Siderophores are iron-chelating extracellular fungal metabolites which may be involved in initiating wood decay. A purified siderophore extract isolated from the brown-rot decay fungus Gloeophyllum trabeum (Pers. ex Fr.) Murr. (Basidiomycetes: Polyporaceae) was found to deter feeding by Formosan subterranean termites, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). This fungus has previously been associated with preferential feeding on decayed wood by subterranean termites, and solvent extracts have been reported to induce termite trail-following, arrestment, and/or aggregation. This is the first report of G. trabeum metabolites or fungal siderophores having a negative behavioral effect on subterranean termites

Chandhoke, Vikas

Goodell, Barry S.

Grace, J Kenneth

Jellison, Jody

Jones, Wendy E.

Proceedings of the Hawaiian Entomological Society

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Vol. 31, December 31,1992 249SCIENTIFIC NOTEEvidence for Inhibition of Termite(Isoptera: Rhinotermitidae) Feeding byExtracellular Metabolites of aWood Decay Fungus1'2J. KENNETH GRACE3, BARRY S. GOODELL4, WENDY E. JONES3,VIKAS CHANDHOKE4, andJODYJELLISON4ABSTRACT. Siderophores are iron<hclaling extracellular fungal metabolites which may beinvolved in initiating wood decay. A purified siderophore extract isolated from the brown-rotdecay fungus Gloeophyllum tmbeum (Pcrs. ex Fr.) Murr. (Basidiomycetcs: Polyporaceae) wasfound to deter feeding by Formosan subterranean termites, Coptotermn formosanus Shiraki(Isoptera: Rhinotermilidae). This fungus has previously been associated with preferentialfeeding on decayed wood by subterranean termites, and solvent extracts have been reportedto induce termite trail-following, arresiment, and/or aggregation. This is the first report of G.Irnbnim metabolites or fungal siderophores having a negative behavioral effect on subterranean termites.Many brown and white-rot wood decay fungi (Basidiomycetes: Polyporaceae) produce low molecular weight (500-1,000 daltons) iron-chelatingextracellular metabolites, or siderophores (Fekete et al. 1989;Jellison etal.1990; Kim et al. 1990). Because cellulase enzymes are too large to diffusefreely through wood cell walls (Cowling and Brown 1969), a non-enzymaticdegradative agent has been proposed to be active in the initial stages offungal decay (Koenigs 1974). In addition to scavenging transition metalsfor fungal metabolism and extracellular metabolism, siderophores may playsuch a role in lignocellulose degradation by preceding enzymes into thewood cell wall (Jellison et al. 1990).Fekete et al. (1989) demonstrated that the brown-rot decay fungusGloeophyllum trabeum (Pers. ex Fr.) Murr. produced phenolate-type siderophores. This fungus also produces compounds inducing trail-following,arrestment and/or aggregation, and preferential feeding by subterraneantermites (Isoptera: Rhinotermitidae) (Esenther et al. 1961; Allen et al.1964; Grace 1989, 1990). The compound (cis,cis,trans)-3,6,8,-dodecatrien-l-ol, originally isolated from G. trabeum infected wood (Matsumura et al.1968), is considered to be the principal component of the trail pheromone'Reported at the 23rd Annual Meeting of the International Research Group on Wood Preservation. Harrogate, U.K., May 1992. This is Journal Series No. 3670 of the Hawaii Institute ofTropical Agriculture and Human Resources.'Financial support provided by USDA-ARS Specific Cooperative Agreement 58-43YM-9-0025.'Department of Knlomology, University of Hawaii, 3050 Maile Way, Honolulu, Hawaii 96822.4Wood Science and Technology, University of Maine, Orono, Maine 04469-0125.250 Proceedings, Hawaiian Entomological Societyof a number of Rhinotermitids, including the Formosan subterraneantermite Coptotermes formosanus Shiraki (Tokoro et al. 1990).In an ongoing investigation of termite-fungal interactions, we evaluatedthe effects of a purified siderophore extract from G. trabeum on C.formosanus feeding behavior. Although it is possible that siderophores mayplay a role in lignocellulosic degradation by both fungi and wood-feedinginsects, filter papers treated with the G. trabeum extract were found to deterfeeding by C. formosanus. Production of semiochemicals having positiveeffects on termite behavior varies with different G. trabeum isolates andculture conditions (Amburgey and Smythe 1977), but induction of negativebehavioral effects by G. trabeum metabolites has not previously been reported.MATERIALS AND METHODSThe purified siderophore extract was prepared as described byJellisonet al. (1990) from G. trabeum grown in low iron stationary liquid culturemedium. The spent culture medium was separated from the mycelium byfiltering through Whatman No. 2 filter paper, concentrated by rotary evaporation, and filtered through an Amicon ultrafiltration unit using a YM2membrane (1000 MW cutoff). Phenolate siderophores were isolated byacidification of the supernatant to pH 2.5 with concentrated HCL, threefold extraction with an equal volume of ethyl acetate, evaporation of theethyl acetate extract to dryness, and resuspension of the residual in deion-ized water. Whatman No. 2 filter papers were soaked to saturation in theaqueous suspension, and air-dried in the dark at ambient laboratory conditions.For bioassays with C. formosanus, the treated filter papers were cut intoca. 1.5 cm squares. Three bioassays were performed: (i) a no-choice(forced-feeding) assay in which only one food source (either a treated oruntreated filter paper) was provided to the termite workers, (ii) a two-choice feeding assay in which each siderophore-treated paper was pairedwith an untreated control paper within the same termite container, and(iii) a siderophore degradation/leaching assay in which siderophore-treated papers were placed on damp sand without termites present for aperiod equivalent to that of the feeding assays.In each assay, siderophore-treated and untreated filter papers wereair-dried, weighed, and placed on ca. 5 ml dry crushed coral sand (sievedto pass a US 20-mesh screen) in a 3.5 cm diameter plastic petri dish, and2.5 ml distilled water was added to the sand. Coptotermesformosanus workerswere collected from traps on the Manoa campus of the University of Hawaii(Tamashiro et al. 1973), and 50 workers (pseudergates) were placed in eachno-choice and two-choice replicate. In the no-choice feeding assay, 5 treatedand 5 untreated papers were evaluated; the two-choice assay was performedwith 10 replicates (each containing both a treated and untreated paper);and 5 treated papers were evaluated for weight loss without termites presentin the siderophore degradation/leaching assay. After 3 days, surviving ter-Vol. 31, December 31,1992 251mites were counted, and the papers were removed, oven-dried (80°C, 24hours), and weighed to determine mass loss due to termite feeding and/orsiderophore degradation/leaching.Due to the large paper weight losses observed in the siderophore degradation/leaching assay (without termites), weight loss data from all threeassays were subjected to analysis of variance (unbalanced ANOVA, PROCGLM), and significantly different mean weight losses separated by Tukey'sStudentized Range Test (SAS Institute 1987).TABLE 1. Weight loss of siderophore-trealed and untreated filter papers placed an dampsand for three days in no-choice (force-feeding) and two-choice (treated vs.untreated) assays with Coplotennrsformosanus workers, and without termite workers present.Mean (i SEM) paper weight loss (ing)*Assay type Siderophore-treated UntreatedNo-choice feeding assay 17.42 £ 0.95 a 8.72 ± 0.62 bTwo-choice feeding assay 18.43 ♦ 0.83 a 8.22 ± 0.51 l>Without termites 17.34 ± 1.04 a —•Mean weight losses within each column are not significantly different (ANOVA, Tukey'sStudentized Range Test, a s 0.05). In no-choice and two-choice assays, termite feeding wasobserved only on the untreated papers.RESULTS AND DISCUSSIONPractically no termite mortality (4% maximum) was observed in eitherthe no-choice or two-choice assays over the 3-day period. Termites wereobserved walking on both treated and untreated papers, and building theircharacteristic carton (consisting of sand, masticated paper, feces and salivary secretions) at the edges of both papers. However, the untreated paperswere almost completely consumed in three days, while only slightly roughened edges, which could indicate very minor feeding, were noted on thesiderophore-treated papers.Termites were fed equally on the untreated papers in the no-choice andtwo-choice assays (Table 1). The greater weight loss of the siderophore-treated papers in these assays was not due to termite feeding, as was confirmed by the equivalent mean weight loss of the treated papers when notermites were present. These weight losses are attributable either to leaching of the G. trabeum extract from the treated papers or siderophore-initi-ated chemical degradation of the cellulose.Although this is the first report of G. trabeum metabolites deterringtermite feeding, certain white-rot wood decay fungi have been reported toinhibit termite attack (Amburgey and Beal 1977). White-rot decay fungialso produce biological chelators (Fekete et al. 1989), which may initiatecellulose depolymerization (Jellison et al. 1990) as well as participate insome lignin degradation reactions. Since termites and decay fungi competefor the same cellulosic resource, secretion of a termite feeding deterrent252 Proceedings, Hawaiian Entomological Societycould benefit the fungus. Conversely, the presence of a high concentrationof fungal siderophores could act as a chemosensory cue to termites thatthe substrate is excessively degraded and therefore nutritionally inadequate. This suggests an intriguing possibility for protecting wood fromtermite attack by artificial stimulation of this behavioral response. Studiesare proceeding to clarify the mechanism of termite deterrence, and therole of fungal and siderophore-mediated lignocellulose degradation infeeding by the Formosan subterranean termite.REFERENCES CITEDAllen, T.C., R.V. Smythe, and H.C. Coppel. 1964. Response of twenty-one termite species toaqueous extracts of wood invaded by the fungus Isiizitn tmbra Pers. ex Fr. J. Econ.Entomol. 57:1009-1011.Amburgey, XL. and R.H. Beal. 1977. White rot inhibits termite attack. Sociobiology 3:35-38.Amburgey, T.L. and R.V. Smythe. 1977. Factors influencing the production of termite trail-following and arrestant stimuli by isolates of Cloeophylium Irabeum. Sociobiology 3:13-25.Cowling, E.B. and W. Brown. 1969. Structural features of cellulosic materials in relation toenzymatic hydrolysis. In G.J. Hajny and E.T. Reese [eds.], Cellulases and Their Applications. Adv. Chem. Ser. 152-187.Esenther, G.R., T.C Allen, J.E. Casida, and R.D. Shencfelt 1961. Termite attractant fromfungus-infected wood. Science 134:50.Fekete, F., V. Chandhoke, and J. Jellison. 1989. Iron-binding compounds produced by wood-decaying basidiomycetes. Appl. F.nviron. Microbiol. 55:2720-2722.Grace, J.K. 1989. Habituation in termite orientation response to fungal semiochemicals.Sociobiology 16:165-182.Grace, J.K. 1990. Effect of antioxidants on eastern subterranean termite (Isoptera:Rhinotermitidae) orientation to fungal extract. Proc. Entomol. Soc. Wash. 92:773-777.Jellison, J., B. Goodell, F. Fekete, and V. Chandhoke. 1990. Fungal siderophores and theirrole in wood biodegradation. International Research Group on Wood PreservationDoc. No. IRG/WP/1442. 1RG Secretariat, Stockholm, Sweden. 16 pp.Kim, Y.S., J. Jellison, B. Goodell, and V. Tracy. 1989. The use of EI.ISA for the detection ofthe degradative fungi Coriolus vmicolar, l'mlia placenta, Letinus Modes and Tymmycespatustris. Proc. Fourth Intern. Conf. on Biotechnology in the Pulp and Paper Industry.34-35. Raleigh, North Carolina.Koenigs, J.W. 1974. Hydrogen peroxide and iron: a proposed system for decomposition ofwood by brown-rot basidiomycetes. Wood Fiber 6:66-80.Tamashiro, M., J.K. Fujii, and P.Y. Lai. 1973. A simple method to observe, trap and preparelarge numbers of subterranean termites for laboratory and field experiments. Em-iron.Entomol. 2:721-722.Tokoro, M., M. Takahashi, K, Tsunoda, and R. Yamaoka. 1989. Isolation and primary structureof the trail pheromone of the termite, Coptotermes formosanus Shiraki (Isoptera:Rhinotcrmilidae). Wood Research 76:29-38.

Evidence for Inhibition of Termite (Isoptera: Rhinotermitidae) Feeding by Extracellular Metabolites of a Wood Decay Fungus

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Evidence for Inhibition of Termite (Isoptera: Rhinotermitidae) Feeding by Extracellular Metabolites of a Wood Decay Fungus

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