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Morphology and phylogenetic placement of Endoconidioma, a new endoconidial genus from trembling aspen

     Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9

S. Hambleton

     Biodiversity (Mycology and Botany), Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, K1A OC6

R.S. Currah

     Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Endoconidioma populi gen. et sp. nov. is described from black subicula on twigs of trembling aspen, Populus tremuloides, in Alberta, Canada. Pycnidium-like conidiomata are produced on twigs and in culture, but, unlike pycnidia, conidiomata of E. populi have a closed peridium and a locule filled with conidiogenous cells that form conidia endogenously. These endoconidia are hyaline, unicellular and released by the dissolution of the peridial cell wall. In addition to endoconidia, mostly two-celled conidia that form blastically from undifferentiated hyphae occur often in culture but are observed only occasionally on Populus twigs. No coelomycetous taxa have been reported to produce endoconidia, and both the morphological features and DNA sequence data demonstrate that Endoconidioma is distinct from the previously established endoconidial genera. Parsimony analyses of portions of the nuclear ribosomal RNA gene (SSU and ITS) suggest that Endoconidioma is closely related phylogenetically to members of the Dothideales and allied anamorphs in Hormonema and Kabatina.

Key words: black yeasts, Dothideomycetes, meristematic fungi, SSU and ITS rDNA, subiculum, taxonomy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In our survey of filamentous fungi occurring on trembling aspen, Populus tremuloides Michx., in Alberta, Canada, we found on apparently dying or dead twigs, peculiar, black subicula bearing pycnidium-like conidiomata and scattered, hyaline unicellular conidia. Also, brown, two-celled conidia were found occasionally on some subicula. Axenic cultures obtained from the subicula formed both pycnidium-like conidiomata and undifferentiated hyphae bearing brown, two-celled conidia. Unlike pycnidia, the peridium was closed entirely and contained a locule filled with conidiogenous cells that produced hyaline, unicellular conidia endogenously (i.e., endoconidia). No endoconidial coelomycetous taxa have been reported previously and the conidiomatal morphology and the presence of blastic conidia excluded this fungus from previously established endoconidial hyphomycetous genera Hyphospora Ramaley and Phaeotheca Sigler et al.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isolation of fungus. – Pieces of bark bearing fungal subicula were removed from the twigs of P. tremuloides under a dissecting microscope, washed thoroughly with sterile distilled water, immersed in 0.5% NaOCl for 0.5–1 min, rinsed with sterile distilled water, and air-dried about 30 min on sterile filter paper in a biohazard hood. The subicula were crushed with forceps in a small amount of sterile distilled water and spread evenly on agar media. The media used for isolation were cornmeal agar (CMA; Difco, Detroit, Michigan), cornmeal agar with dextrose (CMAD, Difco), 2% malt-extract agar (MEA, Difco), potato-dextrose agar (PDA, Difco) and water agar (WA, 3% Bactoagar, Difco). Streptomycin sulphate (Pfizer, Montreal, Quebec) was added, 50 mg per L, to all media. Inoculated plates were incubated at 16–20 C in the dark.

Microscopy. – Cultural and morphological characteristics were examined periodically for up to 3 mo after inoculation. Microscopic observations were made from cultures grown in Petri dishes or in slide cultures (Malloch 1981). For light microscopy of endoconidiogenesis, sporulating conidiomata were removed from 2–3 mo old CMAD or MEA cultures and embedded in Araldite. Thin sections (about 1 µm) of the embedded material were stained with a slightly alkaline solution of toluidine blue (1%) in borax (1%) (Meek 1970).

DNA sequencing and phylogenetic analysis. – Genomic DNA was extracted from mycelium grown on CMAD using an UltraClean^TM Microbial DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach, California). The small subunit (SSU) and internal transcribed spacer region (ITS) for UAMH 10297 and the ITS for UAMH 10298 were amplified and sequenced as described in Hambleton et al (2003), except that an UltraClean^TM PCR Purification Kit (MO BIO Laboratories Inc.) was used to clean PCR products.

BLAST searches of GenBank and preliminary analyses of the SSU data indicated that the closest relatives of Endoconidioma populi included the taxa sampled previously to examine the phylogenetic placement of Scleroconidioma sphagnicola Tsuneda et al (Hambleton et al 2003). Therefore, the sequences for E. populi were added to the SSU and ITS data matrices constructed for that study and aligned by eye. Leotia lubrica (Scop.) Pers. L37536, L. viscosa Fr. AF113715, Microglossum viride (Schrad.) Gillet U46031, and Cudonia confusa Bres. Z30240 (Leotiomycetes), and Peziza badia Pers. L37539, Morchella elata Fr. L37537 and Urnula hiemalis Nannf. Z49754 (Pezizomycetes) served as outgroup for the SSU data matrix. The ITS data matrix was adjusted with the addition of two sequences for E. populi determined in this study and a GenBank sequence (AF182375) derived from an unnamed species of Hormonema Lagerb. & Melin. Two sequences (AF013230 and AF182376) used by Hambleton et al (2003) since have been updated and the new data substituted in the ITS data matrix analyzed here. Sarcinomyces crustaceus Lindner AJ244258 was chosen as out-group based on the SSU results and the degree of ITS sequence divergence observed. The SSU and ITS alignments have been deposited in TreeBASE (accession No. S1122).

Parsimony analyses were performed with PAUP* version 4.0b10 (Swofford 1999) using random step-wise addition of taxa and tree bisection-reconnection (TBR) branch swapping (SSU data matrix) or branch and bound analysis with simple step-wise addition of taxa and TBR branch swapping (ITS data matrix). Gaps were treated as missing data. Bootstrap percentages used to assess support for the branching topologies were determined from 500 (SSU) or 1000 (ITS) resamplings of the data set employing the full heuristic search option.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Endoconidioma Tsuneda, Hambleton & Currah, gen. nov.

Fungus mitosporicus et dematiaceus. Conidiomata subglobosa ad ampulliformia cum peridio clauso et fusco-pigmentato, continentia cellulas conidiogenosas quae parunt endoconidia hyalina et monocellularia; conidia exsoluta dissolutione tunicarum cellularum conidiogenosarum cellular-umque peridialium conidiomae. Conidia blastica, pallide ad fusco-brunnea, formantur etiam modo holoblastico ex hyphis pigmentatis et indiscretis.

Species typica. – Endoconidioma populi Tsuneda, Hambleton & Currah.

Mitosporic dematiaceous fungus. Conidiomata, forming on a black subiculum, subglobose to flask-shaped, consisting of an entirely closed, darkly pigmented peridium and a locule filled with conidiogenous cells. Endoconidia formed endogenously, hyaline, unicellular, released by dissolution of the conidiogenous and the peridial cells of the conidioma. Blastic conidia, mostly two-celled, light to dark brown, produced holoblastically from pigmented, undifferentiated hyphae.

Teleomorph. – Unknown.

Type species. – Endoconidioma populi Tsuneda, Hambleton & Currah

Etymology. – Conidioma producing endoconidia.

Endoconidioma populi Tsuneda, Hambleton & Currah, sp. nov. FIGS. 1–19

View larger version (180K): [in this window] [in a new window]   FIGS. 1–9. Endoconidioma populi on a twig of Populus tremuloides (FIGS. 1–3) and in culture (FIGS. 4–9, UAMH 10297, except Fig. 5, UAMH 10298). 1. Black subiculum (arrows) formed on a small twig. 2. Developing conidiomata from subiculum (arrows). 3. Dematiaceous hypha and mostly two-celled, blastic conidia (arrows). 4. Colony on MEA. 7 wk. 5. Colony on CMAD. 7 wk. 6. Hyphae bearing conidiomatal initials at the colony periphery (arrows). Numerous blastic conidia and conidiomatal initials occur below the periphery along the length of the hyphae (arrowheads). 4 wk on CMAD. 7. Unicellular, hyaline conidia (arrowhead) and mostly two-celled, pigmented conidia (arrow) formed blastically from the hyphae. 4 wk on CMAD. 8. Two-celled blastic conidia (arrowheads) and conidiomatal initials (arrows). 4 wk on MEA. 9. Bipolar germ tubes emerging from two-celled conidia (arrowheads). In some conidia, cell enlargement and subdivision by septation indicate germination (arrow). 4 d on CMAD. Scale bars: 1 = 2 mm; 2, 3, 8, 9 = 15 µm; 4, 5 = 5 mm; 6 = 200 µm; 7 = 30 µm.

View larger version (192K): [in this window] [in a new window]   FIGS. 10–19. Formation of endoconidia by Endoconidioma populi in slide culture (FIGS. 10–13, MEA) or on CMAD (FIGS 14–19). UAMH 10297 except in Fig. 14, UAMH 10298. 10. Two-celled conidia germinating to form secondary conidia (arrowheads). The arrow indicates a multicellular body initial developed from a conidial cell. 2 d. 11, 12. Enlarging multicellular bodies (arrows). The arrows in FIG. 12 indicate subdividing cells. 4 d. 13. Multicellular body releasing endoconidia (arrow). 7 d. 14. Multicellular body developed from hyphal cells (arrow). 4 wk. 15. Conidiomata (arrows) arising from darkly pigmented, thick-walled cells. 4 wk. 16. Conidioma releasing endoconidia (arrow). The arrowhead indicates a mass of thick-walled cells. 2 mo. 17–19. Thin sections of conidiomata. 2 mo. 17. Conidiomata of different developmental stages. 18. Conidioma containing abundant endoconidia (arrow). Peridium is intact (arrowhead). 19. Conidioma releasing endoconidia (arrows). Peridial cells remain intact (arrowhead), except in the areas where conidial release takes place (arrows). Scale bars: 10 = 10 µm; 11 = 15 µm; 12–14 = 5 µm; 15, 19 = 20 µm; 16 = 40 µm; 17, 18 = 30 µm.

HOLOTY PE: A dried culture (UAMH 10297) established from conidia of Endoconidioma populi growing on a twig of Populus tremuloides collected at Whitemud Creek, ca 1 km west of the Northern Forestry Centre, Lansdowne, Edmonton, Alberta, 7 Aug 2001 by A. Tsuneda. A living culture is deposited at the University of Alberta Microfungus Collection and Herbarium (UAMH), Edmonton.

On twigs of Populus tremuloides (FIGS. 1–3): Conidiomata forming on black subicula, subglobose, non-ostiolate, brown, mostly 39–45 x 27–36 µm, containing conidiogenous cells 5–10 x 5–7.5 µm. Endoconidia formed endogenously, smooth, hyaline, unicellular, mostly oblong, obtuse, 3.4–4.5 x 1.7–2 µm. Hyphae smooth, dematiaceous, septate. Conidia from hyphae, mostly two-celled, light to dark brown, ellipsoidal, 7.5–12.5 x 5–10 µm, observed only occasionally.

On MEA: Colonies slow growing, attaining a diam of 9–14 mm in 7 d at 16–18 C, initially mucoid and yeast-like, becoming a hemispherical, raised, carbonaceous mass of thick-walled cells, with a thin peripheral area, consisting mostly of submerged hyphae. Conidiomata, developing on masses of thick-walled cells or directly from hyphal cells, brown to black, subglobose to flask-shaped, non-ostiolate, 37.5–87.5(–200) x 30–55(–105) µm, with a peridium consisting of a single layer of darkly pigmented, somewhat flattened, thick-walled cells. Locule filled with subglobose to oval conidiogenous cells, mostly 4–10 x 5–8 µm. Endoconidia mostly oblong, obtuse, hyaline, unicellular, 3.7–7.5 x 2–3 µm, forming by septation of conidiogenous cells and schizolysis of the septa, released by dissolution of walls of the conidiogenous cells and the peridial cells. Hyphae smooth, subhyaline to brown, septate, 3–12 µm wide, cylindrical, becoming moniliform with age, forming conidia holoblastically. Conidia arising from sides of hyphae, either hyaline or pigmented; hyaline conidia unicellular, cylindrical, mostly 4–8.5 x 3.5–5 µm; pigmented conidia light to dark brown, mostly 2-celled (1- to 4-celled), ellipsoidal, occasionally constricted at the septum, 8.7–16 x 5.5–7.5 µm, often multiplying blastically to form aggregated masses of thick-walled cells.

Teleomorph. – Unknown.

Etymology. – Occurring on Populus.

Additional material examined. – CANADA. ALBERTA: Near Lansdowne, Edmonton, on a twig of P. tremuloides, 3 Nov 2001, A. Tsuneda (UAMH 10298, living culture).

Comments. – Colony morphology of E. populi varies with the culture medium and strain. Unlike the rough, carbonaceous and crustose colonies on MEA (FIG. 4), colonies on PDA are initially creamy white and mucoid, becoming shiny black, wrinkled and rubbery with age, while those on CMAD (FIG. 5) are olivaceous brown and with the mycelium mostly submerged in agar. On PDA the production of blastic conidia (FIGS. 6–8) is most prevalent and both uni-and two-celled conidia often exhibit yeast-like budding. UAMH 10298 forms much fewer blastic conidia than UAMH 10297, and its hyphae are mostly superficial on agar (FIG. 5). Regardless of the medium or strain, however, hyphae at the colony periphery usually lack blastic conidia and bear darkly pigmented conidiomatal initials (FIG. 6, arrows).

Two-celled, blastic conidia show three different forms of germination on agar media: (i) by cylindrical germ tubes; (ii) by forming another conidium (secondary conidiation); and (iii) by continuous divisions of both or one of the two cells resulting in the formation of enlarged, multicellular bodies (FIGS. 9–12). In slide culture, component cells of the multicellular bodies often separate from each other to form endoconidia that subsequently are released by the breakdown of the mother cell wall (i.e., cell wall of the two-celled conidium from which the multicellular body originated) (FIG. 13). Multicellular bodies also develop from hyphal cells and are particularly abundant in UAMH 10298 (FIG. 14). Mature conidiomata usually occur after incubation for 2–3 mo at 18 C (FIGS. 15–19). Release of endo conidia from the conidiomata follows dissolution of peridial cells (FIGS. 16, 19).

DNA sequencing and phylogenetic analysis. – The SSU sequence determined for E. populi UAMH 10297 was 1762 bp in length, and was complete at the 3' end, finishing with the CATTA box that precedes the first internal transcribed spacer. The ITS sequences for UAMH 10297 and 10298 were identical and 499 bp in length. GenBank accession numbers for the newly determined sequences are AY604526 (UAMH 10297, SSU-ITS) and AY604527 (UAMH 10298, ITS).

The alignment for the SSU data matrix comprised 39 taxa and 1652 aligned characters. Of these, 1285 were constant, 210 were parsimony uninformative and 157 were parsimony informative. A bootstrap analysis of the informative characters only was performed and the resulting tree (FIG. 20A) indicated that there was support for the grouping of E. populi with the Dothideaceae (Dothidea Fr. and Stylodothis Arx & E. Müll.; 72% bootstrap) but not with either of the other two endoconidial genera, Hyphospora and Phaeotheca (arrows). In general, relationships among the dothideomycetous taxa included, other than the Pleosporales (with Rhytidhysteron rufulum [Spreng.] Speg. AF201452), were mostly unsupported.

View larger version (48K): [in this window] [in a new window]   FIG. 20. Results of parsimony analyses of rDNA data matrices constructed to examine the phylogenetic affinity of Endoconidioma populi. Names in bold indicate the taxa that are present in both trees. A. The 50% consensus tree based on a bootstrap analysis of the small subunit region resulting from 500 replicates of the full heuristic bootstrap search option with random sequence addition. Arrows indicate endoconidial fungi. The bar at the side of the tree corresponds to the Dothideaceae and Dothioraceae. B. One of 10 equally parsimonious trees based on a Branch and Bound analysis of the internal transcribed spacer region of Endoconidioma populi and related fungi (314 steps, CI = 0.573, RI = 0.788, RC = 0.452). Parsimony bootstrap values, 65% and greater, are given above the branches. An asterisk indicates a teleomorphic species.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Endoconidioma populi is unique in producing non-ostiolate conidiomata filled with conidiogenous cells that subdivide to form endoconidia. Conidia are released from conidiomata by the dissolution of the peridial walls. With regard to endoconidiogenesis, E. populi is similar to the endoconidial hyphomycete Phaeotheca. In species of Phaeotheca, conidiogenous cells subdivide by septation and form two to several daughter cells. Schizolysis of the cross walls results in separation of the daughter cells to form endoconidia that subsequently are released by rupture of the mother cell walls (Tsuneda and Murakami 1985, DesRochers and Ouellette 1994). Results of our comparative ultrastructural study on endoconidiogenesis in E. populi and P. fissurella Sigler et al are reported in a separate paper in this issue (Tsuneda et al 2004). Likewise, in the Hyphospora state of Comminutispora agavaciensis Ramaley, numerous endoconidia develop from multicellular bodies (like the one shown in FIG. 14) and the endoconidia are released by breakdown of mother cell walls (Ramaley 1996). However, ultrastructural details of the developmental process have not been documented for Hyphospora. Endoconidioma also resembles Phaeotheca and Hyphospora in producing slowly expanding, dematiaceous colonies and in reproduction by nearly isodiametric enlargement with subdividing cells, i.e., meristematic growth (de Hoog et al 1999) (FIGS. 11–14). Endoconidioma, however, is distinct from these endoconidial genera in the conidiomatal and endoconidial morphology and the presence of darkly pigmented, two-celled, blastic conidia. Both Phaeotheca and Hyphospora lack conidiomata with peridia. Endoconidia in Phaeotheca are pigmented (Sigler et al 1981, Tsuneda and Murakami 1985, de Hoog et al 1997, Zalar et al 1999a) and are the only form of spore produced, except for the secondary ameroconidia that develop between the endoconidial masses in P. dimorphospora Des-Rochers & Ouellette (DesRochers and Ouellette 1994). Hyphospora lacks blastic conidia (Ramaley 1996, Zalar et al 1999a). Endoconidioma populi produces pycnidium-like conidiomata but unlike typical pycnidia, the peridium is closed with no predefined ostiole and, rather than lining the inner surface, the conidiogenous cells fill the locule and form conidia endogenously. No coelomycetous taxa have been reported to produce endoconidial conidiomata.

Analyses of 5.8S and partial ITS2 rDNA presented by de Hoog et al (1999) indicated that among the melanized, meristematic fungi and black yeasts with an affinity to the Dothideomycetes, Phaeotheca and Hyphospora were not closely related and both were distant from the Dothioraceae. The SSU phylogram of Hambleton et al (2003, FIG. 27) showed that Hyphospora clustered in a large clade comprising the Capnodiales that also included two other dematiaceous hyphomycete genera, Capnobotryella Sugiy. and Hortaea Nishim. & Miyaji, while P. fissurella was on its own branch in a basal position to the Capnodiales and Dothideales. Based on the same sampling of dothideomycetous taxa with E. populi included, our SSU bootstrap tree indicates that the new species is most closely related to members of the genus Dothidea and is phylogenetically distinct from both endoconidial genera Phaeotheca and Hyphospora. The ITS analysis presented here suggests a close relationship for E. populi to the Dothideales sensu stricto, restricted to one family (Dothideaceae) in the classification of Eriksson et al (2003), to genera in the Dothioraceae and to species in Hormonema and Kabatina. A common feature of ingroup taxa is the tendency to produce Hormonema-like cultural synanamorphs. The spermatial state of Dothidea insculpta Wallr. was described in the stromatal coelomycetous genus Asteromellopsis Hess & Müller (Sutton 1980). The genus Kabatina produces acervuli on a range of coniferous hosts and has a Hormonema-like synanamorph (Schneider and von Arx 1966, Butin and Schneider 1976, Hermanides-Nijhof 1977, Sutton 1980, Ramaley 1992). The genus Hormonema, which includes black yeasts characterized by basipetal conidial development from undifferentiated conidiogenous cells on hyphae (Schneider and von Arx 1966, Hermanides-Nijhof 1977), is used for species known to be cultural anamorphs of the ascomycetous genera Sydowia, Dothiora, Pringsheimia Schulzer and Guignardia Viala & Ravaz. Hormonema and some other black yeast genera form endoconidia, but the formation is only occasional and occurs in undifferentiated hyphal cells (Hermanides-Nijhof 1977, de Leo et al 1999, Wollenzien et al 1997, Zalar et al 1999b).

In E. populi, the endoconidial coelomycetous state predominates both on natural substrate and in culture and the Hormonema-like synanamorph (FIG. 7) lacks basipetal conidiation. Peláez et al (2000) detected a potent antifungal compound, Enfumafungin, in a culture of endophytic Hormonema sp. (ATCC 74360, AF182375) and suggested that the fungus could be a cultural synanamorph of an undetermined Kabatina species based on the analysis of the ITS1-5.8S-ITS2 region. However, given the results of our ITS analysis, it is more likely that the antibiotic-producing fungus belongs to Endoconidioma (FIG. 20). Species of Rhizosphaera and Scleroconidioma sphagnicola formed a well-supported sister group to the large clade that included Endoconidioma. However, Rhizosphaera and S. sphagnicola differ from Endoconidioma by the production of pycnidial conidiomata and microsclerotial conidiomata bearing abundant, phialidic conidiogenous cells on the surface, respectively (Tsuneda et al 2000, 2001).

	ACKNOWLEDGMENTS

 
This research was financed in part by a Natural Sciences and Engineering Research Council of Canada Discovery grant (R.S.C) and by a research grant from Tottori Mycological Institute (A.T.). We thank M. Hertwig-Jaksch, for the Latin diagnoses, and K. Seifert, J. Bissett and W.A. Untereiner, for suggestions to improve this manuscript.

	FOOTNOTES

 
Accepted for publication May 10, 2004.

¹ Corresponding author, E-mail: atsuneda{at}ualberta.ca

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