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Mycologia, 96(3), 2004, pp. 572-597.
© 2004 by The Mycological Society of America

Neonectria and Cylindrocarpon: the Nectria mammoidea group and species lacking microconidia


David Brayford

     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

    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMY KEY TO NEONECTRIA...
 DESCRIPTIONS OF THE SPECIES
 LITERATURE CITED
 

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


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMY KEY TO NEONECTRIA...
 DESCRIPTIONS OF THE SPECIES
 LITERATURE CITED
 
The genus Nectria Fr. (Hypocreales: Nectriaceae) is a classic genus of pyrenomycetes in the sense that its species have been known since it was described early in the 19th century, and over the years many species from temperate and tropical regions have been included in it. The stereotypical nectria has a brightly colored perithecium—usually red—that is seated directly on a woody substratum (e.g., Booth 1959Go). 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 1989Go, Rehner and Samuels 1994Go, Rossman et al 1999Go). Many species of Nectria sensu lato have been placed in other genera and families (Rossman et al 1999Go, 2001Go, Schroers 2002Go). Rossman et al (1999)Go and Mantiri et al (2001)Go resurrected Neonectria Wollenw. for former Nectria species that have Cylindrocarpon Wollenw. anamorphs. The only known teleomorphs of Cylindrocarpon are Neonectria. Mantiri et al (2001)Go 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 1993Go, Samuels and Brayford 1990Go, 1993Go, 1994Go). We discuss species of Neonectria that have been or could have been placed in the Nectria mammoidea group of Nectria (Booth 1966Go) 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. 51Go). 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 1993Go) and N. hypoxantha Penz. & Sacc. (Samuels 1976Go), neither of which has been linked to any anamorph.



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FIGS. 48–63. Neonectria species. 48–57. Neo. viridispora A.Y.R. 2690. 48, 49. Median longitudinal section through a mature perithecium. 50. Surface of the perithecial wall showing the hyphal nature. 51. Hyphal hairs, typical of the N. mammoidea group, arising from the stroma. 52, 53, 54. Asci. Asci in 54 immature but showing the apical discharge mechanism. 55–57. Ascospores. 55 is the opical section of 56, which shows warts at the surface of the ascospore. 58–63. Neo. westlandica PDD 32634. Microscopy: DIC except 48, 58, 52 FL; 54, 60 PC. Scale bars: 48, 50 = 100 µm; 49, 51, 53, 58–61 = 50 µm; 52, 54–57, 62, 63 = 10 µm.

 
The Cylindrocarpon anamorphs of these fungi lack microconidia and chlamydospores and for these reasons were included in Cylindrocarpon ‘‘Group 2’’ by Booth (1966)Go. All species included in this group were anamorphs of members of the ‘‘Nectria mammoidea Group’’. Following Booth (1959Go, 1966)Go 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)Go 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 1959Go; FIGS. 17, 24Go, 36, 44Go, 60Go). 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, 6Go) 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. 14Go), Neo. viridispora (FIG. 13Go) and Neo. dumontii (FIG. 11Go) are warted distinctly because of additional surface layers of pseudoparenchymatous cells over the N. mammoidea palisade.



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FIGS. 15–31. Neonectria species. 15–22. Neo. betulae G.J.S. 8012. 15. Perithecia in nature. 16. Median longitudinal section of a mature perithecium showing palisidal nature of the lateral wall. 17, 18. Asci; apex shown in 18. 19–22. Discharged ascospores. 19, 21 in optical section; 20, 22 in surface view showing fine warts. 23–26. Neo. discophora G.J.S. 8018. 23, 24. Median longitudinal section through a perithecium showing the palisidal nature of the lateral wall. 25, 26. Asci with ascospores. Apical discharge mechanism visible in 26. 27–31. Neo. dumontii K.P.D.-CO 7987. 27. Perithecia in nature. 28. Median longitudinal section of a mature perithecium showing the hyphal nature of the wall and large cells at the exterior. 29. Ascus with ascospores. 30, 31. Discharged ascospores. Microscopy: DIC except 16 and 27, stereo microscope and 26, PC. Scale bars: 16, 17, 31 = 50 µm; 18–22, 26, 30 = 10 µm; 23–26, 28, 29 = 100 µm, 27 = 500 µm.

 


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FIGS. 34–44. Nectria fuckeliana. 34, 35. Median longitudinal section through a mature perithecium seated on a stroma. The palisadal nature of the perithecial wall visible in 35. 36. Asci with filamentous apical paraphyses. 37–39. Asci with ascospores. Apical discharge mechanism visible in 37. 40. Discharged ascospores. 34, 35 from G.J.S. 8014; 36–40 from G.J.S. 90-32. 41–44. Neo. trachosa G.J.S. 8009. 41. Perithecia. 42, 43. Median longitudinal section through a perithecium seated on a stroma. Palisadal nature of the perithecial wall with large cells formed at the exterior visible in 43. 44. Part of an ascus with two ascospores. Apical discharge mechanism visible. Microscopy: DIC except 36, PC, and 42, stereo microscope. Scale bars: 34 = 300 µm; 35, 43 = 100 µm; 36 = 20 µm, 37, 38, 40, 44 = 10 µm; 39 = 20 µm, 41 = 1 mm, 42 = 250 µm.

 


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FIGS. 3–14. Perithecia of Neonectria and Nectria species. 3, 4. Neo. betulae G.J.S. 8012. 5, 6. Neo. discophora. 5. G.J.S. 98-132, 6. G.J.S. 5065. 7. Neo. dumontii K.P.D.-CO 7987. 8, 9. Nectria fuckeliana, respectively BPI 551647 and G.J.S. 90-37. 10. Neo. lucida G.J.S. 2965. 11. Neo. trachosa G.J.S. 8009. 12, 13. Neo. viridispora A.Y.R. 2690. 14. Neo. westlandica G.J.S. 83-204. Scale bars = 500 µm except 6, which = 1 mm. All bright field microscopy.

 
Ascospores in the N. mammoidea Group are typically ellipsoidal and hyaline, becoming pale brown at maturity; they are usually 1-septate, but those of N. phaeodisca Rossman are phragmosporous (Rossman 1983Go, Samuels and Brayford 1993Go). 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 1993Go) 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 1959Go, 1966Go). 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 1959Go, 1966Go, Roll-Hansen 1962Go). 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 1959Go, 1966Go), 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)Go for Cylindrocarpon and Neonectria.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMY KEY TO NEONECTRIA...
 DESCRIPTIONS OF THE SPECIES
 LITERATURE CITED
 
Morphological studies. – Perithecia from herbarium specimens were rehydrated briefly in 3% KOH. Anatomy of perithecial sections was observed in water or lactic acid by transmitted light or by epifluorescence in 0.5% w/v Calcofluor (Sigma Chemical Co., St. Louis, Missouri) in 0.4 M sodium phosphate buffer at pH 8.0, using a Zeiss 05 filter set. Measurements of ascospores were made from water, while those of conidia were made from fresh mounts in lactofuchsin, which made conidial septa more easily seen. Cotton blue stain (Le Gal 1947Go: 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 1971Go), potato-dextrose agar (PDA, Booth 1971Go) and on SNAY (SNA, Nirenberg 1976Go, 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)Go. Herbarium designations are taken from Holmgren et al (1990)Go.

Molecular studies. – The sources of the isolates used in this study are given in TABLE IGo. 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 2001Go). Fungal isolates were maintained on 1.5% malt-extract agar (Difco) or potato-dextrose agar (Difco).


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TABLE I. Identification and origin of isolates used in the molecular analysis
 
The mycelia from stored cultures were inoculated into 250 mL V8 broth (V8 juice 200 mL, agar 15 g, CaCO3 3 g, distilled water 1 L), harvested after incubation in the dark at room temperature for 1–2 wk and freeze-dried. Two to five g of freeze-dried mycelium was ground to a fine powder, suspended in 10 mL of extraction buffer (20 mM EDTA; 50 mM Tris-HCl, pH 8; 2% SDS) and incubated at 65 C for 1 h after the addition of proteinase K to a final concentration of 200 µg/mL. Cell debris was removed by centrifugation at 15 000 rpm for 20 min at 4 C, after which the supernatant was transferred to a fresh tube containing 2–2.5 volumes of cold 95% EtOH. Following an extraction with chloroform—isoamyl alcohol (24:1)—the DNA was precipitated with 3 M sodium acetate and 2.5 volumes of 95% (v/v) EtOH (Sambrook et al 1989Go). 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 1994Go) 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 1990Go). DNA sequences were aligned using the computer program MULTALIN version 5.4.1 (Corpet 1988Go) 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 2000Go). 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 1995Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMY KEY TO NEONECTRIA...
 DESCRIPTIONS OF THE SPECIES
 LITERATURE CITED
 
All the isolates yielded a single amplification product range of 539–682 bp in length. Length differences were due mainly to insertions and deletions in two variable regions that alternated with three relatively conserved regions (data not shown).

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. 1Go. 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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FIG. 1. Phylogenetic relationships of species of Neonectria inferred from mit ssu rDNA sequences. This is one of 164 MPTs (L = 367, CI = 0.78, RI = 0.75, RC = 0.58) resulting from a MP analysis employing the branch and bound algorithm in PAUP*. The numbers of nucleotide changes are indicated above the branches. Numbers in parentheses next to species names (where given) identify multiple isolates for a taxon (see TABLE IGo). Measures of sequence divergence for the three clades are indicated.

 
Members of the genus Neonectria included in this study formed a monophyletic clade, with 74% bootstrap support, with respect to outgroups (FIG. 2Go). 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)Go. Clade I (bootstrap 95%) includes four isolates (two species: Neo. neomacrospora and Neo. galligena) having relatively low sequence variation (0.2–3.8%, TABLE IIGo). 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 IIGo). 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 IIGo). Clade III (bootstrap 89%) includes two isolates of Neo. radicicola.



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FIG. 2. Phylogenetic relationships of species of Neonectria inferred from mit ssu rDNA sequences. This is a 50% majority rule consensus of 164 MPTs generated from a heuristic analysis of 752 bp for 21 taxa. Bootstrap values greater than 50% calculated from 100 replications are given about the branches.

 

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TABLE II. Pairwise genetic distances between the different Neonectria mitochondrial small subunit rDNA sequences analyzed. Below diagonal: absolute distances (base substitutions between two sequences). Above diagonal: mean distances (adjusted for missing data)
 
We studied collections and ascospore-derived cultures of Neo. discophora var. discophora from Europe, temperate North America, tropical South America, Indonesia, Jamaica and New Zealand. Within these collections we have noted variation in perithecial morphology, especially perithecial size, formation of a flattened ostiolar disk and in the presence or absence of an outer layer of cells giving a smooth or roughened appearance. However, we have been unable to detect discontinuities in these characteristics or correlations between them and anamorph characteristics. The cultures and anamorph morphologies are uniform, indicating that there is only one species with a very wide distribution.

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. 2Go]) 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. 36Go) 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. 33Go). Perithecia did not form in our cultures.



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FIG. 32. Neonectria discophora, Cylindrocarpon ianthothele var. majus G.J.S. 90-212, anamorph from PSA. FIG. 33Go. Nectria fuckeliana G.J.S. 92-46, acremonium-like anamorph from PSA. Scale bar = 10 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMY KEY TO NEONECTRIA...
 DESCRIPTIONS OF THE SPECIES
 LITERATURE CITED
 
Earlier analysis of sequences of the mit ssu rDNA of a limited but representative set of species of Neonectria and Cylindrocarpon strongly suggested (i) a one-for-one relationship between Cylindrocarpon and Neonectria and (ii) the monophyly of Neonectria/Cylindrocarpon (Mantiri et al 2001Go).

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. 1Go, 3Go) of Booth (1966)Go 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. 2Go), 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 1993Go).

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 1966Go, Roll-Hansen 1962Go), 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)Go 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 1966Go). Roll-Hansen and Roll-Hansen (1979Go, 1980)Go 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 1959Go, 1966Go) 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 1993Go) and Neo. rugulosa (Samuels and Brayford 1994Go) groups form a morphologically intergrading assemblage of taxa. The only clear discontinuity is with the N. radicicola Group (Samuels and Brayford 1990Go), 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 1999Go).

Teleomorphs of Neo. discophora var. discophora and var. rubi, and Neo. lucida are indistinguishable (cf. FIGS. 6 and 11Go). 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.


    TAXONOMY
KEY TO NEONECTRIA SPECIES HAVING NONMICROCONIDIAL CYLINDROCARPON ANAMORPHS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMY KEY TO NEONECTRIA...
 DESCRIPTIONS OF THE SPECIES
 LITERATURE CITED
 

1. Ascospores averaging ≤20 µm long 2
1. Ascospores averaging >20 µm long 12
    2. Perithecial apex with a fringe of saccate cells Neo. coronata (Penz. & Sacc.) Mantiri & Samuels (Brayford and Samuels 1993Go)
    2. Perithecial apex lacking saccate cells 3
3. Ascospores averaging ≥7 µm wide 4
3. Ascospores averaging <7 µm wide 7
    4. Perithecia rough to conspicuously warted; on hardwood trees 5
    4. Perithecia smooth, on conifers or hardwoods 6
5. Perithecia rough but not conspicuously warted; ascospores (12–)15–20(–25) x (5–)7–8(–9) µm, hyaline . Neo veuillotiana (Sacc. & Roum.) Mantiri & Samuels (Brayford and Samuels 1993Go)
5. Perithecia with conspicuous white warts; ascospores 10–16 x 5.5–7.5 µm 8. Neo. viridispora
    6. On conifers; ascospores smooth, hyaline to indistinctly yellow-brown; colony white to buff, typically acremonium- or verticillium-like, microconidia only forming 5. Nectria fuckeliana
    6. On conifers or hardwoods; ascospores spinulose, becoming light but distinctly brown; colony purple, Cylindrocarpon lacking microconidia 2. Neo. discophora
7. Cells at surface of perithecial wall lacking a definite outline, ca. 5 µm across. 8
7. Cells at surface of perithecial wall circular to angular, 20–35 µm diam 11
    8. Causing canker on Rubus; ascospores (11.5–)12.5–17.0(–23.0) x (4.5–)5.5–6.5(–8.0) µm, colony purple. 3. Neo. discophora var. rubi
    8. Usually corticolous or not causing canker on Rubus; ascospores 9–27 x 4–11 µm, colony white, ochreous, tan, purple. 9
9. Ascospores 10 –11 x (3–)4–5 µm, spinulose; perithecia pyriform with a flattened apex Neonectria platycephala (Brayford & Samuels) Samuels & Brayford, comb. nov. ({equiv} Nectria platycephala Brayford & Samuels, Mycologia 85:625. 1993).
9. Ascospores averaging >11 µm long, spinulose or smooth; perithecia globose and apex rounded or mammiform. 10
    10. Cosmopolitan; ascospores (10 –)12–15(–17) x (5.0 –)5.5–6.5(–7.0) µm, colony purple . 2. Neo. discophora var. discophora
    10. Mainly tropical; ascospores (9.5–)11.5–15.7(–21.5) x (4–)5–7(–11) µm; colonies white to tan 6. Neo. lucida
11. Ascospores 15–20 x 6–7.5 µm, conspicuously warted; perithecial wall pseudoparenchymatous throughout. Neonectria verrucospora (Brayford & Samuels) Samuels & Brayford, comb. nov. ({equiv} Nectria verrucospora Brayford & Samuels, Mycologia 85:631. 1993)
11. Ascospores (15–)17.5–20.5(–22) x 6–9 µm, conspicuously warted; perithecial wall ‘‘mammoidea type’’ below a surface region of circular cells 7. Neo. trachosa
    12. Ascospores phragmosporous, (38–)50 –67 x 10 –14 µm. Neonectria phaeodisca (Rossman) Samuels & Brayford, comb. nov. ({equiv} Nectria phaeodisca Rossman, Mycol. Pap. 150:38. 1983; see Samuels and Brayford 1993Go).
    12. Ascospores didymosporous, smaller 13
13. Perithecial apex with a fringe of saccate cells; ascospores (10 –)16–24(–37) x (3.5–)5.5–8.5(–11.0) µm . Neo. coronata (Brayford and Samuels 1993Go).
13. Perithecial apex lacking saccate cells. 14
    14. Perithecia smooth, shining; perithecia elongated with a broad and obtuse apex; ascospores hyaline, typically conspicuously striate, less often smooth, (15–)22–29(–36) x (5–)8.5–10.2 µm Nenectria jungneri (Henn.) Samuels & Brayford, comb. nov. ({equiv} Nectria jungneri Henn., Bot. Jahrb. Syst. 22:75. 1897 (1895); see Samuels and Brayford 1994Go).
    14. Perithecia rough to warted, or nearly smooth but then dull, not shining, globose 15
15. Perithecia pale brown to brown (brick) with ostiolar area dark brown, nearly black; ascospores (23–)26–32(–36) x (5–)8–10(–11) µm; often associated with a chrome-yellow conidial mass in nature Neonectria cinnamomea (Brayford & Samuels) Samuels & Brayford, comb. nov. ({equiv} Nectria cinnamomea Brayford & Samuels, Mycologia 85:167. 1993).
15. Perithecial red to dark red, not associated with yellow conidia. 16
    16. Perithecia smooth; ascospores (19–)21–24(–26) x 7–8 µm; on Betula, Scotland 1. Neo. betulae
    16. Perithecia conspicuously warted. 17
17. Ascospores (24–)25–34(–37) x (7.0–)8.5–11.5(–13.0) µm, New Zealand. 9. Neo. westlandica
17. Ascospores (27–)31–35(–36) x (4–)6.5–8 µm, Colombia. 4. Neo. dumontii


    DESCRIPTIONS OF THE SPECIES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 TAXONOMY KEY TO NEONECTRIA...
 DESCRIPTIONS OF THE SPECIES
 LITERATURE CITED
 
1. Neonectria betulae Brayford et Samuels, sp. nov. FIGS. 3, 4Go.

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, 6Go, 32Go

{equiv} Sphaeria discophora Mont., Ann. Sci. Nat. Bot. II 3: 353. 1835.
= Nectria tasmanica Berk. in Hooker, Botany of the Antarctic Voyage of H.M. Discovery Ships Erebus and Terror, part 3. Flora Tasmaniae 2:279. 1860.
= Nectria mammoidea W. Phillips & Plowr., Grevillea 3: 126. 1875.
{equiv} Creonectria mammoidea (W. Phillips. & Plowr.) Seaver, Mycologia: 188. 1909.
= Nectria nelumbicola Henn., Verh. Bot. Vereins Prov. Brandenburg 40:151. 1898. Fide Höhnel & Weese, Ann. Mycol. 8:467. 1910.
= Nectria umbilicata Henn., Hedwigia 41:3. 1902.
= Nectria mammoidea var. rugulosa Weese, Akad. Wiss. Wien Math.-Naturw. Kl., Abt. 1, 125:552. 1916.
= Nectria mammoidea var. minor Reinking, Zentralbl. Bakteriol., Abt. 2, 94:135. 1936.
= Creonectria discostiolata Chardón, Bol. Soc. Venez. Ci. Nat. 5:341 [241 sic]. 1939.
= Nectria pinea Dingley, Trans. Roy. Soc. New Zealand 79:198. 1951.

Anamorph: Cylindrocarpon ianthothele Wollenw. var. majus Wollenw., Z. Parasitenk. (Berlin) 1:161. 1928. FIG. 32Go

= Cylindrocarpon ianthothele var. minus Reinking, Zentralbl. Bakteriol., Abt. 2, 94:135. 1936.
= Cylindrocarpon ianthothele var. rugulosum C. Booth, Mycol. Pap. 104:25. 1966.
= Cylindrocarpon pineum C. Booth, Mycol. Pap. 104:26. 1966.

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 (1930aGo, FIG. 536; 1930bGo, FIG. 742, as N. mammoidea). Booth (1966Go, FIG. 16Go, as N. mammoidea). Kar and Gupta (1977Go, FIG. 2Go, as N. mammoidea). Dennis (1978Go, 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 1990Go), the better known name N. mammoidea is a later taxonomic synonym of Neo. discophora.

Booth (1966)Go 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.

{equiv} Nectria rubi Osterw., Ber. Deutsch. Bot. Ges. 29:620. 1911.
{equiv} 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 1926Go, from natural substratum).

Illustrations: Wollenweber (1926Go: 59, 526, 527). Booth (1966Go: FIG. 17Go).

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 1991Go). 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)Go was the first to recognize a relationship between this taxon and Nectria mammoidea (Neo. discophora).

4. Neonectria dumontii Brayford et Samuels, sp. nov. FIGS. 7Go, 27–31Go

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, 9Go, 33Go–40Go

= Nectria cucurbitula (Tode : Fr.) Fr. f. abietis Roumeguère, Fungi Gallici exsiccati 1292. 1881, ut ‘‘Nectria cucurbitula Fries f. abietis Sac. [sic].’’
{equiv} 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. 33Go

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)Go, Roll-Hansen (1962)Go, Booth (1966Go, 1979)Go.

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 kn