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1997

Ophiostoma clavigerum is the Mycangial Fungus of the Jeffrey Pine , jeffreyi

Diana Six University of Montana - Missoula, [email protected]

T. D. Paine

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Recommended Citation Six, Diana and Paine, T. D., "Ophiostoma clavigerum is the Mycangial Fungus of the Jeffrey Pine Beetle, Dendroctonus jeffreyi" (1997). Ecosystem and Conservation Sciences Faculty Publications. 38. https://scholarworks.umt.edu/decs_pubs/38

This Article is brought to you for free and open access by the Ecosystem and Conservation Sciences at ScholarWorks at University of Montana. It has been accepted for inclusion in Ecosystem and Conservation Sciences Faculty Publications by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. Mycologia,89(6), 1997, pp. 858-866. © 1997 by The New YorkBotanical Garden, Bronx,NY 10458-5126

Ophiostomaclavigerum is the mycangialfungus of the Jeffreypine beetle, Dendroctonusjeffreyi

Diana L. Six1 Stock, 1982; Wood, 1982). The are sympatric T. D. Paine over portionsof theirrange (Higby and Stock, 1982; Departmentof Entomology, University of California, Wood, 1982). Genetic evidence suggeststhat the Jef- Riverside,California 92521 freypine beetle and the mountain pine beetle have divergedrelatively recently from a common ancestor (Higby and Stock, 1982). Physiologicaltolerances of Abstract: Dendroctonusjeffreyi and D. ponderosaeare the twobeetles to resinchemical constituentsof their sibling species of bark beetles (Coleoptera: Scolyti- respectivehost tree species also support the desig- dae) withfew morphologicaland molecular genetic nation of these two beetles as separate species differences.The two species are believed to have di- (Smith,1963; 1965; Lanier and Wood, 1968). verged relativelyrecently. Dendroctonus jeffreyi colo- In both species, adults of both sexes carrysymbi- D. nizes onlyPinus jeffreyi,while ponderosaecolonizes otic fungiin mycangialocated on the maxillarycar- Pinus but not P. Adult up to thirteen spp., jeffreyi. dines (Whitneyand Farris, 1970; T. D. P., personal beetles of both D. and D. jeffreyi ponderosaecarry sym- observation). Mycangiaare structuresof the integu- biotic in located on the car- fungi mycangia maxillary ment specialized for carryingfungi (Batra, 1963; dines. Dendroctonus was known to ponderosae carry Francke-Grosmann,1967; Barras and Perry, 1971; two and 0. montium,in fungi,Ophiostoma clavigerum Happ et al., 1971). Although bark beetles and the its However,it was not knownwhich mycangia. fungi related ambrosia beetles are frequentlyassociated be carried D. were isolated might by jeffreyi.Fungi with a range of fungipassively disseminated on the from the of over 900 D. jeffreyicollected mycangia exoskeleton, more specific associations are main- froma large portion of its geographic range. Using tained between mycangia and the fungi associated and rates morphology,isozyme phenotypes, growth withthem. at differenttemperatures, all isolates fromD. jeffreyi The relationshipbetween bark beetles and mycan- mycangiawere determined to be 0. clavigerum;0. gial fungi has long been assumed to be mutualistic montiumwas neverisolated fromD. jeffreyimycangia. due to the presence of mycangiaand the prevalence Key Words: Ophiostomamontium, Dendroctonus of these associationsin Scolytidae.The fungusclearly ponderosae,bark beetles, mycangia,isozymes, Cole- benefitsthrough consistent transport to an ephem- optera, Scolytidae eral and relativelyuncommon resource,the phloem of dyingor newly-killedtrees. The benefit(s) gained by the beetle are less clear.It has been suggestedthat INTRODUCTION mycangialfungi may reduce the impactof antagonis- The Jeffreypine beetle, Dendroctonusjeffreyi Hop- tic blue-stainingfungi (Barras, 1970; Franklin,1970; kins, is a exclusivelyattacking Pinus jef- Whitney,1971; Whitneyand Cobb, 1972, but see Ross et assistthe beetles in the de- freyiGrev. & Balf (Wood, 1982). Its geographicrange al., 1992), overcoming is roughlythat of its host tree, extending from the fenses of the host tree (Berryman,1972), alter the southern Oregon/northern California border to chemical or moisture composition of the phloem northernBaja California (Wood, 1982). The moun- (Nelson, 1934; Reid, 1961; Webb and Franklin,1978; tain pine beetle, Dendroctonus ponderosae, a poly- Wagner et al. 1979), or provide nutrientsor some phagous species utilizingup to thirteenPinus spp. other factor essential for the 's development but not P jeffreyi,and D. jeffreyiare considered sib- and reproduction(Barras, 1973; Bridges,1983; Gold- ling species due to their almost-identicalmorpholo- hammeret al., 1990). gies and indistinguishablegallery systems (Higby and Dendroctonusponderosae is knownto carrytwo my- cangial fungi, Ophiostomaclavigerum (Robinson-Jef- Accepted for publicationJuly 14, 1997. frey& Davidson) Harringtonand 0. montium(Rum- 1 Correspondingauthor, present address: Universityof California, bold) von Arx (Whitneyand Farris,1970). The my- of EnvironmentalScience and Department Policy Management, not been Center for Biological Control, 201 Wellman Hall, Berkeley,CA cangial fungus carried by D. jeffreyihas 94720-3112.Email: sixstine(@earthlink.net identifiedpreviously, but a morphologicalsimilarity

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This content downloaded from 150.131.192.151 on Mon, 3 Mar 2014 18:09:39 PM All use subject to JSTOR Terms and Conditions SIX AND PAINE: O. CLAVIGERUMIN D. JEFFREYIMYCANGIA 859 to 0. clavigerumhas been noted (Paine and Hanlon, collected from bark and from pheromone traps in 1994). Significantdifferences in tolerancesto various SawtoothNational RecreationArea, Idaho (Barbara host tree resin components were found between 0. Bentz, USDA Forest Service, Logan, Utah). Brood clavigerumcarried by D. ponderosaeand the morpho- adults,just prior to emergence,were collected from logicallysimilar fungus carried by D. jeffreyi(Paine the bark of infestedtrees by removingbark fromthe and Hanlon, 1994). The chemical environmentspre- entirecircumference of the trunkof treesfrom near sented by the host treesof the twobeetles differsub- soil level to a heightof ca. 2 m. Beetleswere collected stantially,and the fungi may have been selected for fromas manybroods and treesas possible per site in differenttolerances. Pinus jeffreyi,has n-heptane as order to gain a representativesample of mycangial its main resin component, while hosts of D. ponde- fungi.Only at one site were beetles taken froma sin- rosaehave monoterpenesand resinacids as major res- gle tree (Indiana Summit). Live beetles were held in components (Mirov,1929; Smith,1967; Anderson individuallyin microcentrifugetubes on ice and et al., 1969). transportedto the laboratory. Fungi, in general, are morphologicallysimple or- In the laboratory,each beetle was decapitated.The ganisms,and as such, morphologicaldifferences may head was dissectedto remove the maxillarycardines, be slightto nonexistentbetween related species even which contained the fungus-bearingmycangia. The long afteran initialspeciation event occurs (Brasier, cardines were surfacesterilized for two min in mod- 1986). Although the chemical environmentsof the ifiedWhite's solution (Barras, 1970) and submerged mycangialfungi in the host tree differ,the physical into malt agar and Ophiostomaselective agar (malt environmentswithin the host tree's tissuesare simi- agar with100 ppm cycloheximideand 100 ppm strep- lar. Also, both fungi apparentlyremain dependent tomycin) (Harrington, 1981). Once the mycangial upon the beetles for dispersal. Therefore,selection fungibegan to grow out fromthe mycangia,a plug pressure for morphological change may not be of agar containingthe funguswas removedfrom the strong,and indeed, selectionmay act to conserveex- growingedge and placed onto 2% water agar to fa- tant structures(e.g., disseminatedpropa- cilitatehyphal tipping.Fungal myceliagrow sparsely gules) while at the same time actingupon physiolog- on wateragar allowinghyphal tip culturesto be made ical traitsthat affectsurvival and exploitationof the with relativeease. Aftera few days growthon water chemical environmentof the tree. agar, hyphal tips were taken from each subculture There were two objectivesfor this study.First, we and placed on malt agar. wished to identifythe fungusor fungicarried in the Additionalstrains of 0. clavigerum(TABLE I) were mycangiaof D. jeffreyi.Many species of Dendroctonus acquired froma vouchered collection (T. C. Harring- including D. ponderosae,are known to carry more ton, Departmentof Plant Pathology,Iowa State Uni- than one species of mycangialfungus (Whitneyand versity,Ames, IA). Farris, 1970; Barras and Perry,1972; Bridges, 1983; Paine and Birch, 1983). Therefore,although isola- Morphological comparisons.-Conidiophores were tions from several beetles from a single population taken from2-3-wk old culturesof all mycangialiso- yielded a fungusmorphologically similar to 0. clavi- lates, mounted on glass microscope slides in lacto- gerum(T. D. P., unpublished data), it was not known phenol, visuallyinspected througha compound mi- if thiswas the onlyfungus carried byD. jeffreyiacross croscope (200X), and scored forthe presence of the its entire geographic range. Second, we wished to large, clavate,septate conidia typicalof 0. clavigerum compare 0. clavigerum(carried by D. ponderosae) or the smallerunicellular, globular, truncate conidia withthe morphologicallysimilar fungus carried by D. of 0. montium.Ten strains each of 0. clavigerum jeffreyiusing morphology,isozymes, and temperature (from D. ponderosae) and the D. jeffreyimycangial tolerancesto determinewhether these fungal isolates funguswere used for comparisonsof morphological have remained conspecific or have diverged along characters.For each fungustype, at least one isolate withtheir host beetles. Isolates of 0. montiumisolat- was taken directlyfrom a beetle pupal chamber by ed fromD. ponderosaemycangia were also included removinga small clump of conidiophoreswith sterile in the studyto assess its relatednessto the other two forcepswhich were then placed onto Ophiostomase- mycangialfungi. lective media. Conidiophores from the same pupal chamber were also placed directlyon microscope slides,mounted in and used in MATERIALS AND METHODS lactophenol, morpho- metric comparisons. Nine additional mycangialiso- Collectionsand isolations.-Dendroctonusjeffreyiadults lates of each funguswere grown both on malt agar were collected at ten sites,and D. ponderosae,at two and phloem agar (autoclaved Jeffreypine phloem sites from California.Additional D. ponderosaewere chips in 2% agar). All isolatesused in morphological

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TABLEI. Host tree species, substrate,and location of origin of isolates of Ophiostomamontium, 0. clavigerumfrom D. ponderosae,and the D. jeffreyimycangial fungus used in isozymeelectrophoresis and in determiningradial growthrates at differenttemperatures Isolate Host tree Substrate Location

Ophiostomamontium 15 Pinus ponderosa Pupal chamber Poison Lake, Lassen Nat'l Forest,CA 49 P ponderosa Mycangium Poison Lake, Lassen Nat'l Forest,CA 55 P ponderosa Mycangium Poison Lake, Lassen Nat'l Forest,CA 57 P lambertiana Mycangium Bogard Buttes,Lassen Nat'l Forest,CA 69 P lambertiana Pupal chamber Bogard Buttes,Lassen Nat'l Forest,CA 84 P. lambertiana Mycangium Bogard Buttes,Lassen Nat'l Forest,CA 934 P contorta Mycangium SawtoothNat'l RecreationArea, ID 937 P contorta Mycangium SawtoothNat'l RecreationArea, ID 940 P contorta Mycangium SawtoothNat'l RecreationArea, ID 941 P contorta Mycangium SawtoothNat'l RecreationArea, ID 0. clavigerum 998 P contorta Pupal chamber Deadman Summit,Toiyabe Nat'l Forest, CA 999 P contorta Mycangium Deadman Summit,Toiyabe Nat'l Forest, CA 1049 P contorta Mycangium Truckee,Tahoe Nat'l Forest,CA C295a P contorta D. ponderosae Invermero,Br. Columbia, Canada C291 P contorta D. ponderosaegallery WestCastle, Br. Columbia, Canada C294 P contorta From tree Carbondale,Alberta, Canada C293 P contorta D. ponderosaegallery BallyRiver, Alberta, Canada C292 P contorta D. ponderosaegallery Carbondale, Alberta,Canada C132 Pinus sp. Assc'd D. ponderosae California C86 P contorta Unknown Teton Nat'l Forest,WY D. jeffreyimycangial fungus 35A P jeffreyi Mycangium Coon Creek, San BernardinoMtns, CA 39A P jeffreyi Mycangium Fish Creek, San BernardinoMtns, CA 71 P jeffreyi Pupal chamber Butte Creek, Lassen Nat'l Forest,CA 83 P jeffreyi Mycangium Cone Lake, Lassen Nat'l Forest,CA 840 P jeffreyi Mycangium SmokeyBear Flats,Toiyabe Nat'l Forest, CA 841 P jeffreyi Mycangium Deadman Summit,Toiyabe Nat'l Forest, CA 842 P jeffreyi Mycangium Bell Meadows, StanislausNat'l Forest,CA 844 P jeffreyi Mycangium MonitorPass, Toiyabe Nat'l Forest,CA 884 P jeffreyi Mycangium MonitorPass, Toiyabe Nat'l Forest,(A 885 P jeffreyi Mycangium GreenhornSummit, Sequoia Nat'l For- est, CA

a Isolates beginningwith C are fromthe culturecollection of T. C. Harrington.All othersare fromthe collectionof D. L. Six.

comparisons were grown in darkness at room tem- were inspected for color, shape, size, and presence perature (ca. 21 C). The origin of each isolate used and number of septations. in morphological comparisons is listed in TABLE I. The same cultures used for morphological analysis, Isolates 884 and 35A have been deposited in the as well as ten cultures of 0. montium,were also grown American Type Culture Collection (Rockville, MD). on malt agar at 21 C in darkness and observed for Morphological characters of conidiophores pro- differences in cultural appearance, growth rate, and duced on artificial media as well as of those taken conidiophore production. from pupal chambers were measured or noted for Isozyme electrophoresis.-The isolates of 0. montium, each fungus. These characteristics were: color of co- 0. clavigerum, and the D. jeffreyimycangial fungus nidiophore, number of septations of conidiophore, used in morphological and/or cultural comparisons width at base and apex of conidiophore, number and were also used in horizontal starch gel electropho- width of primary metulae, length of conidiogenous resis to compare isozyme phenotypes of the three apparatus, and type of conidial proliferation. Conidia fungi.

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TABLEII. Enzymes,buffers, and stainingprocedures used in starchgel electrophoresisof Ophiostomamontium, 0. claviger- um, and the D. jeffreyimycangial fungus Enzyme (EC number)a Abbreviationb Buffersystems' Stain references' Aconitase (4.2.1.3) ACO Al 1 Aspartateaminotransferase (2.6.1.1) AAT D2 1 Glucose-6-phosphatedehydrogenase (1.1.1.49) G6PD A 1 Glucosephosphateisomerase (5.3.1.9) GPI D 1 Isocitratedehydrogenase (1.1.1.42) IDH E, 1 Leucine aminopeptidase (3.4.11.1) LAP1,2 M4 2 Malate dehydrogenase(1.1.1.37) MDH1,2 D, E 2 Menadione reductase (1.6.99.2) MNR1,2,3 A 1 Peptidase (3.4.13.1) PEP A 3t aCode of the NomenclatureCommittee of the InternationalUnion of Biochemistry(1984). b Multipleenzymes designated in order of decreasinganodal migration. ' A = DiscontinuousTRIS citrate/lithiumborate system(Marty et al., 1984). D = Continuous morpholinecitrate system (Conkle et al., 1982). E = D withpH 8.1 adjusted withmorpholine citrate (Conkle unpubl. in Zambino and Harrington, 1989). M = Continuous TRIS borate EDTA system(Micales et al., 1986). d 1 = Conkle et al. (1982). 2 = Martyet al. (1984). " Filterpaper overlaymethod (Vallejos, 1983).

Myceliumfor enzyme extractionwas obtained by lated onto plates on the same day and then grown placing agar plugs taken from growingcultures of and measured over the same 10-d period. each isolate in 30 mL liquid medium (20 mg malt Productionof the 0. extract,1 mg yeast extract/mL) in 125 mL Erlen- teleomorph.-While clavigerum and 0. montiumare knownto a sexual meyerflasks for 14 d at 21 C (Zambino and Harring- produce stage, the of the D. ton, 1992). Enzymeswere extractedby vacuum dry- teleomorph jeffreyimycangial fungus has not been observed. Production of the teleo- ing the fungal mats, grindingthe mats in liquid ni- was several trogento a fine powder,and then grindingthe pow- morph attempted by pairing sympatric and isolates from D. on der in extractionbuffer (Zambino and Harrington, allopatric jeffreyimycangia slants of were 1989). The resultingliquid extractwas absorbed onto phloem agar. Twenty-sixpairs placed in darkness at room (ca. 21 C) and 4 mm #1Whatman filterpaper wicks (WhatmanIn- temperature checked over 3 mo for the ternational,Ltd., Kent, England) and loaded onto periodically production of ascomata. 10% horizontal starch gels (Sigma starch, Sigma Chemical Co., St. Louis, MO) along with reference Statisticalanalyses.-Morphological charactersof the wicks at two Reference wicks were made positions. 0. clavigerumand D. jeffreyianamorphs were com- fromhomogenates of a singleisolate of 0. clavigerum pared using t-tests.Growth rates among 0. montium, isolated froma mycangiumof an individualD. jeffreyi 0. clavigerum,and the D. jeffreyimycangial fungus at collected at Deadman Summit,Toiyabe National For- fivetemperatures were compared using one-wayanal- est, Mono Co., California.Following electrophoresis, ysis of variance (ANOVA). Mean separations were gels were sliced horizontallyand stained for enzyme done using Sheffe'sF-test. All statisticalprocedures activity.Buffer systems and stainingprocedure refer- were conducted using StatView statisticalsoftware ences are listed in TABLE II. Nine enzymesexhibiting (Abacus Concepts, 1991). 13 putativegene loci thatresolved well were selected for use (TABLE II). Differentelectromorphs were as- sumed to be the resultof differentalleles. RESULTS Growthrates at differenttemperatures.-Growth rates Morphologicalcomparisons.-Both 0. clavigerumand of the three fungiwere assessed using the same iso- the 938 isolatesfrom D. jeffreyimycangial fungus pro- lates used in electrophoresis.Isolates were inoculated duced two distinctconidial types;small obovate, cy- centrallyin petridishes containingmalt agar using 4 lindrical to ellipsoidal, amerospores when cultures mm plugs of agar colonized by activelygrowing cul- were young,and large septateor aseptate broadlyfu- tures. One replicate plate of each isolate was placed siformclavate conidia typicalof 0. clavigerumwhen in the dark at each of five temperatures(5, 16, 21, cultureswere at least 2 wk old, as well as manyinter- 27, and 32 C). Colony diameterswere measured at mediate forms.Ophiostoma montium was neverisolat- 2-, 4-, 6-, and 10-d intervals.All isolates were inocu- ed from D. jeffreyimycangia. Conidiophores taken

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TABLEIII. Morphometriccomparisons [N = 50, mean ,um (SD)] of Ophiostomaclavigerum and the Dendroctonusjeffreyi mycangialfungus Range fromdescriptions of D. jeffreyi 0. clavigerumQ Character 0. clavigerum mycangialfungus A B Length of conidophore incl. conidiogenous apparatus 337.6 (86.8) 620.0 (273.9)b up to 1040 100-300 Width of stipe (base of conidiogenous apparatus) 8.2 (1.6) 7.9 (1.8) 6.0-10.5 Widthof stipe (at site of attachmentto stolon) 7.4 (1.6) 7.5 (1.6) 6.0-7.5 5-12 Number of septationsof stipe 4.5 (2.1) 7.5 (3.2) 3-5 Number of primarymetulae 2.3 (0.5) 2.1 (0.3) 2-3 Widthof primarymetulae 5.6 (1.3) 5.8 (1.1) 4-6 Widthof small conidia 3.0 (0.7) 3.2 (1.0) up to 3.8 1-2.5 Length of small conidia 7.8 (2.8) 7.8 (3.1) up to 12.6 2-4 Widthof large clavate conidia 3.9 (0.4) 4.4 (0.9)b 4.2-5.6 4-6 Length of large clavate conidia 32.5 (12.1) 33.4 (10.9) 35-68 12.5-62.5 Number of septationsof large clavate conidia 1.3 (1.5) 0.84 (1.21) 0-7 a Column A fromRobinson-Jeffrey and Davidson (1968); column B fromUpadhyay (1981). b Size significantlydifferent (P < 0.05), t-test. from pupal chambers exhibited both the monone- gerumassociated with D. ponderosae(Robinson-Jeffrey matous Leptographiumand synnematousGraphium and Davidson, 1968; Upadhyay,1981) (TABLE III). anamorphs, however, Graphium anamorphs were Cultural characteristics.-Cultural of 0. rare. Only mononematous formswere produced in appearances and the D. were culture. Conidiophores removed frompupal cham- clavigerum jeffreyimycangial fungus similar.Both were at first but soon bers and those fromcultures grown on phloem agar very fungi hyaline ca. 4 became to dark brownand then were more complex than those produced on malt (after d) gray black. When held at 21 both to agar. The use of artificialmedia and repeated sub- finally C, fungigrew cover dishes X 15 within 7-9 d. culturinghas been found to quickly decrease the petri (80 mm) montiumcultures were more complexityof the anamorphs of 0. clavigerum(Tsu- Ophiostoma brownish, slower to darken ca. 6 and co- neda and Hiratsuka,1984). Therefore,isolates grown (after d), produced aerial over on phloem agar were used for morphologicalanaly- pious whitish-gray,sterile, felty, mycelium ses. dark, closely appressed, or submersed mycelium. montiumwas also slow to Morphological comparisons of the two fungi are Ophiostoma very produce in culture while 0. summarizedin TABLE III. Conidiophores of both fun- conidiophores (2-4 wk) claviger- um and the of D. gi were similarin appearance and in numberand size mycangialfungus jeffreyiproduced within a few Conid- of primarymetulae. Both fungiproduced brownco- profuseconidiophores just days. ial masses on cultureswere to nidiophoreswhich graded into hyalinemetulae and yellowish whitish-gray and conspicuous for all three fungi. conidiogenous cells and produced conidia by anne- lidic Some to proliferation. conidiophores appeared Productionof the teleomorph.-Nopaired culturesof as produce sympodulateconidia, however, only light the D. jeffreyimycangial fungus placed on phloem was this could not be determined microscopy used, agar slantsproduced ascomata. with certainty.The t-testscomparing measurements of conidia and conidiophores of the two fungi Isozymeelectrophoresis. -Most of the thirteenloci as- showed no significantdifferences between the fungi sayed resolvedwell and were easilyinterpreted. Poly- for most characteristics;however, length of conidio- morphismwas very low in the fungi. Of the twelve phore (including the conidiogenous apparatus), loci assayed,only MDH2 and PEP were polymorphic number of septationsof the stipe,and widthof large in 0. clavigerumand the D. jeffreyimycangial fungus, conidia were significantlydifferent. The significant and AAT, IDH, and PEP in 0. montium(TABIE IV). differencein number of conidiophore septationsis Ophiostomaclavigerum and the mycangialfungus of due to the greater length of the D. jeffreyifungus' D. jeffreyishared alleles at all loci assayed and could conidiophores. Despite differencesfound in these not be distinguishedwith the enzyme systemsused comparisons,all measurements,including width of in this study.Ophiostoma montium, however, shared conidia and length of conidiophore for both fungi, alleles with 0. clavigerum and the mycangialfungus agreed closely with formal descriptionsof 0. clavi- of D. jeffreyiat only six of thirteenloci (TABt E IV).

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25 - TABLE IV. Isozymephenotypes of Ophiostomamontium, 0. A * 0. clavigerum clavigerum,and the D. jeffreyimycangial fungus 20- E Jeffrey pine beetle mycangial fungus O 0. montium 1 5 - D. jeffreyi -r -r mycangial 10 - Locus 0. montium 0. clavigerum fungus 5 - AAT B,C,Da A A 0- ACO A B B 5 16 21 27 32 G-6PD A A A 35 - B GPI A B B 30 - IDH B,C A A - IT LAP1 A A A 25 T LAP2 A - - 20- MDH1 A B B E E 1 5- MDH2 A B,C B,C 1 0- MNR1 A A A 0 MNR2 A A A co 5 - A A A MNR3 0- 21 27 I 32 PEP A,B,C A,B,C A,B,C 5 16 21 27 32 45 aElectromorphs are designated alphabeticallywith the most anodally migratingelectromorph designated A for 40 each enzyme. 35

_ 30 RX/'l While 0. clavigerum and the D. jeffreyimycangial 25 exhibited one band for 0. montium fungus only LAP, 20 . B exhibited two bands. Because hyphal tip cultures of 1 5 haploid mycelia of the fungi were used in all prepa- rations, the two bands observed for LAP in 0. mon- 1 0 'Sk tium could not be due to the presence of heterozy- 5 gotes. Therefore, the two bands are believed to be products of two loci, with one locus not resolving for 5 16 21 27 32 0. clavigerum and the D. jeffreyimycangial fungus, Temperature perhaps due to fixation for a null allele. All three fungi initially appeared to be monomor- FIG. 1. Radial growth (mm) of Ophiostomaclavigerum associated with Dendroctonus 0. as- phic at the MDH loci. Differences were revealed ponderosae, clavigerum sociated with D. and 0. montiumat 5, 16, 21, 27, when different buffer systems were used. With the D jeffreyi, and 32 C, A. After2 days. B. After4 days. C. After6 days. buffer system (TABLE II), a cathodally migrating elec- At 45 mm,isolates had filledthe plates and no furthermea- for MDH2 to be monomorphic tromorph appeared surementswere possible. and present in all 0. montium isolates as well as pres- ent in three D. jeffreyimycangial fungus isolates. How- the sixth the D. mycangial fungus grew ever, when the E buffer system (TABLE II) was used, day, jeffreyi faster at 27 C than did 0. At the enzyme bands of the D. jeffreyimycangial fungus significantly clavigerum. the lower tested (5, 16, and 21 C), few and 0. clavigerum separated and migrated different temperatures differences in rate among the three fungi oc- distances anodally than did the bands resolving for growth curred. The most differences in growth rate 0. montium. With MDH1, a similar phenomenon was striking occurred at 32 C where 0. and the D. observed. Using the E buffer, this locus appeared clavigerum only poorly or not at monomorphic for all three fungi. However, using the jeffreyimycangial fungus grew all, while 0. montium almost as well as at 27 C. D buffer system, two electromorphs were revealed; grew and the D. jeffreyimycangial one for 0. clavigerum and the D. jeffreyimycangial Ophiostoma clavigerum rates were also significantlydepressed fungus, and one for 0. montium. fungus growth below those of 0. montium at 27 C. Growth rates at differenttemperatures.-Growth rates for the three fungi held at five temperatures are sum- DISCUSSION marized in FIG. 1. Growth rates for 0. clavigerum and the D. jeffreyimycangial fungus were similar at most Our results indicate that the mycangial fungus of D. temperatures tested over the 6-d period, except on jeffreyiis 0. clavigerum. Because the sibling species of

This content downloaded from 150.131.192.151 on Mon, 3 Mar 2014 18:09:39 PM All use subject to JSTOR Terms and Conditions 864 MYCOIOCGIA this beetle also carries 0. clavigerum,this indicates as the ascomata of Ophiostomaspp. are small, they that the fungidid not divergeas the host beetles di- may have been overlookedin naturalsituations. verged from a common ancestor and began to ex- The fungiin thisstudy all exhibiteda high degree ploit differenthost trees. The differencesin resin of monomorphism.Low levels of polymorphismcan component tolerances of the two fungimay be due indicate that sexual recombinationis not common to relativelysmall changes in the genome (at even a (Zambino and Harrington,1989; Zambino and Har- single locus) which allow differentphysiological re- rington,1992; Harringtonet al., 1996). It is possible sponses to host tree chemicals,but that are not de- that D. jeffreyiis associated with an asexual lineage tectableusing isozymes,which mostlyresolve neutral that originatedfrom the isolation of a single mating variationin the genome and not variationdue to se- typewith this beetle. Further,it is the asexual state lection. AlthoughPaine and Hanlon (1994) demon- of 0. clavigerumthat is presentin the pupal chamber, stratedthat there were differentphysiological toler- acquired in the mycangium,and transmittedto new ances to host resin constituentsbetween 0. claviger- host trees.If a sexual state is produced, it may form um and the mycangialfungus of D. jeffreyi,only one distantfrom the developingbrood (e.g., in old adult isolate of each funguswas used in the trials.It is pos- galleries) or afterthe beetles have dispersed. Thus, sible thatthere is wide variationin tolerancesamong if a sexual state is formed,it may actuallybe incon- isolatesfrom many populations that was not captured sequential in the life cycle of the fungusrelative to in thatstudy. Although low levels of genetic diversity the primarybeetle vector.If thisis the case, the asex- were observed among strainsin the isozyme assays ual cycle would predominate and a high degree of presented here, the question of variation among monomorphismwould be expected. strainsin physiologicalresponse to host characters remainsunresolved. ACKNOWLEDGMENTS While the fungiappear to be isolated in different host trees by the behavior of theirbeetle hosts,this We thank Barbara Bentz of the USDA Forest Service, Lo- may not actuallybe the case. Ophiostomaspp. pro- gan, Utah, forproviding the D. ponderosaefrom Idaho, and duce propaguleswell-adapted for arthropod dispersal T. C. Harrington,Department of Plant Pathology,Iowa (Malloch and Blackwell,1993). Numerous other in- State University,Ames, Iowa, for providingcultures of 0. sects, including predators, secondary bark beetles, clavigerum.Helpful reviewsof the manuscriptwere provid- and saprophagous species,also inhabittrees killed by ed by J. Daniel Hare and John Trumble, Departmentof of Riverside. these primarybark beetles. These other subcortical Entomology,University California, Funding was in W-187. coinhabitantsmay occasionally transportconidia or provided, part,by Regional Project ascospores among the host treesof these twobeetles where theyare sympatric.Indeed, withsome mycan- LITERATURECITED gial fungi,it has been shown thatwhile beetles carry the asexual formof the fungus,the sexual ascospores Abacus Concepts. 1991. StatViewstatistical software. Aba- maybe carriedby phoretic mites (Moser et al., 1995). cus Concepts,Inc. Berkeley,California Anderson,A. B., R. Rirrerand A. Wong. 1969. 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