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The Pennsylvania State University, Department of Plant Pathology, 301 Buckhout Lab., University Park, Pennsylvania 16802
Elke Lieckfeldt 2
Humboldt-Universität zu Berlin, Institut für Biologie (Genetik), Chausseestr. 117, D-10115 Berlin, Germany
Gary J. Samuels 3
United States Department of Agriculture, Agricultural Research Service, Systematic Botany and Mycology Lab., Rm. 304, B-011A, BARC-W, Beltsville, Maryland, USA 20705
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ABSTRACT |
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A new species, Hypocrea atroviridis, is described for the teleomorph of Trichoderma atroviride. Based on sequences of ITS-1, 5.8S, and ITS-2 regions of the rDNA complex and translation-elongation factor (EF-1
), T. atroviride and H. atroviridis form a well-supported clade within Trichoderma sect. Trichoderma. The conserved anamorphic phenotype of T. atroviride, observed for both conidial and ascospore derived cultures, was only found within that clade. In contrast, the teleomorph phenotype of H. atroviridis was morphologically indistinguishable from H. rufa, the teleomorph of T. viride. This Hypocrea phenotype may, therefore, be considered to be plesiomorphic within Trichoderma sect. Trichoderma, suggesting that genes controlling the expression of the teleomorph and anamorph evolve at different rates and that the genes controlling expression of the teleomorph are more conserved than are those controlling the expression of the anamorph.
Key words: biological control, Hypocreales, ITS rDNA, systematics, translation-elongation factor EF-1
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INTRODUCTION |
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). Trichoderma atroviride has also demonstrated effective biological control activity against postharvest brown rot of stone fruits (Hong et al 1998), Rhizoctonia solani on potato in the field (McBeath et al 1995), and has provided good protection against Fusarium culmorum when applied as a treatment to wheat seed (Roberti et al 2000). More recently, this species demonstrated promise as a biological control agent against the chestnut blight pathogen, Cryphonectria parasitica, which has effectively brought the American chestnut to extinction (Terry A. Tattar website: http://www.bio.umass.edu/micro/chestnut.html). Biological control capability of T. atroviride is due in part to the production of endochitinase enzymes (Mach et al 1999, Kullnig et al 2000) and in part to the production of antifungal antibiotics, including aromatic pyrone antibiotics (Keszler et al 2000) and peptides (Oh et al 2000).
Trichoderma atroviride was first described by Karsten in 1892 from European material, but was subsequently overlooked in Rifai's (1969) monograph of Trichoderma. The species was later reintroduced and redescribed by Bissett (1992) and Samuels et al (2002). Gams and Meyer (1998) epitypified T. atroviride with a collection from Slovenia.
Trichoderma atroviride has been confused in the literature with the superficially similar species, T. harzianum Rifai, which also has smooth, globose to subglobose conidia. This is illustrated by the confusion over the four biotypes of "T. harzianum" associated with the green mold epidemic of commercially produced mushrooms (Muthumeenakshi et al 1994, 1998, Seaby 1996, 1998). Biotype ‘Th 3’ is now recognized to be T. atroviride (Dodd et al 2000), and is not a pathogen (Samuels et al 2002). Confusion over the identity of T. atroviride and T. harzianum is further exemplified by Hermosa et al (2000), who reevaluated biocontrol strains reported as T. harzianum and T. viride Pers. and found four groups, among which was T. atroviride; and by Kullnig et al (2001), who found T. atroviride among strains reported in the biocontrol literature as T. harzianum (ATCC 74058, IMI 206040, ATCC 36042).
Trichoderma atroviride is one of five species of Trichoderma that have green, globose to subglobose conidia. Its smooth conidia distinguished it from T. viride and T. asperellum Samuels et al, both of which have warted conidia (Samuels et al 1999). Trichoderma atroviride is distinguished from T. harzianum and T. aggressivum Samuels & W. Gams in growing very slowly at 35 C on PDA, in having larger conidia, in its more abundant chlamydospore production on CMD, and in its ability to produce a distinctive sweet, or coconut odor in culture (Samuels et al 2002). Trichoderma aggressivum is only known as a parasite in commercial mushroom houses.
The sweet odor that helps in recognizing T. atroviride is the coconut-like odor of the antifungal antibiotic 6-pentyl--pyrone. It is also produced by cultures of the closely related T. viride. Because this odor has not been observed in any species outside of sect. Trichoderma, the many reports of alpha pyrone production by the more distantly related T. harzianum (Cutler et al 1986, 1996, Kalyani et al 2000, Rito-Palomares et al 2000, Rey et al 2001) probably all refer to misidentified strains of T. atroviride or T. viride. It is, therefore, likely that T. atroviride is under-represented in the literature.
Both morphology and DNA sequence data place Trichoderma atroviride in Trichoderma section Trichoderma (Bissett 1992, Kuhls et al 1997, Lieckfeldt et al 1998, 1999). This section has been shown, by sequence analysis of the ITS regions of rDNA, to have been derived from within clade ‘A2’ of the paraphyletic sect. Pachybasium (Kindermann et al 1998). Section Trichoderma s. str. includes the three economically important species T. atroviride, T. viride and T. koningii Oud. as well as the species T. asperellum, recently segregated from T. viride, and H. cf. muroiana Hino & Katumoto (Lieckfeldt et al 1998, 1999, Samuels et al 1999).
To date, there has been only one report of a possible teleomorph for T. atroviride. Bissett (1992) noted a similarity between an ascospore isolate (DAOM 172826) and T. atroviride, but subsequent examination of the isolate proved it to be morphologically distinct from T. atroviride (Samuels unpubl). Examination of the teleomorph of DAOM 172826 revealed it to be similar to H. aureoviridis Plowr. & Cooke, the teleomorph of T. aureoviride Rifai. Over several years we have collected specimens of a Hypocrea that was morphologically H. rufa (Webster 1964) but that produced the morphological species T. atroviride in pure culture. In this study, we examined and compared cultural, morphological, and sequence data of cultures derived from these specimens with cultures of T. atroviride isolated directly from nature. Sequences from the ITS regions of the rDNA complex and partial sequence of the translation-elongation factor (EF-1) gene help to confirm the link between T. atroviride and its Hypocrea teleomorph. A full morphological description of the teleomorph is given.
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MATERIALS AND METHODS |
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) and isolate IFO 31293, deposited as H. muroiana, from Japan, the teleomorph specimen of which was not available for study. Eight isolates representing additional species from the A2 group of T. sect. Trichoderma/Pachybasium group of Kindermann et al (1998), namely, T. asperellum, T. viride, T. koningii, T. hamatum (Bon.) Bain. and T. strigosum Bissett, were included in DNA sequence analysis studies. Two isolates from the T. sect. Pachybasium B group (Kindermann et al 1998), namely T. hamatum and T. aggressivum, plus the T. sect. Longibrachiatum species T. longibrachiatum Rifai were included to root the ITS gene tree. Ascospores of Hypocrea specimens were isolated with use of a micromanipulator onto cornmeal dextrose agar (Difco cornmeal agar + 2% glucose). All isolates were subsequently stored as living cultures on CM (cornmeal agar, Difco Beckton Dickinson, Sparks, Maryland) slopes at 4 C. Color designations (K&W) are taken from Kornerup and Wanscher (1978).
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) were employed for each isolate at an annealing temperature of 55 C to amplify the ITS regions of rDNA using primers ITS 4 and ITS 5 (White et al 1990). Similarly, a section of the translation-elongation factor gene was amplified using primers EF1-728F (Carbone and Kohn 1999) and EF-2 (O'Donnell et al 1998b). PCR products were prepared for direct sequencing with a commercial kit (QIAquick PCR purification kit. Qiagen, Valencia, California). DNA sequence was obtained using the BigDye Terminator Cycle Sequencing kit (Perkin Elmer Applied Biosystems, Foster City, California). Products were analyzed directly on an ABI PrismTM 377 DNA Sequencer (Perkin Elmer Applied Biosystems). Both strands were sequenced for each isolate.
DNA sequences were edited and aligned using the computer program's SEQUENCHER 3.1 (Gene Codes, Ann Arbor, Michigan) and Clustal X 1.81 (Thompson et al 1997), respectively. Alignments were manually adjusted using the program Genedoc 2.5.000 (http://www.psc.edu/biomed/genedoc/). Trees were produced with both neighbor-joining (NJ) and maximum parsimony (MP) analysis for ITS and EF-1 sequence datasets using PAUP 4.0b8 (Sinauer Associates, Sunderland, Massachusetts). The Kimura-2 parameter distance calculation was employed for neighbor-joining analysis. For parsimony analysis, the heuristic search option with 1000 random addition sequences and TBR branch-swapping options were employed. Gaps were treated as a fifth base. Stability of clades was assessed with 1000 bootstrap replications. Ambiguously aligned regions were excluded from EF-1 sequence data analysis. To root the ITS 1 trees, sequences from the two T. sect. Pachybasium B (Kindermann et al 1998) isolates (T. harzianum and T. aggressivum) plus the T. sect. Longibrachiatum isolate T. longibrachiatum were designated as the outgroup. Because the true root of the T. sect. Trichoderma isolates could not be determined from the ITS trees, T. asperellum, T. viride, T. koningii, T. hamatum, and T. strigosum were all designated the outgroup on EF-1 sequence trees.
Morphological examination –
Measurements of teleomorph characters were made from dried herbarium material. Stromata were hydrated briefly in 3% KOH prior to sectioning with a freezing microtome to produce 15-µm-thick sections. The sections were mounted in water on a microscope slide, which was then replaced by lactic acid. Observations and measurements of perithecial and stromal anatomy were made from material mounted in lactic acid. Ascospores and asci were measured from material re-hydrated in a drop of 3% KOH on a microscope slide; the KOH was then replaced by water. Measurements were taken from material either in KOH or in water; there were no differences in the measurements obtained in the respective reagents. Individual characters were observed under the microscope and recorded with SPOT 3.2.4 camera computer software (Diagnostic Instruments Inc., Sterling Heights, Michigan). Characters were subsequently measured from images using the computer program Scion Image 4.0.2 (Scion Corporation, Frederick, Maryland). Where possible, 30 measurements of each parameter for each isolate were made. Mean and range were determined for each character of each isolate. To determine if there were significant differences between EF-1 sequence groups, exploratory data analysis was undertaken using histogram or scatter plots within the computer program SYSTAT 10 (SPSS Inc., Chicago, Illinois).
The sexual compatibilities of some T. atroviride isolates were tested by co-inoculating PDA plates with conidia from two isolates 4 cm apart. Inoculated plates were incubated at 25 C under a 12 h light/dark cycle and examined weekly for the presence of perithecia and/or stromata. Eleven T. atroviride isolates were selected from the four EF-1 gene tree clades and crosses included matings between isolates within a clade and between clades. Perithecia did not form in any of the crosses over a three-month period.
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RESULTS |
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96% bootstrap support for their respective clades.
EF-1 sequences were more variable between isolates than ITS sequences. Of the 532 characters used in EF-1 data analysis (excluding 75 ambiguously aligned characters), 323 were constant, 72 variable characters were parsimony-uninformative and 137 were informative. The sequence alignment is available from Treebase (SN1071). A total of 43 equally parsimonious trees were generated from the heuristic search. EF-1 parsimony trees exhibited a high level of homoplasy between taxa as indicated by CI = 0.633, RI = 0.729 and HI = 0.367. However, the level of homoplasy within the H. atroviridis/T. atroviride in-group itself was low (CI = 0.929, RI = 0.974 and HI = 0.071). Both NJ and MP analysis of EF-1 sequence produced essentially the same tree topology (Fig. 1B
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trees produced similar results but the ITS region, being more highly conserved than the EF-1 gene, did not give the same high degree of resolution as EF-1. As with ITS sequence data, high bootstrap support (98%) was obtained for the H. atroviridis/T. atroviride clade in the EF-1 sequence tree. Furthermore, in the EF-1 tree this branch consisted of >12 changes, making it more robust than its equivalent in the ITS tree. In contrast to the ITS trees, in the EF-1 tree, this clade was further split into four well-supported (83% bootstrap) subclades designated A, B, C, and D. Both the epitype of T. atroviride (CBS 142.95) and the holotype of H. atroviridis (G.J.S. 98-134) were members of subclade A. Hypocrea cultures grouped within the T. atroviride subclades A (4), B (1) and D (1). Differences in the gross morphology of the Hypocrea stromata were detected, but because of the limited number of specimens and the variability in their age and condition, the significance of these differences could not be determined. However, stromata of the four Hypocrea specimens of subclade A were very similar in gross morphology. They tended to be pruinose, semi-effused at first and light brown, but becoming darker and pulvinate with age; neither perithecial apices nor ostiolar openings were visible (Figs. 3–6). In contrast, the stroma of the single specimen of subclade C (C.T.R. 68-1) was slightly tuberculate because of the perithecial apices, and the single specimen of group D (G.J.S. 91-87, Fig. 7) was the only one to have obvious ostiolar openings. We did not observe any micromorphological differences among the various subclades either in teleomorph or in anamorph. Subclades A, C, and D showed no geographic or substrate bias, consisting of isolates from a wide global distribution. Subclade B, however, consisted of all New Zealand isolates, which, with the exception of G.J.S. 95-30, had been isolated from kiwifruit orchards.
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trees. In fact, MP analysis of ITS sequence gave high support for a clade with T. viride (Ve) and T. koningii 3. |
DISCUSSION |
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The distribution of phenotypically indistinguishable T. atroviride strains among four well-supported clades, and the fact that there was geographic bias in only one of those clades (‘B,’ New Zealand), suggests that T. atroviride is a single, somewhat genetically diverse, cosmopolitan species. Clade ‘B’ included only asexual strains from New Zealand, all but one of which was isolated from kiwifruit cultivation. However, New Zealand strains were also found in clades ‘A’ and ‘C.’ The seven cultures derived from Hypocrea ascospores were dispersed among clades ‘A,’ ‘C’ and ‘D,’ and 4 of them clustered in clade ‘A.’ Consequently, there is no doubt that this Hypocrea is the teleomorph of T. atroviride.
The monophyly of the morphological species T. atroviride contrasts to the other sect. Trichoderma morphological species T. viride and T. koningii. The genotypic diversity of these two morphological species, reported by Lieckfeldt and Samuels (Lieckfeldt et al 1998, 1999) was augmented by the sequence data presented here. In the few examples of these species that were included, ITS sequence trees showed two strains of morphological species T. koningii and T. viride to be more closely related to each other than to other strains of T. koningii and T. viride respectively. Furthermore, preliminary EF-1 sequence studies showed the morphologies of both T. koningii and T. viride to be split into three well-supported clades, i.e., T. koningii 1, 2 and 3; T. viride Vb, Vd and Ve (data not shown). A single representative isolate of each of these clades was included in this study for comparison, and the EF-1 tree shows that although the morphological species cluster together, these clusters are not significantly separated (<50% bootstrap). Results of this study suggest that the respective morphologies of T. koningii and T. viride probably represent more than a single species. The taxonomic circumscription of these two well-known species, therefore, remains unresolved and is the subject of continuing study. Taxonomically useful phenotypic characters will be sought in the light of these genotypic differences, as was the case with the Gibberella fujikuroi complex studied by O'Donnell et al (1998a).
The subdivision of the genus Trichoderma into 5 sections based on morphology (Bissett 1992) has been tested and challenged by DNA sequence analysis. The only section that has remained intact, albeit expanded from its original description, is sect. Longibrachiatum, the H. schweinitzii Fr. complex (Samuels et al 1998), which appears to be basal to Trichoderma/Hypocrea and consistently assumes the role of outgroup in phylogenetic studies of the genus. Our ITS sequence results are consistent with those of Kindermann et al (1998), where species T. atroviride, T. viride and T. koningii from T. sect. Trichoderma, and T. strigosum and T. hamatum from sect. Pachybasium cluster together in a well-supported clade. Section Pachybasium Bissett (Bissett 1991), which includes most of the described species of Trichoderma, was shown by Kindermann et al (1998) to be paraphyletic, the species being divided between two well-separated clades, which they designated as ‘A’ and ‘B.’ Species of sect. Trichoderma nested within clade ‘A2’ along with the type species of sect. Pachybasium, T. hamatum. Section Trichoderma includes the nomenclatural type of the genus (T. viride) and must therefore be taken as the ‘type’ section of the genus. However, DNA sequence analyses indicate that this section must include morphologically inconsistent members of ‘Pachybasium A2’ (Kindermann et al 1998). For the purposes of the present discussion we refer to sect. Trichoderma as comprising the narrow group of species that consistently cluster with T. viride, essentially as it was constituted by Bissett (1992).
There may be a geographic bias in the respective distributions of H. atroviride, and H. rufa. Hypocrea rufa/T. viride is not among the most common Hypocrea species, but it is widespread at north temperate latitudes, being found in North America, central and northern Europe and eastern Asia (Doi 1974, Lieckfeldt et al 1999, Liu et al 2000). We have seen no tropical collections of the teleomorph. In contrast H. atroviride/T. atroviride is found at all latitudes and perithecial collections of the teleomorph have been found in Japan, Europe and North and Central America.
Although some of the species aggregates or species proposed, respectively, by Rifai (Rifai 1969) and Bissett (1991) were derived from Hypocrea specimens, most described Trichoderma species are based on direct isolations from soil and other natural substrata, and not from ascospores. The genetic link between Trichoderma and Hypocrea has been known since 1865, when Tulasne and Tulasne linked T. viride to H. rufa by following hyphae from the sexual morph to the asexual morph. An increasing number of named Trichoderma species are now recognized to be anamorphs of Hypocrea (Doi and Doi 1979, Table II).
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description of H. rufa, which parallels closely and amplifies the elegant illustrations provided by Tulasne and Tulasne (1865) for that species, has become the reference point for H. rufa. The anamorph linked to H. rufa by Tulasne and Tulasne (1865), Webster (1964), Doi (1974), Lieckfeldt et al (1999), and Liu et al (2000) falls within the broad concept of T. viride, despite variation in the conidiophores and conidial shape.
Japanese specimens identified by Doi as H. muroiana (NY!) fit the H. rufa stroma phenotype well, and one culture that Doi deposited in IFO (IFO 31293) as H. muroiana is typical T. atroviride. Doi (1974) reported that H. muroiana is the most common species of Hypocrea in Japan but he illustrated three conidial types for the species and the range of variation included in those illustrations comprises T. viride, T. koningii, and T. atroviride. There is no doubt that H. atroviridis occurs in Japan, but it is unlikely to be conspecific with H. muroiana. Hypocrea muroiana was originally found on bamboo in Japan, and in our experience, species that form on bamboo—a large grass—are not found on woody substrata. We have not been able to obtain the type specimen of H. muroiana on loan from TNS, but based on Doi's confused description of H. muroiana, and the likelihood that the bambusicolous H. muroiana is not also lignicolous, we are confident in proposing H. atroviridis as a new species.
Convergent phenotype of the teleomorph was also seen in Trichoderma sect. Longibrachiatum, the H. schweinitzii complex (Samuels et al 1998), where only two, very similar and distinctive Hypocrea teleomorph phenotypes were shared by six holomorphic species. The picture that is emerging is that while teleomorph morphology may be predictive of higher level clades, morphological differences among closely related species are manifested in the anamorph and not the teleomorph. This is not only true of Hypocrea. In Gibberella there is no variation in perithecial morphology and anatomy, and only limited variation in ascospore size and septation, whereas the Fusarium anamorphs have been divided among many species in several sections (Samuels et al 2001). Similarly, perithecia of several species of Hypomyces present few or no taxonomically useful characters, again the species differences being manifested in their Cladobotryum anamorphs (Põldmaa and Samuels 1999). In the Sordariales Réblová (2000) has documented a plethora of character-rich anamorphs for the otherwise rather morphologically conservative Chaetosphaeria. These examples suggest that this may be generally the case in pyrenomycetes and loculoascomycetes.
Evidence points to the existence of independent genes for anamorph and teleomorph morphologies that evolve at different rates. The teleomorph genes appear to be more highly conserved than anamorph genes. Anamorph genes also appear to be more adaptable to environmental conditions and this could explain why some fungi are much more widely dispersed in their anamorphic form, while sexual reproduction is highly limited in place or substratum. For example, perithecia of Leuconectria clusiae (Samuels & Rogerson) Rossman et al are only known from leaves and fruits of Clusia in a few stations in Guyana and Puerto Rico whereas its anamorph, Gliocephalotrichum bulbilium J.J. Ellis & Hesseltine, is nearly panglobal (Rossman et al 1993). Similarly, Hypomyces polyporinus Peck produces its perithecia almost exclusively on basidiomata of Trametes species but its anamorph can be found on a wide diversity of aphyllophores and on Auricularia (Rogerson and Samuels 1993, Põldmaa and Samuels 1999).
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TAXONOMY |
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Anamorph. Trichoderma atroviride P. Karsten, Finl. Mögelsvamp. p. 21. 1892.
Stromata solitary to gregarious, (0.5–)0.9–2.4(–7.0) mm diam, adjacent stromata often fused, irregular in outline, at first thin and with margins attached to the substratum gradually becoming pulvinate to nearly discoidal, surface of stroma at first pruinose and plane, becoming glabrous; perithecial apices broadly tuberculate, ostiolar openings not visible, at first light orange (K&W 5A5), the youngest stromata with a fringe of white hyphae, sometimes becoming darker brown (K&W 6E–F7), not reacting to KOH. Cells at stroma surface in surface view hyphal to angular. Cylindrical hairs to 30 µm long x 3 µm wide, with cell walls ca 1 µm wide sometimes visible at the stroma surface. Stroma surface region in section with a pigmented layer of cells sharply distinguished from the internal tissue, (13–)23–37(–47) µm wide, formed of loosely packed angular cells (3.0–)5.5–9(–16.5) µm in length with walls ca 1 µm (0.4–1.4) thick. Cells immediately below the stroma surface loosely packed hyphal to angularis cells (3.4–)5.4–6.7(–11.5) µm long with walls ca 1 µm (0.3–1.2) thick. Cells below the perithecia compact, hyphal to angular, (3.7–)7.4–10.5(–17.9) µm long, walls ca 1 µm (0.4–1.5) thick. Perithecia globose to subglobose, densely disposed with walls of adjacent perithecia mostly touching, (133–)160–300(–335) µm tall, (100–)130–230(–290) µm diam, ostiolar canal (55–)70–96(–123) µm long, perithecial apex flush with the stroma surface or slightly protruding through the surface region of the stroma. Perithecial apex not differentiated from the cells of the surrounding stroma surface; ostiolar canal periphysate, some periphyses protruding through the ostiole and with enlarged club-shaped tips. Asci cylindrical, (60–)79–90(–105) x (3.6–)4.2–6.2(–7.2) µm; apex slightly thickened, lacking a visible pore; part ascospores uniseriate, ca 8 µm of the base of each ascus empty. Part-ascospores dimorphic, hyaline, thick walled, finely spinulose, distal part globose to subglobose, (2.5–)3.5–5.0(–5.7) x (2.5–)3.2–4.7(–5.7) µm, proximal part oblong to wedge-shaped, (3.0–)3.6–5.3(–6.2) x (2.1–)2.9–4.1(–4.9) µm.
Cultures and anamorph. Optimum temperature on PDA 25–30 C, on SNA 30 C. Colony radius on PDA after 72 h in darkness at 30 C (22–)29–35(–44) mm, at 35 C (1.0–)5.0–7.5 (–11.5) mm. After 96 h at 30 C darkness on CMD, conidia forming in the middle of the colony in an area ca 4 cm diam, uniformly dispersed and not pustulate or in confluent, dense pustules that have a radial arrangement. On PDA after 96 h at 30 C, colony sharply delimited and with a more or less dense central disk within which most conidia form. No pustules observed. Coconut odor typically noticed on CMD and PDA. Branching of conidiophores typically unilateral although paired branches are common. Branches typically arising at 90° or less with respect to the main axis above the point of branching. Phialides (n = 659) (4.2–)7.7–8.0(–15.0) µm long, (1.7–)3.0–3.1(–4.7) µm at the widest point, (1.2–)2.0–2.1(–3.5) wide at the base, L/W = 2.5–2.7, straight or sinuous, sometimes hooked; in whorls of 2–4 phialides, often solitary; the terminal phialide of a whorl and solitary phialides often cylindrical and constricted only below the tip to form a narrow neck; phialides formed below the terminus typically flask-shaped and enlarged in the middle, constricted to the tip and slightly at the base; cells supporting the phialides (1.7–)2.8–2.9(–4.2) µm wide, and at most only slightly wider than the phialide base. Intercalary phialides not observed. Conidia subglobose to ovoidal, (n = 659) (2.7–)3.0–3.8(–5.0) x (2.3–)2.8–3.5(–4.0) µm, lacking a visible basal abscission scar, smooth. Chlamydospores abundant on CMD, globose to subglobose, terminal or intercalary, (5.2–)10.0–10.5(–16.5) µm diam.
HOLOTYPE. FRANCE. PYRENEES ATLANTIQUES: Isle de la Sauveterre de Bearn, elev. 100 m, on decorticated wood, 25 Oct 1998, Samuels & Candoussau (BPI 748312, cultures: G.J.S. 98-134, CBS 110086).
Additional specimens examined. COSTA RICA. Bosque los Niños, 09°00'N, 84°00'W, elev. 0 m, on log, 18 May 1996, S. Huhndorf & F. Fernandez 2508 (BPI 744561, cultures: G.J.S. 96-200, CBS 110018). UNITED STATES. VIRGINIA: Giles County, Cascades Recreation Site, 4 mi N of Pembroke, Little Stony Creek, 37°02'N, 80°35'W, elev. 840 m, on bark, 18 Sep 1991, Samuels et al (BPI 1112858, culture G.J.S. 91-87, CBS 110017). GEORGIA: Rabun County, Chattahoochee Natl. Forest along Little Creek Rd near Double Bridge Creek, 34°59'N, 83°10'W, on decorticated wood, 17 Oct 1990, Samuels & Rossman (BPI 1107171, cultures: G.J.S. 90-134, CBS 110016). INDIANA: Owen County, McCormick Creek State Park, 2 mi E of Spencer, on decorticated log, 15 Aug 1981, Rogerson (NY, cultures C.T.R. 81-50, CBS 110015). WASHINGTON: Larabee State Park Fragrance Lake Trail, 1/2 mi east of Chuckanut Drive, on Ganoderma tsugae on conifer log, 11 Dec 1967, R. Haard (NY, culture: C.T. Rogerson 68-1, CBS 110014).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476 Golm, Germany
3 Corresponding author, gary{at}nt.ars-grin.gov
Accepted for publication April 15, 2002.
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