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Purchased by USDA Forest Service for Official Use

Mycologia, 83(2), 1991, pp. 233-236.@ 1991, by The New York Botanical Garden, Bronx, NY 10458-5126

FATIY ACID ESTERASE PRODUCI10N BY ECfOMYCORRHIZAL FUNGI

B. A. CALDWELL.

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804

M. A. CASTELLANO

U.S. Department of Agriculture, Forest Service, Pacific Northwest Research Station,Forestry Sciences Laboratory, 3200 Jefferson Way, Corva/is, Oregon 97331

R. P. GRIFFITHS

AND

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804

Several reports have indicated the stimulatingeffect of fatty acid-containing materials on thegrowth of ectomycorrhizal fungi. Growth of dif-ferent ectomycorrhizal fungi can be stimulatedby lipids (Schisler and Volkoff, 1977; Fries et aI.,1985), Tween 80 (Straatsma and Bruinsma, 1986),or certain free fatty acids (Lindeberg and Lin-deberg, 1974). Palmer and Hacskaylo (1970) re-ported no growth stimulation using the syntheticlipid triacetin, but this may have been the resultof acetate inhibition (Lindeberg and Lindeberg,1974) or the inability to use acetate as a solecarbon source.

Fatty acid esters are cleaved by enzymes, in-cluding lipases (sensu stricto triacylglycerol acyl-hydrolase, E.C. 3.1.1.3), which can release freefatty acids from several sources, including lipids,phospholipids, sterol esters, waxes, cutin, andsuberin. Although lipolytic activity, frequentlyusing Tween substrates, has been studied in var-ious saprophytic fungi from diverse environ-ments (Das et al., 1979; Egger, 1986; Gessner,1980; Gochenaur, 1984; Zare-Maivan andShearer, 1988) and food materials (e.g., Robertset aI., 1987), few ectomycorrhizal fungi have beenexamined.

Fungal strains (TABLEI) were obtained fromthe USDA Forest Service, Pacific Northwest Re-search Station, Corvallis, Oregon, RWU-4503cuture collection, except for Hebeloma crustu/in-

I Corresponding author.

iforme which was provided by the USDA Agri-cultural Research Service, Horticultural CropsResearch Laboratory, Corvallis, Oregon. Allstrains were maintained on an ectomycorrhizalbasal medium (Pearson and Read, 1975) modi-fied to reduce phosphate levels (Giltrap and Lew-is, 1981) and increase Ca2+concentration (Sierra,1957). The medium consisted of 109 glucose,0.25 g ammonium tartrate, 0.4 g KH2P04, 0.5 gMgS04'7H20, 0.1 g CaCI2'2H20, 50 mg yeastextract, 5 mg ferric citrate, 5 mg MnS04'4H20,4 mg ZnS04. 7H20, and 15 g agar (per liter dis-tilled water). To detect fatty acid esterase pro-duction, a supplement of 1%(v/v) of either Tween20, 40, or 80 was added. The medium was ad-justed to pH 5.5 and autoclaved (121 C, 15 min).Plates were inoculated in duplicate or triplicatewith halves of a 5 mm plug cut from the marginof an actively growing culture. Control plates offungi grown on medium without Tween addedwere used to evaluate possible excretion of oxalicacid that might cause a false-positive reaction.Plates were incubated at room temperature forup to 60 days and periodically examined forgrowth and production of the characteristic haloresulting from the pr~cipitation offatty acid-cal-cium salts (Sierra, 1957).

Of 48 strains tested, 25 produced characteristiccalcium-fatty acid salt halos on one or more ofthe three Tween substrates tested (TABLEI), with17 detected on Tween 20, 25 on Tween 40, and18 on Tween 80. In several cases, Tween inhib-

ited growth from the fungus inoculum plug ontothe agar medium, although absence of growthdid not always correspond to lack of fatty acidesterase (e.g., Arcangeliella parva). Growth sup-pression and lower frequency of esterase detec-

tion using Tween 20 was probably caused by theincreased toxicity of either the Cl2 fatty acid orits Tween ester (Kabara, 1987). This suggests thatcaution be exercised when using this substratealone in screening fatty acid esterase production

234 MVCOLOGIA

TABLEIREAcnON ONTWEENESTERSBYECTOMYCORRHIZALFUNGI

Habi- Reaction on Tweensb

Taxon Strain tat" 20 40 80

A/pova o/ivaceotinetus(Smith) Trappe 8245 (-) (-)Areange/ie//aparva Thiers 9467 M (+) (+) +Austrogautieriasp. 7873 M (-) (+) (+)Byssoporiate"estris' Z30 W + + +Cenococcumgeophi/um Fr. A145AChondrogastersp. 9833 M + + +Cortinariusmagnive/atus (Morse)Thiers & Smith 9537 M + +E/aphomyces sp. 8646 (-)Endogone (immature) S566 (-) (-) (-)Gautieriaeaudata (Harlen.)Zeller & Dodge 8481 M + + +G. cf. graveo/ens 8724 M + + +G. montieo/a Harkness AG-7 M + + +Genabeaeerebriformis(Ha1en.)Trappe 6264 W (+) + +Hebe/oma erustu/iniforme(Bull.ex. St. Am.) Quel. HECR2 (-)Hydnangium carneum Wallroth 9831 (-)Hymenogaster a/bus Berk. & Br. 9832 (-) (+) (-)Hysterangium coriaceum Hesse AH-7 M + + +H. erassirhaehisZeller & Dodge AH-6 M + + +H. gardneriFischer 8405 M + + +H. inflatum Fischer 9787 M (-) + +Hysterangium sp. 4154 M + + +Laccaria bie%r (Maire) Pat. S238 (-)Leceinum seabrum (Bull.ex. Fr.) S. F. Gray 8967 (-) (+) (+)Leucophe/psspinisporaFogel 7435 (-) (-) (-)Me/anogastervariegatus(Vittadini) Tul. & Tul. 8222 (-) (-) (-)Mycoamaranthus auriobisCastellano, Trappe & Malajczuk 4045 M + +Paxillus invo/utus(Butsch: Fr.) Fr. 8969 (-)Pi/oderma eroceum Erikks. & Hjortst 163 M + + +Piso/ithus tinctorius (pers.) Coker & Couch S359Radiigera atrog/ebaZeller 9470 W + + +Rhizopogon c/avitisporusSmith 5412 (-) (-)R. e%ssus Smith S548 (-) (-)R. e//enaeSmith 8974R. evadensSmith 8972 (-)R. hawkeraeSmith A104 (-) (-)R. occident/aisZeller & Dodge 7544 -R. ochraceisporusSmith 9009 (-)R. ochraceorubensSmith 7555 (-)R. reaii Smith 8073R. vinie%r Smith 7534R. vulgaris(Vitt.) M. Lange A56Sarcodonseabrosus(Fr.) Bourd. & Galz. 8727 (-)Trieh%ma magnive/are(peck) Redhead 7088 - +Tuber aestivum Vittadini 491 (-) +T. borchiVittadini S211A (-) + (-)T. uncinatum Chatin 5491 - +

"Habitat: M-forms mat structures; W-associated with decomposingwood.b Reaction: (-) no growth, no esterase reaction; (+) no growth, but esterase positive reaction; - growth with

no esterase reaction; + growth with positive esterase reaction.

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BRIEF ARTICLES

(e.g., Hankin and Anagnostakis, 1975). Oxalateproduction (precipitation of calcium oxalatecrystals) was not detected on any of the controlplates.

Most positive reactions seemed limited to adistinct group of presumptive ectomycorrhiza1fungi that form extensive hyphal or rhizomorphmat structures in forest soils. Of particular in-terest were the results for those ectomycorrhizalfungi, Gautieria monticola, Hysterangium cori-aceum, and H. crassirhachis, that produce thesemat structures in the upper soil layers of Doug-las-fir forests in the Pacific Northwest. The matsformed by these fungi and H. setchellii, whichwas not available for inclusion in this survey, areloci of significantly altered microbial activitiesand soil chemistry (Cromack et al., 1979,1988;Griffiths et aI., 1987, 1990; Sollins et aI., 1981).Hysterangium gardneri forms mats in the litterlayer of Eucalyptus globulusplantations in north-ern California, and H. inj/atum forms mats inassociation with Eucalyptus in Australia. Isolatesof other mat-forming ectomycorrhizal fungistrongly positive for hydrolysis of the Tweenadditives included G. caudata, G. othii, Myco-amaranthus auriobis, Piloderma croceum, andunidentifiable species of Austrogautieria, Chon-drogaster, and Hysterangium. Three ectomycor-rhizal fungi frequently with decomposing wood,Byssoporia terrestris, Genabea cerebriformis andRadiigera atrogleba, also hydrolyzed all threeTween additives.

Some isolates, not associated with mat for-mation or decomposing wood, produced limitedreactions in the presence of Tween additives.These included taxa that are considered late suc-cessional fungi (Dighton and Mason, 1985), e.g.,Leccinum scabrum and Tricholoma species.Dighton and Mason (1985) have suggested suchfungi have higher energy demands than early suc-cessional fungi (e.g., Hebeloma crustuliniforme)and are able to use organic nutrient pools.

The capacity of ectomycorrhiza1 fungi to hy-drolyze fatty acid esters has several potentialbenefits. Assimilated fatty acids could be takenup by the fungus and incorporated into new fun-gal lipids or cleaved into acetate subunits byP-oxidation and used either for energy or bio-synthesis. Energy supplementation, in additionto that normally provided by the host tree, couldbe particularly important for those ectomycor-rhiza1 fungi that form extensive mat structureswith peripheral mycelia distant from the root-

235

fungus interface or for late successional fungi withhigh energy requirements (Dighton and Mason,1985). Alternatively, the acetate subunits or ace-tyl CoA could be directed through different bio-synthetic pathways (Garroway and Evans, 1984)to produce a wide variety of cellular materialsas well as secondary metabolites.

Fatty acid esterases may also influence themineral nutrition of those mat-forming ecto-mycorrhizal fungi in forests where accumulatingdetritus ties up increasing quantities of nitrogenand phosphorus in organic pools. While colo-nizing fresh litter, the initial capacity to penetrateor disrupt wax or suberin barriers would allowhyphal or enzymatic access to otherwise occlud-ed pools of organic nitrogen and phosphorus.

ACKNOWLEDGMENTS

This research was supported by a National ScienceFoundation grant to study the microbial ecology ofectomycorrhizal mat communities in forest ecosys-tems. We also acknowledge the technical assistance ofJane Smith, USDA Forest Service, Pacific NorthwestResearch Station.

Key Words: ectomycorrhizal fungi, fatty acids, ester-ase, lipase

UTERATURE CITED

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-, P. Sollins, W. C. Graustein, K. Speidel, A. W.Todd, G. Spycber,C. Y.Li,andR.L. Todd. 1979.Calciumoxalateaccumulation and soilweatheringin mats of the hypogeous fungus Hysterangiumcrassum. Soil Bioi. & Biochem. 11: 463-468.

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Digbton, J., and P. A. Mason. 1985. Mycorrhizaldynamics during forest tree development. pp. 117-139. In: Developmental biology of higher fungi.Eds., D. Moore, L. A. Casselton, D. A. Wood, andJ. C. Frankland. Cambridge Univesity Press,Cambridge.

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236 MYCOLOGIA

growth of ectomycorrhizal fungi in culture byphosphate. New Phytol 87: 669-675.

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