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Stud Mycol 55(1): 213-226 2006
Copyright © 2006 CBS Fungal Biodiversity Centre
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Calonectria species and their Cylindrocladium anamorphs: species with clavate vesicles

Pedro W. Crous1,*, Johannes Z. Groenewald1, Jean-Michel Risède2, Philippe Simoneau3 and Kevin D. Hyde4

1 Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands
2 CIRAD-FLHOR, Station de Neufchâteau, 97130 Capesterre Belle Eau, Guadeloupe, French West Indies
3 UMR PaVé N°77-Faculté des Sciences, Université d'Angers 2, Bd Lavoisier 49045 Angers cedex, France
4 Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong, SAR China

* Correspondence: Pedro W. Crous, crous{at}cbs.knaw.nl


   Abstract
TOP
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 References
 
The present study compares all known species of Cylindrocladium that have clavate vesicles. Several isolates were obtained from baited soils collected in various parts of the world, while others were associated with leaf litter or symptomatic plant hosts. Isolates were compared based on morphology, as well as DNA sequence data from their β-tubulin and histone gene H3 regions. Cylindrocladium australiense and Cy. ecuadoriae, are described as new species, a decision based on morphology and molecular data. A group of isolates associated with toppling disease of banana in the West Indies is identified as Cy. flexuosum. An epitype is designated for Cy. ilicicola, and a new name, Curvicladiella, proposed to replace the anamorphic genus Curvicladium, which is a homonym.

Taxonomic novelties: Cylindrocladium australiense Crous & K.D. Hyde sp. nov., Cylindrocladium ecuadoriae Crous & M.J. Wingf. sp. nov., Curvicladiella Decock & Crous nom. nov., Curvicladiella cignea (Decock & Crous) Decock & Crous comb. nov.

Keywords Ascomycetes / Calonectria / Cylindrocladium / Hypocreales / leaf spots / soil fungi / systematics


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 References
 
Members of the genus Calonectria De Not. (Ca.) (Nectriaceae, Hypocreales, Ascomycetes) and their Cylindrocladium Morgan (Cy.) anamorphs are commonly associated with a wide range of plant disease symptoms (Crous 2002). The current paper represents the second in a series assessing the taxonomy of species of Cylindrocladium, by integrating morphology with DNA sequence data and sexual compatibility studies (Crous et al. 2004).

Cylindrocladium species with clavate vesicles are well-known pathogens from a wide range of hosts in most subtropical to tropical countries (Crous & Wingfield 1992, Crous et al. 1995, 1997, 1999, 2000, Kang et al. 2001, Crous 2002). In the current study, we obtained numerous isolates of Cylindrocladium from baited soils collected in tropical areas. Further Cylindrocladium isolates were obtained from a biotic complex including root rot fungi and plant-parasitic nematodes associated with toppling disease of banana (Risède & Simoneau 2001). Previous studies have shown that isolates resembling Cy. gracile (Bugn.) Boesew. were pathogenic to banana, and associated with stem lesions, root breakage and toppling disease (Risède & Simoneau 2001, 2004). The aim of the present study was to analyze all available Cylindrocladium strains with clavate vesicles using morphology and DNA sequence analysis of their β-tubulin and histone H3 gene regions in order to resolve the status of Cylindrocladium species with clavate vesicles. A further aim was to identify the Cylindrocladium sp. associated with toppling disease of banana.


   MATERIALS AND METHODS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 APPENDIX
 References
 
Isolates
Isolates were obtained from plant hosts, or baited from soil as explained in Crous (2002). Cultural characteristics and morphology were determined on plates containing 2 % malt extract agar (MEA) (20 g/L), and carnation leaf agar (CLA) [1 % water agar (10 g/L) with autoclaved carnation leaves placed onto the medium] in the other (Gams et al. 1998). Plates were incubated for 7 d at 25 °C under continuous near-UV light, to promote sporulation.

DNA phylogeny
Genomic DNA was isolated from fungal mycelia collected from the plates using the isolation protocol of Lee & Taylor (1990). Two loci were amplified and sequenced as explained in Crous et al. (2004), namely, part of the β-tubulin gene, amplified with primers T1 (O'Donnell & Cigelnik 1997) and CYLTUB1R (Crous et al. 2004); and part of the histone H3 gene using primers CYLH3F and CYLH3R (Crous et al. 2004).

The sequences generated in this study were added to other sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov) and TreeBASE (http://www.treebase.org) and the alignment was assembled using Sequence Alignment Editor v. 2.0a11 (Rambaut 2002) with manual adjustments for improvement made visually where necessary. Sequences for Cylindrocladiella peruviana (Bat., J.L. Bezerra & M.M.P. Herrera) Boesew. and Cylindrocladiella lageniformis Crous, M.J. Wingf. & Alfenas were added to the alignments as outgroups.


Figure 1
Figure 1
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  		Fig. 1. One of 657 most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the β-tubulin sequence alignment. The scale bar shows 10 changes and bootstrap support values from 1000 replicates are shown at the nodes. Thickened lines indicate branches present in the strict consensus tree. The tree was rooted to two Cylindrocladiella species.

 


Figure 2
Figure 2
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  		Fig. 2. One of four most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the histone H3 sequence alignment. The scale bar shows 10 changes and bootstrap support values from 1000 replicates are shown at the nodes. Thickened lines indicate branches present in the strict consensus tree. The tree was rooted to two Cylindrocladiella species.

 
The phylogenetic analyses of sequence data were done using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2002). Phylogenetic analysis of both datasets in PAUP consisted of distance (using the uncorrected "p", Jukes-Cantor and HKY85 substitution models) and parsimony analysis as described in Crous et al. (2004). Sequences were deposited in GenBank (Table 1) and the alignments in TreeBASE (S1508, M2711).


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  		Table 1. Isolates of Cylindrocladium (Calonectria) species studied.

 

Taxonomy
Morphological examinations were made from cultures sporulating on CLA. Structures were mounted in lactic acid, and 30 measurements at x 1000 magnification were made of each structure. The 95 % confidence levels were determined, and the extremes of spore measurements given in parentheses. Colony reverse colours were noted after 6 d on MEA at 25 °C in the dark, using the colour charts of Rayner (1970) for comparison. All cultures studied are maintained in the culture collection of the Centraalbureau voor Schimmelcultures (CBS), Utrecht, the Netherlands (Table 1).


   RESULTS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 APPENDIX
 References
 
DNA phylogeny
Approximately 550 bases of the β-tubulin gene were determined for the isolates indicated in Table 1. The manually adjusted alignment contained 123 isolates (including the two outgroups) and 533 characters including alignment gaps. Of these characters, 220 were parsimony-informative, 60 were variable and parsimony-uninformative, and 253 were constant. Neighbour-joining analysis using the three substitution models, as well as parsimony analysis, yielded trees in which the same clades were supported. In some analyses, for example between the uncorrected "p" and HKY85 substitution models, the basal order of the clades were different (data not shown). Parsimony analysis of the alignment yielded 657 most parsimonious trees (TL = 881 steps; CI = 0.529; RI = 0.853; RC = 0.451), one of which is shown in Fig. 1. Most of these trees resulted from the reordering of taxa within the Cy. colhounii Peerally and Ca. reteaudii (Bugn.) C. Booth clades. All taxa from the same species clustered in well-supported clades, namely Cy. curvisporum Crous & D. Victor (100 % bootstrap support), Cy. hurae (Linder & Whetzel) Crous (98 %), Cy. rumohrae El-Gholl & Alfenas (100 %), Cy. angustatum Crous & El-Gholl (100 %), Cy. graciloideum Crous & M.R.A. Mchau (100 %), Cy. theae (Petch.) Subram. (100 %), Cy. pseudogracile Crous (100 %), Cy. ecuadoriae Crous & M.J. Wingf. (88 %), Cy. flexuosum Crous (100 %), Cy. gracile (Bugn.) Boesew. (91 %), Cy. pteridis F.A. Wolf (99 %), Cy. madagascariense Crous (93 %), Cy. macroconidiale (Crous, M.J. Wingf. & Alfenas) Crous (100 %), Cy. colhounii (96 %), Cy. multiseptatum Crous & M.J. Wingf. (100 %), Cy. acicola Gadgil & M. Dick (100 %) and Ca. reteaudii (80 %). All species represented by a single taxon were placed as unsupported sister taxa to the other clades in the tree. The only exception was Cy. penicilloides (Tubaki) Tubaki, which grouped with the Cy. curvisporum clade with a bootstrap support value of 89 %. Association with support values were also observed between some clades, for example the clades containing Cy. hurae, Cy. rumohrae and Cy. angustatum grouped with a bootstrap support value of 79 %.

Approximately 480 bases of the histone gene were determined for the isolates in Table 1. The manually adjusted alignment contained 115 isolates (including the two outgroups), and for each taxon 425 characters including alignment gaps were analysed. Of these characters, 168 were parsimony-informative, 9 were variable and parsimony-uninformative, and 248 were constant. Neighbour-joining analysis using the three substitution models, as well as parsimony analysis, yielded trees in which the same clades were supported. For distance analysis, the Jukes-Cantor and HKY85 substitution models yielded trees with identical topologies, but the tree obtained from the uncorrected "p" model had rearrangements at the deep nodes when compared with the other two trees (data not shown). Parsimony analysis of the alignment yielded four most parsimonious trees (TL = 917 steps; CI = 0.382; RI = 0.868; RC = 0.331), one of which is shown in Fig. 2. All of these trees resulted from reordering of taxa within the Cy. colhounii clade. As with the β-tubulin tree, taxa from the same species clustered together in well-supported clades (Fig. 2). Clade order was not supported at the deeper nodes.

Taxonomy
Calonectria clavata Alfieri, El-Gholl & E.L. Barnard, Mycotaxon 48: 206. 1993.

Anamorph: Cylindrocladium flexuosum Crous, Syst. Appl. Microbiol.18: 248. 1995.

Macroconidiophores consisting of a stipe, a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, pale brown at base, hyaline, smooth, septate, 60–260 x 5–7 µm; stipe extensions septate, straight to flexuous, 120–450 µm long, 3–4 µm wide at apical septum, terminating in a narrowly clavate vesicle, 4–5 µm diam. Conidiogenous apparatus 70–120 µm long, 25–60 µm wide; primary branches aseptate or 1-septate, 30–65 x 4–6 µm; secondary branches aseptate or 1-septate, 30–50 x 3–6 µm, tertiary and quaternary branches aseptate, 15–30 x 3–5 µm, each terminal branch producing 1–4 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 10–20 x 4–5 µm, apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, (55–)68–75(–95) x (5–)6(–7) µm (av. = 70 x 6 µm), 1-septate (but up to 5-septate at germination), lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime (description based on isolates obtained from Musa).

Specimens examined: U.S.A. Florida, Lake Placid, roots and stems of Callistemon viminalis, 5 Apr. 1978, C.P. Seymour & E.L. Barnard, PREM 51721 holotype of Cy. flexuosum, P078-1543 = ATCC 66389 = STE-U 2536 = CBS 114557culture ex-type, heterothallic mating with P078-1261 = STE-U 2537 = CBS 114666, Florida, Lee County, root debris in non-sterilized peat, 4 Mar. 1978, D. Ferrin, Aug. 1989, N.E. El-Gholl, FLAS F55430, holotype of Ca. clavata. Guadeloupe, Musa sp., J.M. Risède & Ph. Simoneau, Gua12 = CPC 11351, CPC 11352 = CBS 119338, Gua9 = CBS 112676. Martinique, Musa sp., J.M. Risède & Ph. Simoneau, Mar11 = CPC 11349 = CBS 119336, Mar23 = CPC 11348 = CBS 119335, Mar8 = CPC 11347 = CBS 119334. Saint Lucia, Musa sp., SLU2 = CBS 112675, SLU5 = CPC 11350 = CBS 119337.

Cultural characteristics: See Crous (2002).

Substrates and distribution: Musa spp., Guadeloupe, Martinique, Saint Lucia; Callistemon viminalis, and root debris in peat U.S.A. (Florida) (Crous 2002).

Notes: Cylindrocladium flexuosum is known to have conidia that are straight to curved, (44–)50–70(–80) x (4–)5–6 µm (av. = 65 x 5 µm) and 1(–3)-septate. The isolates obtained from Musa differ from the ex-type strains by having conidia that are up to 7 µm wide. Although we originally suspected the Musa isolates to represent an undescribed taxon, they clustered in the same clade as those of Cy. flexuosum. None of the isolates were able to mate, and since its original description, it has not proven possible to reproduce perithecia of Calonectria clavata in culture.


Figure 3
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  		Fig. 3. Cylindrocladium australiense. A. Sporulation on MEA. B–D. Conidiophores on CLA. E–F. Clavate vesicles. G–H. Three-septate conidia. Scale bar = 10 µm.

 
Cylindrocladium australiense Crous & K.D. Hyde, sp. nov. MycoBank MB500864. Figs 3, 4.


Figure 4
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  		Fig. 4. Cylindrocladium australiense. Penicillate conidiophore, clavate vesicles and conidia. Scale bar = 10 µm.

 
Etymology: Named after the country from which it was collected.

Cylindrocladio colhounii simile sed conidiis latioribus, (48–)57–68(–75) x (6–)6.5(–7) µm, distinguendum.

Teleomorph unknown. Conidiophores consisting of a stipe bearing a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, 60–150 x 6–7 µm; stipe extensions septate, straight to flexuous, 300–450 µm long, 2.5–3 µm wide at the apical septum, terminating in a clavate vesicle, (3.5–)5(–6) µm diam. Conidiogenous apparatus 40–80 µm long, and 40–60 µm wide; primary branches aseptate or 1-septate, 15–30 x 5–7 µm; secondary branches aseptate, 12–20 x 5–6 µm, tertiary and additional branches (–6), aseptate, 10–15 x 5–6 µm, each terminal branch producing 1–4 phialides; phialides cylindrical to allantoid, hyaline, aseptate, 10–15 x 3.5–4.5 µm; apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, (48–)57–68(–75) x (6–)6.5(–7) µm (av. = 63 x 6.5 µm), (1–)3-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Megaconidia and microconidia unknown.

Specimen examined: Australia, Queensland, Topaz, Atherton Tablelands, Ficus pleurocarpa, 2 Apr. 2001, C. Pearce & B. Paulus, holotype CBS H-17872, culture ex-type CBS 112954 = CPC 4714.

Cultural characteristics: Colonies fast growing with abundant white aerial mycelium; surface and reverse sienna (13i), with moderate numbers of chlamydospores.

Substrate: Ficus pleurocarpa.

Distribution: Australia.

Notes: This species can be confused with taxa in the Cylindrocladium colhounii Peerally species complex that form 3-septate conidia of similar dimensions, and yellow Calonectria perithecia. It can be distinguished by having wider conidia (48–)57–68(–75) x (6–)6.5(–7) µm than Cy. colhounii [(45–)60–70(–80) x (4–)5(–6) µm], and Cy. madagascariense Crous [(42–)52–58(–65) x (3.5–)4–5 µm]. Another species that needs to be compared to Cy. australiense is Cy. theae (Petch) Subram., which again has larger conidia (65–)70–88(–96) x 5–6(–7) µm, and also forms megaconidia and a Calonectria teleomorph with red perithecia in culture (Crous 2002).

Cylindrocladium ecuadoriae Crous & M.J. Wingf., sp. nov. MycoBank MB500865. Figs 5, 6.


Figure 5
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  		Fig. 5. Cylindrocladium ecuadoriae. A. Sporulation on CLA. B. Conidial packet. C–G. Conidiophores. H. Clavate vesicle. I–K. One-septate conidia. Scale bar = 10 µm.

 

Figure 6
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  		Fig. 6. Cylindrocladium ecuadoriae. Penicillate conidiophores, clavate vesicles and conidia. Scale bar = 10 µm.

 
Etymology: Named after Ecuador, where it appears quite commonly in soil.

Cylindrocladio gracili simile, sed conidiis angustioribus, (45–)48–55(–65) x (4–)4.5(–5) µm, distinguendum.

Teleomorph unknown. Conidiophores consisting of a stipe bearing a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, 60–100 x 5–7 µm; stipe extensions septate, straight to flexuous, 200–300 µm long, 2–3 µm wide at the apical septum, terminating in a clavate vesicle, (3–)4(–5) µm diam. Conidiogenous apparatus 30–100 µm long and wide; primary branches aseptate or 1-septate, 20–30 x 3–5 µm; secondary branches aseptate, 15–25 x 3–5 µm, tertiary branches aseptate, 12–17 x 3–5 µm, additional branches (–7), aseptate, 10–15 x 3–5 µm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–15 x 3–4 µm; apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, (45–)48–55(–65) x (4–)4.5(–5) µm (av. = 51 x 4.5 µm), 1(–3)-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Megaconidia and microconidia unknown.

Specimens examined: Ecuador, soil, 20 Jun. 1997, M.J. Wingfield, holotype CBS H-17871, culture ex-type CBS 111406 = CPC 1635; CBS 111394 = CPC 1628; CBS 111412 = CPC 1648; CBS 111393 = CPC 1627; CBS 111425 = CPC 1657. Brazil, Belém, Cpatu, soil, 1996, P.W. Crous, CBS 111383 = CPC 1587.

Cultural characteristics: Colonies sienna on the surface, and umber in reverse; chlamydospores extensive, dense, occurring throughout the medium, forming microsclerotia, with moderate to extensive sporulation on the aerial mycelium.

Substrate: Soil.

Distribution: ?Brazil, Ecuador.

Notes: When first isolated, isolates of Cy. ecuadoriae were observed to also form a few conidia that were 3-septate when studied on CLA. Presently, however, strains seem to have lost this ability and only form 1-septate conidia. The same phenomenon was also observed in the strain obtained from Brazil (CBS 111383). Although the Brazilian strain clusters close to those obtained from Ecuador, its conidia are somewhat shorter (av. 44 µm) than those from Ecuador (av. 51 µm), and it might very well end up representing a cryptic species closely related to Cy. ecuadoriae.

Cylindrocladium ecuadoriae is morphologically similar to others in the Cy. gracile (Bugn.) Boesew. species complex. Its conidia are (45–)48–55(–65) x (4–)4.5(–5) µm (av. = 51 x 4.5 µm), thus longer and wider than those of Cy. graciloideum Crous & G.R.A. Mchau [(35–)40–48(–60) x 4–5(–6) µm (av. = 45 x 4.5 µm)], narrower than those of Cy. gracile [(38–)40–55(–65) x (3.5–)4–5(–6) µm (av. = 53 x 4.5 µm)], and shorter than those of Cy. flexuosum Crous [(44–)50–70(–80) x (4–)5–6 µm (av. = 65 x 5 µm)]. In the past, isolates of Cy. ecuadoriae were treated as representative of Cy. pseudogracile Crous, which has conidia of similar dimensions of [(40–)53–58(–65) x (3.5–)4–5 µm (av. = 56 x 4.5 µm)]and are 1(–3)-septate. Cylindrocladium ecuadoriae can be distinguished from Cy. pseudogacile based on its lower average conidial length, and the absence of a Calonectria state in culture (Crous 2002).


   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
APPENDIX
 References
 
Several studies in recent years have focused on resolving the taxonomy of Cylindrocladium spp. with clavate vesicles (Crous et al. 1995, 1997, 1999, 2000, Kang et al. 2001, Crous 2002). In a study focusing on taxa with sphaeropedunculate vesicles, Crous et al. (2004) described nine new species from the Cy. floridanum species complex. Contrary to what we expected, only two new species could be resolved from all Cylindrocladium strains with clavate vesicles available to us. Part of the reason for this could be that the this complex has been studied in more detail than that with sphaeropedunculate vesicles, but also that Calonectria teleomorphs are more common among species with sphaeropedunculate vesicles than those with clavate vesicles, causing more variation. Some taxa, for instance Cy. colhounii and Cy. reteaudii, proved to be quite variable for the loci sequenced. However, we believe that it is currently premature to split these species into more taxa, and that additional isolates and more loci will have to be investigated to fully resolve their status.


Figure 7
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  		Fig. 7. Cylindrocladium ilicicola (epitype). Conidiophore, vesicles and conidia. Scale bar = 10 µm.

 
A further aim of the current study was to resolve the Cy. pteridis F.A. Wolf or Cy. gracile-like isolates associated with toppling disease of banana (Risède & Simoneau 2001, 2004). Although we originally expected these strains to represent an undescribed species, we were surprised to find that both β-tubulin and histone H3 datasets placed them in Cy. flexuosum (teleomorph Ca. clavata). This was rather unexpected, as conidia of Cy. flexuosum are [(44–)50–70(–80) x (4–)5–6 µm (av. 65 x 5 µm)] (Crous 2002), while those of the banana isolates are [(55–)68–75(–95) x (5–)6–7 µm (av. 70 x 6 µm)]. Notwithstanding this discrepancy, these isolates clustered together in a clade (100 % support) in both data sets, suggesting that the original ex-type strains, which have narrower, slightly curved conidia, might be atypical for what is commonly seen in this species. Although several attempts have been made over the years to redo the crosses between the two mating testers of Cy. flexuosum, or to mate them with the newly collected isolates from banana, none of the matings proved successful. These findings suggest, however, that the Cy. gracile-like isolates associated with toppling disease of banana should be attributed to Cy. flexuosum, and that the latter species is morphologically more variable than originally expected (Crous et al. 1995).

The description of Cy. australiense from Australia adds yet another species to the Cy. colhounii/madagascariense/theae complex. It appears, however, that there are yet more Australian species awaiting description, as CBS 112957, isolated from Eleaeocarpus angustifolius in Queensland (Table 1), also clustered apart from any known taxon. Vesicles were clavate, and conidia 3-septate, 60–90 x 5–6 µm. We chose not to name this species, as the strain sporulated rather poorly, making it difficult to determine its range of morphological variation on CLA.

Isolates of Cylindrocladium ecuadoriae have until recently been treated under the name Cy. pseudogracile. Given the significant overlap in general conidial dimensions, this is not surprising, as these two species are rather similar, and can only be distinguished once the mean conidial dimensions have been determined. The single Brazilian isolate, CBS 11383, which again has smaller conidia than both Cy. ecuadoriae and Cy. pseudogracile, suggests that there may be yet more cryptic taxa within this complex that need to be resolved.


   APPENDIX
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
References
 
Calonectria pyrochroa(Desm.) Sacc., Michelia 1: 308. 1878.

Anamorph: Cylindrocladiumilicicola (Hawley) Boedijn & Reitsma, Reinwardtia 1: 57. 1950. Fig. 7.

Macroconidiophores consisting of a stipe, a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, up to 70 µm long, 5–6 µm wide; stipe extensions septate, straight to flexuous, 160–210 µm long, 3–4 µm wide at apical septum, terminating in an obpyriform to broadly ellipsoidal vesicle, 5–8 µm diam. Conidiogenous apparatus with primary branches that are aseptate or 1-septate, 15–20 x 3–5 µm; secondary branches aseptate, 10–20 x 3–5 µm, tertiary branches aseptate, rarely observed, 8–15 x 3–5 µm, each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, 9–15 x 3–4 µm, apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, (50–)63–68(–70) x 5(–6) µm, (1–)3-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Megaconidia and microconidia unknown.

Specimens examined: Ireland, Clare Island, Ilex aquifolium, Hawley, K 61269!, holotype of Cy. ilicicola, IMI 76542 isotype. Netherlands, South-East Limburg, Vijlenerbos, Vijlen, Ilex aquifolium, Aug. 1970, H.A van der Aa, epitype designated here CBS H-15110, ex-epitype culture CBS 749.70.

Cultural characteristics: Cultures sterile, white.

Substrate and distribution: See Crous (2002).

Notes: The genus Calonectria is based upon Calonectria pyrochroa (= Ca. daldiniana), which is linked to a Cylindrocladium ilicicola anamorph (Rossman 1979, Brayford & Chapman 1987, Crous 2002). All cultures thus far collected by us, and thought to be representative of Cy. ilicicola, have turned out to represent other species, and hence no authentic cultures of Cy. ilicicola have as yet been obtained. A strain not previously studied by us was recently retrieved from the CBS collection (CBS 749.70). Although the isolate sporulated poorly, it was accompanied by a very good specimen, which proved to be identical to the original holotype collection. We therefore designate this specimen as epitype, thereby obtaining an authentic strain of Cy. ilicicola for further study.

Curvicladiella Decock & Crous, nom. nov. MycoBank MB500866.

Type species: Curvicladiella cignea (Decock & Crous) Decock & Crous

Curvicladiella cignea (Decock & Crous) Decock & Crous, comb. nov. MycoBank MB500867.

Specimens examined: French Guiana, Matoury, first part of the Lamirande trail, on decaying leaf of unknown angiosperm, 23 Jan. 1997, C. Decock FG2240, MUCL 40269 = CPC 1595 = CBS 109167 (ex-type culture); on decaying seed of unknown angiosperm, 20 Jan. 1997, C. Decock FG2158, MUCL 40268 = CPC 1594 = CBS 109168.

Notes: It was recently brought to our attention (J. Bischoff, NCBI), that the generic name "Curvicladium", which was proposed by Decock & Crous (1998) for a anamorphic fungus collected from leaf litter in French Guiana, was already occupied for a species of moss (Enroth 1993). A new name is thus called for, and herewith we propose Curvicladiella Decock & Crous, to replace Curvicladium Decock & Crous (1998).


   Acknowledgments
 
The authors acknowledge collections made by various researchers, without which this study would not have been possible. We are especially grateful to C. Pearce, B. Paulus, H.A. van der Aa, A.C. Alfenas and M.J. Wingfield, who placed numerous collections from diverse locations at our disposal for study. Bart van Asten (CBS) is thanked for the sequencing of isolates.


   References
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 References
 

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