APPLIED MICROBIOLOGY, Oct. 1972, p. 660-662 Vol. 24, No. 4 Copyright ( 1972 American Society for Microbiology Printed in U.S.A. Microbial Metabolites with Insecticidal Properties

M. COLE AND G. N. ROLINSON

Beecham Research Laboratories, Betchworth, Surrey, RH3 7AJ, England Received for publication 14 July 1972

A screen of fungi for insecticidal activity revealed the ability of Aspergillus versicolor to make versimide, methyl-a-(methylsuccinimido)acrylate, a novel contact insecticide. The larvicidal activities of Alternaria tenuis and Fusarium lateritium were found to be due to tenuazonic acid and diacetoxyscirpenol, re- spectively. Thiolutin, cycloheximide, rubratoxin, patulin, trichothecin, an acti- nomycin, and scirpene-producing fungi also had insecticidal activity.

A screen of fungi for insecticidal activity was combined, and 10 ml was evaporated in a 7-cm carried out in view of the need for a new class petri dish. Adult Drosophila were placed in of nontoxic insecticides and the fact that the petri dish, and knockdown was observed metabolites have very diverse biological prop- over a period of 24 hr. The larvicide tests were erties. When we began this investigation, sev- carried out by G. Yeoman at Astra-Hewlett eral fungal metabolites such as the destruxins, Limited, England (now part of Beecham 379X and Y, , pantherine, and Group Limited), and the contact tests by J. tricholomic acid had been described as having Cole of Huntingdon Research Centre, England. insecticidal activity, but difficulty of prepara- Three fungi, Alternaria tenuis, Aspergillus tion, mammalian toxicity, or weak activity versicolor, and Fusarium lateritium were found seems to have precluded further development. to produce insecticidal substances; these were The subject of microbial metabolites has been isolated and identified (Fig. 1). reviewed by Huang and Shapiro (6). The culture of A. tenuis, BRL 837, showed Our screening procedure was carried out as 100% mortality in the larvicide test, but was follows. Fungi were grown in submerged cul- inactive in the contact test. The active prin- ture in 500-ml conical flasks on a rotary shaker cipal was isolated by extraction at pH at 26 C by using two media: (i) 2 and precipitation. It was identified as tenu- 2% malt extract (Oxoid Ltd., London, Eng- azonic acid (11) by comparison with an au- land), 0.3% NH4NO3, 0.1% K2HPO4 (pH 6.5), thentic sample kindly supplied by the late C. deionized water (for contact test) and (ii) 2% E. Stickings. In the larvicide test, the sodium (v/v) corn steep liquor (50% solids content, salt of tenuazonic acid showed a median lethal Garton and Sons, London, England), 1% bacte- concentration (LC50) of 120 jig/ml. Synthetic riological peptone (Oxoid Ltd.), 1% malt ex- material, prepared from DL-isoleucine by M. S. tract, 0.1% K2HPO4 (pH 6.5), deionized water Verrall in these laboratories by using Lacey's (for larvicide test). method (8), showed a similar level of activity. After 5 days, the were tested The culture of A. versicolor, IMI 129,488, for insecticidal activity. Two tests were used, showed 100% knockdown in the contact test at one a larvicide test with Lucilia sericata and 24 hr but was inactive in the larvicide test. the other a contact test using Drosophila me- Activity in shaken flask cultures was erratic lanogaster. For the larvicide test, culture-fil- and was later found to be associated with the trates were freeze-dried and added to sheep instability of the active component in those serum to give a concentration of 25 mg/ml. flasks in which the pH had risen beyond 7.5. First-instar larvae were cultivated in this By using the contact test as an assay system, medium, and mortality was observed at 48 hr. the active component was isolated (Beecham For the Drosophila contact test, a dichloro- Group Limited, British patent 1,187,070, 1970) methane extract was made of the entire fermen- as a brown oil from neutral dichloromethane tation sample at acid (pH 2), neutral, and alka- extracts and was purified further by chroma- line (pH 9.5) pH values. The extracts were tography and molecular distillation to give a 660 VOL. 24, 1972 NOTES 661

Culture Metabolite Insecticidal activity

Tenuazonic acid Alternaria a-Acetyl- y -sec-butyltetramic acid LC50120 pg/mi. tenuis 48 hr. 1st instar 0 BRL 837 N larvae of OH > / \ CH3 Lucilia sericata

N

Versimide Aspergillus Methyl-a-(methylsuccinimido) acrylate 100% knockdown versicolor 0 at 5)Jg/sq. cm. IMI 129.488 glass. 4 hr. N! oFOCH~melanogaster~~~~~~Drosophila /OCH3

Diacetoxyscirpenol Fusarium 4. 15-Diacetoxyscirp-9-en-3-ol LC50 7.5 pg/m lateritium %' 48 hr. 1st instar 0 IMI 140.879 OH larvae of 0 Lucilia sericata

CH3.CO.O.CH2 O.CO CH3

FIG. 1. Insecticidally active fungal metabolites. pale, straw-colored, viscous liquid. The struc- against D. melanogaster in the contact test, a ture of this material was shown to be 100% knockdown was obtained at a loading of methyl( +)-a-(methylsuccinimido)acrylate and 5 Ag/cm2 in 3 to 4 hr. By the oral route in mice was named versimide (3). Synthetic racemic it has a median lethal dose of 92 mg/kg when material, prepared by A. G. Brown and T. C. administered in 10% dimethyl sulfoxide. This Smale of our laboratories, showed a somewhat latter result was obtained by the toxicology lower level of activity than versimide, whereas unit of Beecham Research Laboratories. a synthetic preparation of the methyl ester of The culture of F. lateritium, IMI 140,879, pencolide (2), a related metabolite, was inac- was inactive in the contact test but gave 100% tive. mortality in the larvicide test. The latter was Other strains of A. versicolor, IMI 129,489, used to follow the isolation of the active com- 94,159, and 96,228 were also found to produce ponent from the culture filtrate. Solvent ex- versimide, yields being in the range 1 to 5 traction with isobutylacetate at neutral pH fol- pg/ml. Tests carried out by R. Sutherland and lowed by chromatography on silica gel columns M. Richards of our laboratories showed that yielded a colorless crystalline solid. This solid versimide has essentially no antibacterial ac- has an LC50 of 7.5 Ag/ml against L. sericata at tivity but is active against certain fungi, no- 48 hr and was also active as a moth-proofing tably Trichophyton mentagrophytes (minimal agent (Beecham Group Limited, Belgian inhibitory concentration, 3 Ag/ml). Versimide patent 771,902, 1972). has a low order of toxicity to a wide range of Two assistants involved in the isolation adult insects by contact and vapor action; work developed localized rashes on the face, 662 NOTES APPL. MICROBIOL. reminiscent of the irritant action of the scir- An unidentified metabolite, MM 4460, iso- penols (1). Chromatography column fractions lated in these laboratories from a strain of showing insecticidal activity were dried down, Aspergillus ochraceous, BRL 731, was found to dissolved in a mixture of ethanol, Methyl Cel- have an LC50 of 50 qg/ml against larvae of L. losolve, and Tween 80 (45:45: 10, v/v), and sericata. However, from physical and chemical applied to the shaved back of a guinea pig. In data, this metabolite does not appear to be the this test, which was carried out by J. M. same as aspochracin which is known to be Dewdney of our laboratories, there was a de- produced by A. ochraceous and reported by layed but marked inflammatory reaction with Myokei et al. (9) as having activity against severe erythema, and, by 72 hr, central ne- silkworm larvae. crosis and lateral thickening of the skin. It would thus seem that anti-insect anti- Further support for the compound being a biotics occur widely, and it appears that larvi- scirpenol came from the observation that ma- cidal activity is more frequently encountered terial from the insecticidally active fractions than contact activity. This may be because the had weak antifungal activity (Geotrichum sp. larvae have continuous oral and topical con- agar diffusion test) and showed inhibitory ac- tact with the test substances during a rapid tivity towards the extension of wheat coleop- growth phase. In the contact test, the com- tile tips in sucrose (1). Elemental analysis, pound must penetrate the thin cutin layer of molecular weight, infrared, and nuclear mag- the tarsi before it can exert its effect, and it netic resonance spectra, carried out by A. E. seems that relatively few substances in mi- Bird of our laboratories, confirmed that the croorganisms have this property coupled with active compound was diacetoxyscirpenol (4, 5, intrinsic activity. 10), systematic name, 4,15-diacetoxyscirp-9- We thank M. V. Hart and S. Box of the Microbial Bio- en-3-ol (1). A sample of this material, pur- chemistry Laboratory, D. Butterworth, M. S. Verrall, and G. chased from Makor Chemicals, Israel, was Hanscomb of the Microbiological Pilot Plant, and E. la found to have the same level of larvicidal ac- Croix of the Entomology Laboratory for information on their tivity as that shown for the compound isolated aspects of the work. from culture IMI 140,879. LITERATURE CITED Column chromatography of solvent extracts 1. Bamburg, J. R., W. F. Marasas, N. V. Riggs, E. B. of submerged cultures of Myrothecium verru- Smalley, and F. M. Strong. 1968. Toxic spiroepoxy caria, IMI 72,753, Myrothecium roridum IMI compounds from Fusaria and other hyphomycetes. 60,912, Cephalosporium crotocinigen, IMI Biotechnol. Bioeng. 10:445-455. 112,775, and Trichothecium roseum, BRL 328, 2. Birkinshaw, J. H., M. G. Kalyanpur and C. E. Stick- ings. 1963. Studies in the biochemistry of microorga- showed larvicidal activity in fractions which nisms. 113. Pencolide, a nitrogen-containing metabo- also had antifungal or irritant activity, sug- lite of Penicillium multicolor Grigorieva-Manilova and gesting that verrucarins, roridins, crotocin, and Poradielova. Biochem. J. 86:237-243. tricothecin may also have larvicidal 3. Brown, A. G. 1970. Versimide, a metabolite of Asper- activity. gillus versicolor. J. Chem. Soc. C p. 2572-2573. For tricothecin and trichothecolone, this was 4. Dawkins, A. W. 1966. Phytotoxic compounds produced confirmed when samples, kindly supplied by by Fusarium equiseti. II. The chemistry of diacetoxy- Ewart Jones, were found to have LCO values scirpenol. J. Chem. Soc. C p. 116-123. of about 100 jtg/ml against L. sericata larvae. 5. Dawkins, A. W., J. F. Grove, and B. K. Tidd. 1965. Di- acetoxyscirpenol and some related compounds. Chem. Support for the roridins being insecticides Commun. 2:27-28. comes from the work by Kishaba et al. (7) on 6. Huang, H. T., and M. Shapiro. 1971. Insecticidal ac- the antifeeding substance 379X which, from tivity of microbial metabolites, p. 79-112. In D. J. D. the chemical and Hockenhall (ed.), Progress in industrial microbiology, physical properties, appears vol. 9. J. & A. Churchill Ltd., London. to be identical to roridin A. 7. Kishaba, A. N., D. L. Shankland, R. W. Curtis, and M. An examination of other microbial metabo- C. Wilson. 1962. Substances inhibitory to insect lites isolated as antibacterial or antifungal feeding with insecticidal properties from fungi. J. substances revealed L. sericata larvicidal ac- Econ. Entomol. 55:211-214. 8. Lacey, R. N. 1954. Derivatives of acetoacetic acid. VII. tivity, but not contact activity, in thiolutin a-Acetyltetramic acids. J. Chem. Soc. p. 850-854. (LC5o 32 ,ug/ml), an actinomycin type of sub- 9. Myokei, R., A. Sakurai, Ching-Fun Chang, Y. Kodaira, stance (LCSO 15 ,ug/ml), cycloheximide (LC5o 5 N. Takahashi, and S. Tamura. 1969. Structure of gg/ml), rubratoxin A (LCso 18 and rub- Aspochracin, an insecticidal metabolite of Aspergillus ,gg/ml), ochraceous. Tetrahedron Lett. 9:695-698. ratoxin B (LC5o 200 gg/ml); the last two sub- 10. Sigg, H. P., R. Mauli, E. Fleury, and D. Hauser. 1965. stances were kindly provided by M. 0. Moss of Structure of diacetoxyscirpenol. Helv. Chim. Acta. the University of Surrey. Patulin, although 48:962-988. showing no activity in the larvicide test, did 11. Stickings, C. E. 1959. Studies in the biochemistry of microorganisms. 106. Metabolites of Alternaria tenuis show weak activity in the contact test (90% Auct.: the structure of tenuazonic acid. Biochem. J. knockdown in 24 hr at 10 ,ug/cm 2). 72:332-340.