Toxicity ofrapeseed Meal-amended Soil to Wirewonns, Limonius californicus (Coleoptera: Elateridae)I

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1 Toxicity ofrapeseed Meal-amended Soil to Wirewonns, Limonius californicus (Coleoptera: Elateridae)I Leslie R. Elberson, Vladimir Borek, Joseph P. McCaffrey and Matthew J. Morra Department of Plant, Soil and Entomological Sciences University of Idaho Moscow, Idaho USA J. Agric. Entotl1ol. 13(4): (October 1996J ABSTRACT The acute toxicity of soil amended with rapeseed (Brassica napu.s L.) seed meal to wireworms, Limonius californicus (Mannerheim) (Coleoptera: Elateridae), was determined in laboratory bioassays. Wireworms were exposed to rapeseed seed meal at concentrations from glkg soil for 24 h. Probit analysis estimated a LC so value of glkg soil 7 d after treatment. The LC so value decreased to gfkg soil 21 dafter treatment with no further change in mortality up to 35 d. Brassica napus tissues show potential lor reducing populations of L. cali{omiclls. However, applications to soil for insect pest management would require plant material with even higher levels of isothiocyanate-pl'oducing glucosinolate to provide useful biological fumigation activity. KEY WORDS Coleoptera, Elateridae, Limonius californicus, rapeseed, Brassica spp., glucosinolates, isothiocyanates, biofumigation Soil fumigation is commonly used for controlling a wide array of soilborne diseases, nematodes, insects and weeds. Due to environmental and health conce1us, and the prohibitive cost of reregistration, the arsenal of fumigants available for pest management is being reduced. A leading soil fumigant, methyl bromide, will be phased out in the United States by the year 2000 under the direction of the Environmental Protection Agency and the 1990 Clean Air Act (White 1994). The future availability of alternative fumigants is questionable as individual states eliminate their use. For example, in California, the registrations for Telone 0,3 dichloropropene) and Vorlex (methyl isothiocyanate + 1,3-dichloropropene) were canceled in 1990 and 1991, respectively, and chloropicrin is currently facing suspension (White 1994). These concerns and actions have spurred interest in the development of alternative control strategies for soilborne pests. Current research involves the incorporation of plant-generated toxicants, acting as biofwnigants, into the soils. For I Accepted for publication 5 July

2 324 J. Agric. Entomol. Vol. 13, No.4 (1996) example, glucosinolate hydrolysis products, specifically the isothiocyanates, are naturally occurring allelochemicals produced by plants within the order Capparales, most commonly within the genus Brassica (Kjaer 1976). Isothiocyanates have been shown to mediate herbivory of phytophagous insects, affect insect development, and show insecticidal and nematocidal properties (Lichtenstein et al. 1964, Wolfson 1982, Louda & Rodman 1983, Lazzeri et ai. 1993, McCloskey & Isman 1993, Borek et al. 1995). Damage to Brassica spp. plant tissue results in enzymatic degradation of glucosinolates by myrosinase (thioglucoside glucohydrolase, E.C ) to a variety of biologically active compounds (Underhill 1980). More than 20 different aliphatic and aromatic isothiocyanates have been identified among degradation products of glucosinolates in Brassica napus L., B. hirta Moench, B. cam.pestris L., B. juncea L., or B. nigra (L.) Koch (Spencer & Daxenbichler 1980, Brown et al. 1991). Incorporation of plant tissues containing glucosinolates into soil has been associated with reduction or inhibition of soilborne pests (Papavizas 1966, Chan & Clase 1987, Brown et al. 1991, Mojtahedi et al. 1991). It is thus possible that Brassica tissues could act as a source for glucosinolates and result in the release of isoth.iocyanates as sou fumigants. Therefore, the acute and chronic effects of rapeseed (B. napus) seed mea] amended soil were evaluated in laboratory bioassays against Limonius californicus (Mannerheim) (Coleoptera: Elateridae), a serious and widespread wireworm pest attacking potatoes, sugar beets, and small grains in the Pacific Northwest ofthe United States. Materials and Methods LimonillS californicus larvae were collected in April 1993 from an alfalfa field in Walla Walla County, Washington, and in May of 1994 from fallow fields near IGmberly and Twin FaJls, Idaho. Wireworms were placed in plastic vials with soil from which they were sieved and held at 4 C until preconditioning. Preconditioning followed a procedure modified after that described by Williams et a1. (1993). \Vireworms were removed from a cooler, placed individually in or plastic vials with Latahco silt loam soil at 15% soil moisture, and provided with at least two germinated wheat seeds (germination slarted 2 d earlier). Characteristics of Latahco soil (Argiaquic Xeric Argialboll, ph 6.10) were reported by Borek et al. (1994). Vials were held in the dark at 23 ± 2 C for approximately 72 h. Dead or obviously weakened larvae were discarded. The remaining wireworms were individually weighed immediately before the bioassays were conducted. Voucher specimens were deposited at the W. F. Barr Entomological Museum, University of Idaho, Moscow, Idaho. Defatted rapeseed seed meal, a by-product from seed pressed for industrial qua.lity oil, was obtained by extracting the oil from machine-harvested mature seed of B. napus (Brown et al. 1991, Brown & Morra 1995). Ground B. napus ('Dwarf Essex') seed meal at eight specified doses and detoxified seed meal at an application equal to the highest dose of' the original meal were added to Latahco silt loam soil and mixed thoroughly. Seed meal for the control treatment was detoxified through hyd.rolys.is ofglucosinolates by saturating the meal with water (3.0 mt H 2 0/g mea!), covering, venting to allow escape of

3 ELBERSON et nl.: Toxicity orrapeseed Seed Meal~amended Soil to Wirewonlls 325 volatiles for 48 h, and air drying. Previously published analyses or glucosinolate content in hydrolyzed rapeseed seed meal compared to intact rapeseed seed meal revealed elimination of most glucosinolates by this technique (Brown & Morra 1995). A treatment consisting of Latahco soil without any meal also was used. Sixteen grams of soil-meal mixtures or unamended soil was placed in 20-ml glass scintillation vials. Individual wireworms were placed approximately midway in the soil within the vial and 4-12 ml of deionized water was added to bring the soil-meal mixture to saturation. Vials were capped and placed in the dark at 23 ± 2 C for 24 h. Wireworms were removed from the vials, placed in clean vials with intact Latahco soil at 15% moisture, and provided with at least two germinated wheat seeds. Because wireworms may appear dead after exposure to fumigants, but subsequently recover (Lehman 1933), mortality was assessed 7 d after exposure by observing each wireworm for movement. Surviving wireworms were weighed, again placed in sou, and provided with wheat seeds. This procedure was repeated at 7-d intervals for 5 wk. Wireworms assessed as dead were treated identicauy to surviving wireworms and not disposed of until they began to decompose. The bioassay design incorporated two runs or eight treatment levels of rapeseed seed meal, and two controls, detoxified meal and unamended soil. Each treatment within a run had 10 replications for a total or 20,"eplications. Descriptive statistics and probit analyses were conducted by using PROC UNIVARIATE and PROC PROBIT (normal model procedures), respectively (SAS Institute 1993). The procedure for analysis of silyl derivatives of glucosinolates in rapeseed seed meal was adapted from Raney & McGregor (990) by using benzylglucosinolate (glucotropaeolin) and allyl glucosinolate (sinigrin) as standards. The mixture of glucosinolate silyl derivatives was analyzed by Gas Liquid Chromotography (GLC) (HP 5890A, Hewlett Packard. Avondale. Pennsylvania) by using a DB-5 capillary column (30 m X 250 pm pm film, J&W Scientific, Folsom, California) and a temperature program of 15 C/min from to 300 C. Glucosinolates were detected and identified with a mass spectrometric detector (HP 5972, Hewlett Packard) on the basis of published spectra and quantified using published response coefficient.s (Fenwick et a , Raney & McGregor 1990). To analyze isothiocyanates released from hydrolyzed glucosinolates, intact rapeseed seed meal was hydrolyzed for 24 h in 25 ml of 0.01 M sodium phosphate burrer, ph 6.5, and degradation products were continuously extracted using a mixtul e of acetone: dichloromethane:pentane (l:2:1, vol:vol:vol) (Borek et a , Brown et al. 1994). The organic phase was separated by centl"ifugation and dried 24 h with anhydrous Na2S04. Isothiocyanates, together with other co-extracted products were analyzed by GLe (as previously described), using a temperature program from 35 to 250 C at looc/min. Isothiocyanates in the chromatogram were identified with a mass spectrometric detector (as previously described) by using their characteristic ion fragments.

4 326 J. Agric. Entomol. Vol. 13, No.4 (1996) Results and Discussion Rapeseed seed-meal amendments of g/kg caused 15%-95% mortality of wireworms 7 d after treatment (Table 1) with a corresponding LCso of glkg soil (Table 2). Mortality increased slightly during the 35-d observation period, and the LC so value decreased to glkg soil at 21 d, with no change in mortality occurring during the last 14 d of observation. No larval mortality occurred within detoxhied rapeseed seed-meal treatments. Mean wireworm weights were not significantly different among all treatments (F= 0.94; df= 9,189; P= 0.49 and F= 3.26; df= 1,189; P= 0.07). These results support previous findings that the primary toxic effect of rapeseed seed-meal soil amendments is due to organic isothiocyanates (Chew 1988, Borek et al. 1995, McCaffrey et al. 1995) because removal of isothiocyanates by glucosinolate hydrolysis eliminated the toxic effects of rapeseed seed meal on wireworm larvae. Few studies have investigated the toxic effects of glucosinolate degradation products against soil insects. Williams et al. (1993) showed lethal and sublethal effects on L. californicus wireworms resulting from soil amendments of allyl isothiocyanate. Brown et a1. (1991) showed that wireworms were repelled by the presence of rapeseed seed meal in soil, but it was not toxic at the dose of 30 glkg soil. The findings of Brown et al. (1991) are consistent with results of the present study, which used the same variety of meal and in which the minimum dose resulting in toxic effects was 41.7 glkg soil (Table 1). Ionic thiocyanate, another important glucosinolate degradation product, was found to be essentially nontoxic and had no deterrent effects on wil'e\vol'ms in a soil environment (McCaffrey et al. 1995). These results further support the contention that of the degradation products, isothiocyanates are the major toxicants. Lethal concentrations of rapeseed seed meal incorporated into the soil against larvae of the black vine weevil, Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae), were 19.3 glkg (LC 50 ) and 86.9 glkg soil (LCgo) (Borek et ai. in press). These values are six times less than those reported for wireworm larvae in the present study, indicating a major difference in the susceptibility of the two species to toxic en-ects of the meal. Glucosinolates identified in the rapeseed seed meal with concentrations equal to or greater than 0.2 pmollg of meal are shown in Table 3. Conversions to }Jg/g are included to help illustrate the glucosinolate content in the meal. Rapeseed seed meal hydrolysis at ph 6.5 produced over 35 compounds that appeared in the chromatogram. Those identified as isothiocyanates are listed in Table 4. Analysis of the B. nupus seed meal used in these trials revealed that the level of recoverable isothiocyanates was about 15% (difference between quantities in Tables 3 and 4) of what would be expected, had there been total conversion of glucosinolates. This low production is likely due to side reactions of isothiocyanates with pt'oteins and other relevant chemicals that convert them into nontoxic products, i.e., nitriles, oxaz.oledinethiones, and indolyl derivatives (Chew 1988). Plant tissues containing lower concentrations of protein, such as root, leaf, and stem tissues, may thus release more isothiocyanates because of less deactivation by protein even though glucosinolate concentrations may not be as high as in seed meal

5 ELBERSON et a1.: Toxicity of Rapeseed Seed Meal-amended Soil to Wirewonns 327 Table 1. Mortality of wireworms (Limonius californicus> exposed to rapeseed seed meal~amendedsoil. Dose a Mortality (%) at days after treatment (g/kg) O.Ob ~TwC!nty replications per dose. ContTols treated with intact soil. Isothiocyanates are not equal in their potency. For example, methyl, propyl, allyl, phenyl, benzyl, and 2-phenylethyl isothiocyanates were all found to be toxic to eggs of the black vine weevil in laboratory tests; however, aromatic M isothiocyanates (phenyl, benzyl, 2 phenylethy1) were considerably more toxic than aliphatic (methyl, propyl, allyl) isothiocyanates (Borek et al. 1995). In addition, the content and composition of glucosinolates producing isothiocyanates vary between tissue types. Root tissues of some Brassica spp. contain relatively high concentrations of 2-phenylethyl glucosinolate (Brown & Morra in press), which was found to be the most toxic of the isothiocyanates tested against black vine weevil eggs (Borek et al. 1995). Our results suggest that lethal doses (kg/ha) of currently available rapeseed seed meal or other tissues, necessary to produce high enough levels of isothiocyanates for effective control of' wirewol'ms in field applications, may be too high for practical use. For example, to obtain 90% mortality of L. cali{ornicus, the soil concentration of rapeseed seed meal would need to reach approximately 50%. Further research should include the development of Brassica spp. containing higher levels of specific isothiocyanate~producing glucosinolates and lower protein content in their tissues for greater isothiocyanate release efficiency. These cultivars would be specifically for use as cover crops with subsequent plow-down. Biofurnigation has potential in soil pest control if the appropriate material is available.

6 328 J. Agric. Eotomol. Vol. 13, No.4 (1996) Table 2. Toxicity of rapeseed seed meal (glkg of soil) to Limonius californicus. Days no Slope", SE LC W LC 50 LCW after treatment {95% FL} C (95% FL) C (95% FL) C X 2b ± ( ) ( ) ( ) ± ~n '" Number ofwircworm!:l used in bioassay. Chi-square goodness-of lil Lests (Hobcrtson & Preisler 1992); df= 6. cfiducial limits. ( ) ( ) ( ) Table 3. Glucosinolate content in rapeseed seed meal {Brassica napus L., 'Dwarf Essex'} determined by gas liquid chromatographic analysis of silyl derivatives. Glucosinolate pmoljg Allyl" 3-Butenyl" 4-Pentenyl" 2-Hydroxy-3-butenyl 2-HydJ"Oxy-4-pentenyl Phenethyl" 3-Indolylmethyl 4-Hydroxy-3-indolylmethyl Total Clucosinolatcs producing isothiocyanates aner hydrolysis.

7 ELBERSON el at: Toxicity ofrapeseed Seed Mcal amended Soil to Wireworms 329 Table 4. Glucosinolate degradation products from rapeseed seed meal determined by gas liquid chromatographic analysis of organic extracts. Isothiocyanates (ltc) l'moug Ally! ITC Butenyl ITC PentenyllTC 3.8 Phenethy! ITC 0.9 Acknowledgment Funding...as provided by USDAlCSRS as part of the Water Quality Initiative (grant no ) and USDA cooperative agreements ( ) of the Western Regional rpm Special Grants of the Advanced Materials from Renewable Resources Program (93 COOP l 9543). Published with the approval of the Agricultural Experiment Station, University of Idaho, as journal Article No References Cited Borek, V., M. J. Morra, P. D. Brown & J. P. McCaffrey Allclochemicals produced during sinigrin decomposition in soil. J. AJ"rric. Food Chern. 42: Borek, V., L. E. Elberson, J. P. McCaffrey & M. J. Morra. 19!)5. Toxicity of nliphatic and aromatic isothiocyanates to eggs of the black vine weevil (Coleoptera: Curculionidae). J. Econ. Entomol. 88: Borek, V., L. Eo Elberson, J. P. McCaffrey & M. J. Morra. In press. Toxicity of rapeseed meal and methyl isothiocyanate to larvae of the black vine weevil (Coleoplcra: Curculionidae). J. Econ. Entomol. Brown, P. D. & M. J. Morra Glucosinolate containing plant tissues as bioherbicides. J. Agric. Food Chern. 43: Brown, P. D. & M. J. Morra. In press. Hydrolysis products of glucosinolatcs in Brassica "OPu.s tissues as inhibitors ofseed germination. Plant Soil. Brown, P. D., M. J. Morra, J. P. McCaffrey, D. L. Auld & L. Williams, m Allelochemicals produced during glucosinolatc dcf:,'tadation in soil. J. Chern. Eoo!. 17: Bro\.Vll, P. D., M. J. Morra & V. Borek Gas chromat.ography of nl1elochemicals produced during glucosinolate degradation in soil. J. Agric. Food Chern. 42: Chan, M. K. Y. & R. C. Close Aphanomyces root rot of peas 3. Control by the use ofcruciferous amendments. N. Z. J. Agric. Res.30: Chew, F. S Biolob';cal effects of glucosinolates, pp H. C. Cutler [Ed.l, Biologically active natural products: potcntinl usc in agriculture. The American Chemical Society, Washington, D.C., ACS Symposium No pp. Fenwick, G. R., J. Eagles, R. Gmelin & D. Rllkow The mass spectra of glucosinolates and desulphoglucosinolates. Biomcdic. Mass. Spectrom. 7:

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