaining large enough numbers of scale in the lab. This resulted in a lower number of trials with fewer replicates than desired. In both Institute for Sustainable Horticulture October 14 Page 13

experiments, treatment TER447 appeared to have the greatest effect on reducing the number of live crawlers on stems. CRANBERRY TIPWORM Common Name: Cranberry tipworm Scientific Name: Dasineura oxycoccana Order: Diptera Family: Cecidomyiidae The cranberry tipworm larva feeds on the growing tips of cranberry uprights. Past research has shown this feeding damage to result in a decreased number of flowering upright, and thus, a lower yield. This pest is relatively new to the Fraser Valley, but has now become wide-spread throughout the region and is of great concern to growers. Control options are currently limited in both conventional and organic cranberry production. Fig. 1 Fig. 11 Fig. 12 Figure 1 Tipworm eggs deposited on inner leaves of terminal buds. Figures 11 and 12. The larvae are maggot-like, reach 1.5-2. mm when fully grown, and undergo three larval instars. They are initially clear but develop a milky coloration in the second instar (Fig. 11). In the third and final instar, the larvae undergo an orange color (Fig. 12). Fig. 13 Fig. 14 Fig. 15 Institute for Sustainable Horticulture October 14 Page 14

Figure 13, 14, 15. The e are 2. mm long (Left photo), initially orange but darken as they get closer to adult emergence. The e are enclosed within a white silken cocoon which remains in the cranberry tip (Center photo). The adults are gray and tiny only 2. mm long (about 1/1 the size of a mosquito). Adult females have a reddish abdomen. LIFE CYCLE The cranberry tipworm overwinters in the larval stage within the leaf litter. The adults emerge in mid- May when the cranberry buds have opened to provide an ideal habitat for oviposition. The larval stage lasts approximately a week, depending on factors such as temperature and population density (number of larvae per upright). The stage lasts three days with the adults living four to six days. The cranberry tipworm undergoes continuous generations throughout the entire season until early September when the larvae descend to the ground to overwinter. BIOASSAYS D1-Contact and Residual Toxicity of TER 397 and TER 447 on second instar larvae of Dasineura oxycoccana METHOD One second instar larva was placed on the top of a cranberry shoot which did not contain tipworm. The cranberry shoots containing one second instar larvae were then placed on a paper towel and sprayed with the formulations using a small spray bottle. Afterwards all shoots and larvae were placed into the cups; one second instar larva and one shoot in each cup. The cups were filled ¾ with tap water and crystal soil to prevent foliage dehydration. Another cup was inverted over the first one to maintain humidity and sealed with parafilm for attachment. Each treatment contained ten replicates. The control was sprayed with tap water. Mortality of the second instar larvae was determined under a dissecting microscope 6 days after treatment. Institute for Sustainable Horticulture October 14 Page 15

Percent mortality RESULTS The highest mortality of larvae after 6 days was observed in the group treated with formulation TER447 (75% of treated 2 nd instar were observed to be dead). Mortality was also found in the TER397 treated group (5% of treated 2 nd instar were observed to be dead). No larvae were observed to be dead in the control group (Graph 14). 8 6 4 397 447 Control Graph 11. Percent of tipworm mortality (2 nd instar larvae) 6 days after treatment (direct contact) D2-Contact toxicity of TER 397 and TER 447 on tipworm eggs METHOD Cranberry shoots infested with tipworm eggs were collected in the field. Each upright growing tip was opened carefully to ensure it contained only one egg (eggs were removed or added if need be). These uprights were then treated with 2µl (by syringe directly on the top of the upright) of product or tap water in the case of the control. All shoots were placed into the cups. The cups were filled ¾ with tap water and crystal soil to prevent foliage dehydration. Another cup was inverted over the first one to maintain humidity. Five replicates for each treatment were done. Mortality was determined under a dissecting microscope after 7 days. RESULTS During the assessment of this experiment many upright were observed to be empty (no dead eggs, larvae, egg, damage, larvae, or e) (Graph 12) and some contained more than one larva. As each upright was intended to contain one egg, these results were unexpected. The empty uprights may represent eggs which died and disintegrated, or uprights in which the egg had completed its life-cycle from larvae to adult (midge). Hidden eggs in the uprights may have also been present and contributed to uprights containing more than one larva. There was a lot of upright disturbance in this method as inner leaves were opened to place or remove tipworm eggs (to achieve one egg per upright). This disruption Institute for Sustainable Horticulture October 14 Page 16

Upright percent may have had a result on the microclimate within the cranberry upright; which may impact eggs. In response to these inconclusive results, a new methodology with less physical manipulation of uprights was incorporated in subsequent trials. 8 6 4 Control 397 447 Graph 12. Tipworm uprights assessed 7 days after treatment. The highest developmental stage was recorded for each upright D3 (a) and (b) - Contact toxicity of TER 397 and TER 447 on all tipworm stages METHOD The objective of this experiment was to determine the effect of treatments on undisturbed uprights containing tipworm, which would mimic more closely a field situation. Cranberry shoots were collected randomly from a field containing high levels of tipworm and brought to the lab. Uprights with no visual damage were selected at random for each treatment group; undamaged uprights typically contained tipworm at the early stages (egg or 1 st instar stage). It was known from previous monitoring that the majority of uprights collected from this field contained tipworm. The uprights were treated with 3µl of each formulation applied directly inside the unfolded leaves of the upright growing tip with a syringe. All shoots were placed into drink cups. The cups were filled ¾ with tap water and crystal soil to avoid foliage dehydration. Another cup was inverted over the first one to maintain humidity, and sealed with parafilm to attach the two cups. Control was sprayed with tap water. Ten replicates for each formulation were used. Mortality and upright damage was determined under a dissecting microscope after 7 days. The most advanced stage observed within the upright was recorded (some upright contained multiple tipworm at different stages). This trial was repeated twice. Institute for Sustainable Horticulture October 14 Page 17

Percent of uprights containing tipworm damage Upright percentage RESULTS Some variability was observed between the two bioassays, but overall trends were consistent. No dead larvae were observed in the control group in either trial. In comparison, dead larva were observed in both trials in the neem treatment groups (TER447-1 st bioassay (%), 2 nd bioassay (6%); TER397-1 st bioassay (%), 2 nd bioassay (%)). When looking at uprights with surviving tipworm, the larvae in the control group appeared to be more developed (larger) with more tipworm at the e and 3 rd instar stage (Graphs 13 and 15). Both neem formulations may have had an effect on upright feeding as a lower percentage of upright feeding damage was observed in these groups (Graphs 14 and 16). Some uprights containing only dead larvae were observed to have tipworm feeding damage. 1 st Bioassay D3(a) 9 8 7 6 5 4 3 1 Control 397 447 Graph 13. 1 st bioassay D3(a)- Tipworm upright assessment after 7 days. The highest developmental stage was recorded for each upright inspected 8 6 4 397 447 Control Graph 14. 1 st bioassay D3(a). Percent of uprights containing tipworm damage 7 days after treatment Institute for Sustainable Horticulture October 14 Page 18

Percent of uprights contianing tipworm damage Upright Percentage 2 nd Tipworm Bioassay D3(b) 9 8 7 6 5 4 3 1 Control 397 447 Graph 15. 2 nd bioassay D3 (b). Tipworm upright assessment 7 days after treatment. The highest developmental stage was recorded for each upright inspected 8 6 4 397 447 Control Graph 16. 2nd bioassay D3 (b). Percent of uprights containing tipworm damage 7 days after treatment D4- Contact toxicity of TER 447 and Movento 24 SC Insecticide on all tipworm stages The objective of this experiment was to compare the best neem formulation, TER447 (4g/64 ml) with Movento (4ml/ha)(the industry standard chemical contro), to compare their ability to control tipworm on undisturbed uprights. Institute for Sustainable Horticulture October 14 Page 19

Upright percent METHOD Cranberry shoots were collected randomly from a field containing high levels of tipworm and brought to the lab. Uprights which did not contain damage were selected for the trial (because they contained eggs and 1 st instar larvae). The uprights were treated with 3ul of each formulation and applied directly inside the unfolded leaves in the tip of the upright with a syringe. Three uprights were place per cup. All shoots were placed into the cups. The cups were filled ¾ with tap water and crystal soil to prevent foliage dehydration. Another cup was inverted over the first one to maintain humidity, and sealed with parafilm to attach the two cups. Control was sprayed with tap water. Eight replicates were used for each formulation. Larval stage and mortality were assessed under a dissecting microscope after 7 days. The most advanced larval stage was recorded for each upright assessed. RESULTS All uprights in the control treatment contained larvae at the 3 rd instar (23%) or stage (77%). In contrast, no uprights in the Movento treatment contained live larvae however, 25% of uprights were empty (no larvae, eggs or e) and 75% of uprights contained dead larvae. In comparison to these results, 63% of theter447 treated uprights contained dead larvae and 25% of uprights were empty. Only 12 % of uprights contained larvae (3 rd instar only) in the 447 group (Graph 17). 9 8 7 6 5 4 3 1 Control Movento 447 Graph 17. Tipworm upright assessment after 7 days with the Movento and TER447 treatment. The highest developmental stage was recorded for each upright inspected. Institute for Sustainable Horticulture October 14 Page

DISCUSSION A much lower rate of empty cranberry uprights were observed when using the undisturbed methodology to apply treatments. Survival and development in the control was also observed to be high and consistent with this experimental design. When testing formulations TER447 and TER397 on tipworm eggs and larvae, both appeared to induce death and reduce the rate of development. Of the two neem products, formulation TER 447 consistently caused higher mortality and reduced rates of development in all trials (lower percentage of uprights containing tipworm at the 3 rd instars and e stage). Further experiments should be conducted with a similar methodology in the field to determine the effect of these products on cranberry tipworm in a natural environment. Although lower levels of feeding damage were observed on neem treated uprights in experiments D3 (a) and D3 (b) (Graph 14 and 16), some feeding damage was still present. The presence of feeding damage in uprights containing only dead larvae indicates that tipworm larvae may have feed on uprights before death. When compared to the industry standard for cranberry tipworm control (Movento), the TER447 formulation showed a similar percent of uprights containing dead larvae (Graph 17). These results were interesting as Movento is the only effective product for cranberry tipworm, but is restricted on berries intended for the export market. Due to the restrictions on current products used for cranberry tipworm in both convention and organic productions, further testing should be done to determine the efficacy of these neem formulations within cranberry fields. PROJECT DISCUSSION AND CONCLUSION As a result of these preliminary trials with neem formulations TER 447 and TER 397, it appears that both products are toxic to all three cranberry pests studied in this project, with higher efficacy likely with TER 447. We may have observed not only mortality but negative effects on development of immature stages of all three insects. In addition to these results, improvements in methodology were made to conduct product testing on fireworm, scale and tipworm pests. Methods developed for holding these pests and applying treatments should aid in future product testing with these diverse insects. The promising results of this preliminary study should be followed up to further explore the value of these products for commercial cranberry growers and encourage Terramera to consider an early registration in cranberry. The results for Scale and Tipworm were particularly encouraging since control options are so very limited for these pests. Field testing of these products will better demonstrate the actual effectiveness of these new formulations however. Testing these products at varying concentrations in the lab with accompanying small scale field trials would be the next step. This report will be shared with Terramera as it will give them some direction about fine tuning a product for cranberry use, which would include the desirability of a formulation which could be applied via Institute for Sustainable Horticulture October 14 Page 21

chemigation. Current and future organic cranberry growers would benefit immensely from a new control product for all three pests. Conventional growers would also benefit from a new product for tipworm and scale as current control options are very limited and projected to become even more limited in the future. APPENDIX A RAW DATA Fireworm A. 1-Indirect Contact and Residual Toxicity of TER 397and TER 447 on cranberry blackheaded fireworm (Rhopobota naevana) larvae Bioassay with small instar larvae (2 nd 3 rd ) Institute for Sustainable Horticulture October 14 Page 22

1 st Bioassay (24 hours) Treatments Number of larvae Dead % Mortality Formulation 397 1 Formulation 447 1 Control 1 2 nd Bioassay (6 and 84 hours) Mortality 6 hours and 84 hours after treatment: Treatments Number of larvae 6 hours Larvae Dead % Mortality 6 hours 84 hours Larvae Dead % Mortality 84 hours Formulation 397 6 3 5 4 67 Formulation 447 6 4 67 4 67 Control 6 1 17 1 17 Institute for Sustainable Horticulture October 14 Page 23

A. 2- Direct contact toxicity of TER 397 and TER 447 on cranberry (2 nd - 3 rd instar) blackheaded fireworm (Rhopobota naevana) larvae. Treatments Number of larvae 65 hours Larvae Dead % mortality Formulation 397 1 6 6 Formulation 447 1 1 Control 1 1 1 B.1 Indirect contact and residual toxicity of TER 397and TER 447 on cranberry (1 st -2 nd ) black-headed fireworm (Rhopobota naevana) larvae. Results after 24 hours: Treatments Number of larvae Dead % Mortality Formulation 397 1 Formulation 447 1 Control 1 Results 6 hours after treatment: Treatments Number of larvae Dead % Mortality Formulation 397 1 1 1 Formulation 447 1 4 4 Control 1 1 1 Institute for Sustainable Horticulture October 14 Page 24

After 8 days: Treatments Number of larvae Dead % Mortality Formulation 397 1 3 3 Formulation 447 1 9 9 Control 1 1 1 B. 2 Indirect contact and residual toxicity of TER 397and TER 447 on cranberry (3-5 th instar) blackheaded fireworm (Rhopobota naevana) larvae. Results 24hours after treatment: Treatments Number of larvae Dead % Mortality Formulation 397 1 % larvae didn t make tents and were on the filter paper indicating possible repellent action and deterrence of feeding Formulation 447 1 % the larvae made tents Control 1 % Institute for Sustainable Horticulture October 14 Page 25

Results 6hours after treatment: Treatments Number of larvae Dead % Mortality Formulation 397 1 % larvae didn t make tents and were on the filter paper indicating possible repellent action and deterrence of feeding Formulation 447 1 3 3% the larvae made tents Control 1 % Scale C. 1- Indirect contact and Residual toxicity of TER 397 and TER 447 on dearness scale. Treatments Number of shoots Number of shoots with shelled scales % of shoots with shelled scales Formulation 397 1 7 7 Formulation 447 1 5 5% Control 1 9 9% Institute for Sustainable Horticulture October 14 Page 26

C. 2-Contact toxicity of TER 397 and TER 447on dearness scale crawlers. 1 st Bioassay (a) Treatments Number of stems with one stationary female scale Number of stems with dead female stationary scale % of stems with dead female stationary scale Formulation 397 1 3 3 Formulation 447 1 1 % Control 1 % 2 nd Bioassay (b) Treatments Number of stems with one stationary female scale Number of stems with dead female stationary scale % of stems with dead female stationary scale Formulation 397 1 6 6 Formulation 447 1 1 Control 1 Institute for Sustainable Horticulture October 14 Page 27

3 rd Bioassay (b) Treatments Number of stems with one stationary female scale Number of stems with dead female stationary scale % of stems with dead female stationary scale Formulation 397 1 8 8 Formulation 447 1 1 Control 1 Tipworm D. 1-Contact and Residual Toxicity of TER 397 and TER 447 on second instar larvae of Dasineura oxycoccana Treatments Number of larvae Dead % Mortality Formulation 397 8 4 5% Formulation 447 8 6 75% Control 8 % D. 2-Contact toxicity of TER 397 and TER 447 on tipworm eggs Cups Control 397* 447* 1 eggs No eggs, larvae or 2 larvae -1 nd instar 2 2 s No eggs, larvae or No eggs, larvae or Institute for Sustainable Horticulture October 14 Page 28

3 1 larvae-2 nd instar and 1 larvae 3 rd instar No eggs, larvae or No eggs, larvae or 4 2 larvae- 3 rd instar No eggs, larvae or No eggs, larvae or 5 No eggs, larvae or No eggs, larvae or No eggs, larvae or D.3- Contact toxicity TER 397 and TER 447 on all tipworm stages 1 st bioassay (a) Cups Control 397* 447* 1 1 larva 1 st instar and 3 larvae 3 rd instar 1 larvae 3 rd instar No eggs, larvae or 2 2 larvae 3 nd instar 3 eggs dead 1 larvae 2 nd instar 3 2 larvae 2 nd instar 1 larvae 2 nd instar 2 larvae 3 rd instar 4 1 1 larvae 2 nd instar No eggs, Larvae or 5 1 larvae 3 rd instar and 3 6 No eggs, larvae or 1 larvae 2 nd instar and 1 larvae 3 rd instar No eggs, larvae or 2 dead 1 st instars No eggs, larvae or e 7 No eggs, larvae or 5 e No eggs, larvae or e 8 2 larvae 3 rd instar 1 larva 2 nd instar and 1 larva dead 1 larva dead 9 2 e 1 larva 3 rd instar 1 2 nd instar 1 1 2 larva 3 rd instar No eggs, larvae or Institute for Sustainable Horticulture October 14 Page 29

e 2 nd bioassay (b) Cups Control 397* 447* 1 No eggs larvae or. 2 e No eggs, larvae or 2 4 e 2 e 1 dead 1 st instar 3 1 e Eggs dead No eggs, larvae or 4 1 e 1 st instar dead 1 dead egg 5 No eggs larvae or No eggs, larvae or e No eggs, larvae or 6 3 e 2 e 1 dead egg 7 4 3 rd instars 1 3 rd instar 2 e 8 1 e No eggs, larvae or e 9 2 e No eggs, larvae or e 1 1 e No eggs, larvae or e 1 dead egg and 1 st instar 3 dead 1 st instar 1 dead 2 nd instar D. 4- Contact toxicity of TER 447 and Movento 24 SC Insecticide on all tipworm stages Cups Control 447 Movento 1 2 s No eggs, larvae or No eggs, larvae or 2 3 s 1 dead egg No eggs, larvae or Institute for Sustainable Horticulture October 14 Page 3

3 1 1 egg dead and 1 3 eggs dead 4 2 larvae 2 nd instar and 2 s 5 1 larvae 2 nd instar and 4 larvae 3 rd instar No eggs, larvae or 2 eggs, 2 larvae 1 st and 1 larvae 2 nd instar dead 1 larvae 1 st and 2 larvae 2 nd instar dead 1 egg and 1 larva 1 st instar dead 6 3 larvae 3 rd instar 1 egg and 1 larva 1 st instar dead 7 1 3 larvae 2 nd instar dead, 1 larva 3 rd and 1 1 egg and 2 larvae 3 rd dead 5 eggs and 1 larvae 2 nd instar dead 8 1 larvae 2 nd instar and 3 s 1 larva 3 rd instar 1 dead egg Funding for this project has been provided by Agriculture and Agri-Food Canada and the BC Ministry of Agriculture through the Canada-BC Agri-Innovation Program under Growing Forward 2, a federal-provincial-territorial initiative. The program is delivered by the Investment Agriculture Foundation of BC. Institute for Sustainable Horticulture October 14 Page 31