8447
Natural Toxins Nat. Toxins 7: 337-341 (1999)
SHORT COMMUNICATION
Relative Inhibition of Insect Phenoloxidase by Cyclic Fungal Metabolites from Insect and Plant Pathogenst
Patrick F. Dowd* USDA, Agncultural Research Service, National Center for Agricultural Utilization Research, Bloactlve Agents Research Unit, 1815 N, University St, Peoria, IL 61604, USA
ABSTRACT The fungal metabolite kojic acid, which is produced by Aspergillus and Penicillium species fungi that may be pathogens of both Insects and plants, was a significant inhibitor of phenoloxidase of different representative beetle and caterpillar Insed species. Fusaric acid and picolinic acid, produced by Fusanum spp., were also significant Inhibitors of phenoloxidase, while dipicolinic acid and beauvericin were Ineffective at concentrations tested. PrevIous reports of the ability of kojic and fusaric acid to inhibit defensive enzymes of plants suggest that these compounds may be important in allowing the producing fungi to be pathogens of both Insects and plants Published in 1999 by John Wiley & Sons, Ltd. Key words: kOjic aCid; fusanc aCid; plcolinlc aCid; AsperglJlus; Fusarium
INTRODUCTION plants (Bossi. 1960) and arthropods (Dowd, 1988), while picolinic and dipicolinic acids inhibits enzymes from Cyclic fungal metabolites that can act as chelators or other sources (Hachisuka et al.. 1965; Fonnagel and ionophores are produced by many species of fungi that Freese. 1968; Mann and Byerrum, 1974). The ability of can potentially attack both plants and arthropods. Kojic some of these fungal aromatic acids to inhibit defensive acid produced by several species of AsperRillus and enzymes in insects and plants suggests an adaptation that Penicillium (Cole and Cox. 1981). which can be allows the same species of fungi that produce them to be pathogenic on plants (Frisvad and Samson. 1991) and potential pathogens of diverse organisms such as insects InSCW, (Brooks and Raun. 1965: Madelin. 1963: and fungi (Dowd, 1994). This multihost adaptation has Steinhaus. 1949). Fusaric. picolinic and dipicolinic acids also been suggested for proteolytic enzymes produced by an: produced hy several species of Fusarium (Turner. insect and plant pathogens (Clarkson and Charnley. 1971: Turner and Aldridge, 1983: Bacon el 01.. 1996). 1996). The present repon indicates that these cyclic which can also be pathogenic on insects (C1aydon and metabolites differentially inhibit phenoloxidase (mono Grove. 1982) or plants (Turner. 1971: Turner and phenol oxidase. catechol oxidase E.c. 1.1 0.3.1), the Aldridge. 1983: Bacon el of.. 1996: Desjardins. 1992). enzyme responsible for wound healing and pathogen Bcauvericin is a cyclic peptide produced by different species of Beaul'eria and Fusarium (Turner. 1971: Turner and Aldridge. 1983: Krska el 01., 1996). Although of generally low toxicity to venebrates. invenebrates or 'Correspondence to: P. F. Dowd, USDA, Agricultural Research Service. National Center for Agricultural Utilization Research, plants. compared to other secondary metabolites pro Bloactlve Agents Research Unit, 1815 N. University St, Peoria, IL duced by the same fungal species (Cole and Cox. 1981: 61604, USA. Bacon el al.. 1996: Desjardins, 1992: Dowd. 1992: 'ThiS article IS a US Government Work and is in the public domain Khachatourians, 1991). some of these fungal cyclic In the USA. metabolites can be effective enzyme inhibitors. Kojic Disclaimer. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the acid inhibits oxidative enzymes from both plants and standard of the product, and the use of the name by USDA implies anhropods (Saruno el al.. 1979: Chen el 01.. 1991 a.b: no approval of the product to the exclusion of others that may also Dowd. 1988. 1994: Dowd el of.. 1994: Lee and Anstee. be SUitable. 1995). Fusaric acid also inhibits oxidative enzymes from Received 10 December 1999; Accepted 10 March 2000
Published In 1999 by John Wiley & Sons, Ltd 338 DOWD encapsulation in insects, further supporting the role of Table 1. Inhibition of S. frugiperda hemolymph and cuticular some of these compounds in the insect pathogenic phenoloxidase by fungal cyclic metabolites process. 'k Inhibition of phenoloxidase Inhibitor Hemolymph Cuticle MATERIALS AND METHODS Fusaric acid 10-3 M 38.7 ± 2.2 a 34.6 ± 5.2 a Insects Picolinic acid 10-3 M 16.2 ± 3.5 b 41.2 ± 6.5 a Dipicolinic acid 10-3 M -23.8 ± 14.4 be -8.6± 2.6 c Fall armyworms (Spodoptera frugiperda (1. E. Smith». 3 corn earworms (Helicoverpa zea (Boddie») and Freeman Beauvericin 10- M -51.5 ± 5.5 c -9.8 ±6.5 c Kojic acid 10-3 M 88.2 ± 1.2 d 80.7 2.1 d sap beetles (Carpophilus freemani Dobson) were reared ± Kojic acid 10-4 M 40.8 ± 1.8 a 36.6 ± 1.9 a at 27 ± 10c, 40 ± 10 % r.h. and a LJ 4:0 I0 photoperiod on pinto bean-based diet (Dowd. 1988: Oowd and Weber. Values are means ± SE. Values in columns followed by the same letter 1991). Cigarette beetles (Lasioderma serricorne (F.) are not significantly different at p < 0.05 by analysis of variance. were reared at 27 ± 1°C, 60 ± 10 % r.h. and a LJ4:D1O light:dark photoperiod on a flour. maize meal. brewer's yeast mix (Oowd. 1989a). Greenhouse whitefly adults L. serricome and C. freemani. and 0.03-0.045 mg ml- I (Trialellrodes vaporariorum (Westwood) were obtained for T. vaporariorum. from infested tobacco in the greenhouse c. I week after emergence. Enzyme Assays The reaction mixture for phenoloxidase activity consisted Enzyme Extraction of 700 Jll of room temperature buffer: 100 JlI of 10-2 M Last instar caterpillar larvae were used as an enzyme kojic acid (in buffer), fusaric acid (in ethanol), picolinic source of both hemolymph and cuticle phenoloxidase. acid (in ethanol), beauvericin (in ethanol) or dipicolinic which can differ in types of phenoloxidase properties and acid (in 50 % ethanol) with appropriate solvent controls: activity (Marmaras el al.. 1996). To collect hemolymph. 100 JlI of 0.2 % L-DOPA (L-3. 4-dihydroxyphenyl prolegs were clipped off and hemolymph was allowed to alanine) in water; and 50-100 JlI of enzyme source. All drip into test tubes held at O°c, Approximately 50 III chemicals were obtained from Aldrich Chemical Co.. of hemolymph could be obtained from one individual. Minneapolis, MN, USA or Sigma Chemical Co.. St The hemolymph was diluted 1:9 with pH 7.-L 0.1 M Louis. MO. USA. The oxidation ofDOPA was monitored phosphate buffer. and frozen solid at -:woC in order to at 470 nm for 10 min using a Perkin-Elmer Lambda 4B rupture hemocytes and free the phenoloxidase (Brook spectrophotometer equilibrated to 30°C. Assays were run man el at., 1989: Dunphy. 199 I ). The material wa~ then at least in duplicate on at least two separate occasions. thawed to a liquid and centrifuged at 10000 xx for 5 min Assays were initially run with S. frugiperda to determine at 4°C, The supernatant was further diluted I: 10 and used the most active compound (kojic acid). Kojic acid was 3 in assays. Protein was from c. 0.1 :25-0.:250 mg mI" I In then tested as an inhibitor at 10- M against the other the different assay series using the Bio-Rad proteIn insect enzyme sources. reagent and instructions (Bio-Rad. Richmond. CA. USA). The cylinder of cuticle behind the last pair of RESULTS AND DISCUSSION true legs and in front of the last pair of prolegs wa~ spilt lengthwise. and gut. fat body and Malplghian tuhub At 10-1 M. kojic acid inhibited phenoloxidase activity by were removed. The cuticle strip was rin~ed In the fall armyworm «(Spodoptera frugiperda) cuticle and phosphate buffer. and then two strips were homogenlled hemolymph by c. 80 %. At 10-4 M. significant inhibition in :2 ml of the buffer with a ground glas~ h0l1111!-!ellller. of S. frllgiperda cuticular and hemolymph phenoloxidase The homogenate was centrifuged at 10000 xg lor:; mIn. activity by kojic acid (35-40 %) was again noted (Table and the supernatant was removed. The supernatant was I I. Both sources showed about the same susceptibility to diluted I: 10 for assays: protein content in a~~ay ~ was t. inhibition by kojic acid. Kojic acid was also a highly O. 175-0.320 mg ml- I using the Bio-RaLl kit. effective inhibitor of phenoloxidase activity in other Due to the small size of the other insects. whole body species of Lepidoptera and Coleoptera tested. but was preparations were used. One hundred las! instar L. much less effective against adult whiteflies (Table 2). serricorne and C. freemani larvae and T. mporariorum Levels of inhibition of S. frllgiperda hemolymph adults were chilled and homogenized in :250--500 III of phenoloxidase by kojic acid (Dowd et aI., 1994; present the phosphate buffer. Homogenates were Olherv.:ise study) are similar to those reported for S. lirroralis treated as for the caterpillar preraratHm~. Final protein hemolymph (Lee and Anstee. 1995). The levels of concentration in assays wa~ c. O.lJ-1.1 mg ml I lor inhibition for insects were similar to those reported for
Published in 1999 by John Wiley &. Son,. Ltd Nat. Toxins 7: 337-341 (1999) INHIBmON OF INSECT PHENOlOXIDASE BY FUNGAL METABOLITES 339 lobster polyphenoloxidase when similar concentrations aromatic acids to function as chelators. which causes it of kojic acid were used and DL-DOPA was used as a to complex with metal ions of these enzymes (Dowd. substrate (Chen et al.. 1991 a). A mixed type of inhibition 1988. 1994). At the molecular level this complexing was noted with kojic acid and polyphenoloxidase from appears to result in interference of oxygen uptake and white shrimp. spiny lobster and grass prawn (Chen et al.. reduction of o-quinones that may be produced (Chen et 1991 b). Kojic acid also inhibits monophenol mono al., 1991b). oxygenase (tyrosinase: E.c. 1.14.18. I) from mushroom The main defense reaction of insects against fungi is (Sarono et al.. 1979). kidney D-amino acid oxidase (E.C. the encapsulation response. which involves hemocyte 104.3.3; Klein. 1953). different plant catechol oxidases phenoloxidase (Hajek and 5t Leger. 1994). an enzyme (polyphenoloxidases: (E.c. 1.10.3.1: Chen et al.. that is critical to the process (Marmaras et at.. 1996). The 1991 a.b). plant peroxidase (E.C. 1.11.1.7: Dowd. 1994). present study indicates that kojic. fusaric. and picolinic insect unspecific monooxygenase (E.C. 1.14.14.1: Dowd. acids are all capable of inhibiting the enzyme involved in J988) and insect phenoloxidase (Dowd et at.. J994: Lee the hemocyte encapsulation response. as well as wound and Anstee. 1995). healing by the cuticle (a site of penetration for the fungi). Fusaric and picolinic acids were significantly less In addition to direct enzyme inhibition. the insect active than kojic acid at the same concentration against S. phenoloxidase also requires Ca2~ ions for activation frugiperda phenoloxidase (Table 1). Dipicolinic acid was and activity. although this varies among insect species inactive to slightly stimulatory for hemolymph phenol (Brookman et al.. 1989; Dunphy. 1991). The aforemen oxidase. while beauvericin was strongly stimulatory for tioned chelating/ionophore activity of these compounds hemolymph phenoloxidase (Table I). Picolinic acid was suggest that this is an additional way that they can inhibit less active towards hemolymph phenoloxidase compared insect phenoloxidase. as well as plam peroxidase. which to cuticle phenoloxidase. Fusaric acid inhibits oxidative also requires Ca2~ ions (Gaspar et al.. 1982). Although enzymes such as mammalian dopamine B-hydroxylase dipicolinic acid and beauvericin were sometimes stimu 2 (monooxygenase: E.C. I. 14. I7.1: Hidaka. 197 I). mam latory to the phenoloxidase. ability to interact with Ca -r malian tyrosinase (Nagatsu et al.. 1972). plant catechol ions may ultimately result in enzyme inhibition. How oxidase (Bossi. 1960) and insect unspecific monooxy ever. their ability to enhance phenoloxidase activity genase (Dowd. J998). Picolinic acid inhibits oxidative suggests the insects have partially adapted to pathogens enzymes such as bacterial aldolase (aldose I-dehydro that produce these compounds. genase: E.C. 1.1.1.121: FortnageJ and Freese. 1968) and Kojic acid is produced relatively early overall in solid plant quinolic acid phosphoribosyl transferase (E.C. culture and in much higher quantities compared to 2.4.2.19: Mann and Byerrum. 1974). Fusaric acid also aflatoxin. a more toxic secondary metabolite (Lee et al.. synergizes the toxicity of fungal metabolites. insecticides 1986). This early production suggests that kojic acid is and plant allelochemicals (Dowd. J988. I989b). Dipil:o important in the infection process. compared to highly hnic acid inhibits bacterial glucose dehydrogenase (E.C. toxic metabolites such as aflatoxin. which may ultimately 1.1.99.10: Hachisuka et al.. 1965) but stimulates maize kill the insect. Quantities of kojic acid produced in liquid NADPH oxidase (dehydrogenase: E.C. 1.6.99. I: Tyagi et culture can be in the 100 g kg - I range (Wilson. 197 I ). £1/.. 19R7). Presumably inhibition of metalloenzymes. suggesting high quantity production could also occur in such as unspecific mono-oxygenase. peroxidase and insects at a level at least comparable to that tested in the phenoloxidase are due to the ability of the fungal present study. Although there are no reports for kojic acid concentration in infected insects to the author's knowl edge. aflatoxin has been reported at 0.05 ~mole per A. Table 2. InhiblllOn of msect whole body. hemolymph or ~utl~ular ,tim'us infected Bombyx mori larva (Ohtomo et at.. 1975). phenolmiua,e hy 10 'M kojic a~id Assuming a larva weighs 0.5 g and has the density of water (which is conservative). the concentration of 'if Inhibitiun uf phenoh'>.lda,,: 4 aflatoxin B j would be 1.0 x 10- M. Even assuming In'l'cl Hemolymph Cuticle Whole hod;. kojic acid was only produced at the same rate as aflatoxin. S. .1rugl[Jerdv 88.2 ± 1.2 a 80.2:::: 2.1 a NO this would still put its concentration in the insect at a level H :<'{/ 83.8 ± 0.7 a 89.0 ± 0.4 ;J NO biologically relevant to concentrations tested in the L .\('rnCIJl"llc NO NO 1)6./ ::: 3.0 a present study. C. freemvlli ND ND 88...+:::: 0.1) a The relatively short. three-step biosynthetic pathway T \'{/[Jorvriorum NO ND 19.0:::: 4.2 b for production of kojic acid from glucose (Wilson. 1971) suggests isolation and cloning of biosynthetic genes is a Value, are means::: SE. Value, in the same column followed by ulfterent leiters are signiticantly different at {' < !l.U:; by analysis of possibility for increasing the virulence of insect patho \anance. gens that do not produce this compound. Determining the l\'D =nOI determmed metabolic pathway difference for picolinic and dipico-
Published in J999 by John Wiley & Sons. Ltd. Nar. Toxins 7: 337-341 (1999) 340 DOWD
linic acid production. and altering it to favor picolinic Dowd PE McGuire MR. Behle RW. Vega FE. Richard JL. Shasha BS. acid by mutation or genetic engineering. may enhance the Banelt RJ. Nonon RA. Beland GW. Duvick JP. Miller 10. Kennel 19\j~ virulence of insect pathogens that produce dipicolinic D 1994. Insect IPM for mycotoxin control in midwest com: studies. In AjlalOxin Eliminarion Workshop 1994. Robens JF ledl. acid (which includes Beauveria bassiana and Paecilo United States Depanment of Agriculture: Beltsville MD: 68. myces fumoso-roseus (leI Ltd. 1983 l. The ability of the Dowd PF. Weber CM 1991. A labor-saving method for rearin o a corn fungal aromatic acids to inhibit defensive enzymes in sap beetle Carpophi/us freemani Dobso~ (Coleoptera: Nitidulidae J. both plants and insects helps explain the ability of on pinto bean-based diet. J Agric Enromo! 8: 149-153. Dunphy GB. 1991. Phenoloxidase activity in the serum of two species different strains of the producing organisms to be both of insects. the gypsy moth. Lymanrria dispar (Lymantriidae) and the plant and insect pathogens. greater wax moth. Galleria mellonella (Pyralidael. Coml' Binchem PhysioI98B:535-538. Fonnage! P. Freese E 1968. Inhibition of aconitase bv chelation of ACKNOWLEDGEMENTS transition metals causing inhibition of sporulatio~ in Bacillus I thank C. M. Anderson. A. J. Hartter and D. A. Lee for subri/is. J Bioi Chern 243:5289-5295. Frisvad Jc. Samson RA 1991. Filamentous fungi in foods and feeds: technical assistance and M. R. McGuire and R. A. Norton ecology. spoilage and mycotoxin production. In Handbook or for comments on earlier drafts of this manuscript. Applied Mycology. vol 3. Foods and Feed.<. Arora DK. Mukerji KG. Manh EH (eds). Marcel Dekker. London: 31-68. Gaspar T. Pene! C. Thorpe T. Greppin H 1982. Peroxithlses: /970- REFERENCES 1980. A Survey of Their Biochemica! and Physiological Ro!es in Higher Planrs. University of Geneva: Geneva. Hachisuka Y. Tochikubo K. Murachi T 1965. Inhibitory effect of Bacon CW. Poner JK. Nored WP. Leslie JF 1996. Production of dipicolinic acid on the anaerobic oxidation of glucose by the cell fusaric acid by Fusarium species. Appl Envtrlm Mlcro/JIlJ!62:4039 free extract from vegetative cells ofB. subri/is. NaTUre 207:221-222. 4043. Hajek AE. St Leger RJ 1994. Interactions between fungal pathogens Bossi R 1960. Ober die Wirkung der Fusannsaure auf die Poly and insect hosts. Ann ReI' Enromo/ 39:293-322. phenoloxydase. Phwoparho! ~ 37:27:--:-1 t> Hidaka H 1971. Fusaric (5-butylpicolinicJ acid. an inhibitor of Brookman JL. Ratcliffe NA. Rowley AF 19H9 Studies on the dopamine II-hydroxylase. affects serotonin and noradrenaline. activation of the prophenoloxida.~e system of InseCh hy bacterial Nalllre 231:54-55. cell wall components. J Insecr PhYSIlJ! 34::-:-7-:-45 ICI Ltd 1983. Unpublished data cited in Turner WB. Aldridge DC, Brouks DL. Raun ES 1965. Entomugenuus fungi trum cum Insects In FunRal Merabolires Academic Press: New York. Iowa. J Im'err ParllO! 7:79-81 n. Khachatourians GG 1991. Physiology and genetics of entomopatho Chen JS. Wei C. Rolle RS. Otwell WS. Balaban MO. Marshall MR genic fungi. In Handbook of Applied Myco!ogy. vol ~. Humans. 1991 a. InhibilOry effect of kojiC acid on some plan! and cruslacean Animals and Insecrs. Arora DK. Ajello L. Mukerji KG leds). Marcel polyphenol oxidases. J ARric Food Chem 39: 1:-96-140 I Dekker: New York; 613-663. Chen JS. Wei C. Marshall MR 1991 b. Inhlblllon mechanl\m, of kujlC Klein JR 1953. Inhibition ofD-amino acid oxidase by aromatic acids. J acid on polyphenol oxidase. J Agnc Food Chl'nl 39: I HY7 -I
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Sarono R. Kato F. Ikeno T 1979. Kojic acid. a tvrosinase inhibitor from Tyagi VV. Henke RR. Farkas WR 1987. Activation of an NADPH Aspergillus albus. Agric Bioi Chem 43:133"7-1339 oxidase from maize by dipicolinic acid. Phytochemistry 26:91:' Steinhaus EA 1949. Prlllcipies of InseCT Patholog\. McGrav. Hill: 915. New York. Wilson BJ 1971. Miscellaneous AsperRiIlus toxins. In Microbial Turner WB 1971. Fungal Metabolites. AcademiC Press: New York. Toxins. vol VI. Fungal Toxins. Ciegler A. Kadis S. Ajl 5J (eds). Turner WB. Aldridge DC 1983. FunRal Metabolites II. Academic Academic Press: New York: 207-295. Press: New York.
Supplied by the U.S. Department of Agriculture, National Center for Agricultural Utilization Research. Peoria, Illinois.
Published 10 1999 by John Wiley & $on,. Ltd Nat. Toxins 7: 337-341 (1999)