Historic, archived document Do not assume content reflects current scientific knowledge, policies, or practices. , Er iu, ee ne esis i] @) cominenct = Effect of Lauricidin and “=” roesseviee Ethylenediaminetetraacetic rectacneree §6©XACId on Growth of Experiment Station reseacnnore Nine Hymenomycetous Fungi PNW-411 Regs: GC: Y. Liand Paul E. Aho acs ST PANICE YVDEDIMASArr eranriny EXPERIMENT STATION | STATION LIBRARY COPY enone a oe en Abstract Growth of nine wood-decaying basidiomycetes was measured on media containing 10, 100, and 1,000 parts per million (p/m) Lauricidin with or without 0.1 percent ethylenediaminetetraacetic acid (EDTA). EDTA alone significantly reduced the growth of all fungi tested. Lauricidin at 1,000 p/m significantly retarded the growth of all fungi except two: Ganoderma applanatum and Armillariella mellea. The addition of EDTA to 1,000 p/m Lauricidin completely inhibited Echinodontium tinctorium, Fomitopsis officinalis, and Perenniporia subacida. These compounds show promise as consti- tuents of tree-wound dressings. Keywords: Decay fungi (wood), lipids, fatty acids, wounds, tree injury. Introduction Lauricidinl’ is the trade name of monolaurin with more than 90- percent monoester attached to the first glycerol hydroxyl group. This compound has shown remarkably high activity against oral streptococci and actinomycetes and has been incorporated into products used to prevent dental caries in humans (Kabara and others 1978). In vitro growth of Heterobasidion annosum (Fr.) Bref. and Phellinus weirii (Murr.) Gilb. was inhibited by Lauri- cidin (Li and Kabara 1978). A preliminary field test indicated that Lauricidin applied on stump surfaces of western hemlock (Tsuga heterophylla (Raf.) Sarg.) could prevent colonization by H. annosum (Nelson and Li 1980). 1/ Lauricidin is a compound available from Med-Chem Laboratories, Monroe, Michigan. Trade names are included for information only and do not imply endorse- ment by the U. S. Department of Agriculture. Cc. Y. LL is microbiologist and PAUL E. AHO is research plant pathologist, Forestry Sciences Laboratory, 3200 Jefferson Way, Corvallis, Oregon 97331. Intensive forest management usually requires repeated stand entries with logging equipment. Aho and others (1983) have shown that up to 50 percent or more of the residual (crop) trees were injured during thinning in young true fir stands in northern California. About 14 percent of the board-foot volume of the wounded trees was lost to decay after only 13 years. Wounds on trees in recreation and urban areas may lead to exten-— Sive decay and tree failure, resulting in property damage and injury or death to people. Recent studies have shown that commonly used wound dressings do not prevent invasion by bacteria and fungi that cause discolor- ation and decay (Shigo and Wilson 1977). Shigo and Wilson (1971) suggested that an effective dressing should protect wounds not only from invasion by decay fungi, but also from the bacteria and nondecay fungi that are often the pioneer invaders of exposed wood and contribute to discoloration and decay. Because Lauricidin is relatively inexpensive, is not toxic to animals and higher plants, and inhibits both bacteria and fungi, it is a potential component of tree-wound dressings. We tested the inhibitory effects of Lauricidin alone and combined with EDTA in vitro on nine hymenomycetous fungi. EDTA was added because it increases the solubility of Lauricidin and the permeability of microbial cells to Lauricidin (Shibasaki and Kato 1978). Materials and The fungi used in this experiment were Armillariella mellea Methods (Fr.) Karst., Coniophora puteana (Fr.) Karst., Echinodontium tinctorium E. and E., Fomitopsis pinicola (Fr.) Karst., F- officinalis (Vill. ex Fr.) Bond. et Sing., Ganoderma applanatum (Pers. ex Wallr.) Pat., G. tsugae Murr., Phellinus pini (Fr.) Pilat, and Perenniporia subacida (Pk.) Donk. Cultures of these fungi were provided by the Center for Mycology Research, Forest Products Laboratory, Madison, Wisconsin. Cultures had been maintained on malt or potato dextrose agar since 1968. Inocula of the fungi obtained were from colonies grown on malt agar in petri plates at 25 °C until they reached 25 to 30 mm in diameter. Lauricidin was added to malt agar and malt agar containing 0.1 percent EDTA to give concentrations of 10, 100, and 1,000 p/m. Controls were malt agar and malt agar containing 0.1 percent EDTA. The media were autoclaved at 15 1b pressure for 15 minutes, then adjusted to pH 5.4 with sterile 0.1 N NaOH. Petri dishes, 90 mm in diameter, were filled with 20 ml of medium. Four replicate plates were inoculated with F. officinalis, G. applanatum, G. tsugae, and Phellinus pini; five replicate plates were inoc-— ulated with C. puteana, E. tinctorium, F. pinicola, and Perenniporia subacida. Radial growth of A. mellea mycelia was difficult to measure because irregularly shaped colonies de- veloped. We therefore measured the dry-weight gain of A. mellea after growing it in tubes 25 mm outside diameter x 200 mm, each containing 80 ml of medium. A plug of inoculum 4 mm in diameter was taken from the edge of colonies of each fungus and inverted in the center of each agar plate or tube. Inoculated plates and tubes were incubated at room temperature (22 to 24 °C). Because of variations in growth rate, radial mycelial growth was measured at three intervals: 12 days for C. puteana, F. pinicola and Perenniporia subacida; 27 days for Phellinus pini, F. officinalis, G. applanatum and G. tsugae; and 37 days for E. tinctorium. Dry weight of A. mellea colonies was determined after incuba- tion for 26 days. Colonies were removed from warm agar, washed in water, and ovendried at 80 °C for 48 hours. The experiment had a completely random factorial design. Unfor- tunately, some of the fungi grew quickly to the maximum size permitted by the petri dishes; thus, potential growth beyond this size was unknown. Data for these cultures were eliminated from the analyses. A one-way analysis of variance was used for the unbalanced data. The remaining fungi were analyzed in the pre- scribed manner. Individual differences in both formats were analyzed using Tukey's multiple comparison technique. Results and The effects of Lauricidin and EDTA on radial mycelial growth are Discussion shown in figure 1. Growth of C. puteana, F. pinicola, and Perrenniporia subacida on media with 10 p/m Lauricidin was sig- nificantly lower than for controls, and growth was further significantly decreased on concentrations of 100 and 1,000 p/m. Growth of F. officinalis in 10 p/m Lauricidin did not differ sig- nificantly from the control but was significantly less on 100 and 1,000 p/m than on controls or 10 p/m Lauricidin. Ganoderma applanatum, G. tsugae, and Phellinus pini on 1,000 p/m Lauri- cidin grew significantly less than on controls and on 10 and 100 p/m Lauricidin. Growth of Echinodontium tinctorium did not differ on 10, 100, and 1,000 p/m Lauricidin. Except for P. pini at 1,000 p/m Lauricidin combined with EDTA, the other Lauri- cidin-EDTA combinations significantly decreased growth of all fungi compared to Lauricidin alone. Lauricidin at 1,000 p/m plus EDTA significantly reduced mycelial growth of F. pinicola and G. applanatum, and completely inhibited the growth of E. tinctorium, F. officinalis, and Perenniporia subacida. Ganoderma tsugae failed to grow on media with EDTA or EDTA- Lauricidin combinations. EDTA alone significantly reduced the growth of all fungi. Lauricidin alone, however, increased the growth of A. mellea. Legend Lauricidin alone === —e@ Lauricidin + EDTA 40 Coniophora puteana Perenniporia subacida 40 Phellinus pini Ganoderma applanatum a a a 40 Echinodontium tinctorium b b ab, a Ganoderma tsugae 20 Diameter of fungal colony (mm) Cc Cc — — Cc ees we we ac Fomitopsis officinalis 40 Fomitopsis pinicola a o 20 ave wad 7 THIS 6) : . . . . S x 0 0 10 100 1000 0 10 100 1000 Level of Lauricidin (p/m) Figure 1. Effect of Lauricidin and EDTA on mycelial growth of eight hymenomycetous fungi. Means not sharing a common letter significantly differ at the 95—-percent confidence level with the Tukey test. Legend Lauricidin alone =——=— —= Lauricidin + EDTA 180 Armillariella mellea 0 10 100 1000 Weight of mycelium and rhizomorph (mg) Level of Lauricidin (p/m) Figure 2.--Effect of Lauricidin and EDTA on growth of Armil- lariella mellea. Means differ significantly at the 95-percent conf i- dence level with the Tukey test. These data indicate that EDTA enhances the antifungal activity of Lauricidin on C. puteana, E. tinctorium, F. pinicola, F. officinalis and Perenniporia subacida, as was shown for Phellinus weirii and Heterobasidion annosum in earlier studies (Li and Kabara 1978). EDTA either inhibited or reduced fungal growth. The mechanism by which these compounds inhibit growth of fungi is unknown. Shibasaki and Kato (1978) reported that bacte- rial cells treated with EDTA released lLipopolysaccharides from the outer cell membrane, allowing monolaurin to penetrate easily into the inner membrane, a primary site for its antibacterial action. Some wood-destroying fungi were reportedly inhibited by metal- complexing agents, such as EDTA, because essential elements are not available for metal-requiring fungal enzymes (Highley 1975, Mandels and Reese 1963). Bohne (1973) reported that chelation of metal elements by EDTA can inhibit deoxyribonucleic acid synthesis. Metric Equivalents Literature Cited Our study indicates that growth of most test fungi was reduced Significantly, and four were completely inhibited by one or more of the treatments. Two important invaders of tree wounds, G. applanatum and F. pinicola, are the least affected by Lauri- cidin and EDTA. Their growth was unaffected or only delayed. Malt agar is an excellent growth medium for these fungi, how- ever, and the size and type of inoculum used is probably more effective than that found under natural conditions. Lauricidin and EDTA show promise for interfering with the growth of decay fungi. These compounds may also effectively inhibit germination of basidiospores on the surface of wounds (Nelson and Li 1980). Further testing in combination with other chemi- cals or at higher concentrations is necessary, however, to es-— tablish these compounds as attractive alternatives to the use of petrochemicals in forest or urban environments. Field tests on wounds are also needed to establish what works in practice. 1 millimeter (mm) = 0.039 inch 1 millimeter (mm) = 0.001056 quart (U.S. liquid) Aho, Paul E.; Fiddler, G.; Srago, M. Logging damage in thinned, young-growth true fir stands in California and recommendations for prevention. Res. Pap. PNW-304, Portland, OR: U.S. Depart- ment of Agriculture, Forest Service, Pacific Northwest Forest and Range Experiment Station; 1983. 8 p. Bohne, F. Metabolism and toxicity of therapeutic chelating agents: II. Effect on DNA synthesis in intestinal crypt cells of the rat. Biol. Abstr. 56: 4088; 1973. Highley, T. L. Inhibition of cellulases of wood decay fungi. Res. Pap. FPL-247, Madison, WL: U.S. Department of Agricul- ture, Forest Service, Forest Products Laboratory; 1975. 8 p. Kabara, J. J.; Lynch, P.; Krohn, K.; Schemmel, R. The anti- cariogenic activity of a food-grade lipid — Lauricidin.: In: Kabara, J. J., ed. Symposium on the pharmacological effect of lipids. Champaign, IL: The American Oil Chemists’ Society; O73 Chapter ses pen Zo- S60 binGey a sKkabara due st hects Of Laurieidin on Fomes annosus and Phellinus weirii. In: Kabara, J.J., ed. Symposium on the pharmological effect of lipids. Champaign, IL: The American Vil Chemists’ Society; 1978: chapter 5. p. 45-49 Mandels, M.; Reese, E. T. Inhibition of celluloses and B-glucosidases. In: Reese, E.T., ed. Advances in enzymic hydrolysis of cellulose and related materials. New York: Pergamon Press; 1963: 115-157. Nelson, E. E.; Li, C. Y. Preliminary test of two stump surface protectants against Fomes annosus. Res. Note PNW-363, Portland, OR: U.S. Department of Agriculture, Forest Service, Pacific Northwest Forest and Range Experiment Station; 1980. () fee Shibasaki, I.; Kato, N. Combined effects on antibacterial activity of fatty acids and their esters against gram- negative bacteria. In: Kabara, J.J. ed. Symposium on the pharmacological effects of lipids. Champaign, IL: The Amer- ican Oil Chemists' Society, 1978: chapter 2. p. 15-24. Shigo, A. L.; Wilson, C. L. Are tree wound dressings beneficial? Arborist's News 36: 85-88; 1971. Shigo, A. L.; Wilson, C. L. Wound dressings on red maple and American elm: effectiveness after five years. J. Arboric. Si eOl— 87/2) 19) 7.- The Forest Service of the U.S. Department of Agriculture is dedicated to the principle of multiple use management of the Nation’s forest resources for sustained yields of wood, water, forage, wildlife, and recreation. Through forestry research, cooperation with the States and private forest owners, and management of the National Forests and National Grasslands, it strives — as directed by Congress — to provide increasingly greater service to a growing Nation. The U.S. Department of Agriculture is an Equal Opportunity Employer. Applicants for all Department programs will be given equal consideration without regard to age, race, color, sex, religion, or national origin. Pacific Northwest Forest and Range Experiment Station 319 SW. Pine St. PO. Box 3890 Portland, Oregon 97208