An illustrated guide to the members of the ascomycete order Hypocreales that are known to occur in the south-eastern states of the United States, including North and South Carolina, Tennessee and Georgia is presented. Species were selected mainly based on records in the United States National Fungus Collections (BPI). These states surround the Great Smoky Mountains National Park. The members of the Hypocreales are among the most numerous, and certainly most conspicuous of the microfungi. The order also includes some of the most economically important fungi. This guide is intended for individuals who are participating in All Taxa Biological Diversity studies of the Great Smoky Mountains National Park as well as other interested professionals and amateurs. Short descriptions and color illustrations of 101 species and two varieties in nineteen genera are provided. Keys to genera and species are included.
The ascomycetous order Hypocreales and its anamorphs include fungi of economic importance ranging from virulent plant pathogens and producers of powerful antibiotics to effective agents of biological control and sources of potent mycotoxins. The Hypocreales is one of the orders recognized by Eriksson (2005) in the Sordariomycetes of the Ascomycota.
In the last thirty years progress toward understanding the systematics of the Hypocreales has been made through descriptive accounts centred around the genera Bionectria, Calonectria, Hypocrea, Hypomyces and Nectria. Careful study of hypocrealean fungi has revealed relationships among species that are based on suites of correlated characters including those of the anamorph. Combined with phylogenetic analyses based on DNA sequence data, these serve as the basis for revised generic concepts, especially for those species previously classified as Nectria sensu lato. The revised genera for three of the four major families of the Hypocreales, specifically the Bionectriaceae, Hypocreaceae, and Nectriaceae, has been accounted for in Rossman et al. (1999). The order also includes the Clavicipitaceae and Niessliaceae (Samuels & Barr, 1998) but species of the latter family are not included here. This book includes descriptions and illustrations of one-hundred and one species and two varieties in twenty genera of hypocrealean fungi in the Bionectriaceae, Clavipitaceae, Hypocreaceae and Nectriaceae that are known to occur in the states that include and surround the Great Smoky Mountains National Park. We have included two representative genera of the Clavicipitaceae, viz. Cordyceps and Neobarya, although updoubtedly many more species of this family occur in the Great Smoky Mountains National Park.
Members of the Bionectriaceae, Clavicipitaceae, Hypocreaceae and Nectriaceae are associated with a variety of substrata ranging from living and decaying plant material, dung, and soil to fungi, insects, and, occasionally, animals and humans. Although often unrecognized as such, a majority of hypocrealean fungi are mycoparasitic or mycosaprobic and are extremely versatile in their abilities to exploit fungal substrata (Gams et al., 2004). In some species the fungicolous nature is conspicuous with ascomata developing on other fungi, as, for example, Cosmospora episphaeria on old carbonous black pyrenomycetes, species of Hypomyces parasitizing mushrooms, or Nectriopsis violacea growing on myxomycetes. Less conspicuous are the hypocrealean fungi that occur on rotting wood, but actually are necrotrophic on the fungal hyphae in the wood. These include many of the biocontrol fungi in the Hypocrea-Trichoderma complex, such as T. virens and T. harzianum and Bionectria ochroleuca often encountered as its anamorph, Clonostachys rosea (Schroers, 2001). A number of hypocrealean species occur on lichens such as species of Pronectria (Rossman et al., 1999). Although primarily fungicolous, the genus Cosmospora also includes insecticolous species such as C. flammea, having in common the ability to degrade chitin as a substratum (Rossman et al., 1999).
Aggregations of ascomata of species in the Nectriaceae are often found in quantity erupting through the bark of recently killed woody substrata. Some species appear to function as endophytes residing harmlessly in the healthy plant but sporulating profusely following the death of the host. Trichoderma species, the anamorphs of Hypocrea, and Clonostachys species, the anamorphs of Bionectria, recently have been found to dominate the endophyte mycota of trunks of Theobroma gileri, a wild cacao, in Ecuador (Evans et al., 2003). Despite their primarily saprobic nature, many hypocrealean fungi, especially members of the Nectriaceae, are facultative and virulent plant pathogens, causing serious problems on crop plants, often encountered in the anamorph. These include the Cylindrocladium anamorphs of species of Calonectria, causing twig dieback of Ilex and potato tuber rot (Crous, 2002), Fusarium sambucinum, anamorph of Gibberella pulicaris, cause of hop canker and root rot of many crops, and other species of Gibberella and related anamorphs, including F. oxysporum (Samuels et al., 2001), Nectria cinnabarina, often seen as the Tubercularia anamorph, causing coral spot of fruit and hardwood trees (Sinclair et al., 1987), and Neonectria coccinea var. faginata, cause of beech bark disease (Sinclair et al., 1987).
STROMA.—A stroma is any vegetative tissue that subtends or surrounds the ascomata. The stroma may be pseudoparenchymatous, consisting of hyphae that have lost their hypha-like structure, prosenchymatous, consisting of cells that retain their hyphal integritty, or reduced to a hyphal subiculum. The structure of an individual stroma may vary from prosenchymatous to pseudoparenchymatous forming distinct regions. Placement of the ascomata within the stroma and location of the stroma within the substratum are characteristic of certain genera. Within the Bionectriaceae, the stroma is often lacking or reduced to a subiculum as in Nectriopsis. Within the Hypocreaceae, most genera have ascomata embedded in a more or less extended pseudoparenchymatous stroma. The stroma may be large and expansive as in Hypocreopsis, in which the stromata are lobed and spreading up to 20 mm diam across the substratum. In Hypocrea the stroma may range from 1–3 mm or more in diameter with or without a stipe. In some species of Hypomyces the stroma may completely cover and obliterate the hymenium of the host, particularly those occurring on members of the Agaricales, or the stroma may be a thin subiculum beneath which the host fungus can still produce viable basidiospores. Within the Nectriaceae the stroma may be inconspicuous or absent as in most species of Cosmospora, or it may be basal, consisting of a pseudoparenchymatous pad of tissue giving rise to two or more ascomata. The pseudoparenchymatous basal stroma is often continuous with the outer region of the ascomatal wall and is frequently associated with the anamorph, as in Nectria sensu stricto, in which the basal stroma is initially associated with a sporodochial, synnematal or pycnidial anamorph.
ASCOMATA AND ASCOMATAL WALL STRUCTURE.—Ascomata are generally light- to bright-coloured, soft-textured, uniloculate perithecia that are hyaline, white, pale yellow, orange, red, to purple, or brown. For all genera the ascomatal colour is slightly darker in dried specimens. Descriptions given here are based on the colour of rehydrated ascomata. The nature of the ascomatal pigmentation, specifically the reaction in 3% KOH or 100% lactic acid, correlates with other characteristics useful in defining relationships within genera and families. In general, species of the Nectriaceae react with KOH darkening to a blood-red colour or purple and turn yellow in lactic acid while species in the Bionectriaceae do not react with KOH or lactic acid. Within the Hypocreaceae, some species of Hypomyces and Hypocrea have ascomata and/or stromal tissues that become red in KOH, e.g. Hypomyces lactifluorum and Hypocrea pallida. In the Hypocreaceae the significance of this colour reaction does not extend above the species level.
Within the Bionectriaceae and Nectriaceae ascomatal wall structure is often useful in defining genera, and this structure correlates with other morphological characteristics of both the teleomorph and anamorph. The ascomatal wall of hypocrealean fungi generally consists of one to three regions of cells with each region of two to four cell layers. The outer region is usually composed of angular to globose cells with walls that may or may not be thickened. The inner region is almost always of thin-walled, hyaline cells elongated parallel to the centrum. In the Hypocreaceae, the ascomatal wall features are generally the same in all genera and thus are not used as defining characters. In the Bionectriaceae and Nectriaceae the structure of the wall may be characteristic of the genus, e.g., the ascomatal wall of Hydropisphaera consists of large, thin-walled, globose cells. In Neonectria, the cells near the ascomatal surface are thick-walled and flattened, forming a textura epidermoidea that results in a varnished appearance. In some species of this group an additional outer layer of loose cells obscures the textura epidermoidea, yet the distinctive structure is evident in longitudinal sections. Some genera have characteristic features on the surface of the ascomata. In certain species of Bionectria, the ascomata have large wart-like projections on the surface, and the outermost walls of the outer wall cells are greatly thickened on the outside edge, a characteristic described as capitate. Other genera have more subtle ascomatal structures such as the large, loose, globose outer wall cells of Calonectria and Haematonectria (Rossman et al., 1999).
Centrum characters, such as the presence/absence of the apical paraphyses and the periphyses lining the ostiole, are similar for all hypocrealean fungi exclusive of the Clavicipitaceae, in which apically free paraphyses form. The ostiolar canal is always periphysate in perithecial members of the Hypocreales. Interthecial elements in hypocrealean fungi are, by definition, apical paraphyses; these may often appear as deliquescing strands in mature ascomata, although they may be present as thin-walled, inflated cells as they go through the process of deliquescing (Rossman et al., 1999).
ASCI AND ASCOSPORES.—The asci of hypocrealean fungi are unitunicate. They may or may not have a ring at the apex. The presence of an apical ring generally correlates with ascospore size and shape and is useful only in defining species. In hypocrealean species having relatively short ascospores, i.e. less than 20 µm, the asci frequently have an apical ring; species having elongate or large ascospores generally lack an apical apparatus. In many hypocrealean species the relatively short ascospores are forcibly discharged. In others the asci deliquesce, releasing the ascospores into the centrum. As the soft-textured ascomata periodically dry out, and swell up again when rehydrated, aided by the gelatinous, deliquescing apical paraphyses, the ascospores are extruded en masse through the ostiole and appear as a cirrus or column emerging from the ascomatal apex. Neither the apex nor any other part of the ascus or centrum reacts with iodine.
Within the Bionectriaceae, Hypocreaceae and Nectriaceae, ascospores are typically one-septate, ellipsoid to fusiform with rounded apices, although there is some variability. Ascospores may be non-septate as in Neocosmospora. As the result of disarticulating, bicellular ascospores, the part-ascospores of some species of Hypocrea appear globose. After disarticulating the part-ascospores may be the same size and shape termed monomorphic or the two parts of the part-ascospores may have a slightly different shape and size termed dimorphic. The ascospore apices of many species of Hypomyces are apiculate. Ascospore colour is generally hyaline to golden-yellow or golden-brown, as in Cosmospora in the Nectriaceae, although Viridispora and some species of Hypocrea in the Hypocreaceae have green ascospores. Ascospore ornamentation in the hypocrealean fungi is variable, ranging from smooth to verrucose or striate, and is sometimes characteristic of a genus. For example, ascospores of species of Hypomyces are often verrucose to tuberculate; in Cosmospora ascospores tend to be verrucose and golden-brown; in Hydropisphaera, the ascospores are generally striate. Traditionally the genus Nectria sensu lato has included species with one-septate ascospores. However, in Nectria sensu stricto ascospores are variable in size and septation, ranging from one-septate in N. cinnabarina to ellipsoid and transversely septate in N. coryli to very long and transversely septate in N. cucurbitula or muriform in N. pseudotrichia. Within the Clavicipitaceae ascospores are generally cylindrical and filiform. Neither germ slits nor germ pores are known in the Hypocreales.
ANAMORPHS.—The anamorphs of the Bionectriaceae, Clavicipitaceae, Hypocreaceae and Nectriaceae are primarily phialidic, producing hyaline or bright-coloured conidia, although there are some exceptions. Conidiomata range from non-existent to acervular, pycnidial or synnematal. Conidia may be aseptate or septate, ranging from having one or multiple, transverse septa to muriform, and are generally hyaline or slightly yellow to yellow-brown or green, but not brown or black. Although emphasis is placed here on teleomorphic states, hypocrealean fungi are commonly encountered as anamorphs. For some species, the anamorphic state appears to have a much wider geographic distribution than the teleomorph. Asexual states of most genera in the Bionectriaceae are reduced, often described as acremonium-like, although there are exceptions such the anamorphs of Bionectria classified in Clonostachys (Schroers et al., 1999; Schroers, 2001). Anamorph genera connected to teleomorphs in the Hypocreales include Cladobotryum (Gams & Hoozemans, 1970; Rogerson & Samuels, 1994), Gliocladium (Seifert, 1985), Stilbella (Seifert, 1985), and Trichoderma (Bissett, 1984, 1991 a, b; Gams & Bissett, 1998; Chaverri & Samuels, 2003; Samuels et al., 1998). The greatest diversity of anamorphs occurs in the Nectriaceae including the following pairs of teleomorph/anamorph genera: Calonectria-Cylindrocladium (Crous, 2002); Nectria-Tubercularia (Seifert, 1985) and Neonectria-Cylindrocarpon (Brayford et al. 2004).
To examine specimens of hypocrealean fungi, a drop of water was placed directly on the ascomata for a few minutes. Ascomata are routinely mounted in 3% KOH. Cotton blue in lactic acid is often helpful in revealing surface ornamentation of ascospores. The test for colour reaction of the ascomata was made by placing a drop of 3% KOH and 100% lactic acid directly on the rehydrated ascomata or mounting the rehydrated ascomata in these media. If the wall of the ascomata changes colour usually to a dark blood red or purple, this is indicated as KOH+. If the wall of the ascomata becomes yellow in lactic acid, this is indicated as lactic acid becoming yellow. Longitudinal median sections were made using a freezing microtome. To make sections, ascomata were picked off the substratum, rehydrated briefly in water, placed on a freezing stage, and mounted in Tissue-Tek (Miles, Inc., Elkhart, IN). Sections of ascomata and stromata were approximately 15 µm thick.
Cultures of fresh specimens were obtained by the isolation of single or mass ascospores in the laboratory. Recently collected fresh or air-dried specimens were rehydrated with water. Several ascomata were placed in a drop of sterile water in the well of a hanging drop slide. The ascomata were smashed with a needle, releasing asci and ascospores into the water, and stirred vigorously in order to distribute the centrum contents evenly. The drop of water with asci, ascospores and remnants of the ascomata was placed on a plate of agar using a sterile micropipette. Firm cornmeal dextrose agar with antibiotics (Difco cornmeal agar + 2% dextrose and 2 mg/L each of streptomycin, tetracyclin and neomycin) was used for primary isolation. The drop was spread over the surface of the agar plate using a sterile blunt glass rod. Plates were incubated overnight at room temperature. The next day the agar surface was examined using a 50× binocular dissecting microscope with transmitted light. Germinated single or mass ascospores with subtending agar were picked out of the agar with a fine insect pin and transferred to agar plates and tubes. Several ascospores were placed in a drop of cotton blue in lactic acid for observation of germination and to check the identity of the isolated ascospores. Alternatively, cultures were isolated using a micromanipulator as described in Kendrick (1979). Cultures derived from mass and single ascospores were placed on cornmeal agar slants (lacking dextrose) in screw top tubes and placed in a cold room (10ºC), in 10% glycerine in cryovials, in vapour phase of liquid nitrogen, or in 10% glycerine in cryovials at –80º C. Specimens from which cultures were obtained, dried cultures, and living cultures were deposited at BPI or NY. Abbreviations used for herbaria are those of Holmgren et al. (1990).
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