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Hirsutella uncinata, a new hyphomycete from Australia

     Biodiversity Theme (Mycology and Botany), Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa, Ontario, K1A 0C6 Canada

	ABSTRACT

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Hirsutella uncinata, collected on follicles of Hakea sp. in New South Wales, Australia, is described as a new anamorph species. The fungus produces stiff, erect, verticillate conidiophores with up to 10 whorls of hooked, phialidic conidiogenous cells that have rough-walled necks. The conidia are subfusiform, flattened on one side and produced in groups of 2–3 in a dense slimy ball at the end of each phialide. Parsimony analysis of aligned internal transcribed spacer rDNA sequences suggests that H. uncinata is closely related to the Cordyceps sinensis complex (Clavicipitaceae, Hypocreales), which includes species with morphologically similar anamorphs.

Key words: Clavicipitaceae, Cordyceps sinensis, internal transcribed spacers, phylogenetics, taxonomy

	INTRODUCTION

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The hyphomycete genus Hirsutella Pat. includes about 65 species (Hodge 1998), some that are pathogenic to insects, mites and other invertebrates. The species of this genus have unusual phialides that taper into a long narrow neck, and produce usually only 1–3 conidia in a dense terminal sphere of slime. In some species, more than one neck emerges from the basal part of the conidiogenous cell; in others, the neck branches to form a polyphialide. Many of the known species produce synnematous conidiomata, but several species lack conidiomata. Minter and Brady (1980) revised the taxonomy of 12 mononematous species and distinguished them by the wartiness of the conidia and the phialides, characters of the phialide neck (helical versus straight, multiple versus single necks, monophialidic versus polyphialidic), the arrangement of conidiogenous cells on conidiophores, the pigmentation and dimensions of microscopic structures. Additional mononematous species were described by Minter et al (1983), Evans and Samson (1982), Samson et al (1980), Samson and Evans (1985) and Fernández-García and Evans (1990). The mononematous species of Hirsutella include H. thompsonii F.E. Fisher, an important pathogen of the citrus-rust mite Phyllocoptruta oleivora Ashm. and other eriophyoid mites (van der Geest et al 2000) and the anamorph of Cordyceps sinensis (Berk) Sacc., the famous "caterpillar fungus" of traditional Chinese herbal medicine (Liu et al 2001).

The known teleomorphs of Hirsutella species are classified in Cordyceps (Fr.) Link, Ophiocordyceps Petch, Torrubiella Boudier (Morris 1963, Samson et al 1982, Hywel-Jones 1994, 1995a, , Hywel-Jones b) and Cordycepioideus Stifler (Ochiel et al 1997, Suh et al 1998). Comparatively little DNA sequence data derived for taxonomic purposes has been published specifically on Hirsutella, but its phylogenetic affinities are well established in the Clavicipitaceae, Hypocreales. Several other anamorph genera are associated with Cordyceps, many of which also have species with phialidic conidiogenous cells, such as Akanthomyces Lebert, Gibellula Cavara, Harposporium Lohde, Isaria Pers., Paraisaria Samson & B.L. Brady, and Tolypocladium W. Gams (Hodge 2003).

In 1999, a striking species of Hirsutella emerged from follicles (fire-resistant, woody, nut-like fruits) of Hakea sp. (Proteaceae) collected in New South Wales, Australia, and incubated 1–2 wk several months later in a damp chamber. The phialides and conidial masses clearly identified it as a species of Hirsutella, with a conidiophore branching pattern and hooked phialides that easily distinguished it from all known species. This paper presents a description, illustration and preliminary phylogenetic analysis of this new species.

	MATERIALS AND METHODS

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Morphological examination. – For isolations, malt yeast agar (MYA: 10.0 g Difco Bacto malt extract, 2.0 g Difco yeast extract, 20.0 g Difco Bacto agar, 1000 mL distilled water). For morphological studies, cultures were grown on oatmeal agar (OA), Blakeslee’s malt extract agar with trace metal solution (MEA, made with Difco Bacto malt extract, Sparks, Maryland) and Synthetischer nährstoffarmer agar with filter paper (SNA) (Samson et al 2000) in the dark at 25 C, or in a 12 h:12 h light:dark cycle on a room temperature incubation bench fitted with fluorescent and near UV lights. Color codes and capitalized color names refer to Kornerup and Wanscher (1978). Means and standard errors are presented for some measurements. Cardinal temperatures were determined by growing the culture in incubators at 5 C increments, from 5 to 40 C, on cornmeal agar (CMA) (BBL, Sparks, Maryland) in the dark, with three colonies at each temperature.

DNA extraction, DNA sequencing and phylogenetic analyses. – DNA extraction, purification, amplification and sequencing methods were similar to those described by Hambleton et al (2003), using primers NS1, NS4, ITS1, ITS4 (White et al 1990), except that UltraClean Microbial DNA Isolation and UltraClean PCR Purification kits (Mo Bio Laboratories Inc., Solana Beach, California) were used for DNA extraction and cleaning of PCR products, and an ABI PRISM^® 3700 DNA Analyzer (Applied Biosystems, Foster City, California) was used for sequencing. Reference sequences for the small subunit rDNA (18S) and internal transcribed spacer (ITS) were chosen from GenBank using BLAST searches. After initial alignments and parsimony analyses, sequences that either were relatively distant from the Hirsutella species, or identical sequences from different accessions of individual species, were discarded. GenBank accession numbers for ITS sequences are shown on FIG. 1. These reference sequences originated in studies by Chen et al (2001), Liu et al (2001), Nikoh and Fukatsu (2000) and Gernandt and Stone (1999). Alignments were calculated using Clustal W as implemented in MegAlign 5.05 (DNAStar, Madison, Wisconsin), and adjustments were made visually using Se-Al v.1.01a1 (Rambaut 1996). Parsimony analysis of alignments were performed with PAUP* 4.0b10 (Swofford 2003) using heuristic searches with uninformative characters removed. Searches with simple stepwise addition were run, and for the ITS analysis 1000 replicates of random stepwise addition also were run. Bootstrap and jackknife analyses were run with 1000 replications using simple stepwise addition.

View larger version (23K): [in this window] [in a new window]   FIG. 1. One of 36 equally parsimonious trees based on a heuristic analysis of the internal transcribed spacer of Hirsutella uncinata and related species (95 steps, CI 0.625, RI 0.741, RC 0.460, HI 0.379). "H" after a Cordyceps name indicates species with known Hirsutella anamorphs. The single pair of numbers over the long branch indicates the bootstrap and jackknife support received for the C. sinensis complex. Bold lines indicate the parts of the tree with 100% consensus support.

	RESULTS

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Cardinal temperatures. – Radial growth after 4 wk on CMA, 5 C 1 mm, 10 C 2 to 3 mm, 15 C 4 to 5 mm, 20 C 8 to 9 mm, 25 C, 9 to 10 mm, no growth at 30, 35 or 40 C. Sporulation occurred at all temperatures where growth occurred.

Phylogenetic analysis. – Analyses of the small subunit rDNA (SSU) sequences confirmed that H. uncinata was related to the teleomorph genus Cordyceps and supported its classification in Hirsutella. The topology of these trees was similar to the more resolved ITS tree discussed below and ultimately the SSU analysis was used only to select an outgroup for the ITS analysis. Therefore, the results are not shown here but the NS1-4 SSU sequence has been deposited in GenBank as AY365469. A consideration of the phylogenetic distribution of the various anamorph genera affiliated with Cordyceps is beyond the scope of this paper, but preliminary investigations based on ITS phylogenies were presented by Liu et al (2002).

The ITS alignment included 570 characters, 45 of which were informative (TreeBase study S1004, matrix M1696). Although the most closely related species were chosen on the basis of the 18S analyses, the Clustal W alignment of the data set required considerable adjustment. Parsimony analyses resulted in 36 equally parsimonious trees of 95 steps, one of which is shown as FIG. 1. The different trees were the result of reorganization of taxa within the C. sinensis complex, which was the only group with significant bootstrap support. Cordyceps japonica Lloyd was chosen to root the ITS analysis based on its position in the 18S analysis; this is one of the Cordyceps species parasitic on other fungi. Hirsutella uncinata is a member of a clade of Cordyceps species including three other species of mononematous Hirsutella anamorphs. The new species was sister of the C. sinensis complex, with C. robertsii (Hook.) Berk. occupying a basal position. Comparatively little ITS data is publicly available for Hirsutella species or allied Cordyceps species, so the significance of these relationships is difficult to assess. However, these data support the classification of H. uncinata in this anamorph genus, and suggest that if a teleomorph exists, it would be similar to the members of the Cordyceps sinensis complex.

	TAXONOMY

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Hirsutella uncinata Seifert & Boulay, sp. nov. FIGS. 2–8

View larger version (111K): [in this window] [in a new window]   FIGS. 2–8. Photomicrographs of Hirsutella uncinata holotype, DAOM 229256. 2. Habit of conidiophores. 3. Conidia. 4. Conidiophore, showing verticillate branching and downward directed hyphae at the base. 5. Detail of phialides and conidial masses. 6–8. Polyphialides. 2, 4–5 from living material, 3, 6–8 from dried material. Scale bars = 50 µm for FIG. 2, 10 µm for FIG. 4, and 10 µm in FIG. 5 for FIGS. 3, 5, 6, 7, 8. FIGS. 2, 3 and 4 are composites.

Conidiophores macronematous, scattered and single, or in pairs or caespitose groups, erect or nodding, stiff, hyaline, 200–400 µm tall, the stipe 8–10 µm wide at the base, tapering to 4.0–5.0 µm wide where the fertile zone begins, walls up to 1.0 µm thick at the base, sterile for one-half to two-thirds of the length, and then with (1–)3–10 whorls of conidiogenous cells 14.5–28 µm apart, usually without branches of the main axis, sometimes with a single branch giving rise to a lateral verticillate structure, with downwards directed hyphae sometime branching from the basal cell and anchoring the conidiophore to the substrate. Conidiogenous cells phialidic, 21–36(–50) µm long (mean = 29 ± 1.3, n = 25, measured as maximum linear distance of the curvature away from junction with the stipe rather than to the conidiogenous aperture), in whorls of 3–6(–9), sometimes in pairs, hyaline, uncinate, curving up to 180° and then with the conidiogenous aperture facing the stipe, with a smooth-walled, broad base 2.5–4.0 µm wide, tapering more or less uniformly to a rough-walled to echinulate, curved, cylindrical neck, conidiogenous aperture about 1.0 µm wide, periclinal thickening inconspicuous, collarettes not seen; polyphialides present on some conidiophores, evident as straight or curved lateral necks 4.0–7.5 x1.0 µm or entirely new secondary phialides emerging from the primary phialide; phialides sometimes with an internal septum; the terminal phialide on the conidiophores often uncurved. Conidial mass bright white under the dissecting microscope, hyaline with the compound microscope, 7–11 µm diam, containing 1–3 conidia. Conidia 5.5–7.0 x3–4 µm (mean = 6.4 ± 0.1 x3.7 ± 0.1, n = 13), in face view subfusiform or subclavate, in side view flattened on one side, with a truncate base about 1 µm wide, and a rounded or somewhat pointed apex, hyaline, produced in an ellipsoidal, globose or limoniform slimy mass. In addition to the macronematous conidiophores, repent conidiophores spread sparsely over the substrate, with irregularly scattered single, paired or whorled phialides and conidia identical to those described above.

Colonies on MEA after 6 wk at room temperature: 22–24 mm diam, planar, with no aerial mycelium and then Golden Yellow to Orange (5BC6–8) or Brown (6E8), or with relatively dense, felty aerial mycelium around the inoculum and then Orange Grey (5B2–4), the surface dense and rubbery, reverse concolorous with obverse, often dark brown below inoculum. Colonies on OA after 6 wk at room temperature: 30–40 mm diam, white, planar, with low, sparse white aerial mycelium, sometimes with a denser ring of white aerial mycelium near the margin, reverse grayish in center, white in a ring at the margin. Repent conidiophores spreading sparsely on both media; erect conidiophores similar to those on the natural substrate mostly arising from the inoculum block, microscopically similar to those produced in vivo, but usually with fewer whorls of phialides, and often fewer phialides per whorl.

Cardinal temperatures. – Minimum <5 C, optimum 25 C, maximum <30 C.

Etymology. – uncinatus, derived from the Latin uncus, meaning "hooked", describing the shape of the phialides.

Material examined. – Australia. New South Wales: Mount Tomah Botanical Garden, S33°32.4', E150°25.4', ca 1100 m elevation, on woody follicles of Hakea sp. on the ground, dried after incubation in a moist chamber for 1–2 wk, K. A. Seifert No. 1316, 17 Aug 1999 (HOLOTY PE DAOM 229256, ex-type culture deposited in Canadian Collection of Fungal Cultures under the same number, ISOTY PE DAR).

	DISCUSSION

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Hirsutella uncinata is recognized easily on the natural substrate by its stiff conidiophores with multiple whorls of uncinate phialides. The conidiophores in culture appear less regularly verticillate and rather lax compared to those on the natural substrate. In common with some of the mononematous species of Hirsutella described by Minter and Brady (1980), the holotype material is sparse but the distinctive morphology is diagnostic. All other species of Hirsutella grow on insects, mites or nematodes, but we were unable to detect an invertebrate host for H. uncinata. The substrate, a follicle of a Hakea sp., is dense and woody and not amenable to slicing or subsurface probing. Seeds of these plants are high in protein and attractive to a variety of insects (Lamont and Groom 1998), and we assume that the true substrate of H. uncinata was larvae hidden by the solid tissues of the follicle.

The phylogenetic analyses of the SSU and ITS sequences of H. uncinata confirm that it is classified appropriately in Hirsutella and suggest a sister relationship with the Cordyceps sinensis complex. As illustrated by Liu et al (1989), H. sinensis has similar verticillate conidiophores and rough-walled phialides to H. uncinata. The microscopic dimensions of the phialides are similar, but the phialides of H. sinensis are not uncinate and polyphialides have not been reported. The conidia of H. sinensis are described and drawn as reniform and up to 14 µm long, longer and differently shaped from those of H. uncinata. Because H. sinensis is reported only from culture, we are unable to compare its wild-type morphology with that of H. uncinata. Liu et al (1989) reported an optimal temperature of growth for C. sinensis as 15–20 C, with significant inhibition at 25 C, which correlates with its occurrence in the high Himalaya, whereas optimal growth of H. uncinata occurs at 25 C, consistent with its temperate home in the Blue Mountains of Australia. Given the close relationship among representatives of the C. sinensis complex (including C. multiaxialis Zang & N. Kinjo and C. nepalensis Zang & N. Kinjo) in our ITS analysis, the relatively distant position of H. uncinata and its other closest neighbor, C. robertsii, further supports the recognition of our fungus as a distinct species.

Of the mononematous species of Hirsutella considered by Minter and Brady (1980), and those described subsequently, only three other species, H. verticillioides Charles, H. guyana Brady & Minter and H. necatrix Minter, Brady & R.A. Hall have conidiophores with verticillate conidiogenous cells. Of these, only H. necatrix has the multiple whorls along the central conidiophore axis so characteristic of H. uncinata, but it has shorter, more or less ampulliform phialides lacking a terminal hook. The conidiogenous cells of H. verticillioides usually are conspicuously polyphialidic, whereas those of H. guyana have a characteristic constriction in the phialide necks and only occasionally are polyphialidic. There are no published sequence data for these species.

	ACKNOWLEDGMENTS

 
We are grateful to Dr Brett Summerell, Royal Botanic Garden, Sydney, Australia, for arranging the field trip during which this fungus was collected. Dr Sarah Hambleton kindly advised us on details of DNA sequencing and phylogenetic analysis. We appreciate helpful presubmission reviews by Sarah Hambleton and John Bissett.

	FOOTNOTES

 
Accepted for publication January 15, 2004.

¹ Corresponding author. E-mail: seifertk{at}agr.gc.ca

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