6470 Emomol. expo appl. 57: 23-28, 1990. © 1990 Kluwer Academic Publishers. Primed ill Belgium. 23 f'm'l::m~ bf U. 8. Dept. 0 4.~..cu..!tt.'U-e fOiJi u~ Insecticidal properties of Lactarius fuliginosus and Lactarius fumosus Patrick F. Dowd & Orson K. Miller I Northern Regional Research Center, A.R.S., U.S.D.A., Peoria, IL 61604, U.S.A.; 1 Department of Biology, Virginia Polytechnic Institute & State University, Blacksburg, VA 24061, U.S.A. Accepted: !Vlay I, 1990 Key words: Heliothis zea, Oncopeltlls fasciatus, chemotaxonomy, chromenes Abstract Acetone and ether: acetone extracts of the mushrooms Lactarius fuliginosus (Fr. ex Fr.) Fr., L. fumosus fumosus Peck and L.fumosus.fumosoides (Smith and Hesler) Smith and Hesler were toxic to the corn earworm, Heliothis zea (L.), while water extracts were inactive. Ether: acetone extracts of L. fuliginosus and L. fumosus fumosus were toxic to the large milkweed bug, Oncopeltusfasciatus (L.), and in some cases caused precocious development. Profiles of compounds separated chromatographically and visualized with chromene reagents, literature reports ofchromenes from L. fuliginosus, and known insecticidal/anti­ hormone effects of chromenes suggest that chromenes may be responsible for the activity of some of the extracts. Introduction exuding a milky fluid and/or color change reactions (Ramsbottom, 1954), which could be a The ability of higher plants to produce secondary warning reaction. Several species of Lactarius metabolites that serve a defensive role is well contain sesquiterpene lactones that deter insects recognized (Whittaker & Feeny, 1971). Ana­ from feeding (Nawrot et al., 1986). Other species, logously, the secondary metabolites produced by such as the European Lactariusfuliginosus (Fr. ex fungi are also thought to play a defensive role Fr.) Fr., do not contain these compounds. How­ (J anzen, 1977; Wicklow, 1984, 1988). The secon­ ever, L. jidiginosus does contain a variety of dary metabolites of several molds and entomopa­ chromenes (Conca et al., 1981; Allievi et al., thogenic fungi are reported to have insecticidal 1983). Chromenes from higher plants may be toxic properties, but information on insecticidal activity to insects (e.g. Proksch et al., 1983) or cause anti­ of other groups of fungi is limited (Wright et al., hormone effects that induce precocious meta­ 1982). Interestingly, one ofthe oldest examples of morphosis (e.g. Bowers, 1976). Thus, any a fungus-derived insecticidal compound is chromenes produced by L. jidiginosus or related muscarine, produced by the fly agaric mushroom fungi may also negatively affect insects. (Amanita muscaria) (Dickenson & Lucas, 1983). This paper investigates the insecticidal and an­ Recently, an increasing number of mushrooms tihormone effects of L. jidiginosus extracts. Ex­ have been found to contain insecticidal tracts ofthe closely related North American L. jil­ compounds. Among these are species ofLactarius mosus var. jimlOsus Peck and L. jill110SUS var. fil­ (Russulaceae), which react to wounding by mosoides (Smith and Hesler) Smith and Hesler 24 (Hesler & Smith 1979) were included for com­ and the supernatant was removed. This process panson. was repeated twice more with 5 ml of each sol­ vent. The pelleted material was dried under nitro­ gen between extractions with different solvents. Materials and methods Solvents were evaporated from the extracts by freeze-drying (water extracts) or under a stream of Mushrooms. A dried herbarium specimen of nitrogen (other extracts). The resulting yield of L. fuliginosus was kindly supplied by A. J. P. Oort viscous materials was weighed, and used for fur­ (# 14928, collected 9-9-66 from open woods of ther testing. Pinus, Fagus, Betula, Alies, and Piceas with Vac­ cinium myrtillus on peaty soil, located in Swieta Chromene detection. In order to facilitate com­ Katarzyna, Golina Wilkowska, Poland). Two parisons with previously reported chromenes different samples of caps from L. fumosus were from L. jitliginosus (Conca et al., 1981; Allievi obtained by O. K. Miller, Department of Botany, et al., 1983), an additional separation step on Virginia Polytechnic Institute and State Univer­ standard silica plates with a solvent system of sity: Lactarius fumosus v. jimlOsoides, # 22 728, hexane: ethyl ether, 3: 1, was used for the collected 11-18-86 from woods with Sitka ether: acetone extracts. Five ,al of a 10 mgjml spruce, Western hemlock, Port Orford cedar and solution ofeach extract was spotted on the plates, Chinkapin oak from Patrick Point State Park in and they were developed ca. 9 cm. A vanillin rea­ Humbolt County, CA; and Lactarius jimlOsus v. gent was used to specifically detect chromenes jimlOsus, #81744, collected from Highland Re­ (Conca et al., 1981). creation Area, Oakland County, Michigan, 8-27-72 by A. H. Smith Bioassays. The H. zea larvae were assayed as neonate larvae by previously reported methods Insects. Larvae of corn earworm (Heliothis zea) (Dowd, 1988a, b). Extracts were dissolved into were reared on pinto bean-based diet (Dowd, either acetone or water, and blended into pinto 1987), and adults were fed with 10~~ sucrose. bean-based diet at a rate of 250 ppm wet wt Insects were reared at 27 ± 1 0 C, L14: D 10 pho­ (1000 ppm dry wt). Diets were sectioned and ad­ toperiod, and 40 ± 10% Lh. Large milkweed bug ministered to at least 40 individual larvae for each (Oncopeltus fasciatus) adults (sunflower-adapted extract (Dowd, 1988a, b). Mortality was recorded strain) were obtained from Carolina Biological after 2, 4, and 7 days, and insects were weighed Supply, and reared on sunflower seeds and water after 7 days. at 22 ± 1 0 C, ambient light conditions, and Potential antihormone effects of the extracts 40 ± 10'/0 Lh. were examined with second instar (ca. 2 mg) nymphs ofO. fasciatus by a method based on that Chemicals. Precocene II and vanillin were from of Bowers (1976). In order to increase the sensi­ Sigma Chemical Co. All solvents were ofspectro­ tivity of the assay, the method was modified to scopic grade. increase the exposure to the treated surface. Due to the change in assay methodology, and un­ Extraction of jimgi. Approximately one-gram known response of this strain of milkweed bugs, samples of caps were ground in a smooth glass the assay was calibrated using precocene II. mortar and pestle and sequentially extracted with Twenty-ml glass vials were used as assay cages. ether: acetone (1: 1) (to initially remove chrom­ Sunflower seeds (sufficient quantity for ad libitum enes as per Bowers (1976)), hexane, acetone, and feeding) were placed in the bottom ofthe vials. A water. Ten ml of solvent was added to the ma­ sheet offilter paper, impregnated with the extracts terial, the suspension was vigorously vortexed for was inserted into the vials so that the side of the 1 min, the suspension was centrifuged at 1200 g, vial was entirely covered. The top of the vial was 25 covered with organdy, and a vial of water con­ Table 2. Toxicity of Lactarius spp. extracts to Heliothis zea taining a cotton wick was inverted over the cloth larvae after 7 days to provide water. The filter paper (4.5 x 7.5 cm) Treatment Mortality (~.~) Weight (mg) was impregnated by adding a solution of 20 mg material (ether: acetone fungal extracts) or doses Control (acetone) 0.0 44.9 ± 3.0 ranging from 2 to 0.1 mg of precocene II in 2 ml Control (water) 0.0 55.6 ± 2.4 of acetone over a sheet of waxed paper. The sol­ 14928 (ether: acetone) 52.5* 13.3 ± 1.6* vent was evaporated in the hood. Differences in 14928 (acetone) 37.5* 14.1 ± 1.8* toxic responses were analyzed by chi square ana­ 14928 (water) 0.0 60.0 ± 3.0 lysis (mortality or percent precocious) or by linear 22728 (ether: acetone) 50.0* 14.7 ± 1.8* contrast analysis of variance (weights or days to 22 728 (acetone) 21.6* 20.0 ± 2.1 * adult), which are appropriate analyses for these 22728 (water) 10.3 56.5 ± 3.6 types of data (SAS Institute, 1985). 81 744 (ether: acetone) 12.5* 29.6 ± 1.8* 81744 (acetone) 2.5 31.0± 1.7* 81744 (water) 0.0 62.3 ± 2.0 Results \Vater extracts were dissolved n water, other extracts were dissolved in acetone, 14928 = Lactatills jztligillosuS, Yields of materials. The greater part of the ex­ 22728 = L. jzllnosus v. jzmlOsoides, 81744 = L. jzllnoslis v. tractables were obtained in the initial extraction jzmlOslis. Weights are means ± standard errors of survivors with ether: acetone in most cases (Table 1). The of mortality assays. Values in columns followed by an '*' are significantly different from solvent controls at P < 0.05 by chi =11= 14928 sample generally yielded the greatest square (mortality) or linear contrast analysis of variance amount of material in all extractions. Due to the (weights) (SAS Institute, 1985). low yield of the hexane extract for all three samples, they were omitted from bioassays of the caterpillars so that enough material would be extracts were less active. The water extracts of all available for chromatographic comparisons. three samples were not toxic but caused enhanced growth, perhaps due to the presence of additional Results ofbioassays. The ether: acetone and ace­ nutrients. tone extracts of =11= 14928 and =11= 22 728 were the most toxic extracts to larvae of H. zea (Table 2). Table 3. Effects ofdifferent doses ofprecocene II on second Significant mortality and reductions in rates of instar milkweed bug nymphs development ofthe survivors occurred with these ~o) extracts. The =11= 81744 ether: acetone and acetone Dosage Mortality Weight Precocious ( (~~ ) (mg) 4th 5th Table 1. Yields of extracts from Lactarius spp. 20.0 mg 100.0* 1.0 mg 50.0* 12.9 ± 2.6* 30.0* 20.0* Source Yield of isolates (mg) 0.5 mg 31.6* 7.0 ± 1.4* 0.0 47.4* 0.2 mg 0.0 29.2 ± 2.8* 0.0 88.2* 14928 22728 81744 0.1 mg 0.0 49.8 ± 3.8 0.0 11.1 0.0 mg 0.0 42.4 ± 2.3 0.0 0.0 Powder 1040 1190 930 Ether: acetone (1: 1) 342.5 100.3 146.7 Weights are means ± standard errors of survivors.