A Floral Fragrance, Methyl Benzoate, is An Efficient Green Pesticide

Over-reliance on synthetic pesticides in insect pest control has caused widespread public and scientific concerns for human health and the environment, especially since many insect pests have already developed resistances to conventional pesticides and Bt products. For this reason, there is a considerable interest in development of alternative control methods for insect pest management. Based on laboratory studies, we report that methyl benzoate (MB), a naturally-occurring compound in many plants, may possess toxicity against various stages of a variety of insect pests, including the brown marmorated stinkbug, Halyomorpha halys, diamondback moth, Plutella xylostella, and tobacco hornworm, Manduca sexta, as well as the spotted wing drosophila, Drosophila suzukii. Based on our laboratory toxicity data, MB was at least 5 to 20 times more toxic than the conventional pyrethroid (β-cyfluthrin), sulfur & pyrethrin mixture, and some organic commercial products available on the market against H. halys, P. xylostella, and M. sexta, eggs. Because MB is considered an environment-friendly, it has great potential to be used as an alternative tool to synthetic pesticide for insect pest management in crop production, thereby, reducing threats to natural ecosystems and human health caused by over-application of conventional synthetic pesticides.


Results
Toxicity of MB to D. suzukii. MB, at a concentration of 1%, exhibited potent toxicity against D. suzukii when blueberries were exposed to D. suzukii four days prior to treatment ("pre-infested"). Exposure to MB resulted in 100% mortality as no larvae and pupae had developed nor adult flies emerged after 12 days incubation at room temperature ( Comparison of MB to 'minimum risk' pesticides against D. suzukii. Of all the compounds tested, MB exhibited the most toxicity against D. suzukii (Fig. 2). MB exhibited complete mortality and no adult flies survived after two days exposure to pre-treated blueberries (N = 6, F = 10.691; df = 6,35, p < 0.0001). All other essential oils ('minimum risk pesticides') tested did not show significant toxicity, when compared to the control. Following further incubation at ambient (room) temperature for 10 days, no adults emerged and significantly fewer pupae developed from MB-treated berries when compared to the blank control or other essential oil  The toxicity of MB is concentration dependent. After two days exposure to pre-treated blueberries, MB exhibited potent activity against adult D. suzukii at 1% and 5% concentrations. Little activity at 0.5% and no significant activity at 0.1% concentrations were observed (Fig. 4). (N = 6, F = 12.151; df = 4,25, p < 0.0001). Following further incubation at room temperature for 10 days, no adults emerged and significantly fewer pupae developed from 1% and 5% MB treated berries comparing to the blank control (Fig. 5 Ovicidal toxicity of MB and commercial pesticides. The ovicidal action of MB was compared to several commercially available organic pest control products ( Table 2). The evaluations were conducted by measuring hatchability in direct spray bioassay on three species of eggs, including H. halys, M. sexta, and P. xylostella. Our results indicated that the MB had potent ovicidal effects with an LC 50 value at 0.020 mg/cm 2 and LC 95 value at 0.048 mg/cm 2 on H. halys (Table 3). A lower concentration of MB (0.0637 mg/cm 2 active ingredient) was needed to reach 100% egg mortality for H. halys comparing to the other products used in the study (Table 4). At 0.0318 mg/cm 2 , MB was as potent as deltamethrin (0.0019 mg/cm 2 ), ζ-cypermethrin (0.0223 mg/cm 2 ), carbaryl (0.0080 mg/cm 2 ), pyrethroid (β-cyfluthrin, 0.1592 mg/cm 2 ), sulfur/pyrethroid (sulfur/pyrethrin, 0.6525 mg/cm 2 ), and one of the organic essential oil products (2-phenethyl propionate, clover oil, rosemary oil, and thyme  oil, at 0.3979 mg/cm 2 ). Commercially available pesticides, λ-cyhalothrin (0.0016 mg/cm 2 ), and acetamiprid, (0.0004 mg/cm 2 ) and another organic essential oil product tested containing rosemary oil and peppermint oil (0.0637 mg/cm 2 ) were almost ineffective. MB not only exhibited excellent ovicidal toxicity, but also had contact nymphicidal effect against H. halys nymphs ( Table 1).
The MB was also ovicidal against M. sexta eggs at 0.0637 mg/cm 2 dose with an LC 50 value at 0.015 mg/cm 2 and LC 95 value at 0.060 mg/cm 2 (Table 3). It was significantly better than the mixture of bifenthrin & ζ-cypermethrin (0.0239 mg/cm 2 ) and an essential oil product containing 2-phenethyl propionate, clover oil, rosemary oil, and thyme oil (0.3979 mg/cm 2 ) ( Table 4).
For P. xylostella, MB demonstrated potent ovicidal activity at a dose as low as 0.0032 mg/cm 2 , with an LC 50 value at 0.001 mg/cm 2 and LC 95 value at 0.005 mg/cm 2 (Table 3). Interestingly, the carbaryl was one of the most effective compounds against H. halys egg at 0.0080 mg/cm 2 , but it was one of the most ineffective ovicidal compounds against P. xylostella (Table 4).

Discussions
This current study demonstrates that methyl benzoate (MB) is an efficient green pesticide against invasive insect pest D. suzukii, and several other agricultural pests. MB not only effectively prevented D. suzukii from oviposition and inhibited subsequent larvae/pupae development, but also caused complete mortality of adult flies on pre-and post-treated blueberries at a concentration as low as 1%. Moreover, MB possessed ovicidal activity against several different species of eggs, when compared to some commercially available pesticides. On the basis of toxicity data, MB was five times more toxic than the conventional pyrethroid (β-cyfluthrin), 20 times more toxic than sulfur & pyrethrin mixture, and 12 times more toxic than one of the organic commercial products (2-phenethyl propionate, clover oil, rosemary oil, and thyme oil) against H. halys eggs. Neither γ-cyhalothrin nor acetamiprid exhibited ovicidal toxicity against H. halys at tested doses. For M. sexta and P. xylostella, similar toxic results were obtained, but P. xylostella appeared to be more sensitive to MB treatment. To reach 100% egg mortality, only 0.0064 mg/cm 2 was needed, which was 10 times less than H. halys and 20 times less than M. sexta eggs needed for the same results.
In nature, many plant species emit a great amount of VOCs into atmosphere, which are related to plant ecology, physiology, and atmospheric chemistry 16,17 . Some of these VOCs may act as defensive compounds against insect herbivores and plant pathogens; while others may act as chemical signals involved in plant-plant, plant-animal, and plant-microorganisms interactions 18 . MB naturally occurs as an aroma and scent of many plants 19 , including flowers 20,21 and fruits [22][23][24][25][26][27] , and plays important roles in plant communication with the surrounding environment.  Particularly, MB is one of the more abundant scents emitted from petunia, Petunia hybrid and snapdragon, Antirrhinum majus, functioning as a long-range attractant to lure bees for pollination [28][29][30][31] . MB has also been used by many insect species as a semiochemical that carries a message for purpose of communication between individuals of the same species (intraspecific) or between different species (interspecific) 32 . Moreover, MB is known for its sweet, balsamic, spicy, and heady floral odor; and it has been used as a fragrance ingredient and preservative in many personal care applications, such as shampoos, shower products and face/neck care, liquid soaps, mouthwash, perfume, hair colorants and cosmetics 33 . MB is of low to moderate human toxicity by ingestion and inhalation 34,35 , and it is approved by the US Food and Drug Administration (21 CFR 172.515) 36 37 for food use as a food-grade flavor ingredients. While MB is also considered environment-friendly, slowly biodegrading in the atmosphere 38 , it would still need to be registered as a pesticide with the EPA. To the best of our knowledge, the pesticidal activity of MB has not been previously reported. Overall, our research findings demonstrated that the methyl benzoate was an effective green pesticide against some invasive species, especially, H. halys and D. suzukii, with low concentration and high mortality; therefore, providing an alternative tool to synthetic pesticides for insect pest management in crop production.

Methods
Chemicals. Methyl 39 . Eggs were collected weekly and hatched in plastic Petri dishes with a water vial, and after molting to second-instars, the nymphs were transferred to the ventilated plastic cylinders for the remaining four instars 39 . Adult males and females were separated 1 or 2 days after emergence and subsequently maintained in different containers. The colony of M. sexta was originally obtained from the University of Arizona, Tucson, AZ, reared, and maintained on an artificial wheat germ diet 40 in an insectary located in the same USDA, Beltsville facility at 24 °C and 40% RH. Eggs and young larvae were covered by glass trays. Older larvae were kept in ventilated  plastic boxes (27 × 17.5 × 10 cm, BioQuip Inc, Rancho Dominguez, CA). Adults were kept in screened cages (45.75 × 45.75 × 45.75 cm, BioQuip). After small tomato plants introduced into the screened cages for 3-4 days, deposited eggs on the plants were removed by hand. The P. xylostella colony was obtained from Benzon Research, Carlisle, PA, reared, and maintained on an artificial wheat germ diet 41 at the same USDA facility. Eggs and larvae were put in closed cardboard cups (236 mL, 8.9 cm diameter, 5.7 cm height, Solo Cup Company, Lake Forest, IL) and kept in a Percival incubator at 25 °C, 34% RH, under a 16 L:8 D photoperiod in the same insectary. Adults were maintained in screened cage (30.5 cm × 30.5 cm × 30.5 cm, BioQuip Inc). Eggs were deposited on aluminum foil strips (approx. 5.0 × 30.5 cm) dipped in cabbage juice and collected after 3-4 days.
The D. suzukii colony was provided by Rutgers University, originally obtained from D. suzukii-infested blueberry (Vaccininum corymbosum cv. Bluecrop) fruits in Burlington County, New Jersey. The colony was reared on cornmeal diet 42  Toxicity of MB to D. suzukii. To investigate the acute toxicity of MB against D. suzukii, the bioassays were performed according to a published procedure 43 with modifications. A total of 100 blueberries were placed in a plastic cage (30 × 30 × 30 cm) and infested by 100 mixed sex adults D. suzukii for 4 days. After removing all insects, half of pre-infested blueberries were dipped in100 mL MB aqueous emulsion at 1% concentration, while other half of pre-infested blueberries was dipped in deionized water (DI) water as a control. The blueberries were subsequently placed in two separate Petri dishes and allowed to air dry for 2 hr. After drying, the blueberries were stored separately in two plastic cups (32 oz) with ventilated lids and incubated at ambient room temperature on the bench top for 12 days. At this time, the presences of adult D. suzukii were recorded, and the berries were dissected to assess development of any larvae and pupae. The experiment was repeated six times.

Comparison of MB to 'minimum risk' pesticides against D. suzukii.
To compare the pesticidal properties of MB to other essential oils (considered 'minimum risk pesticides') against D. suzukii, a series of bioassays were conducted according to the published procedure 43 with modifications. 10 blueberries were separately dipped in either 100 mL MB, or other essential oil aqueous emulsions (1%) Blueberries dipped in 100 mL DI water served as controls. The blueberries were placed in different Petri dish bottoms and allowed to air dry for 2 hr. After drying, the treated blueberries were placed in plastic cups (32 oz) with ventilated lids, and 10 adult (males and females) D. suzukii were introduced into each cup. Mortality of the D. suzukii was examined after 48 hr. After removing all insects, the blueberries were maintained at room temperature on the bench top for another 10 days. Present of adults were subsequently assessed and development of larvae and pupae was further inspected by dissection of the berries.
Toxicity of MB to H. halys nymphs. The contact mortality bioassays were carried out in scintillation glass vials (20 mL, Wheaton Scientific Product, Millville, NJ), following published procedures with modifications 44 . Filter paper was cut into round pieces (2.4 cm diameter). MB was added to acetone to give different concentrations (0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.0, 4.0%). 50 μ L of each solution was applied to the filter papers, and the filter papers were dried for 1 min before being placed in the bottom of the vials. A small piece of green bean (1 cm) was put on the filter paper in each vial as food source. Different stages of H. halys nymphs were introduced into the vials and then the vials were capped with loosen cotton balls to prevent the nymphs from escaping. For each stage, 30 nymphs were used for each concentration of MB and nine different concentration solutions (including blank control) were tested. In total, 270 nymphs were tested for each stage. Due to the size and weight of H. halys increased significantly from first to fifth instar, the number of nymph tested per vial was decreased from 10 to 2 accordingly so that they had enough space and could move freely in the vial. For example, for the first instar, 10 nymphs were tested pre vial (3 vials per concentration); for the second and third instars, 5 nymphs were tested per vial (6 vials per concentration); for the fourth instar, 3 nymphs were tested pre vial (10 vials per concentration); and for the fifth instar, 2 nymphs were tested pre vial (15 vials per concentration). The vials were maintained in a fume hood and mortality was assessed after 24 hr. Mortality data was subjected to probit analysis using PoloPlus for LC 50 , LC 95 with 95% confidence intervals calculation. In all tests acetone acted as the control. Ovicidal toxicity of MB and commercial pesticides. Aqueous solutions of MB at varying concentrations (0.00, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.0, 4.0%) and commercially available pesticides (see Table 2) were tested against several insect species. Eggs (10 for H. halys and M. sexta, 100 for P. xylostella) were collected and then placed on filter paper in Petri dishes. Different aqueous solutions were sprayed on the surfaces of the eggs three times (~ 0.5 mL total) using glass spray bottles (Amber glass with spray top, 30 mL) to completely cover the treatment areas (egg and filter paper). Then Petri dishes were covered with lids and maintained in a fume hood for 10 days. After this time, the Petri dishes were then inspected for presence of nymph or larvae, and the number of unhatched eggs, if any. An acetone spray was used as a control. The experiment was repeated three times.

Data analysis.
Comparisons of different treatments were analyzed using one-way ANOVA followed by Ryan-Einot-Gabriel-Welsch F test (SPSS 10.0 for Windows) 45 for significance at α = 0.05). Some data that ranges from being heavily positively skewed distribution (skewness = 1.37-2.38); therefore, log transformations were performed to remedy non-normality prior to the statistical analysis. PoloPlus software (LeOra Software, Berkeley, CA) was used to conduct probit analysis for mortality data, and LC 50 and LC 95 with 95% confidence intervals were estimated.