Insecticidal Properties of Lactarius Fuliginosus and Lactarius Fumosus

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. Some Hexane 24.8 16.4 12.7 escapes occurred, and values have been adjusted ac Acetone 68.6 26.0 27.6 cordingly. Values in columns followed by an '*' are signifi Water 79.9 208.8 61.5 cantly different from solvent controls at P < 0.05 by chi square analysis (mortality and precociousness) or linear con 14928 = Lacterius fuligillosus, 22728 = L. flllllOSUS V. fll trast analysis of variance (weights of survivors) (SAS Insti lI1osoides, 81744 = L. jzmlosus v. jzIlIlOSUS. tute 1985). 26

Table 4. Effects of Lactarius spp. ether: acetone extracts on milkweed bug nymphs

Treatment Mortality (~!o) DTA Adult weight (mg) Precocious (%)

Control 0.0 18.9 ± 0.4 42.2 ± 2.3 0.0 14928 50.0* 17.8 ± 0.5 42.9 ± 3.3 30.0* 22728 . 5.3 19.2 ± 0.4 47.5 ± 1.5 0.0 81744 25.0 19.3 ± 0.7 45.4 ± 3.3 37.5*

DTA = days to adult, means ± standard errors, weights are also means ± standard errors of survivors. Values in columns followed by an '*' are significantly different from controls at P < 0.05 by chi square analysis (mortality and precociousness) or linear contrast analysis of variance (DTA and weights) SAS Institute, 1985). 14928 = L. fuliginosus, 22728 = L. jimlOsus v. jil/nosoides, 81744 = L. jil/nosus v. jil/nosus.

The precocious effects caused by different few ofthe precocious nymphs showed the charac doses of precocene II often occurred at the same teristic enhanced wing development described doses that caused mortality (Table 3). Significant previously (Bowers 1976). The rest developed mortality occurred within 2 days at doses of 2.0, adult abdominal reproductive features without 1.0 and 0.5 mg/paper, and the latter two doses obvious changes in wing morphology. The num caused a significant increase in the numbers of ber of segments in the tarsi (sometimes reported precocious insects. The 0.2 mg dose resulted in a to change in these insects when treated with significant number of precocious nymphs, but precocenes (Bowers, 1976)) was also unaffected. caused no mortality. Sample =11= 22 728 had no significant effect on the Similar to the effects noted with doses of 1.0 milkweed bug nymphs at the doses administered. and 0.5 mg/paper of precocene II, precocious effects caused by ether: acetone extracts of Presence ofchromenes. The vanillin reagent pro samples # 14928 and #81744 cooccurred with duced a reddish-orange reaction with the significant levels of mortality (Table 4). Only a chromene precocene II. Spots of a similar colour were obtained from the ether: acetone extracts from all three fungi (Table 5). The profiles ofthese apparent chromenes, as visualized by the vanillin Table5. Compounds with a positive reaction to the vanillin reagent (ether: acetone extract) reagent, were also similar for the different samples. All three extracts had spots of Rf 0.222, Rf Precocene II Chemical source 0.500, and 0.833. A unique spot of RfO.722 was present from the L. fuliginosus extract, one of 14928 22728 81744 Rf 0.311 for the L. f fumosus, extract, and two of 0.167 +, f RfOA11 and 0.666 for the L.ffumosoides ex 0.222 +, s +, S +, s tract. 0.311 -f', f 0.411 +,f 0.500 +,f +, f +,f Discussion 0.555 + 0.666 +, f 0.722 +,f The precocious responses seen when the 0.833 +,f + + ether: acetone extracts of the Lactarius spp. were administered to the O.fasciatus nymphs suggest + = Positive, clear reaction, - = no reaction, s = smeared that compounds related to the higher plant spot, f = faint spot. 14928 = Lactarius jitliginosus, 22728 = L. jimlOsus v. jimzosoides, 81744 = L. jil/nosus v. derived precocenes could be present, but positive jimlOsus. identifications are necessary to confirm this sup- 27

position. The Rf values of the compounds that for these lactones. Certainly these are not the only reacted with the vanillin reagent from the possibilities. ether: acetone extracts corresponded to some At least some of the extracts from all of the values for chromenes previously reported from samples were toxic when fed to the H. zea larvae. L.fuliginosus (Conca et al., 1981; Alleivi et al., The levels used in the bioassays (250 ppm wet 1983). Because the color reactions of the spots weight, 1000 ppm dry weight) were equal to or less were similar to that of precocene II, this lends than (especially for the ether: acetone extracts) further support to the contention that they could the levels that naturally occur in the dried caps. be chromenes. Chromenes that are structurally This information suggests that the Lactarius may related to plant-derived precocenes have been re be naturally protected from many insects. ported from samples of L. fuliginosus and L. pi Although H. zea is unlikely to be a natural enemy cinus Fries (Conca et al., 1981). Thus, it is not of these fungi, many flies (e.g. Mycetophilidae, surprising that these extracts would have pre Sciaridae) and beetles (e.g. Erotylidae, Endomy cocious effects on O.fasciatus nymphs. It should chidae, Mycetophagidae) are predators of be pointed out that Conca et al. (1981) also re mushrooms (Bruns, 1984), and could signifi ported chromenes in L. picinus, which is in the cantly undermine the survival of unprotected same subgenus (Plinthogali) as the other species of mushrooms. In spite of the presence of insec Lactarius examined in our study. It is difficult to ticidal compounds in the cap of L. fumosus var. induce Lactarius spores to germinate so that fumosoides, they are still preyed upon by larvae of DNA hybridization studies can be performed to unspecified species (Hesler & Smith, 1979). This determine sibling species relationships (Miller, predation suggests that the larvae ofsome species 1982). Chromene comparisons in these and re have been able to overcome the chemical defences lated species may be a useful tool in determining of L. fumosus fumosoides. appropriate taxonomic relationships. The extracts also contained compounds that were toxic to H. zea. Precocenes are not reported Acknowledgements to induce true precocious metamorphosis in Le pidoptera (Staal, 1986). However, precocenes We thank the late A. J. P. Oort for specimens. We may be toxic to various Lepidoptera at high doses also thank C. Weber and K. Riedesel for technical (Bowers, 1977), including to H. zea (Wisdom assistance, and T. C. Nelsen for suggestions on et al., 1983). A variety of other chromenes from appropriate statistical analyses. higher plants, such as encecalin (Proksch et al., The mention of firm names or trade products 1983; Klocke et al., 1985), eupatoriochromene, does not imply that they are endorsed or recom and 6-vinyl-7-methoxy-2, 2-dimethyl chromene mended by the U.S. Department of Agriculture (Klocke et al., 1985) can cause mortality without over other firms or similar products not men precocious effects to insects. Thus, it is possible tioned. that chromenes may be responsible for some of the acute toxicity. The chemicals responsible for the toxicity of Resume other extracts to H. zea are unknown. The fruiting bodies ofthe genus Lactarius also commonly pro Propribes insecticides de Lactarius fuliginosus et duce a variety of sesquiterpene lactones, which L. fumosus are reported to be toxic to insects (Nawrot et al., 1986), but these compounds appear to be absent L'activite insecticide d'extraits solubles du cham in L. fuliginosus and related species (Conca et al., pignon europeen L. fuliginosus Fr. et des especes 1981; Allievi et al., 1983). However, the two americaines etroitement apparentees L. fumosus varieties of L. fumosus have not been examined var. fumosus Peck et L. fillnosus var. fUlllosoides 28

Smith & Hesler a ete exammee. Les extraits a allelochemicals to the corn earworm, Heliothis zea (Lepid l'acetone et al'ether: acetone des 3 champignons optera). J. Chern. Eco!. 15: 249-254. ont ete toxiques pour Heliothis zea L., mais non les Hesler, L. R. & A. H. Smith, 1979. North American Species of Lactarius. University of Michigan Press, Ann Arbor: extraits aqueux. Les extraits ether: acetone de 841 p. L. fuliginosus et de L. fumosus var. Fumosus ont Janzen, D. H., 1977. Why fruits rot, seeds mold, and meat ete toxiques pour Oncopeltus fasciatus F. et ont spoils. Am. NAt. II I: 691-713. induit dans quelques cas une metamorphose pre Klocke, J. A., M. F. Balandrin, R. P. Adams & E. Kingsford, coce. Les metabolites secondaires etaient presque 1985. Insecticidal chromenes from the volatile oil ofHemi zoniafitchii. J. Chern. Eco!. II: 701-712. aussi semblables entre L.fumosus var.fumosus et Miller, O. K., Jr., 1982. Taxonomy of ecto and ectendo L.fumosus var.fumosoides qu'entre ces 2 varietes mycorrhizal fungi. pp.91-101. In: Schenck, N. C. (ed.), et L. fuliginosus. Les chromenes peuvent etre res Methods Manual on Mycorrhizae. American Phytopa ponsables d'une partie de l'activite biologique thological Society, New York. contre les insectes. Nawrot, J., E. Bloszyk, J. Harmatha, N. Ladislav & B. Dohdan, 1986. Action of antifeedants of plant origin on beetles infesting stored products. Acta Entomo!. Bohemoslov. 83: 327-35. References Proksch, P., M. Proksch, G. H. N. Towers & E. Rodriguez, 1983 Phototoxic and insecticidal activities of chromenes Allievi, c., M. DeBernardi, F. Demarchi & G. Mellerio, 1983. and benzofurans from Encelia. J. Natural Products 46: Chromatographic analysis of 2,2-dimethylchromene deri 331-334. vatives. J. Chromatog. 261: 311-314. Ramsbottom, J., 1954. ivIushrooms and Toadstools: A Study Bowers, W. S., 1976. Discovery of insect antiallatotropins. of the Activities of Fungi. Collins, London: 306 p. pp.394-408. In: Gilbert, L. 1. (ed.), The Juvenile Hor SAS Institute, 1985. SASISTAT Guide for Personal Com mones. Plenum Press, New York. puter, Version 6. SAS Institute, Cary, North Carolina: Bowers, W. S., 1977. Anti-juvenile hormones from plants: 378 p. chemistry and biological activity. pp. 129-156. In: ivIarini Staal, G. B., 1986. Antijuvenile hormone agents. Ann. Rev. Bettolo, G. B. (ed.), Natural Products and the Protection Entomo!. 31: 391-429. of Plants. Elsevier. New York. Whittaker, R. H. & P. P. Feeny, 1971. Allelochemicals: Bruns, T. D., 1984. Insect mycophagy in the Boletales: fungi Chemical interactions between species. Science 171: vore diversity and the mushroom habitat. pp. 91-129. In: 757-770. Wheeler, Q. & M. Blackwell (eds.), Fungus-Insect Re Wicklow, D. T., 1984. Adaptation in wild and domesticated lationships: Prospectives in Ecological Evolution, Colum yellow-green aspergilli. pp. 78-86. In: Kurata, H. & Y. bia University Press, New York. Ueno (eds.), Toxigenic Fungi - Their Toxins and Health Conca, E., M. DeBernardi, G. Fronza, M. A. Girometta, G. Hazard. Elsevier, New York. Mellerio, G. Vidari & P. Vitta-Finzi, 1981. Fungal metabo Wicklow, D. T., 1988. Metabolites in the coevolution of lites 10. New chromenes from Laclarills !lIliginoslIs and fungal chemical defence system. pp.173-201. In: Laclarills pici/llls Fries. Tetrahedron Lett. 22: 4327-4330. Pirozynski, K. A. and D. L. Hawksworth (eds.), Co Dickinson, C. & J. Lucas, 1983. The Encyclopedia of evolution of Fungi with Plants and Animals. Academic Mushrooms. Crescent Press, New York: 280 p. Press, New York. Dowd, P. F., 1987. A labor-saving method for rearing the Wisdom, C. S., J. T. Smiley & E. Rodriguez, 1983. Toxicity driedfruit beetle (Coleoptera: Nitidulidae) on a pinto bean and deterrency of sesquiterpene lactones and chromenes based diet. J. Econ. Entomo!. 80: 1351-1353. to the corn earworm (Lepidoptera: Noctuidae). J. Econ. Dowd, P. F., 1988a. Synergism of aflatoxin BI toxicity with Entomo!. 76: 993-998. the co-occurring fungal metabolite kojic acid to two cater Wright, V. E, R. F. Vesonder and A. Ciegler, 1982. IvIyco pillars. Entomo!. Exper. App!. 47: 69-71. toxins and other fungal metabolites as insecticides. Dowd, P. F., 1988b. Fusaric acid: A secondary fungal meta pp. 559-583. In: Kurstak, E. (ed.), Microbial and Viral bolite that synergizes the toxicity of cooccurring host Pesticides. Dekker. New York.