Armillaria Nabsnona, a New Species from Western North America
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Mycologia. 88(3), 1996, pp. 484-491. Armillaria nabsnona, a new species from western North America Thomas J. Volk1 pleiomorphic species with a wide host range and dis- Harold H. Burdsall, Jr. tribution. Herink (1973), among others, suspected Mark T. Banik that this single species might actually be a species Center for Forest Mycology Research, Forest Products complex. However, since basidioma morphology is Laboratory, Forest Service, United States Department of difficult to study because of overlapping and appar- Agriculture, One Gifford Pinchot Dr., Madison, 2 ently inconsistent traditional characters, other ave- Wisconsin 53705-2398 nues of research were pursued. Hintikka (1973) de- veloped a technique that allowed determination of Abstract: Armillaria nabsnona is characterized mor- mating types in Armillaria. Using a modification of phologically and described as a new species. It is com- this method, Korhonen (1978) was able to distin- patible with tester strains of North American biolog- guish five European biological species (EBS). The ical species (NABS) IX. Using restriction fragment technique depended on growing single spore isolates length polymorphism (RFLP) of the polymerase together in a petri dish and observing the change or chain reaction (PCR) products amplified from the lack of change in colony morphology. Single spore intergenic spacer (IGS) region of the ribosomal isolates of Armillaria species are generally white and DNA, we also demonstrate an association of cultures fluffy, but when fusion of compatible mating types of specimens with tester strains of NABS IX. Armil- occurs, the coalesced colonies become dark brown, laria nabsnona appears to be restricted to several spe- appressed, crustose, and sometimes produce rhizo- cies of hardwood trees in the states of Washington, morphs, depending on nutritional and cultural con- Oregon, California, Idaho and Alaska, and in the Ca- ditions. If the single spore isolates are from different nadian province of British Columbia. Basidiomata species, the colonies will not grow together and will have been found in both the fall and spring. remain white and fluffy (Anderson and Ullrich, Key Words: Agaricales, Basidiomycotina, NABS 1982). The five EBS have been characterized mor- IX, biological species, systematic, Tricholomataceae. phologically, and appropriate names have been given (see Watling, Kile, and Burdsall, 1991). Anderson and Ullrich (1979) applied the tech- INTRODUCTION niques used by Korhonen to isolates collected from widely distributed locations in North America and The genus Armillaria (Fr.: Fr.) Staude has undergone demonstrated that what had been considered as Ar- significant revision in the past twenty years. The ge- millaria mellea in North America was actually 10 dis- nus once accommodated any white-spored agaric tinct biological species (North American biological with broadly attached gills and an annulus (Volk & species or NABS), some of which are compatible with Burdsall, 1995). With Agaricus melleus Vahl: Fr. now EBS (Anderson et al., 1980; Anderson, 1986). Anoth- accepted as the type species (Donk, 1962; Watling, er, apparently rare, biological species, NABS XI, was Kile, and Gregory, 1982), Armillaria has a much nar- recognized by Morrison et al. (1985). Three of the rower circumscription, including only those white- northeastern-occurring NABS were described and spored wood-inhabiting agarics with broadly attached given names by Bérubé and Dessureault (1988, to decurrent gills and forming macroscopic black to 1989). A tentative key to macromorphological iden- reddish-brown rhizomorphs. tification of Armillaria species has been published Until the late 1970s Armillaria mellea (Vahl: Fr.) (Burdsall & Volk, 1993). For additional nomenclatur- Kummer was considered by most researchers to be a al details see Volk and Burdsall (1995). Accepted for publication January 2, 1996. The cumbersome nature of the mating type meth- 1 Current email addresses [email protected], burdsall@ od of species identification has prompted a search facstaff.wisc.edu,/s=m.banik/[email protected] for other techniques for identifying collections. Sev- 2 The Forest Products Lab is maintained in cooperation with the eral methods have been employed with varying de- University of Wisconsin-Madison. This publication was written and prepared by U.S. government employees on official time, and it is grees of success including isozyme analysis (Morrison therefore in the public domain and not subject to copyright. et al., 1985; Bérubé, 1994), cultural characteristics 484 VoLK ET AL.: ARMILLARIA NABSNONA 485 (Rishbeth, 1986), immunology (Burdsall and Banik, from various areas on the Olympic Peninsula of 1990) and molecular biological techniques (Harring- Washington, USA, in October 1993. Compatibility ton and Wingfield, 1995). Of these, molecular bio- with tester strains was determined according to the logical techniques seem to hold the most promise in methods of Hintikka (1973), Korhonen (1978) and terms of quick, accurate diagnosis from a variety of Anderson and Ullrich (1979) using second genera- test materials. Barrington and Wingfield (1995) re- tion tester strains that we developed by testing against cently reported a technique involving restriction frag- the original bank of tester strains of Anderson and ment length polymorphisms (RFLPs) of the riboso- Ullrich (1979). Specific strain information is avail- mal DNA intergenic spacer (IGS) region amplified able from the authors upon request. To determine by the polymerase chain reaction (PCR). They were sexuality of this species, pairwise crosses of ten single able to separate all North American species of Ar- basidiospore isolates from TJV-93-188 were done on millaria except for A. calvescens and A. gallica, which 1.5 % malt extract, 2 % agar medium (MEA). All cul- are apparently very closely related (Anderson and tures were incubated at 24 C. Stasovski, 1992). In Barrington & Wingfield (1995), Basidiomata were examined microscopically with digestion of the IGS region of NABS IX with the re- phloxine in 3 % KOH or in Melzer’s solution. Colors striction endonuclease Alu I yielded a unique band- in quotation marks are from Ridgway (1912). All ing pattern that distinguished it from the other spe- specimens and cultures (single spore and tissue iso- cies. This technique may prove invaluable for iden- lates) are deposited in the Center for Forest Mycol- tifying collections of this and other species in the fu- ogy Research (CFMR) Madison, Wisconsin, USA, ex- ture, and thus we examined it in conjunction with cept where noted. Other herbarium abbreviations the formal description of this species. are taken from Holmgren et al. (1990). In spite of being a known entity, NABS IX has re- mained morphologically undescribed and not validly Molecular studies.—Eighteen tissue isolates of NABS named. The species has been reported from Idaho IX and 2 single spore isolates of the original NABS (Anderson and Ullrich, 1979), California (Jacobs et IX testers (Anderson & Ullrich, 1979; TABLE I) were al., 1994), Alaska (Shaw & Loopstra, 1988), and Brit- analyzed for RFLPs in the IGS region using the tech- ish Columbia (Morrison et al., 1985). The species has nique of Barrington and Wingfield (1995). Fungal also been reported from Connecticut (Wargo, 1988), material for PCR was obtained from mycelial mats Newfoundland (Warren, 1994) and Japan (Moham- grown on cellophane overlaying enriched medium 2 med et al., 1994), but these identifications are incor- (EM, Larsen et al., 1992). Approximately 25 mm of rect, as noted later in this work. mycelial mat was ground with 500µL TE (10 mM The major difficulty in naming and describing Tris, pH 8, 1 mM EDTA) in a sterile 15 mL ground NABS IX has been finding specimens, and particu- glass tissue homogenizer and centrifuged at 14000g larly, an appropriate type collection, partially because for 10 min. The supernatant was diluted 1 to 100 in of the long lag period (up to two months) between TE and 5µL of this was used as template DNA in collection of the basidiocarps and the determination PCR. The remaining PCR reaction mixture is as fol- of the NABS by mating studies. To resolve this prob- lows; 1.25 units Taq polymerase (Promega, Madison, lem, methodical collection, culture, and molecular WI), reaction buffer supplied with the enzyme (final studies were initiated in the fall of 1993 using large concentrations 50 mM KCl, 12.5 mM Tris-HCl, pH-9, numbers of Armillaria basidiomata from the Olympic 0.1 % Triton X-100), 1.5 mM MgCl, 200 uM each Peninsula of Washington. Identification was con- dNTP, and 0.2 uM of each primer in a volume of firmed by compatibility with tester strains, and the 25µL. The primers used were LR-12R (5’ CTGA- morphological features of the basidiomata studied ACGCCTCTAAGTCAGAA 3’) and O-1 (5’ AGTCCT- and characterized. The literature was also searched ATGGCCGTGGAT 3’) (Operon Technologies, Ala- (Volk and Burdsall, 1995) to determine if there was meda, CA). The reaction mixture was overlain with an existing name for this fungus. Type specimens of a drop of mineral oil. Thermocyler (Perkin-Elmer) northern temperate Armillaria species were also ex- parameters were as follows: One cycle of 93 C for 3 amined. No appropriate name was found, so the fun- min, 53 C for 2 min and 72 C for 3 min followed by gus is described here as a new species. 29 cycles of 93 C for 1.5 min, 53 C for 2 min and 72 C for 3 min. This was followed by elongation at 72 C for 10 min. Following amplification, 3 µL of the un- MATERIALS AND METHODS purified reaction product was digested for 16 h at 37 Cultural and morphological studies. — Isolation of sin- C with 5 U Alu I (GIBCO BRL, Gaithersburg, Mary- gle basidiospores was made according to Darmono land) mixed with the appropriate amount of buffer and Burdsall (1992) from fresh basidiomata collected supplied with the enzyme in a total volume of 20 µL. 486 MYCOLOGIA The digestion products were separated on a 4 % aga- rose gel in tris-acetic acid-EDTA (TAE) buffer (pH 8) at 2.4 V/cm for 3-4 h.