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International Mycological Institute, Bakeham Lane, Egham, Surrey, England TW20 9TY
Barry M. Honda
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6 Canada
Feky R. Mantiri
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6 Canada
Gary J. Samuels 1
United States Department of Agriculture, Agriculture Research Service, Systematic Botany and Mycology Laboratory, Room 304, B-011A, 10300 Baltimore Ave., Beltsville, Maryland 20705-2350
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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Neonectria (Hypocreales: Nectriaceae) species having Cylindrocarpon anamorphs that lack microconidia and chlamydospores include: Neo. discophora var. discophora, Neo. discophora var. rubi, stat nov. et comb. nov., Neo. lucida, comb. nov., Neo. viridispora, sp. nov. and Neo. westlandica, comb. nov. Perithecia of these species are red and perithecial anatomyis of the N. mammoidea type, with a palisade of hypha-like cells in the outer perithecial wall. These species occur on recently dead or dying trees. Perithecia of Neo. betulae, sp. nov and Neo. dumontii, sp. nov. are anatomically and biologically similar to those of Neo. discophora. The only known culture of Neo. betulae remained sterile, while Neo. dumontii has not been cultured; their anamorphs are presumed to be Cylindrocarpon. Analyses of mit ssu rDNA sequences indicate that Neonectria/Cylindrocarpon is monophyletic. Within the genus, species having N. mammoidea type perithecia are paraphyletic. Most species cluster with Neo. discophora, but Neo. westlandica and Neo. trachosa are basal to a clade that includes species that do not have a N. mammoidea-type perithecium. Nectria fuckeliana clusters independently of Neonectria and Nectria. Although reported to have a Cylindrocarpon anamorph, fresh ascospore isolates of N. fuckeliana did not produce Cylindrocarpon macroconidia but produced acremonium- or verticillium-like anamorphs. A key to nectriaceous species of Neonectria that have Cylindrocarpon anamorphs that lack microconidia and chlamydospores and/or that have a N. mammoidea type perithecial wall anatomy is presented. New combinations are proposed for other species formerly included in Nectria that have non-microconidial Cylindrocarpon anamorphs: Neonectria cinnamomea, Neo. jungneri, Neo. platycephala, Neo. phaeodisca and Neo. verrucospora.
Key words: forest pathology, Hypocreales, mitochondrial rDNA, Nectria, Nectriaceae, phylogeny, systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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). Closer consideration of the diversity that has been included in Nectria, along with DNA sequence analysis has shown the genus to be polyphyletic. Nectria now is restricted to its type, N. cinnabarina (Tode : Fr.) Fr., and a relatively small number of similar species (Rossman 1989, Rehner and Samuels 1994, Rossman et al 1999). Many species of Nectria sensu lato have been placed in other genera and families (Rossman et al 1999, 2001, Schroers 2002). Rossman et al (1999) and Mantiri et al (2001) resurrected Neonectria Wollenw. for former Nectria species that have Cylindrocarpon Wollenw. anamorphs. The only known teleomorphs of Cylindrocarpon are Neonectria. Mantiri et al (2001) used mitochondrial small-subunit ribosomal (mit ssu rDNA) sequences with a selected group of species to show that Cylindrocarpon, and therefore Neonectria, is monophyletic.
In the present paper we continue describing or re-describing species of Nectria s. lat. that have Cylindrocarpon anamorphs (Brayford and Samuels 1993, Samuels and Brayford 1990, 1993, 1994). We discuss species of Neonectria that have been or could have been placed in the Nectria mammoidea group of Nectria (Booth 1966) because of their perithecial wall anatomy. Throughout this paper, these abbreviations are used: N. = Nectria, Neo. = Neonectria.
Perithecia of species of Neonectria that have an N. mammoidea wall structure often arise from a characteristic stroma, here termed the N. mammoidea-type stroma. In this stromal type, compacted hyphae grow within cells of the cortex. Densely packed, discrete hyphae, ca 3.5 µm wide, arise from these stromal cells and extend perpendicular to the substratum, surrounding the perithecial base (see FIG. 51). Such a stroma is seen rarely in species that do not have a N. mammoidea wall, such as Nectria vermispora Samuels & Brayford (Samuels and Brayford 1993) and N. hypoxantha Penz. & Sacc. (Samuels 1976), neither of which has been linked to any anamorph.
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. All species included in this group were anamorphs of members of the ‘‘Nectria mammoidea Group’’. Following Booth (1959, 1966) this group is centered on N. mammoidea Phill. & Plowr., a later taxonomic synonym of N. discophora (Mont.) Mont. (= Neo. discophora [Mont.] Mantiri & Samuels). In addition to N. discophora, Booth (1966) included N. mammoidea var. rubi, N. fuckeliana, N. lucida and N. westlandica. The new species Neo. betulae, Neo. dumontii and Neo. viridispora can be added to these. Perithecia of Neo. betulae and Neo. dumontii are anatomically and biologically similar to those of Neo. discophora, but cultures of the former remained sterile and we have not had the opportunity to culture the latter. Perithecia of all of these species are red. They often are yellow or orange when young, darkening to red and brown, or sometimes near black with age and are red in KOH and yellow in lactic acid.
Members of the N. mammoidea Group most distinctively possess characteristic perithecial walls. In longitudinal section the wall is seen to comprise a layer of hyphae that have thickened walls and typically are arranged radially, giving the appearance of a palisade (Booth 1959; FIGS. 17, 24, 36, 44, 60
). This wall structure appears in squash mounts as an intertwined layer of thickened hyphal elements, rather than as a pseudoparenchymatous structure. As a result of the uniform palisade layer, the perithecia of N. discophora typically appear smooth and shining (see FIGS. 5, 6) but, in some collections or species, an additional outer layer of globose cells covering the perithecia gives a roughened, frosted or pitted appearance. The perithecial walls of N. westlandica (FIG. 14), Neo. viridispora (FIG. 13) and Neo. dumontii (FIG. 11) are warted distinctly because of additional surface layers of pseudoparenchymatous cells over the N. mammoidea palisade.
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, Samuels and Brayford 1993). The ascospores usually are smooth but often are spinulose. Typically the developing ascospores are surrounded by a thin (<1 µm) sheath, contraction of which may result in the ornamentation. Ascospores with a distinctly warted, tuberculate or striate ornamentated sheath such as found in the Neo. veuillotiana (Sacc. & Roum.) Brayford & Samuels Group (Brayford and Samuels 1993) have not been observed in taxa of the N. mammoidea Group. Discharged ascospores are typically pale yellow-brown and are usually spinulose. The remains of apical paraphyses are usually not conspicuous among mature asci.
The Cylindrocarpon anamorphs of Group 2 (the N. mammoidea Group) form round-ended, slightly arcuate macroconidia but lack microconidia and chlamydospores (Booth 1959, 1966). However, we have found some exceptions to this generalization. Neonectria betulae has a typically N. mammoidea perithecial anatomy, and colony characteristics closely resemble Neo. discophora, but cultures lack conidia, even when freshly isolated. Nectria fuckeliana is reported to produce a Cylindrocarpon anamorph with abundant microconidia (Booth 1959, 1966, Roll-Hansen 1962). This species could be assigned to the Neo. coccinea Group due to its microconidial production and colony characteristics and by the fact that perithecia of all ages occur in a single cluster (Booth 1959, 1966), but it is included here because of its N. mammoidea-like perithecial anatomy.
Taxa in this group occur on a wide range of living or recently dead, woody plant substrata, including palms, gymnosperms and dicotyledonous plants; less frequently they occur on herbaceous tissue. Neonectria discophora var. rubi is known only from Rubus species where it causes a canker. Perithecia are typically superficial, solitary or clustered on a characteristic, weakly developed basal stroma as was described above. Perithecia typically do not collapse when dry but sometimes become cupulate.
To test the monophyly of Cylindrocarpon and Neonectria we sequenced the mit ssu rDNA gene for representative species. This expands work reported by Mantiri et al (2001) for Cylindrocarpon and Neonectria.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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: 0.5 g cotton blue in 30 g lactic acid, boiled, filtered over 24 h) was used to reveal ascospore ornamentation. The three kinds of compound microscopy are abbreviated: Nomarsky differential contrast (DIC), phase contrast (PC), fluorescence (FL).
Single ascospores were isolated on cornmeal dextrose agar (CMD, Difco cornmeal agar plus 2 g/L dextrose) with the aid of a micromanipulator. Characteristics of cultures were recorded from potato-sucrose agar (PSA, Booth 1971), potato-dextrose agar (PDA, Booth 1971) and on SNAY (SNA, Nirenberg 1976, with filter paper + 0.1% yeast extract). Plates were incubated at 20 C in a 12 h light/dark cycle of daylight fluorescent strip lamps and black light tubes emitting nUV (366 nm). Colony growth was measured on 90 mm diam Petri dishes containing PSA, 5 mm deep, inoculated with a 4 mm diam mycelial plug after 10 d. Isolates were tested for chlamydospore production by incubating 10 mm square blocks of culture taken from SNAY into 10 mL of sterile water for 2–3 wk at 20 C.
The most frequently cited collectors and their cultures are abbreviated: Kent P. Dumont (K.P.D.), Richard P. Korf (R.P.K.), Amy Y. Rossman (A.Y.R.), and Gary J. Samuels (G.J.S.). Many Dumont collections were grown in pure culture from ascospores and originally were held in the culture collection of C.T. Rogerson (C.T.R., The New York Botanical Garden); those cultures now are preserved at BPI.
Color terminology follows Rayner (1970). Herbarium designations are taken from Holmgren et al (1990).
Molecular studies. –
The sources of the isolates used in this study are given in TABLE I. Among the 22 isolates sampled, nine isolates were sequenced by the authors and the sequences for the other isolates were derived from our previous studies (Mantiri et al 2001). Fungal isolates were maintained on 1.5% malt-extract agar (Difco) or potato-dextrose agar (Difco).
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). NaAc was added to a final concentration of 0.3 M before ethanol precipitation, as an exception in DNA extraction of Neo. betulae and Neo. viridispora. The precipitated DNA was pelleted by centrifugation, washed with cold 70% EtOH and resuspended in 50–200 µL TE buffer.
A portion of the mit ssu rDNA coding region was defined by primers NMS1 and NMS2 (Li et al 1994) and amplified by the polymerase chain reaction (PCR). Reactions were accomplished in a volume of 50 µL containing approximately 20–60 ng fungal genomic DNA, 200 µM each of dNTP (Perkin-Elmer, Norwalk, Connecticut), 1.5 mM MgCl2, 10 µM each primer, 1x reaction buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl) and 2.5 U Taq DNA Polymerase (Gibco BRL, Burlington, Ontario) and overlain with 50 µL light paraffin oil. Thermal cycling parameters used were an initial denaturation at 95 C for 2 min, followed by 34 cycles consisting of 95 C for 1 min, 56 C for 1 min and 72 C for 1 min and a final extension at 72 C for 4 min.
PCR products were purified with Nucleotra®PCR (Macherey-Nagel, Duren, Germany). All sequences were verified by sequencing both complementary strands using both primers NMS1 and NMS2. Most of PCR products were sequenced employing the ABI PRISMTMDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, following the manufacturer’s protocol (Applied Biosystem, Foster City, California). Some of the purified PCR products were sequenced using a Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Cleveland, Ohio) following the manufacturer’s protocol. Contiguous sequences were assembled by the ESEE software package (Cabot 1990). DNA sequences were aligned using the computer program MULTALIN version 5.4.1 (Corpet 1988) available on the internet (see http://www.toulouse.inra.fr/multalin.html) followed by manual adjustment.
The aligned sequences were analyzed using the maximum-parsimony method (MP) found in PAUP* beta version 0b10 for Windows (Swofford 2000). A branch and bound search was conducted using these settings: Addition sequence was set to furthest, and zero length branches were set to collapse to yield polytomies. Bootstrap support for internal branches was evaluated from 100 full heuristic searches using tree bisection-reconstruction branch swapping with these settings: MulTrees saved, and steepest descent option not in effect. Groups with a frequency greater than 50% were retained in the bootstrap consensus tree. Neighbor-joining analyses also were performed to explore the effects of different substitution models with transition: transversion biases set to 1:3 and 1:10. Gaps were treated as missing in all analyses.
Fusarium inflexum (GenBank U34519), Nectria cinnabarina and Calonectria sp. were chosen as outgroups because of their apparent close relationship to Neonectria (Rehner and Samuels 1995).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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The total length of the sequence alignment was 752 positions. Of these, 208 bp were variable and 113 bp (15.03%) were parsimony informative. One of the 164 most-parsimonious trees (MPTs) 357 steps in length (L) generated by branch and bound search algorithm, with consistency (CI), retention (RI) and rescaled consistency (RC) indices of 0.78, 0.75 and 0.58, respectively, is shown in FIG. 1. The topologies of the distance trees with transition:transversion biases weighted at 1:3 and 1:10 were identical to the tree with no transition:transversion bias (data not shown).
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). The ingroup consisted of three clades, designated I–III. Clade designations correlate with the Cylindrocarpon grouping based on cultural and morphological characteristics proposed by Booth (1966). Clade I (bootstrap 95%) includes four isolates (two species: Neo. neomacrospora and Neo. galligena) having relatively low sequence variation (0.2–3.8%, TABLE II). Clade II (bootstrap 95%) includes 11 species: Neo. betulae, Neo. coronata, Neo. discophora, Neo. jungneri, Neo. lucida, Neo. phaeodisca, Neo. rugulosa, Neo. trachosa, Neo. veuillotiana, Neo. viridispora and Neo. westlandica. Percent sequence dissimilarity among these species ranged from 0.9 to 7.6% (TABLE II). In this clade, the first five species mentioned above formed a subclade with ver y strong bootstrap support (100%). Sequence variation among species in this subclade ranged from 0.9 to 4.3% (TABLE II). Clade III (bootstrap 89%) includes two isolates of Neo. radicicola.
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One of the species originally included in the in-group, Nectria fuckeliana was positioned in outgroups in all analyses. This species formed a clade (with 72% bootstrap support [FIG. 2]) together with Nectria cinnabarina and Calonectria sp. The collections of N. fuckeliana that we cultured and studied are indistinguishable from the type specimen of the species in all respects. Although N. fuckeliana, a species that occurs on Abies, Larix, Picea and Pinus, is almost indistinguishable from Neo. discophora in its perithecial anatomy and morphology, it is atypical in the N. mammoidea Group. Unlike Neo. discophora and all other Neonectria species in which apical paraphyses disintegrate early and ascospores are pale tan, apical paraphyses of N. fuckeliana remain as copious narrow filaments among mature asci (see FIG. 36) and the ascospores are hyaline when discharged. We did not observe macroconidia in any of the fresh ascospore isolates from the specimens of N. fuckeliana. Rather, these isolates produced slowly growing, slimy colonies with abundant unicellular conidia from acremonium-or verticillium-like conidiophores (FIG. 33). Perithecia did not form in our cultures.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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).
This earlier work also suggested that perithecial anatomy in Neonectria is not as indicative of relationships among species as is the anamorph, a conclusion strongly supported in the present study. Once again, Cylindrocarpon Group 1 (macroconidia, microconidia, no chlamydospores), Group 2 (macroconidia, no microconidia, no chlamydospores) and Group 3 (microconidia, macroconidia, abundant chlamydospores) (respectively I, II and III in FIGS. 1, 3) of Booth (1966) are shown to be monophyletic. Species of groups 1 and 3, respectively, are homogeneous in their biology and perithecial morphology and anatomy. Although Group 2 is monophyletic (FIG. 2), there is diversity in perithecial morphology and anatomy such that representatives of the clade have been included in different groups of Nectria, viz. the Nectria mammoidea and Nectria veuillotiana Groups (Brayford and Samuels 1993).
That Nectria fuckeliana did not cluster in Neonectria was as much a surprise as its lack of Cylindrocarpon macroconidia. Cylindrocarpon cylindroides Wollenw. var. tenue Wollenw. is reported to be the anamorph of N. fuckeliana (Booth 1966, Roll-Hansen 1962), but according to Booth and Roll-Hansen many isolates do not produce macroconidia or the ability to produce macroconidia may be lost after some time in culture. Even when our isolates were fresh we did not observe macroconidia. Roll-Hansen (1962) also reported that the species produces perithecia in culture, but none formed in either of our two cultures.
A northern temperate species, N. fuckeliana, is reported to be a wound parasite of conifers and to be associated with dieback (see Booth 1966). Roll-Hansen and Roll-Hansen (1979, 1980) found that N. fuckeliana invaded wounds of Picea abies more frequently than any of the several other fungi that they surveyed but that it had little or no effect on the wood. The results of analysis of the mit ss rDNA sequences and the absence of macroconidia in our cultures lead us to question the link between N. fuckeliana and C. cylindroides var. tenue. Although N. fuckeliana is neither Nectria nor Neonectria, we prefer to maintain it in Nectria pending further work with additional isolates. The possibility that more than one species falls under what has been identified as N. fuckeliana/Cylindrocarpon cylindroides var. tenue should be considered. Resolution of this question will require additional collections from conifer forests.
The stromatic nature of perithecial formation on host tissues, with perithecia of different ages present on the same stroma, the hyaline ascospores, the colony pigmentation and microconidial anamorph characteristics of N. fuckeliana are more characteristic of Neo. coccinea, a species that produces abundant microconidia, than of Neo. discophora. However, perithecial anatomy in N. fuckeliana shows strong similarity to that of Neo. discophora and its relatives. Nectria fuckeliana therefore may form a link between the Nectria coccinea and Nectria mammoidea Groups (sensu Booth 1959, 1966) and indicate that there is no clear-cut demarcation between the two groups. Indeed, the other Nectria groups with Cylindrocarpon anamorphs, viz. the Neo. veuillotiana (Brayford and Samuels 1993) and Neo. rugulosa (Samuels and Brayford 1994) groups form a morphologically intergrading assemblage of taxa. The only clear discontinuity is with the N. radicicola Group (Samuels and Brayford 1990), which has clearly different perithecial anatomy, more similar to that of Calonectria de Not. than to the other Neonectria/Cylindrocarpon groups.
Although the N. mammoidea perithecial anatomy is distinctive and most species that possess the anatomy are closely related species of Neonectria, the phylogenetic importance of the wall anatomy is evidently limited. In addition to N. fuckeliana, this anatomy also is found in Calostilbe striispora (Ellis & Everh.) Seaver (anamorph = Calostilbella calostilbe Höhn.), a species that is not obviously related to Neo. discophora (see Rossman et al 1999).
Teleomorphs of Neo. discophora var. discophora and var. rubi, and Neo. lucida are indistinguishable (cf. FIGS. 6 and 11). Neonectria discophora and N. lucida can be distinguished only when their anamorphs are known. Cultures of Neo. discophora are purple, while those of Neo. lucida are white to tan. DNA sequence analysis presented here indicates that Neo. discophora and Neo. lucida are distinct but closely related, thus supporting our decision to separate the two taxonomically.
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TAXONOMY KEY TO NEONECTRIA SPECIES HAVING NONMICROCONIDIAL CYLINDROCARPON ANAMORPHS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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DESCRIPTIONS OF THE SPECIES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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. Perithecia globosa, 350–475 µm diam, glabra, rubra. Cellulae superficiales perithecii globosae, 15–20 µm diam. Asci anguste clavati, 100–135 x 10–15 µm, apice anulo refractivo instructi. Ascosporae ellipsoideae vel fusiformes, (19–) 21–24(–26) x 7–8 µm, bicellulares, minutae spinulosae, hyalinae vel pallide luteo-brunneae. Culta in agaro PDA lente crescit, mycelio aerio albo, reversum purpureum. Conidia absunt.
HOLOTYPUS: SCOTLAND. Argyllshire: Cowall Peninsula, Argyll Forest Park, N end of Loch Goil, vicinity of Locqoilhead, along trail to Rob Roy’s Cave, elevation 20–100 m, on bark of Betula sp., 12 Apr 1992, leg. D. Brayford & G.J.S. (8012) (G.J.S. culture 92-47: BPI 802663, IMI 352566).
Etymology: Betulae refers to the host genus, Betula.
Anamorph: None formed in culture.
Mycelium not visible on host. Perithecia solitary to gregarious in groups of a few, superficial, globose, 350–475 µm diam, nonpapillate, apex with a dark, areolate area 90–135 µm diam; dark red with ostiolar area near black, uniformly dark red in 3% KOH, yellow in 100% lactic acid, not collapsing when dry, smooth, appearing slightly pitted. Cells at surface of perithecial wall circular, 15–20 µm diam with walls 1 µm thick. Perithecial wall 40–50 µm wide, comprising three regions: outer region a single layer of circular, 15 µm diam cells with walls 1 µm thick, unpigmented; middle region ca 15 µm wide, of intertwined, branched, ca 3.5 µm wide hyphal elements arranged ± perpendicular to the perithecial surface, walls ca 1.5 µm thick, pigmented; inner region 7–10 µm wide, cells adjacent to the middle wall region intertwined, with small, ± elliptic lumina, walls ca 1.5 µm thick, pigmented, cells of this region becoming progressively more fusiform, compressed; walls thinner, unpigmented toward the perithecial locule. Perithecial apex a disk formed of vertically oriented hyphal elements with rounded tips, gradually merging with the periphyses and continuous with the middle region of the perithecial wall. Asci narrowly clavate, 100–135 x 10–15 µm, apex with a conspicuous refractive ring, 8-spored. Ascospores ellipsoid to fusiform, (19–)21–24(–26) x 7–8 µm, 1-septate, not constricted at the septum, finely spinulose, with a 1 µm wide sheath, colorless or very pale yellow-brown, uniseriate with overlapping ends, filling each ascus.
Characteristics in culture. – Colonies on PSA slow growing, 20 mm diam after 10 d, aerial hyphae white, cottony, colony surface violet; reverse with purple diffusing pigment. Cultures remaining sterile. Colonies grown on autoclaved strips of Betula bark also lacking conidia.
Habitat: Bark of Betula sp.
Distribution: Scotland.
Commentary: Although a Cylindrocarpon anamorph did not form in ascospore isolates of these specimens, there is little doubt that this species belongs to the Nectria mammoidea Group due to its perithecial wall anatomy and violaceous pigment in PSA cultures. The N. mammoidea-type of anatomy of the perithecial wall is difficult to infer from crush mounts because of the superficial layer of circular cells but is suggested by the general perithecial form. This species is distinctive in the group because of its large ascospores and because of its apparent lack of anamorph.
2. Neonectria discophora (Mont.) Mantiri & Samuels, Can. J. Bot. 79:339. 2001 var. discophora FIGS. 5, 6, 32
Sphaeria discophora Mont., Ann. Sci. Nat. Bot. II 3: 353. 1835. Creonectria mammoidea (W. Phillips. & Plowr.) Seaver, Mycologia: 188. 1909.
Anamorph: Cylindrocarpon ianthothele Wollenw. var. majus Wollenw., Z. Parasitenk. (Berlin) 1:161. 1928. FIG. 32
Mycelium not visible on host. Perithecia solitary to gregarious or cespitose in groups of a few, superficial or with base partially immersed in substratum on a minute stroma, stroma at first producing conidia, having long, unbranched hyphal hairs, often in fissures in bark, globose, (260–)300–550(–670) µm diam, nonpapillate or with a broad, mammiform apex 150–200 µm diam, not collapsed when dry; red to dark red and appearing almost black, smooth, often shining; ostiolar area often darker red than the perithecial body, uniformly dark red in 3% KOH, yellow in 100% lactic acid. Cells at surface of perithecial wall lacking definite outline, appearing to be intertwined hyphae with lumina irregular in shape, 2–2.5 µm in greatest dimension, walls 2–4 µm thick. Perithecial wall 30–50 µm wide, of two intergrading regions; outer region 20–30 µm wide, continuous over the perithecium to form a uniform palisade of hyphal cells, cells distinctly hyphal, perpendicular to surface of perithecium, lumina <1 µm wide and tips rounded, walls 2–2.5 µm thick; inner region of perithecial wall 10–20 µm wide, cells lacking a definite outline but with long axis parallel to surface of perithecial wall, wall of cells 2.0–2.5 µm thick, cells increasingly more compacted, thin-walled toward the perithecial locule; perithecial apex of vertically elongated cells, continuous with the lateral perithecial wall, forming a disk around the ostiolar opening, cells increasingly narrower and merging with the periphyses within. Stroma arising from cortex of host, cells angular to circular in outline, ± vertically oriented, walls 2–2.5 µm thick, continuous with cells of outer region of perithecial wall. Asci cylindrical, (68–)72–95(–120) x 7–10(–15) µm, apex with a refractive ring, 8-spored. Ascospores ellipsoidal, (10–)12–17(–20) x (4–)5–8(–9) µm, equally 2-celled, not constricted at the septum, spinulose, light brown, uniseriate with overlapping ends, filling each ascus.
Characteristics in culture. – Colonies 20–30 mm diam after 14 d at 20 C on PSA. Colony surface slimy; aerial mycelium sparse or floccose, initially white, darkening to blood/bay or dark violet. Colony reverse strongly pigmented blood color or purpleslate with pale vinaceous margin. Slimy sporodochia usually abundant on colony surface and in aerial mycelium, often forming in concentric rings. On SNA or SNAY growth sparse, lacking pigment, white/buff sporodochia forming on agar surface. Conidiogenous cells formed apically on densely, irregularly branching clusters of cells borne laterally on broad, thick-walled, pale brown vegetative hyphae. Conidiogenous cells cylindrical, 12–18 x 3–4 µm, with apical thickening and collarette. Macroconidia (2–)3–5(–7) septate, 43–73 x 4–6 µm, cyindrical, but often broader in the upper half, uniformly cur ved or more strongly curved at the tip, with apical cell rounded and basal cell rounded to truncate. Microconidia sparse or absent. Chlamydospores not formed. Most strains developing fertile perithecia in single-spore cultures, suggesting homothallism; this ability may decline in old isolates.
Habitat: Bark of twigs and branches of recently dead trees, less frequently on palm trunks.
Distribution: Cosmopolitan.
Illustrations: Wollenweber (1930a, FIG. 536; 1930b, FIG. 742, as N. mammoidea). Booth (1966, FIG. 16, as N. mammoidea). Kar and Gupta (1977, FIG. 2, as N. mammoidea). Dennis (1978, Pl. 33 C, as N. mammoidea).
HOLOTYPE: CHILE. Juan Fernandez, sur cortice arborum, date unknown, Bertero 1700 (PC!; ?ISO-TYPE, PC!).
Additional representative specimens examined (out of ca 70): AUSTRALIA. TASMANIA: locality unknown, [on bark], Archer (K, HOLOTYPE of N. tasmanica; PAD, ISOTYPE of N. tasmanica). VICTORIA: Otway Ranges, vicinity of Lorne, Angahook-Lorne State Forset, along track from Blanket Leaf Picnic ground to Cora Lynn Cascades, along Cora Lynn Creek, elevation 200–350 m, primarily Eucalyptus forest, on twigs of recently dead tree, 28 Aug 1999, G.J.S. 8650 (BPI, culture G.J.S. 00-71); Tarra Valley, Tarra-Bulga National Park, between Yarram and Traralgon, Tarra Rainforest Walk, on bark of recently fallen Eucalyptus sp., 22 Aug 1999, G.J.S. 8561 (BPI 746774). BRAZIL. SANTA CATHARINA: Blumenau, (on decorticated wood), date not known, A. Moeller (S, herb Sydow; HOLOTYPE of Nectria umbilicata). ‘‘CENTRAL AMERICA.’’ isolated from soil and inoculated onto apple ‘‘faule’’, 1934/1935, S5360 (B ex BBA, probable HOLOTYPE of N. mammoidea var. minor). VENEZUELA. ARAGUA: forests at Rancho Grande, on deadwood, elevation 1200 m, 30 Apr 1938, C.E. Chardón 2617 (CUP, HOLOTYPE of Creonectria discostiolata). ENGLAND. CAMBRIDGESHIRE: Brandon, on Pinus sylvestris stump, 16 Oct. 1958, J. Rishbeth (IMI 074992). DEVON: Dartmoor Forest, on Picea sitchensis, 25 Jul 1975, C.C. Gulliver (IMI 195455). SUSSEX: Dunford House, Midhurst, on Pinus sp., 25 May 1959, C. Booth (IMI 076785). YORKSHIRE: Clapham, Larix sp., 3 May 1963, C. Booth (IMI 100408); Buttercombe Woods, Stamford Bridge, on Pinus sp. cone, 17 Sep 1961, W.G. Bramley (IMI 089845 Thornton Dale, on Pinus laricis, 21 May 1955, W.G. Bramley [IMI 062653]); Cloughton Woods, Yorkshire, ?Pinus sp., 17 Apr. 1955, J. Webster & C. Booth (IMI 059891). FRANCE. PYRENÉES ATLANTIQUES: forest vicinity of Laruns, on ?Corylus avellana, 26 Aug 1978, G.J.S. 78-67 & G. Roux (PDD 38803); Isle de la Sauveterre de Bearn, elevation 100 m, on Populus nigra, 25 Oct 1998, G.J.S. & F. Candoussau (BPI 748310, culture G.J.S. 98-132). GUYANA. Cuyuni-Mazaruni Region, VII; Mazaruni Subregion, VII-2, along Koatse R, ca. 2 km E of Pong R., ca. 5 h walk S of Chinoweing Village, 05º28'N, 60º04'W, elevation 600–650 m, on terminal branchlets of recently dead tree, 28 Feb 1987, G.J.S. et al 4952C (G.J.S. culture 87-49: BPI 747057, BRG, NY); Base of Mount Wokomung, ca. 5.5 km walk NE of Kopinang Village, in legume-dominated forest, 05º05'N, 59º49'W, elevation 720 m, on bark of recently fallen tree, 27 Jun 1989, G.J.S. et al 6269a (G.J.S. culture 89-57: NY). INDONESIA. NORTH SULAWESI: Eastern Dumoga-Bone National Park, at confluence of Toraut and Tumpah rivers, vicinity of Project Wallace Base Camp, 00 º34'N, 123º57'E, 211 m, on twig, Oct–Nov 1985, G.J.S. 2222A (G.J.S. culture 85-179, NY, IMI 329113); Gn. Muajat, Danau Alia, 00º45'N, 124º25'E, 1400 m, on bark, 26 Oct. 1985, G.J.S. 2399 (NY), 2421A (BO, NY). JAMAICA: Border between Portland and St. Andrew parishes: Traveler’s Rest, Silver Hill Gap, 25 mi marker from Kingston, on wood, 8 Jan 1971, R.P.K. et al CUP-MJ 726 (CUP, NY); PORTLAND PARISH: along trail to Silver Hill Gap, near Woodcutter’s Gap, vicinity of Newcastle, on wood, 9 Jan 1971, R.P.K. et al CUP-MJ 760 (NY). NEW ZEALAND. AUCKLAND: Waitemata City, Waitakere Ranges, Anawhata Road, Chateau Mosquito Track, on bark of ?Hoheria populnea, 30 Oct 1973, J.M. Dingley et al (PDD 32631); Piha Road, Cowan Track, on Ripogonum scandens, 4 Jun 1983, G.J.S. 83-125 & A.Y. Rossman (PDD 44247). BAY OF PLENTY: Whakarewarewa, ex Pinus radiata, Sep 1949, G.B. Rawlings (HOLOTYPE of N. pinea, PDD 7510; ISOTYPE IMI 050395). COROMANDEL: Thames County, along Kauaeranga R., NE of Thames, on bark of Beilschmiedia tawa, 8 Mar 1973, J.M. Dingley et al (PDD 30629). GISBORNE: Urewera National Park, Lake Waikaremoana, track to Lake Waikareiti, on Nothofagus sp., G.J.S. 85-62 & L.M. Kohn (PDD 50072). NORTHLAND: Hokianga County, Waipoua Forest, vicinity of Te Matua Ngahere kauri tree, on Freycinetia baueriana subsp. banksii, 31 May 1982, G.J.S. 82-93 et al (PDD 43190, IMI 329104). WAIKATO: Waitomo, on bark, 26 Apr 1983, G.J.S. 83-206 et al (PDD 46410, IMI 326258). WESTLAND: Mount Aspiring National Park, Haast Pass, ca 30 km E of Haast Junction, Roaring Billy Forest Walk, on bark of ?Podocarpus sp., 28 Apr 1985, G.J.S. 85-35 & L.M. Kohn (PDD 50047); Franz Josef, track to Lake Wombat, on bark of Fuchsia excorticata, 10 Apr 1983, G.J.S. 83-188 & R.H. Petersen (BPI 1109329, PDD 46365). SCOTLAND. ARGYLLSHIRE: Cowal Peninsula, Cowal Peninsula, Argyll Forest Park elevation 50–100 m, ca 10 km N of Dunoon, on branchlets of Larix sp., 11–13 Apr 1992, G.J.S. & D. Brayford 8007 (G.J.S. culture 92-36: BPI 802651); Benmore Estate, on Pseudotsuga menziesii bark and wood of stump, 30 Mar 1935, J. Ehrlich & C.J.C. Chesters, 30.iii.0935, (IMI 052091, Herb. J. Ehrlich 1667); on Pinus ?sylvestris bark of log, 30 Mar 1935, J. Ehrlich & C.J.C. Chesters (IMI 022815, Herb. J. Ehrlich 1669); Benmore Forestry Estate, near Dunoon, on Picea excelsa log, 5 Apr 1935, J. Ehrlich & C.J.C. Chesters (IMI 217845); Kincardine, Maryculter, Pinus sylvestris, 13 Sep 1979, D.W. Minter (IMI 256525). PERTHSHIRE: Braco, wood on Crieff Road, on Picea excelsa log, 31 Mar 1935, J. Ehrlich & C.J.C. Chesters (IMI 217844). THAILAND. Khao Yai National Park, vicinity of Park Headquarters, trail, on bark of recently dead tree, 2 Aug 1997, G.J.S. 8266 & P. Chaverri (G.J.S. culture 97-66, BPI 745816; BioTek, Bangkok). UNITED STATES. NORTH CAROLINA: Great Smoky Mountains National Park, on bark of ?Quercus sp., 14 Oct 1990, G.J.S. 90-46 & A.Y. Rossman (BPI 1107126); Jackson County, Nantahala National Forest, Bull Pen Road to Chattoga Run, Ellicott Rock Trail from Fowler Creek, on recently dead Acer rubrum, 28 Sep 1989, G.J.S. 89-132 et al (BPI 1107267, IMI, NY). PENNSYLVANIA: Mercer County, Grove City, in former city dump at foot of Gilmore Street, on Acer sp., 14 Oct 1972, G.J.S. (C.T.R. culture 72-372, NY). VENEZUELA. ARAGUA: Forests at Rancho Grande, elevation 1200 m, on deadwood, 30 Apr 1938, C.E. Chardón 2617 (CUP, HOLOTYPE of Creonectria discostiolata); Parque Nacional Henri Pittier, Parque Nacional Henri Pittier, Rancho Grandé Biological Station, trail to Guacamayo, ca 10º21'N, 67º41W, elevation 1250–1400 m, 4 Dec 1990, on bark, G.J.S. et al 7933 (G.J.S. culture 90-212: BPI 842123, NY, VEN). DISTRITO FEDERALE: ca 13 km NE of Colonia Tovar on road between Colonia Tovar and El Tigre, on bark, 29 Jul 1972, K.P.D.-VE 6531 et al (C.T.R. culture 72-168: NY); vicinity of El Portachuelo, NE of Colonia Tovar, on bark of tree, 1972, K.P.D.-VE 6618 et al (Rogerson culture 71-187: NY). MERIDA: Parque Nacional Sierra Nevada, above Tabay, Qda. Coromoto, La Mucuy 08º36'N, 71º02'W, elevation ca 2000 m, on bark, 20, 23 Nov 1990, G.J.S. et al 7244A (G.J.S. culture 90-155: BPI, VEN). MONAGAS: SE of Caripe, Hacienda Las Acacias, La Carmelita, on stem of Cecropia sp., 17 Jul 1972, K.P.D.-VE 5253 et al (C.T.R. culture 72-147: NY). SUCRE: NW of Irapa, trail between Los Pocitos and the peak of Cerro Humo, on palm, 12 Jul 1972, K.P.D.-VE 4769 et al (C.T.R. culture 72-90: NY). TRUJILLO: Parque Nacional Guaramacal, ca 10 km SW of Batatal, La Defensa, along Rio Saguas, Campamiento Granja Bocono, in forest along trail to water source, 09º18'N, 70º10'W, elevation 2000 m, on palm fruit, 20, 23 Nov 1990, G.J.S. et al 7447 (BPI 744858, VEN). YARACUY: N of Nirgua, in mountains, on palm, 7 Jul 1971, K.P.D.-VE 1536 et al (NY, VEN).
Commentary: As discussed previously (Samuels et al 1990), the better known name N. mammoidea is a later taxonomic synonym of Neo. discophora.
Booth (1966) described the Cylindrocarpon anamorph of Neo. discophora (as N. mammoidea) and also recognized several varieties of the species. Of these, we accept only var. rubi (see below).
3. Neonectria discophora var. rubi (Osterw.) Brayford et Samuels, stat. nov. et comb. nov.
Nectria rubi Osterw., Ber. Deutsch. Bot. Ges. 29:620. 1911. Hypomyces rubi (Osterw.) Wollenw., Phytopathology 3: 224. 1913. Anamorph: Cylindrocarpon ianthothele var. ianthothele
Wollenw., Ann. Mycol. 15:56. 1917.
Perithecia morphologically and anatomically indistinguishable from those of Neo. discophora var. discophora, formed on cankers at the roots and crowns of diseased Rubus canes; ascospores ellipsoid, 1-septate, (11.5–)12–16(–23) x (4.5–)5.5–6.5(–8.0), becoming pale brown, finely spinulose at maturity.
Characteristics in culture: Colonies on PSA 15–35 mm diam after 14 d at 20 C. Aerial mycelium floccose, white or honey-colored, becoming dark purple. In some strains, aerial mycelium sparse or absent, colonies slimy. Colony reverse-pigmented dark vinaceous to vinaceous, in some cases unpigmented or with unpigmented sectors. Abundant slimy sporodochia formed on the agar surface and in the aerial mycelium; sporodochia buff, pale violet or dark purple, often forming in concentric rings. In some strains fertile ascocarps developing, especially at the colony margins of mature cultures. On SNA or SNAY hyphal growth diffuse and spreading through the agar, unpigmented, with scattered buff sporodochia forming on the agar surface. Conidiogenous cells cylindrical, 10–25 x 2.0–3.5 µm, with apical thickening and collarette, formed apically on irregularly branching clusters of cells, often borne on broad, thick-walled hyphae. Macroconidia curved, fusoid, 3–5 septate, 36–70 x 4–7 µm, with rounded apical and basal cells. Sparse microconidia occasionally found but normally absent. Chlamydospores absent (but illustrated by Wollenweber 1926, from natural substratum).
Illustrations: Wollenweber (1926: 59, 526, 527). Booth (1966: FIG. 17).
Habitat: Associated with cankers on roots and crowns of Rubus idaeus and R. fruticosus.
Distribution: England, New Zealand, Northern Ireland, Scotland, Switzerland, Venezuela.
Specimens Examined: ENGLAND. locality and date unknown, on Rubus sp, Herb. J. Ehrlich 1518 (IMI 22841). KENT: on Rubus idaeus cv. Glen Clora, 13.v.1983, G. Thorpe (IMI 277659). BATHEASTON: on Rubus fruticosus, iv.1870, Herb. Broome (IMI 52177). SURREY: Merstham, host unknown, viii.1928, R.V. Harris (IMI). NORTHERN IRELAND. locality unknown, on Rubus idaeus, 10.viii.1984, comm. R.S. McIlwaine (IMI 288100). SCOTLAND. Loch Lomond, Inchcailloch, on Rubus fruticosus, R. Watling (IMI 180609). SCOTLAND. Locality unknown, 6.x.0973, collector unknown, (IMI 52183, Herb. J. Ehrlich 1642, perithecia on oat agar, ex Rubus idaeus); locality unknown, on Rubus idaeus, McIntosh, det. N.A. Alcock (IMI 52181, Herb. J. Ehrlich 1640); INVERNESSHIRE: Beauly, Beaufort Castle, Rubus idaeus, 9 Oct 1925, A. Alcock (IMI 324717), PERTHSHIRE: Alyth, on Rubus idaeus cv. Glen Moy, 17 May 1988, B. Williamson, (IMI 325503); Tayside, Kimimuir, Angus, Path Head Farm, on Rubus idaeus cv. Glen Moy, 10 Jun 1988, B. Williamson (IMI 325504); Angus, Newton Park Farm, Rubus idaeus cv. Glen Moy, 10 Jun 1988, B. Williamson (IMI 325505, IMI 325506, IMI 325507). NEW ZEALAND. NELSON: Tapawera, Rubus idaeus, 9 Jan 1949, P.R. Fry (IMI 50403); Tapawera, on Rubus idaeus, 1956, J.M. Dingley (IMI 70017).
Cultures studied (all ex Rubus idaeus): UNITED KINGDOM. ENGLAND: location unknown, R.M. Nattrass (IMI 113917 = CBS 177.27); Plymouth, Wembury, 20 Jun 1978, A.U. Chapman (IMI 229976, 288100, 324717, 325503, 325504, 325505, 325506, 325507, 325509). SCOTLAND: location unknown, 1929, H.W. Wollenweber (IMI 113919, CBS 241.29). SWITZERLAND. 1911, A. Osterwalder (IMI 113918).
Commentary: This fungus has been associated with a distinctive basal canker of cultivated Rubus sp. on several occasions in Europe since 1911 (Brayford 1991). The host specificity and pathogenic status of the fungus remain unclear, but the recent disease outbreaks in Scotland have been correlated with wind damage to the hosts followed by waterlogging. This suggests that the fungus is either a secondary pathogen or only able to cause disease on severely stressed host plants. There is overlapping in the respective anamorphs of Neo. discophora var. discophora and var. rubi, but the tendency in strains from Rubus is to have smaller ascospores, smaller macroconidia and slower colony growth than those of the type variety. Due to the association of var. rubi with a characteristic plant disease, which is unusual for species in this Neonectria group, and in the absence of DNA sequences for var. rubi, we maintain it as a distinct taxon.
Weese (1912) was the first to recognize a relationship between this taxon and Nectria mammoidea (Neo. discophora).
4. Neonectria dumontii Brayford et Samuels, sp. nov. FIGS. 7, 27–31
Perithecia solitaria vel gregaria, globosa, 750–1250 µm diam, areolata vel discoidea, rubra, stromati immerso insidentia. Asci anguste clavati, (157–)161–238(–250) x (11.7–)13.7–20.0 µm, anulo apicali refractivo praeditii. Ascosporae fusiformes, (27.0–)30.7–35.2(–36.0) x (5.5–)6.5–7.7(–8.0) µm, 1-septatae, laeves, hyalinae vel pallide bubalinae. Status anamorphicus ignotis.
HOLOTYPUS: In Colombia, K.P.D.-CO 7987 (NY).
Etymology: Refers to Kent P. Dumont, formerly of the New York Botanical Garden, in recognition of the many collections of the Hypocreales that he made during his career.
Anamorph: not known.
Mycelium on host not visible. Perithecia solitary or gregarious to caespitose in clusters of 4–5, superficial, seated on an immersed stroma with stromal cells growing within cortical cells of host, globose, 750–1250 µm diam, perithecial apex with a flattened disk, often appearing areolate, not collapsing when dry, red, with a darker red apex, uniformly dark red in 3% KOH, yellow in 100% lactic acid; smooth or minutely roughened to scaly. Cells at surface of perithecial wall circular, 20–30 µm diam, walls 2–3 µm thick. Perithecial wall 65–90 µm wide, comprising three intergrading regions: outer region, to 30 µm wide, including scales, consisting of one or two layers of circular, cells with lumina 15–25 µm diam, walls ca 2.5 µm thick and pigmented; middle region ca 35 µm wide consisting of highly intertwined elements with walls ca 4 µm thick and meandering lumina; inner region 20–30 µm wide, cells with elliptic to fusoid, 5–20 µm long, 2–3 µm wide lumina and walls 1.5–2 µm thick, pigmented in cells at the exterior, unpigmented in cells toward the locule. Perithecial apex formed of vertically elongated hyphal elements with walls ca 2.5 µm thick; elements continuous with the middle region of the perithecial wall and protruding through the outer region, becoming narrower and merging with the periphyses within. Asci narrowly clavate, (157–)161–238(–250) x (11.7–)13.7–20.0 µm, apex with a refractive ring; 8-spored, ascospores biseriate above, uniseriate below, filling each ascus. Ascospores fusiform, (27.0–)30.7–35.2(–36.0) x (5.5–) 6.5–7.7(–8.0) µm, with one median septum, not constricted, smooth, colorless or very light tan, sometimes appearing to have a narrow sheath.
Habitat: Wood, tree fern rachis.
Distribution: Colombia (Boyacá, Cundinamarca). HOLOTYPE: COLOMBIA. DEPARTAMENTO BOYACÁ: Along Tunja-Ramiriquí-Páez Road, vicinity of kilometer post 20 from the crossing of the Tunja-Nueva Colón Road, on wood, 14 Sep 1976, K.P.D.-CO 7987 Buriticá & Umaña (NY, isotype IMI 352114).
Additional collection: COLOMBIA. DEPARTAMENTO CUNDINAMARCA: Between kilometer posts 29–30 from Zipaquirá, on the Zipaquirá-Pacho Road, elevation ca 10 000 ft, on tree fern, 9 Jun 1976, K.P.D.-CO 4491 et al (NY, IMI 352115).
Commentary: This species is morphologically similar to Neo. westlandica but differs in having larger ascospores and a poorly developed N. mammoidea palisade in the lateral wall. Neonectria westlandica is found in New Zealand, whereas Neo. dumontii is known only from Colombia. We have not had an opportunity to grow this species in pure culture but predict that its anamorph will be a species of Cylindrocarpon.
5. Nectria fuckeliana C. Booth, Mycol. Pap. 73:56. 1959. FIGS. 8, 9, 33–40
Nectria abietis Sacc. [ut Sac.] in Roumeguère, Rev. Mycol. (Toulouse) 3:23. 1 Apr 1881, nom. nud.
Anamorph: reported to be Cylindrocarpon cylindroides Wollenw. var. tenue Wollenw., Zeitschr. Parasitenk. 1:153. 1928. FIG. 33
Mycelium not visible. Perithecia in cespitose clusters of few to many, often with developing and mature perithecia in the same cluster, superficial on an erumpent and often extensive orange stroma, often with perithecia scattered on the stroma, globose, (235–)276–346(–404) µm diam, nonpapillate; red to dark red with ostiolar area sometimes darker and ostiolar area of young perithecia appearing slightly umbilicate and viscid, uniformly red in 3% KOH and yellow in 100% lactic acid, not collapsing when dry; smooth and oftening shining. Cells at surface of perithecial wall lacking a definite outline, rather appearing as intertwined hyphae with thickened walls and circular lumina when seen in optical section. Perithecial wall (G.J.S. 8014) 75 µm wide at the widest part, near the perithecial apex, comprising three regions; outer region 50–60 µm wide, of 6 µm wide hyphal elements with walls 1.5 µm thick and lumina 1 µm wide arranged ± perpendicular to the perithecial surface in a N. mammoidea palisade; middle region ca 15 µm wide, of intertwined hyphal elements; elements in section appearing ± elliptic with lumina ca 7.5 µm long and 3 µm wide, walls 1.5–2 µm thick and pigmented, intergrading to the outer and inner regions; inner region of elongated, fusiform cells 10–15 µm long x 3–6 µm wide and walls 1.5–2 µm thick and pigmented, becoming progressively thinner and unpigmented toward the perithecial locule. Perithecial apex formed of a central disk ca 100 µm diam of narrow elements; elements arising from the inner region of the perithecial wall, merging with periphyses; perithecial apex surrounded by hyphal elements of the outer region of the perithecial wall. Cells of the stroma distinctly pseudoparenchymatous, 15–20 x ca 10 µm, walls ca 3 µm thick, pigmented, merging with the middle region of the perithecial wall at the perithecial base. Asci cylindrical to narrowly clavate, (80–)86–100(–120) x 7–11 µm, apex with a refractive ring but frequently appearing simple; 4–8-spored, ascospores uniseriate with overlapping ends to biseriate and clustered in the upper half of the ascus. Ascospores ellipsoidal, (11.0–)12.5–15.5(–19.7) x (4.0–)4.7–6.0(–7.0) µm, 1-septate, septum median, spinulose to smooth, hyaline to indistinctly yellow brown. Pseudoparaphyses persisting among mature asci as ca 3 µm wide, branched and anastomosing filaments with no free ends.
Characteristics in culture: Colonies on PSA 15–30 mm diam after 10 d, irregular or spidery in form, with white/buff aerial mycelium, sometimes sparse, the colony being slimy due to abundant sporulation in small droplets and pustules. Colonies weakly pigmented sienna (pale brown) or rust near the inoculum block but mostly white to buff; reverse with diffuse luteous pigment fading to white at the colony margin. No odor apparent. Red stromatic structures, possibly protoperithecia, present in some strains. On SNAY colonies spidery and spreading, with sparse aerial mycelium but abundant sporulation in slimy droplets. Conidiogenous cells scattered, sessile or on sparsely branched cells, often branching at right angles, cylindrical or tapering slightly, mostly 30–70 x 1.5–3.5 µm, with a single apical locus, periclinal thickening and indistinct collarette. Total length of conidiogenous cell and supporting hyphae might be >100 µm. Microconidia abundant, in slimy droplets throughout the colony, hyaline, 0(–1) septate, ellipsoidal to cylindrical, sometimes clavate, with indistinct basal abscission scar, mostly 3–7 x 2–4 µm. Macroconidia and chlamydospores not observed.
Illustrations: Richter and Gerlach (1958), Roll-Hansen (1962), Booth (1966, 1979).
Habitat: On bark of conifers including Abies, Larix, Pinus and Picea.
Distribution: Circumboreal.
Type: ‘‘Oestereich (Nassau)’’: ‘‘ad Pinorum ramos aridos’’, Fuckel (Herbier Barbey-Boissier 855 ex Fungi rhenani 983 as Nectria cucurbitula, ISOTYPES: NY! BPI 551647!).
Additional specimens examined: BOHEMIA. on Abies picea, 1912, J.E. Kabát (Fungi bohemici, BPI 551629); on Abies excelsa, Nov 1908, F. Bubák (Fungi bohemici, BPI 551626). CANADA. NEW FOUNDLAND: Bay of Islands, Coal River, on Abies, 14 Feb 1886, the Rev. A.C. Waghorne (BPI 551622). EUROPE 5 Sep 1925, on dead bark of Pinus sp., H.W. Wollenweber 7541 (ex herb J.H. Faull ‘‘cause of decay of various conifers’’, BPI 551645). AUSTRIA. Niederösterreich: Sonntagberg vicinity of Rosenau, on Abies, Jun [year unknown], P.P. Strasser (Kryptogamae esx. 965, BPI 551620). SILESIA: Carlsbrünn, on bark of Abies, Aug 1911, Niessl (BPI 551621). FRANCE. ‘‘Bas porte du Rhone a Lyon’’, on bark of ‘‘Abies pectinata’’, Jul 1880, J. Therry (C. Roumeguère, Fungi Gallici exsiccati 1292 N. cucurbitula f. abietis; BPI, CUP, FH, PC) GERMANY: ‘‘Harz im Walde zwischen Braunlage und Schierke’’, on bark of Abies excelsa, 27 Aug 1904, P. Sydow (Sydow, Mycotheca germanica 323: BPI); ‘‘Botanischen Garten’’, on cut branch of Picea excelsa, Apr 1883, P. Sydow (Sydow, Mycotheca Marchica 472, as Nectria cucurbitula: BPI); ‘‘Marchia: Hortus Berolinensis’’, on bark of Picea excelsa, May, P. Magnus (Rabenhorst, Fungi europaei et extraeuropaei 4258, as Nectria cucurbitula: BPI); Bergisches Land: Waldbröl., on Picea branches, 11 Sep 1931, A. Schumacher (Flora des Rheinlandes 575, BPI 551618); Berlin, on Picea excelsa, 20 Mar 1883, P. Magnus (BPI 551634, as N. cucurbitula). Saxony: ‘‘Neckendorfer Thal pr. Islebiam’’, on bark of Picea excelsa, Apr 1875, J. Kunze ( Johs. Kunze, Fungi selecti exsiccati 105, as ‘‘Nectria cucurbitula f. Piceae excelsae [Poir.] Link’’: BPI 551636). Frankfurt am Main, on deadwood and bark, date not known, Auerswald (BPI 551617); Franconia: vicinity of Sugenheim, on bark of pine, date not known, Rehm (Rabenhorst, Fungi europaei 1235 as Nectria cucurbitula: BPI); Harz: im Walde zwischen Braunlage und Schierke, on bark of Abies excelsa, 27 Aug 1904, P. Sydow (Sydow, Mycotheca germanica 323, BPI 551627); Westfalia: Kr. Siegen, Wald am Tunnel bei Burgholdinghausen, on Picea excelsa, 30 Apr 1952, A. Ludwig (BPI 551635). NEW ZEALAND. DUNE-DIN: Tokoiti Forest, on Pinus radiata, 22 Jul 2002, M. Dick (BPI; culture NZFS 891 = G.J.S. 02-60); second collection, NZFRI-M 4613, 21 Nov 2001, R. Thun (BPI 842427, culture G.J.S. 02-61 = NZFS 839). SOUTHLAND: Dipton Forest, on Pinus radiata, 20 Nov 2001, M. Dick (NZFRI 4814, BPI, culture G.J.S. 02-67. SCOTLAND. Glenbriers Forest, on Picea excelsa, 27 Mar 1935, J. Ehrlich & N. Wilson 1675 (PDD 11626 ex IMI 022814). Cowal Peninsula, Argyll Forest Park, N end of Loch Goil, vicinity of Locgoilhead along trail to Rob Roy’s Cave, elevation 20–100 m, 12 Apr 1992, on trunk of Abies sp., D. Brayford & G.J.S. 8014 (G.J.S. culture 92-46: BPI 802662, IMI 352568); second collection, on trunk of recently fallen Larix sp., G.J.S. 8016 (BPI 802657, IMI 352570; culture G.J.S. culture 92-42). Pebbleshire, on Picea sitchensis, Apr 1926, A.E.S. McIntyre (BPI 551642). SWEDEN. Scania, Boekeberg, on Picea excelsa stump, 24 Apr 1934, Ehrlich & Kemner (BPI 1113363 ex K 52084); Uppsala, on conifer stump, 22 Jun 1912, C.L. Shear (BPI 550780). VÄSTERBOTTEN: Umea par., between Yytteröda and Heleneborg, mixed forest near the road to Vindeln, on Picea abies bark, 27 Oct 1975, O. Eriksson 751027-1 (BPI 745076 ex UME 27078). SWITZERLAND. KT. GRAUBUNDEN: vicinity of Zuoz, along Ova d’Arpiglia, elevation 1700 m, on branchlets of Picea sp., 6 Sep 1990, G.J.S. 90-31 (BPI 1107109, IMI 342667); Landwasser Valley, between Monstein and Wiessen, elevation 1000–1347 m, on Picea sp., G.J.S. 90-32 (BPI 1107110, IMI 342668). UNITED STATES. MAINE: Eustis, on Picea rubra, 19 Jul 1935, J.R. Hansbrough (BPI 551640); Crawford, on Picea rubra, 17 Oct 1936, J.R. Hansbrough (BPI 551639); second collection (BPI 551638); Hulls Cove, Mount Desert, on Abies balsamea, 24 Apr 1935, Gillespie (BPI 551625); Westbrook, on Abies balsamea, Jul 1897, P.L. Ricker (Flora of Maine. Cryptogams, BPI 551623). NEW YORK: Hamilton County, 7th Lake Region, on Abies balsamica, Aug 1919, Stork (BPI 551624).
Commentary: The anamorph of this species is reported to be Cylindrocarpon cylindroides var. tenue (Roll-Hansen 1962, Booth 1966). Although microconidia are always abundant from acremonium- or verticillium-like conidiophores, macroconidia are said to be sparse or absent in many strains, especially after some time in vitro. Macroconidia are cylindrical, straight or slightly curved with bluntly rounded ends and no obvious basal abscission scar, 1–7 septate, mostly 3–5 septate, 30–80 x 4.5–6.5 µm. Chlamydospores not observed. Fertile perithecia were obtained by Roll-Hansen (1962) by pairing isolates, indicating heterothallism. None of our ascospore cultures ever produced macroconidia. The absence of macroconidia in combination with the results of DNA sequencing presented here leads us to suggest that the connection between N. fuckeliana and the Cylindrocarpon is not correct.
There is no description on the label of the Roumeguère exsiccata, so that this specimen could not be taken to be the place of description of a new form, N. cucurbitula f. abietis. However, there is clear reference to ‘‘Sacc. Mich. 1. p. 409.’’ where there is a valid description of the form. Thus we take the form to have been published validly by Saccardo in Michelia. ‘‘Nectria abietis Sac.’’ is given as a species in the nomenclator in the Roumeguère reference cited above. This name is not accompanied by a description or reference to a description and, thus, is taken to be a newly but invalidly published nomen nudum based on the Roumeguère exsiccata.
Symptomless infection of Picea stems by Nectria fuckeliana was discussed by Roll-Hansen and Roll-Hansen (1980). A canker disease of Abies concolor in California and Oregon caused by N. fuckeliana was described by Schultz and Parmeter (1990).
6. Neonectria lucida (Höhnel) Samuels et Brayford, comb. nov. FIGS. 10, 45–47
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Nectria lucida Höhnel, Akad. Wiss. Wien Math. Naturw. Kl., 1 Abt., 118:289. 1909.
Anamorph: Cylindrocarpon lucidum Booth, Mycol. Pap. 104:21. 1966. FIGS. 45–47.
Mycelium not visible. Perithecia 430–480 µm diam, gregarious in groups of 2–5, globose, nonpapillate but with apex sometimes slightly flattened and mammiform, not collapsing when dry, red, with darker red ostiolar area, uniformly dark red in 3% KOH, yellow in 100% lactic acid; wall smooth and shining, superficial on an immersed stroma; hyphal hairs arising from the surface and growing around and nearly as long as the perithecial height, hairs 6–7 µm wide, unbranched, septate, wall 1.0–1.5 µm thick, pale yellow. Cells at surface of perithecial wall lacking a definite outline, hyphal, cells ca. 4 µm wide, walls 1.5–2.0 µm thick, lumina narrow. Perithecial wall 20–30 µm wide, of two regions; outer region of short hyphal elements in a palisade with long axis perpendicular to perithecial surface; outer region continuous over the perithecium to form a uniform palisade of hyphal cells; cells of inner region lacking a definite outline but with long axis parallel to surface of perithecial wall, cells increasingly more compacted and thin-walled toward the perithecial locule; perithecial apex of vertically elongated cells and continuous with the lateral perithecial wall, forming a disk around the ostiolar opening, cells increasingly narrower and merging with the periphyses within. Asci cylindrical, 63–72 x 6.5–10.0 µm, apex with a refractive ring, 8-spored. Ascospores ellipsoidal, (10–)12–15(–17) x (5.0–)5.5–6.5(–7.0) µm, equally 2-celled, not constricted at the septum, becoming finely spinulose, pale brown, uniseriate with overlapping ends, filling each ascus.
Characteristics in culture: Colonies 20–30 mm diam after 10 d on PSA. Colony surface slimy to felty; aerial mycelium typically sparse to felty, white to buff or a shade of brown (umber, bay, chestnut, dark brick, sepia). Colony reverse white to dark brown shades. Slimy sporodochia usually abundant on the colony surface and in the aerial mycelium, often forming in concentric rings. Conidiogenous cells formed apically on densely, irregularly branching clusters of cells borne laterally on otherwise undifferentiated vegetative hyphae. Conidiogenous cells cylindrical, 12–20 x 4.5–5.5 µm, with apical thickening and collarette. Macroconidia cyindrical, but often broader in the upper half, uniformly curved or more strongly curved at the tip, with apical cell rounded and basal cell rounded to truncate. Macroconidia of most isolates 5-septate, 60–70 x 5–6 µm; in a second group macroconidia 70–90 x 6–7 µm (G.J.S. 71-105, G.J.S. 71-49, G.J.S. 72-71, G.J.S. 85-27, G.J.S. 86-178); in a third group macroconidia 70–80 x 9.5–10.0 µm (G.J.S. 72-180, G.J.S. 86-336, G.J.S. 87-50, G.J.S. 89-71, G.J.S. 90-166). Microconidia absent. Chlamydospores not formed. Perithecia forming in single ascospore cultures of at least some collections, suggesting homothallism.
Habitat: Bark of twigs and branches of recently dead trees, less frequently on lianas.
Distribution: Indonesia ( Java), Japan, New Zealand, tropical America and Caribbean region ( Jamaica).
HOLOTYPE: INDONESIA. JAVA. Tjibodas, Tjiburrum, an lebenden Zw., 1907–1908, Höhnel (FH-Höhnel 2899!).
Additional representative specimens examined (out of 30): FRENCH GUIANA. ORSTOM research area ‘‘ECEREX’’, kilometer 16 on road between Sinnamary and St. Elie, on decaying stem of Philodendron sp., 20–29 Feb, 1 Mar. 1986, G.J.S. 3987 (CAY, IMI, NY); Saül, Circuit Grand Fossé, 03 º60'N, 53º20'W, elevation 300–350 m, on bark of living liana, 10 Feb. 1986, G.J.S. 3621 (NY, IMI 325249); along trail from Saül to Mount Galbao, ca 15 km SW of Saül, Camp 2, elevation 600 m, on twigs of recently fallen tree, 14, 18, 29 Jan 1986, G.J.S. 2965 & J. Boise (NY). GUYANA. Cuyuni-Mazaruni Region, VII; Mazaruni Subregion, VII-2, along Koatse R, ca 2 km E of Pong R., ca 5 h walk S of Chinoweing Village, 05º28'N, 60º04'W, elevation 600–650 m, on bark of live mimosoid legume, 28 Feb 1987, G.J.S. 4976 et al (BPI 842128, BRG, IMI 325856, NY). Mount Wokomung, on ridge leading NW toward summit, 
–1 h walk from base camp, in well drained forest at lower elevation to wet forest with palms and tree ferns at higher elevation, elevation 1150–1300 m, on twig of recently fallen tree, 1 Jul 1989, G.J.S. 6380 (G.J.S. culture 89-71, BPI 842129, NY). JAMAICA. HANOVER PARISH: Dolphin Head Mountain, vicinity of Askenish, elevation ca 1000 ft, 22 Jan 1971, on bark, R.P.K. et al CUP-MJ 934 (C.T.R. culture 71-107: NY). ST. ANDREW PARISH: vicinity of Kingston, along Cane River and slope of Good Hope Mountain, elevation 1000 ft, on bark, 12 Jan 1971, R.P.K. et al CUP-MJ 821 (C.T.R. culture 71-49: CUP, Jamaica, NY). NEW ZEALAND. SOUTHLAND: Catlins State Park, Lake Wilkie, on bark of tree, 18 Apr 1988, G.J.S. 85-27 et al (PDD 50050). PUERTO RICO. Caribbean National Forest, Luquillo Mountains, trail to El Toro from Route 186, on bark of recently Cecropia sp., 24 Feb 1996, G.J.S. 8098, H.-J. Schroers & D.J. Lodge (G.J.S. culture 96-35, BPI 475543). UNITED STATES. NEW YORK: Pack Forest, on tree stump, 25 Sep 1971, D. Malloch (C.T.R. culture 71-379, NY). VENEZUELA. AMAZONAS: Cerro de la Neblina, along Río Mawarinuma, just outside Cañon Grande, vicinity of Neblina base camp, 00º50'N, 66º10'W, elevation ca 140 m, on bark of recently dead tree, 27 Apr 1984, G.J.S. 1645 (NY, VEN). BÓLIVAR: ca 118 km S of El Dorado on El Dorado-Sta. Elena Road, trail up N facing slope of Ueitepui from former military camp Ciento Veinticinco, on vine, 5 Aug 1972, K.P.D.-VE 6941 et al (C.T.R. culture 72-180: NY). MIRANDA: Parque Nacional El Avila, S facing slope of La Silla, vicinity of refugio ‘‘No te apures’’, on bark, 30 Jun 1972, K.P.D.-VE 3878 et al (C.T.R. culture 72-71: NY). MERIDA: Parque Nacional Sierra Nevada, above Tabay, Quebrada Coromoto, Estacion La Mucuy, 08º36'N, 71º02'W, elevation 2300 m, on bark, Nov 1990, G.J.S. 6763 et al (G.J.S. culture 90-166: BPI 842125, VEN). MONAGAS: vicinity of Caripe, ‘‘Cueva del Guacharo’’, around canker on living tree, K.P.D.-VE 5319 et al (NY). TRUJILLO: Parque Nacional Guaramacal, on road between Bocono and Guaramacal, 8.4 km from Batatal-Bocono Road, 09º15'N,70º13'W, elevation 2350 m, on bark, 22 Nov 1990, G.J.S. 7409 et al (BPI 842127, VEN, G.J. S. culture 90-180).
Commentary: The tan or brown colonies distinguish Neo. lucida from Neo. discophora. However, there is considerable variation in macroconidial size in Neo. lucida. Macroconidia in some collections are the same size as those in purple isolates (Neo. discophora var. discophora); these are interpreted as C. lucidum sensu stricto (Booth 1966). However, macroconidia in other collections are considerably larger and might indicate the existence of distinct taxa within what we currently regard as Neo. lucida.
7. Neonectria trachosa Samuels et Brayford, sp. nov. FIGS. 11, 41–44, 64
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Anamorphosis Cylindrocarpon sp.
Coloniae in agaro PSA post 10 dies temperatura 25 C 10–20 mm diam attingentes. Mycelium aerium floccosum, album vel bubalinum, reversum etiam album vel bubalinum. Microconidia non formantur. Macroconidia cylindrica vel modice fusiformia, latissima in tertio distali, incurvata, (1–)3–5(–7) septata, plerumque 3-septata et (32–)38–52 (–60) x (5.5–)6–7(–7.5) µm. Apex et basis rotundata. Chlamydosporae absentes.
HOLOTYPUS: BPI 802661.
Mycelium not visible. Perithecia solitary to gregarious in groups of 10 or fewer, superficial, globose, 460–540(–600) µm diam, nonpapillate, apex with a dark, slightly sunken, 85–125 µm diam disk; red to dark red with ostiolar area near black, uniformly dark red in 3% KOH, yellow in 100% lactic acid, not collapsing when dry; smooth to slightly roughened. Cells at surface of perithecial wall circular, 20–25 µm diam with walls 1.5–2 µm thick. Perithecial wall ca 100 µm wide, comprising three regions; outer region ca 45 µm wide, cells elliptic to circular, 15–25 µm diam, walls ca 1.5 µm thick; middle region ca. 25 µm wide, of intertwined and branched hyphal elements arranged ± perpendicular to the perithecial surface and with walls 2–3 µm thick; inner region 25 µm wide, cells adjacent to the middle region intertwined with lumina ± elliptic and up to 15 µm long, walls ca 3 µm thick and pigmented but becoming progressively thinner and unpigmented toward the perithecial locule. Perithecial apex a small disk of parallel hyphal elements continuous with the inner region of the perithecial wall and protruding through outer regions. Cells of the stroma pseudoparenchymatous, 10–15 µm diam, and walls pigmented, 1.5 µm thick. Asci cylindrical, 100–140 x 9–11 µm, apex with conspicuous refractive ring; 8-spored, ascospores uniseriate, filling each ascus. Ascospores ellipsoid, (15.0–) 17.5–20.5(–22.0) x (6.0–)6.7–8.7(–9.0) µm, 1-septate, not constricted at the septum, conspicuously warted, with a thin sheath, colorless.
Characteristics in culture: Colonies on PSA 10–20 mm diam after 10 d, with floccose, white aerial mycelium, remaining white to buff at maturity; colony reverse white to buff, lacking purple or dark brown pigmentation. Growth on SNAY sparse and weak. Conidia forming in slimy droplets and small, white to buff sporodochia, arising from monophialides, scattered and on irregularly branching clusters of cells, 10–20 x 2–4 µm, cylindrical, sometimes swollen and constricted near the apex, with periclinal thickening and indistinct collarette. Microconidia not observed. Macroconidia (1–)3–5(–7) septate (mostly 3, one 8-septate conidium observed), 3-septate: (32–)38–52 (–60) x (5.5–)6–7(–7.5) µm, 4-septate: (44–)47–55 (–60) x 6–7.5 µm, 5-septate: (50–)53–63(–65) x 6–7.5 µm, 6-septate: (48–)55–72(–80) x 6–7.5 µm, 7-septate: 63–78 x 7 µm, cylindrical to slightly fusoid, curved, less so near the base, with bluntly rounded apical and basal cells, lacking a distinct abscission scar. Chlamydospores not seen. No odor. Perithecia not observed in culture.
Habitat: Conifer bark.
Distribution: Scotland, known only from the type.
HOLOTYPE: SCOTLAND. ARGYLLSHIRE: Cowall Peninsula, Argyll Forest Park, ca 5 km S of Strachur along River Cur, vicinity of Glenbranter Village, Lauder Broadleaves Walk, elevation ca 50 m, on conifer bark, 12 Apr 1992, D. Brayford & G.J.S. 8009 (BPI 802661, IMI 352560; culture G.J.S. 92-45).
Commentary: Neonectria trachosa is characterized by its large ascospores, the well-developed, uneven region of circular cells at the exterior of the perithecial wall that gives the wall a slightly roughened aspect, and white colonies on agar. The perithecial wall of Neo. westlandica, a species known only from New Zealand, also has a well-developed outer region of circular cells, but perithecia of this species are coarsely warted and ascospores are distinctly larger.
8. Neonectria viridispora Samuels et Brayford, sp. nov. FIGS. 12, 13, 48–57, 65
Perithecia usque ad quinquagintena caespites formantia, globosa vel late pyriformia, 300–375 µm high, 300–375 µm diam, rubra, albis verruculosis praedita, basi immersa, ad apicem discoidea. Asci 80–100 x 7–10.5 um, anulo apicali praediti. Ascosporae ellipsoideae vel fusiformes, 10–16 x 5.5–7.5 µm, 1-septatae, spinulosae vel laeves, viridescentes.
Anamorphosis: Cylindrocarpon sp. Coloniae in PSA cultae post decem dies temperatura 20 C 20–25 mm diam attingentes, albae, reverso sanguineae vel umbrinus, vel bubalinae ad margini. Microconidia nulla. Macroconidia (3–)4–5-septata, (52–)58–67–71) x 6.0–6.5 µm, cylindrica vel in tertio superiore paullo latiora, basi quasi recta sed apicem versus incurva, cellula apicali rotundata, nonnumquam turgida, cellula basali rotundata vel applanata, pedicello distincto carenti. Chlamydosporae haud observatae.
Holotypus: BPI 842122.
Mycelium white, arachnoid, formed around perithecial bases. Perithecia forming around small cankers, cespitose in groups of up to 50, base immersed in a barely erumpent stroma, globose to broadly pyriform, 300–375 µm high, 300–375 µm wide, apex discoidal, ca 125 µm diam; red but appearing white from large white warts, not changing color in 3% KOH, yellow in 100% lactic acid, not collapsing when dry, or sometimes collapsing by lateral pinching; strongly warted, warts formed of cells at the surface of the perithecial wall. Cells at surface of perithecial wall textura epidermoidea, walls 2–3 µm thick, fine pores joining lumina of adjacent cells. Perithecial wall 30–40 µm wide, comprising three regions: outer region uneven, of disrupted, globose, dead cells that form the white layer on the perithecial surface; middle region 15–20 µm wide, of intertwined hyphae with walls 1.5–2 µm thick; inner region 7–10 µm wide, of flattened, compressed cells with thin, non pigmented walls. Perithecial apex a disk formed of thick-walled hyphal elements arranged in a palisade with elements ca 5 µm wide and walls 1.5–2 µm thick and pigmented; elements becoming progressively narrower toward the ostiolar canal and there merging with the periphyses. The stroma of compact, septate, unbranched, parallel hyphae with rounded ends arranged perpendicular to the surface of the host and up to 250 µm long x 5–7 µm wide. Asci narrowly clavate, 80–100 x 7.0–10.5 µm, apex with a refractive ring, ascospores uniseriate to partially biseriate. Ascospores ellipsoid to fusiform, 10–16 x 5.5–7.5 µm, 1-septate, septum median, spinulose to smooth, hyaline becoming green.
Characteristics in culture: Colonies on PSA 20–25 mm diam after 10 d, with white, floccose aerial mycelium. Reverse blood-colored to umber, fading through bay to a buff margin. Ochreous pigment spreading through the medium. Honey to buff slimy sporodochia forming abundantly on agar surface, most commonly around the inoculum block. Macroconidia also forming in slimy, colorless droplets and clusters in the aerial mycelium. Colonies on CMA 35–40 mm diam after 10 d, with sparse, floccose aerial mycelium and sepia to dark brick pigment in reverse. Macroconidia produced in honey to buff sporodochia forming around the inoculum block and in colorless, slimy droplets scattered in the aerial mycelium. Colonies on SNAY unpigmented, sparse and spreading, lacking aerial mycelium. Some broad (8–20 µm) vegetative hyphae present, with pale brown pigmented walls and conspicuous septa. Perithecia forming commonly around the inoculum block and buried in the agar, scattered singly or in small clusters, red, globose/ovoid, with warted walls, a discoidal apex, and exuding blue-green ascospores. Microconidia absent. Macroconidia forming abundantly on the agar surface in buff-colored slimy masses. Conidiogenous cells cylindrical, 10–20 x 3.0–4.5 µm, with periclinal thickening and an indistinct collarette, borne apically on irregularly branching cells arising from broad vegetative hyphae or in small scattered clusters in the aerial mycelium. Macroconidia (3–)4–5 septate, (52–)58–67(–71) x 6.0–6.5 µm, cylindrical or slightly broader in their upper third, almost straight at the base but becoming more curved toward the apex. Apical cell rounded, sometimes becoming swollen. Basal cell rounded or flattened, lacking a distinct pedicel. Cells of old macroconidia commonly becoming inflated, globose, but not developing thick walls. Chlamydospores not observed.
Habitat: On bark of Ochroma lagopus (Bombacaceae).
Distribution: Ecuador, known only from the type collection.
HOLOTYPE: ECUADOR. vicinity of Santo Domingo, on Ochroma lagopus, 19 Aug 1991, C.S. Hodges s.n. (BPI 842122, isotype IMI 350698, ex type culture A.Y.R. 2690, CBS 102162).
Commentary: Neonectria viridispora is characterized by its red perithecia that are covered in nature with coarse, white warts, and by its green ascospores. Perithecia formed in culture are warted, but the warts remain red. This species is referred to the Nectria mammoidea Group (Booth 1959) because of its characteristic stroma, Cylindrocarpon anamorph, pigmented spinulose ascospores and perithecial wall that is formed of intertwined, thick-walled hyphae.
Green ascospores are unusual in the Nectriaceae, known previously only from Viridispora alata (Samuels) Samuels & Rossman, V. penicilliferi (Samuels) Samuels & Rossman, and V. diparietispora ( J.H. Miller et al) Samuels & Rossman, all of which have Penicillifer van Emden anamorphs (Polishook et al 1991, Samuels 1989, Rossman et al 1999). None of these fungi has been included in a molecular phylogenetic analysis, but based on perithecial anatomy and the anamorph. They do not appear to be closely related.
9. Neonectria westlandica (Dingley) Samuels et Brayford, comb. nov. FIGS. 14, 58–63, 66
Nectria westlandica Dingley, Trans. Roy. Soc. New Zealand 79:201. 1951. Anamorph: Cylindrocarpon sp.
Mycelium not visible. Perithecia solitary to gregarious in groups of 3–4, superficial or with base slightly immersed in a basal stroma, globose, 450–550 µm diam, nonpapillate; brownish red with nearly black ostiolar area, uniformly red in 3% KOH, yellow in 100% lactic acid, not collapsing when dry; strongly warted, warts to 90 µm high. Cells at surface of perithecial wall and warts conspicuously circular, 15–40 µm diam, walls ca 2 µm thick. Perithecial wall 45–55 µm wide, comprising three regions; outer region continuous with warts, consisting of a single layer of circular cells 15–18 µm diam with walls 3.5 µm thick; middle region 18–25 µm wide, cells with hyphal characters and arranged perpendicular to the surface of the perithecium; inner region 10–18 µm wide, cells flattened, walls 1.5–2 µm wide, pigmented, becoming progressively more compressed and thin-walled toward the locule. Perithecial apex with a disk ca 150 µm diam, formed of vertically oriented hyphal elements ca 3.5 µm wide at tip, gradually merging with the periphyses and continuous with the middle region of the perithecial wall. Nectria mammoidea stroma sometimes present. Asci clavate, 110–130 x 17–23 µm, apex with an obscure refractive ring; 8-spored, ascospores biseriate above, uniseriate below, filling each ascus. Ascospores ellipsoid to fusiform, (23.5–)25.0 –33.5(–37.0) x (7.2–)8.5–11.5(–13.0) µm, 1-septate, not constricted at the septum, finely spinulose, colorless to pale yellow-brown, with a 1 µm wide sheath.
Characteristics in culture: Colonies on PSA slow growing, 10–25 mm diam after 10 d, either flat and slimy or with floccose, white aerial mycelium. Colony reverse white, cream or vinaceous buff. Strong purple or brown pigment not observed. Conidia forming in slimy clusters on the colony surface and in the aerial mycelium. In some strains a strong actinomycetous odor formed. On SNA or SNAY colonies 35 mm diam after 21 d, lacking pigment, mycelium dense and compact rather than diffuse and spreading. Aerial mycelium sparse, sometimes aggregated in tufts. Small slimy, buff sporodochia forming scattered on the agar surface. Conidia also forming in slimy droplets in the aerial mycelium. Conidiogenous cells borne in small groups scattered on the mycelium or sometimes on long (110 µm), unbranched or sparingly branched conidiophores, each branch terminating in a single phialide. Phialides cylindrical, 14–27 x 3–5 µm, with periclinal thickening and a small collarette at the apex. Microconidia not observed. Macroconidia (0–6) mostly 3–4 septate, (41–)47–66(–76) x (5.5–)5.7–7.5(8.0) µm, fusiform, evenly curved or slightly more curved near the apex, base rounded or truncate. Chlamydospores not observed.
Habitat: Bark of dicotyledonous trees, less frequently on bark of gymnospermaceous trees.
Distribution: New Zealand.
Descriptions and illustrations: Dingley (1951, plate 24, FIG. 7).
HOLOTYPE: NEW ZEALAND. SOUTH ISLAND. Westland: Weheka, on Olearia avicenniaefolia, Dec 1946, J.M. Dingley (PDD 5129!, ISOTYPE IMI 15560!).
Additional specimens examined: NEW ZEALAND. NORTH ISLAND. Auckland: Waitemata City, Waitakere Ranges, Anawhata Road, Chateau Mosquito Track, on bark of Hoheria populnea, 30 Oct 1973, J.M. Dingley & G.J.S. 73-242 & B. Segedin (PDD 32634, IMI 352112); Waitakere Ranges, Huia, Parau Track, on bark of Metrosideros sp., 23 Oct 1980, G.J.S. 80-156A & P.R. Johnston (PDD 41421, IMI 255610). Taranaki: Mount Egmont National Park, Puniho Track, on Pseudopanax crassifolia, 25 Apr 1983, G.J.S. 83-204, P.R. Johnston & R.H. Petersen (PDD 46408, IMI 352113). SOUTH ISLAND. Westland: N of Hari Hari, Lake Ianthe State Forest, on bark of Dacryocarpus cupressinum, 21 May 1983, G.J.S., T. Matsushima & A.Y. Rossman (G.J.S. culture 83-156; PDD 46336, IMI 326254); Mount Aspiring National Park, Haast Pass, ca. 70 km E of Haast Junction, Cameron Flat, on root of indet. tree, 29 Apr 1985, G.J.S. & L.M. Kohn (G.J.S. culture 85-45; PDD 50055).
Commentary: Dingley (1957) reported microconidia in cultures of Neo. westlandica but none were observed in any of our single-ascospore isolates. The geographical ranges of Neo. westlandica and Neo. discophora overlap in New Zealand. Although the two species are closely related, they are easily distinguished by the coarsely warted perithecia and larger ascospores of Neo. westlandica. Their respective Cylindrocarpon anamorphs also are morphologically similar, but the colonies of Neo. discophora are faster growing and have strong purple slate diffusing pigment on PSA, whereas those of Neo. westlandica have little or no pigment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: gary{at}nt.ars-grin.gov
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMY
KEY TO NEONECTRIA...DESCRIPTIONS OF THE SPECIESLITERATURE CITED |
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