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© 2002 Plant Management Network.
Accepted for publication 23 May 2002. Published 10 June 2002.

Toxicity of Indian Mustard and Allyl Isothiocyanate to Masked Chafer Beetle Larvae

Ryan R. P. Noble, Stephanie G. Harvey, and Carl E. Sams, Department of Plant Sciences and Landscape Systems, The University of Tennessee, 369 Ellington Plant Sciences Building, Knoxville, TN 37996

Corresponding author: Carl E. Sams. carlsams@utk.edu

Noble, R. R. P., Harvey, S. G., and Sams, C. E. 2002. Toxicity of Indian mustard and allyl isothiocyanate to masked chafer beetle larvae. Online. Plant Health Progress doi:10.1094/PHP-2002-0610-01-RS.

Abstract

Alternatives for control of soil-inhabiting pests are needed due to the phase-out of methyl bromide. One possible alternative is using the pesticidal properties of compounds released by macerated Brassica tissues. In this study, larvae of masked chafer beetles (Cyclocephala spp.) were placed in soil amended with Brassica juncea L. (PI 458934) tissue. Allyl isothiocyanate (AITC) levels were positively correlated to larval mortality, with the 8% B. juncea treatment resulting in 100% larval mortality with an average AITC concentration of 11.4 mg per liter of soil atmosphere. Although B. juncea produces high levels of AITC, the mass of tissue required for significant insecticidal activity against Cyclocephala spp. also is high, between 4 and 8% of soil mass for this plant accession.

Introduction

Methyl bromide is an important soil fumigant against soil-inhabiting insects, nematodes, fungi, and weeds in strawberry, tomato, and nursery stock. In accordance with the U.S. Clean Air Act amended to the Montreal Protocol, methyl bromide use will be banned by 2005 (20). Therefore, it is imperative that alternative controls be developed. One alternative control is to use the pesticidal compounds released by macerated Brassica tissues. This control alternative recently has been referred to as biofumigation (1).

The toxicity of Brassica tissues is derived from glucosinolates that are converted to isothiocyanates, organic cyanides, oxazolidinethiones, and ionic thiocyanates by the enzyme myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) in solution (4,6,10). The isothiocyanates are the primary products formed from the degradation of glucosinolates (2,3). Allyl isothiocyanate (AITC) is the most toxic compound formed from allyl glucosinolate hydrolysis in B. juncea L. (12,14), and possibly the most important for biofumigation. To determine the effectiveness of biofumigation, the lethal concentration of these toxic compounds must be evaluated relative to percentage mortality of the pest species. The plant mass of B. juncea required as a soil amendment then can be determined.

Previous studies have demonstrated the insecticidal potential of Brassica species. Brassica napus L. has been shown to decrease populations of black vine weevil larvae (2) and wireworms (9) in laboratory experiments. Masked chafer beetle larvae (Cyclocephala spp. Latreille) (Fig. 1) are a common soil-inhabiting pest of turf grasses (7), agronomic crops including corn and soybean (17), and some ornamental plants. The damage they cause is similar to damage caused by larvae of other scarabaeid pests, including the Japanese beetle (Popillia japonica Newman) and European chafer (Rhizotrogus majalis Razoumowsky).

Fig. 1. Lateral view of a masked chafer beetle larva (click image for larger view).

In this study, different levels of fresh B. juncea tissue and AITC were added to soil containing masked chafer beetle larvae to simulate biofumigation. The objectives were to determine the concentration of AITC released from a cultivar of B. juncea and to determine the concentration required to kill masked chafer beetle larvae, as a model soil-inhabiting pest.

Larval Mortality Experiment

Fig. 2. Jar used with SPME (solid phase microextraction) for AITC (allyl isothiocyanates) collection (click image for larger view).

Proportional amounts of B. juncea PI 458934 (USDA-ARS, Ames, Iowa) root, stem, and leaf tissue were combined at the ratio of 1:4.9:6.1, respectively, and macerated for 30 s using a food processor. This ratio was previously determined to be the average proportion of root, stem, and leaf in B. juncea from weighing of random samples. Leaf tissue was taken from the youngest fully developed leaf, stem tissue was taken from the main stem at least 10 cm from the soil surface, and root tissue was taken from the largest lateral roots. Brassica juncea tissue was collected before each experiment from a field plot at the University of Tennessee Plant Sciences Farm, Knoxville. Plants were 10 to 12 weeks old when harvested. Fresh B. juncea tissue was incorporated at treatment levels of 1, 2, 4, and 8% of soil mass (g/g) with 335 g of Waynesboro clay loam soil (air dried, sieved to 2 mm, with an in-jar bulk density of 1.17 g cm^-3). Waynesboro clay loam soil was selected because it is typical of agricultural areas in eastern Tennessee. Bermudagrass (Cynodon dactylon [L.] Pers.) root tissue (2.5 g) was added as a larval food source, along with 55 ml of deionized water to approximate 60% field capacity. Materials were mixed manually with a spatula in a plastic container for 30 s and then placed in sealed 490-ml glass jars (Fig. 2). Using similar methods, we developed other treatments: untreated soil; tomato (Lycopersicon esculentum Mill. cv. 'Trust') tissue at 8% of soil mass (g/g); 3.9-, 7.5-, and 23.7- mg-per-liter AITC standards; and a 20% CO₂ soil atmosphere. AITC standards (Sigma-Aldrich Corp., St. Louis, MO) were pipetted onto a 1-cm² piece of Whatman 541 filter paper, which was placed in the soil 4 cm from the bottom of the jar. The CO₂ treatment represented the build-up of potentially lethal CO₂ through plant degradation and microbial respiration. The 20% CO₂ soil atmosphere was higher than the 2 to 5% CO₂ concentration observed in our laboratory under normal conditions (unpublished data) and was included to determine whether CO₂ build-up in the other treatments was responsible for larval mortality. The CO₂ atmosphere treatment was adjusted over time. Initially the jar contained air with 20% O₂ and 0% CO₂. This was adjusted volumetrically to 15% O₂ and 5% CO₂ at 8 h, 10% O₂ and 10%CO₂ at 24 h, and 2% O₂ and 20% CO₂ at 36 h. The 8% tomato tissue treatment was used to indicate that typical plant breakdown products such as ethanol, Z-3-hexanol, and CO₂ were not responsible for larval mortality. Jars were stored in darkness at 20ºC with relative humidity inside an incubator maintained at 40% as monitored with a humidity sensor. The experiment was replicated three times.

Masked chafer beetle larvae were collected in October, 1998, from under Bermudagrass in Blount County, Tennessee. Five larvae (2.0 to 2.5 cm in length) were placed 5 cm deep in the soil in each treatment jar. A Teflon tube (length 14.5 cm, inside diameter 6 mm) was drilled with 32 holes and capped with a rubber septum. This tube was inserted through the lid and into the soil to allow for collection of volatiles in the soil atmosphere. Samples were collected from all treatment jars at 0.25, 4, 8, 24, and 48 h using a solid-phase microextraction device (SPME) (Supelco, St. Louis, MO). AITC was quantified by gas chromatography (Hewlett-Packard 5890 Series II) with a flame-ionization detector. Peak areas of known concentrations provided a standard curve that determined AITC in mg per liter of soil atmosphere.

After 48 h, jars were unsealed to allow equilibration to atmospheric conditions, and 10 ml of deionized water were added to ensure that larvae would not desiccate before data collection. Based upon previous work by Elberson et al. (9), we assessed mortality of masked chafer grubs after 7 days. Larvae were removed from the soil and placed under bright lights. Larvae that remained motionless for 15 min were considered dead. Dead larvae usually were dark and desiccated. Regression analysis (19) was used to determine the relationship between AITC concentrations and grub mortality. Significant differences among treatments were statistically determined using an analysis of variance (18).

We observed no larval mortality in the control treatments, including untreated soil, soil amended with 8% tomato tissue, and the high CO₂ soil atmosphere (Table 1, Fig. 3).AITC was not detected in these treatments. Mortality of grubs in the untreated control, CO₂ control, and tomato control differed significantly from mortality of grubs in the B. juncea treatments and AITC controls. AITC concentration at 0.25, 4, 8, and 24 h was positively correlated to larval mortality (Fig. 4). The mortality of grubs in the B. juncea treatments were not significantly different (P < 0.05) from those in the AITC controls, indicating that AITC is the predominant toxin from B. juncea responsible for masked chafer beetle larval mortality (Fig. 5). At the conclusion of the experiment, the filter paper used in the AITC controls was intact, suggesting that ingestion was not vital to toxicity. This provides further evidence that the AITC vapor from both the filter paper and the Brassica plant material was responsible for larval mortality.

Table 1. Mean mortality of masked chafer beetle larvae and mean allyl isothiocyanate (AITC) concentrations over time.

a Numbers are means of three replications.

b Standard errors of the means were 16.49 for larval mortality and 1.57 for AITC, respectively.

c Percentage is expressed as fresh tissue weight per gram of soil.

* Denotes significance at 4 h for contrasts against non-AITC controls (P < 0.05). Indicates no data were gathered at these time intervals.

Fig. 3. Live larvae from soil amended with 8% tomato tissue (click image for larger view).		Fig. 4. Average allyl isothiocyanate (AITC) concentrations produced by incorporation of 1, 2, 4, and 8% Brassica juncea tissue at 4 h vs. average larval mortality at 7 d (click image for larger view).

Fig. 5. Dead larvae from soil amended with 8% Brassica juncea tissue (click image for larger view).

AITC from plant tissues decreased from the time of introduction, whereas the AITC standard peaked at 4 h before declining (Table 1). AITC standards required more time to reach equilibrium in the soil atmosphere. The macerated B. juncea samples were thoroughly mixed with soil, and gases were evenly distributed from introduction. Only trace amounts of AITC were detected in any treatments after 48 h.

The results at 4 h were used for generating the equation describing the relationship between larval mortality and AITC concentrations (Fig. 4). AITC concentrations produced from the Brassica treatments were positively correlated with masked chafer beetle larval mortality (R² = 0.99).

The amount of B. juncea tissue required to produce enough AITC to achieve greater than 50% mortality is between 4 and 8% of soil mass for this plant accession, with the production of AITC at 4.0 to 11.4 mg per liter soil atmosphere. According to Duke (8), B. juncea can produce 12 tons per ha of leaf tissue. Based on the B. juncea ratio of root:stem:leaf tissue, this is approximately 24 tons of total plant tissue. Twenty-four metric tons is approximately 1.1% of soil mass for a hectare furrow slice. Therefore, cultivation rates are 3.6 to 7.3 times lower than the application rates necessary to achieve 50% mortality of masked chafer beetle larvae.

The variations in mortality and AITC concentration from B. juncea treatments may have occurred due to the high variability of glucosinolate content of fresh plant tissues. The plant maturity and the age of individual leaves and roots may affect glucosinolate concentrations and, in turn, AITC production. Josefsson (13) found higher concentrations of AITC in the roots and leaves than in other regions of the plant tissue. The use of homogenous freeze-dried mustard material helps reduce variation in glucosinolate levels (16). Other variables, including temperature, moisture, soil texture, and pH, may affect concentration of AITC (15,16).

Tilling a winter cover crop of B. juncea into the soil and covering with plastic mulch (to help retain volatile compounds) is a potential method of biofumigation (Figs. 6 and 7). Although cost may be prohibitive in standard row-cropping systems, there is little additional cost in plastic culture systems. In high-return greenhouse production systems biofumigation may prove economically feasible. However, at present production rates, B. juncea could not produce enough concentration of AITC to kill masked chafer beetle larvae. The lethal concentrations of AITC required for other pests such as fungi and bacteria are lower, so these pests may be easier to control with this treatment (5,11). For biofumigation to be successful against larger soil-inhabiting pest species such as masked chafer beetle larvae, Brassica species could be selected or developed with higher concentrations of glucosinolates, or increased production of biomass.

Fig. 6. Brassica juncea grown as a winter cover crop for biofumigation (click image for larger view).		Fig. 7. After tilling Brassica juncea into the soil, soil is formed into beds and covered with plastic mulch (click image for larger view).

Currently, to achieve required biomass using available cultivars, larger quantities of B. juncea tissue could be grown in one area, harvested, and incorporated into the desired site at much greater concentrations than could be grown using Brassica as a cover crop. In present production systems, only 30 to 40% of the total land area is fumigated, usually the rows or beds to be planted. Biomass could be harvested from the remaining 60 to 70% of land and amassed into the desired planting regions, increasing AITC concentration and the effectiveness of biofumigation. Greenhouse soils and planting beds could benefit from similar applications. Biofumigation using B. juncea could be combined with other pest management strategies, such as solarization, manure additions, and other chemical additives, to enhance pest control in an integrated pest management scheme.

Acknowledgements

We thank Jennifer Ammons, Craig Charron, Charles Pless, Andrew Price, and Egwani Farms Golf Course for their assistance.

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