Toxicity of Neem-Derived Insecticides to Various Life Stages of The ELM Leaf Beetle

Larval bioassays

Elm leaf beetle eggs were field collected from the Ft. Collins, Colorado area in June of 1991. The eggs were placed in Petri dishes, sealed with Parafilm and maintained in a growth chamber (photoperiod 16 L:8 D) at 25 ± 1 °C. Sufficient number of eggs were collected to allow a uniform age of 48 to 72 h post-eclosion for each of the three instars.

Serial dilutions ranging from 1:80 to 1:640 were prepared with Margosan-O® (0.3% azadirachtin, Grace-Sierra Crop Protection Company, Milpitas, CA). Freshly collected Siberian elm (Ulmuspumila) leaves were dipped for 5 seconds and allowed to air dry (approximately 30 minutes). The larvae were then placed onto the foliage in petri dishes and sealed with Parafilm. Fifteen 1st and 2nd instar larvae and twelve 3rd instar larvae were placed in each petri dish, respectively. There were six replications per dilution for each instar. Mortality was recorded at 48, 96 and 216 hr. In addition, mortality of 3rd instar was monitored through 13 days (312 hr) to determine if the treated larvae would develop into adults. Fresh untreated Siberian elm leaves were placed in the Petri dishes after 48 hr. The larvae were maintained in an incubator (photoperiod 16 L:8 D) at a constant 25± 1 °C for the duration of the study. Differences in larval mortality between concentrations were analyzed by ANOVA and means were separated by the Student-Newman-Keuls multiple range test (P < 0.05) (SAS 1988).

Antifeedant activity was assessed by painting both surfaces of one-half of a freshly collected Siberian elm leaf with an azadirachtin treatment. Four concentrations of Margosan-O® (1:80,1:160, 1:320 and 1:640) were evaluated for all three instars. The remaining half of each treated leaf was then treated with water. Larvae were placed on the midrib of the leaf which separated the treated and untreated halves of the leaf. The larvae were maintained in a Parafilm-sealed petri dish (photoperiod 16 L:8 D) at a constant 25± 1 °C. After 48 hr, larval location was recorded as off leaf, on treated or untreated portion of leaf.

Ovicide testing

Eleven elm leaf beetle egg masses (24-48 hr old) were flagged on field grown Siberian elm trees. Six of the egg masses were treated with Bio-Neem® (0.3% azadirachtin, 1:100 dilution, Ringer Corporation, 9959 Valley View Road, Eden Prairie, MN 55344) and five egg masses were treated with water. All treatments were sprayed to the point of run-off on the evening of 10 May, 1992. Eggs were allowed to remain on the trees until hatch was completed. On 11 June, they were returned to the laboratory and percentage of egg hatch assessed. A total of 107 untreated eggs and 140 treated eggs were counted in the trial.

Field trials

Field trials were conducted during 1993 on mature elm trees (Ulmus sp.) located in a municipal park in Denver, Colorado. The field trial was conducted on 15 June 1993, when elm leaf beetle eggs had just begun to hatch and minimal injury had occurred to the leaves. A single treatment of Azatin EC® (3% azadirachtin, 16 fl. oz/100 gal, Agri Dyne Technologies, Inc., Salt Lake City, UT 84108) was applied to run-off. The treatment was applied with a commercial hydraulic sprayer (Meyers Corp. #7360, maximum output 60 gpm). Single trees were considered individual plots, with a buffer tree separating each treatment tree to avoid effects from drift. There were six replications arranged in a randomized complete block design. Plots were evaluated 26 June, 14 and 26 July 1993 by determining the percent of leaves with feeding injury from terminals collected from the four cardinal points of each tree. This involved use of injury rating of 0-10 (0 = no damage, 1=1-10% of leaves with injury, 2 = 11-20% of leaves with injury, etc.) similar to that used by Brewer (2). Differences in leaf injury between treatments were determined using the Student-Newman-Keuls Test (P < 0.05) (SAS 1988).

Larval bioassays

3rd Instar

After 96 hr, mortality among all 4 concentrations was not significantly different than the control (Table 1). After 312 hr, mortality (97.2% to 100%) for all four concentrations was significantly higher than the control.

Developmental disorders were observed in all three instars 48 hr afterfeeding on the azadirachtin treated foliage. The first and second instar larvae that survived showed precocious development. In the process of ecdysis, the larvae were observed to have difficulty in extracting themselves from their exoskeleton. Many 1 st and 2nd instar larvae were found with head and thorax protruding from the old exoskeleton with the abdomen still attached. The azadirachtin also appeared to accelerate the maturation of an instar. The early stages of an instar, which are typically dark in color, would begin to prematurely take on the extensive yellowing of the latter stage of the same instar.

Several growth abnormalities were observed in all azadirachtin treated third instars. Third instar larvae were noticeably smaller than those larvae from the control group. The azadirachtin also appeared to accelerate the maturation of the third instar. Pre-pupal third instar larvae normally curl into a C-shape prior to pupating. This behavior appeared very early in the third instar stage for those larvae which fed on the treated foliage at all concentrations. Third instar larvae also developed a noticeable bulging of the thorax. After 13 days, only 7 larvae (all were still in the pre-pupal stage) from the treated group survived. Only 1 larva actually completed pupation and emerged as an adult. This adult was pink in color as opposed to olive green and black. In the control group, 50 third instar larvae out of the original 72 larvae pupated to adults.

In the antifeedant study, larvae of all instars showed evidence of avoidance of azadirachtin-treated foliage after 48 hr (Figure 1). Few larvae remained on the treated leaf surfaces, and most which did were found along the mid-rib. A minimum amount of treated foliage was consumed.

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Figure 1.

Location of Instar I, II, and III elm leaf beetle larvae 48 hours after being given a choice of leaf position on Margosan-O® (Al 0.3% azadirachtin) treated or untreated foliage. Total of six replications.

Ovicide testing

Treatment of elm leaf beetle egg masses with Bio-Neem® indicated ovicidal activity (Table 2). Treated masses contained large numbers of eggs that darkened within days of treatment and failed to hatch. Overall, 25.0 percent of the treated eggs did hatch, in comparison to 76.6 percent of those in the water check treatment. However, the majority of egg hatch occurred in only one of the six treated egg masses (33 of 35 eggs hatched). Furthermore, larvae observed to emerge from neem-treated eggs usually died shortly after hatch.

Field trials, neem insecticides

Field applications of the neem-derived insecticide, Azatin®, achieved excellent control of elm leaf beetle larvae (Table 3). Nearly complete control of feeding was observed throughout the season on treated trees. A small second generation of beetles prevented assessment of the persistence of these treatments. However a slight increase in foliage damage did occur during midsummer on treated trees, suggesting that persistence on foliage was limited to control of first generation larvae.

Commercial neem formulations that contain azadirachtin have been found to be effective against all three larval instars of the elm leaf beetle. This allows for an expanded application window forthe arborist. The ovicide study indicates that neem formulations which contain azadirachtin also may have potential as an ovicide. Azadirachtin’s low mammalian toxicity and short persistence give the arborist a safe alternative to currently used broad-spectrum insecticides.