Calonectria species and their Cylindrocladium anamorphs: species with clavate vesicles.

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 beta-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.

INTRODUCTION

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 ).

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 , 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

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 ). 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 ); and part of the histone H3 gene using primers CYLH3F and CYLH3R (Crous ).

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.

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).

Table 1.

Isolates of Cylindrocladium (Calonectria) species studied.

Species	Isolate number1,2	β-tubulin3	Histone H33	Host	Country	Collector
Ca. avesiculata (Cy. avesiculatum)	ATCC 38226; CPC 2373T	AF333392	DQ190620	Ilex vomitoria	U.S.A.	S.A. Alfieri
Ca. clavata (Cy. flexuosum)	CBS 112675; SLU2	DQ190547	DQ190621	Musa sp.	Saint Lucia	J.M. Risède
	CBS 112676; Gua9	DQ190548	DQ190622	Musa sp.	Guadeloupe	J.M. Risède
	CBS 114557; CPC 2536T	AF333396	DQ190623	Callistemon viminalis	U.S.A.	N.E. El-Gholl
	CBS 114666; CPC 2537T	DQ190549	DQ190624	—	U.S.A.	N.E. El-Gholl
	CPC 11347; Mar8	DQ190550	DQ190625	Musa sp.	Martinique	J.M. Risède
	CPC 11348; Mar23	DQ190551	DQ190626	Musa sp.	Martinique	J.M. Risède
	CPC 11349; Mar11	DQ190552	DQ190627	Musa sp.	Martinique	J.M. Risède
	CPC 11350; SLU5	DQ190553	DQ190628	Musa sp.	Saint Lucia	J.M. Risède
	CPC 11351; Gua12	DQ190554	DQ190629	Musa sp.	Saint Lucia	J.M. Risède
	CPC 11352	DQ190555	DQ190630	Musa sp.	Martinique	J.M. Risède
Ca. colhounii (Cy. colhounii)	CBS 111205; CPC 1330	DQ190556	—	—	Indonesia	M.J. Wingfield
	CBS 111367; CPC 1339	AF232851	DQ190631	Eucalyptus sp.	Indonesia	M.J. Wingfield
	CBS 112739; CPC 4082	DQ190557	DQ190632	Eucalyptus grandis	U.S.A.	M.J. Wingfield
	CBS 112948; CPC 4669	DQ190558	DQ190633	—	U.S.A.	—
	CBS 112951; CPC 4717	DQ190559	DQ190634	Eleaeocarpus angustifolius	Australia	C. Pearce & B. Paulus
	CBS 114036; CPC 10718	DQ190560	DQ190635	Rhododendron sp.	U.S.A.	C.S. Hodges
	CBS 114660; CPC 2407	DQ190561	DQ190636	Arachis pintoi	Australia	D. Hutton
	CBS 114695; CPC 2424	DQ190562	DQ190637	A. pintoi	Australia	D. Hutton
	CBS 114704; CPC 2422	DQ190563	DQ190638	A. pintoi	Australia	D. Hutton
	CBS 293.79	DQ190564	DQ190639	—	Indonesia	—
	CPC 681	AF232852	DQ190640	Soil	Thailand	M.J. Wingfield
	CPC 705	AF232854	DQ190641	Soil	South Africa	M.J. Wingfield
	CPC 1237	AF232853	DQ190642	Eucalyptus sp.	South Africa	M.J. Wingfield
Ca. gracilipes (Cy. graciloideum)	CBS 111040; CPC 1159	DQ190565	DQ190643	—	Colombia	M.J. Wingfield
	CBS 111141; CPC 1211T	DQ190566	DQ190644	Eucalyptus sp.	Colombia	M.J. Wingfield
	CBS 115674; CPC 1153	AF333406	DQ190645	Soil	Colombia	M.J. Wingfield
Ca. gracilis (Cy. pseudogracile)	AR2677T	AF232858	DQ190646	Manilkara sp.	Brazil	—
	CBS 111284; CPC 1483	DQ190567	DQ190647	—	Brazil	P.W. Crous
	CBS 114454; CPC 1588	AF232864	DQ190648	Soil	Brazil	P.W. Crous
Cy. hawksworthii	CBS 111870; MUCL 30866; CPC 2405T	AF333407	DQ190649	—	Mauritius	A. Peerally
Ca. indusiata (Cy. theae)	ATCC 48895; CPC 2383	AF232861	—	Rhododendron cv. Kingfisher	U.S.A.	—
	CBS 111399; CPC 1620	DQ190568	DQ190650	—	Ecuador	M.J. Wingfield
	CBS 111705; CPC 1713	DQ190569	DQ190651	Rumohra adiantiformis	U.S.A.	J.Y. Uchida
	CBS 114175; CPC 1712	DQ190570	DQ190652	Strelitzia nicolaii	U.S.A.	J.Y. Uchida
	CBS 114684; UFV 16A; CPC 2446	AF232862	DQ190653	Rhododendron sp.	U.S.A.	N.E. El-Gholl
Ca. leguminum (Cy. leguminum)	CBS 728.68; IMI 299578T	AF389837	DQ190654	—	—	M.B. Figueiredo
Ca. macroconidialis (Cy. macroconidiale)	CBS 114880; CPC 307T	AF232855	DQ190655	Eucalyptus grandis	South Africa	P.W. Crous
	CPC 413	AF232856	DQ190656	Eucalyptus sp.	South Africa	P.W. Crous
Ca. madagascariensis (Cy. madagascariense)	CBS 114539; CPC 2321	AF333416	—	Soil	Madagascar	J.E. Taylor
	CBS 114571; CPC 2253	DQ190571	DQ190657	—	Madagascar	P.W. Crous
	CBS 114572; CPC 2252T	DQ190572	DQ190658	—	Madagascar	P.W. Crous
	CPC 2322	AF333417	—	Soil	Congo	J. Roux
Ca. multiseptata (Cy. multiseptatum)	CBS 112682; CPC 1589T	DQ190573	DQ190659	Eucalyptus sp.	Indonesia	M.J. Wingfield
	CPC 1602; CMW 4054	AF210866	—	Eucalyptus sp.	Indonesia	M.J. Wingfield
Ca. reteaudii (Cy. reteaudii)	CBS 112143; CPC 3200	DQ190574	DQ190660	Eucalyptus sp.	Vietnam	M.J. Dudzinski & P.Q. Thu
	CBS 112144; CPC 3201T	AF389833	DQ190661	E. camaldulensis	Vietnam	M.J. Dudzinski & P.Q. Thu
	CBS 112146; CPC 3213	AF389835	DQ190662	E. urophylla	Australia	B. Brown
	CBS 112147; CPC 3214	AF389830	DQ190663	E. camaldulensis	Vietnam	M.J. Dudzinski & P.Q. Thu
	CBS 112149; CPC 3216	AF389832	DQ190664	E. urophylla	Australia	M. Ramsden
	CBS 112151; CPC 3202	DQ190575	DQ190665	E. urophylla	Australia	C. Hanwood
	CBS 112153; CPC 3205	AF389831	DQ190666	E. camaldulensis	Vietnam	M.J. Dudzinski & P.Q. Thu
	CBS 112155; CPC 3210	AF389834	DQ190667	E. pellita	Australia	P.Q. Thu & K.M. Old
	CBS 112634; CPC 4233	DQ190576	DQ190668	Xanthorrhoea australis	Australia	T. Baigent
	CBS 112955; CPC 4716	DQ190577	DQ190669	Ficus pleurocarpa	Australia	C. Pearce & B. Paulus
	CBS 113582; CPC 516	AF389846	DQ190670	Eucalyptus sp.	Thailand	M.J. Wingfield
	CBS 113583; CPC 759	AF389847	DQ190671	Eucalyptus sp.	Madagascar	P.W. Crous
	CBS 113925; ATCC 16550; IMI 114953; CPC 2366	AF389843	DQ190672	Scoiopendrium sp.	—	—
	CBS 582.50; CPC 3701	AF389836	DQ190673	Seedling of Hibiscus sabdariffa	Indonesia	K.B. Boedijn & J. Reitsma
	CPC 3217	AF389829	DQ190674	E. camaldulensis	Vietnam	M.J. Dudzinski & P.Q. Thu
Ca. rumohrae (Cy. rumohrae)	CBS 111431; UFV 218; CPC 1716T	AF232871	DQ190675	Rumohra adiantiformis	Panama	A.C. Alfenas
	CBS 114529; CPC 1603	AF232873	DQ190676	Adiantum sp.	Netherlands	R. Pieters
	UFV 215; CPC 1831	AF232872	DQ190677	Rumohra adiantiformis	Panama	A.C. Alfenas
Cy. penicilloides	CBS 174.55; CPC 2388T	AF333414	—	Prunus sp.	Japan	Tubaki
Ca. pteridis (Cy. pteridis)	CBS 111778; UFV 43; CPC 2451	AF232859	DQ190678	—	Brazil	J.C. Dianese
	CBS 111793; ATCC 34395; CPC 2372T	DQ190578	DQ190679	Arachnoides adiantiformis	U.S.A.	—
	CBS 111867; CPC 2447	DQ190579	DQ190680	Pinus caribaea	Spain	T.L. Krugner
	CBS 111871; UFV 10-A; CPC 2443	DQ190580	DQ190681	Pinus sp.	Spain	T.L. Krugner
	CPC 2190	AF232860	DQ190682	Eucalyptus sp.	Brazil	P.W. Crous
	CPC 2869	AF333415	—	Eucalyptus sp.	Brazil	P.W. Crous
	CPC 11284	DQ190581	DQ190683	E. grandis	Brazil	A.C. Alfenas
	CPC 11285	DQ190582	DQ190684	E. grandis	Brazil	A.C. Alfenas
	CPC 11286	DQ190583	DQ190685	E. grandis	Brazil	A.C. Alfenas
	CPC 11435	DQ190584	DQ190686	Eucalyptus sp.	Brazil	A.C. Alfenas
	CPC 11569	DQ190585	DQ190687	Eucalyptus sp.	Brazil	A.C. Alfenas
	CPC 11618	DQ190586	DQ190688	Tillandsia wagneriana	Netherlands	C.F. Hill
	CPC 11619	DQ190587	DQ190689	Guzmania wittmackii	Netherlands	C.F. Hill
	CPC 12072	DQ190588	DQ190690	—	New Zealand	C.F. Hill
	CPC 12114	DQ190589	DQ190691	Drosera sp.	Netherlands	J. Dijksterhuis
Cy. acicola	CBS 114812	DQ190590	DQ190692	Diffuse leaf lesions of Phoenix canariensis	New Zealand	H. Pearson
	CBS 114813	DQ190591	DQ190693	Diffuse leaf lesions of P. canariensis	New Zealand	H. Pearson
	CPC 10898T	DQ190592	DQ190694	Pinus sp.	New Zealand	M. Dick
Cy. angustatum	CBS 112133; P99-1321; CPC 3152	DQ190593	DQ190695	Tillandsia capitata	U.S.A.	R.M. Leahy
	CBS 114544; P99-0454; CPC 2347T	AF207543	DQ190696	T. capitata	U.S.A.	N.E. El-Gholl
	CBS 114766; CPC 4522	DQ190594	DQ190697	T. tricolor	Guatemala	C.F. Hill
	CBS 114767; CPC 4523	DQ190595	DQ190698	T. tricolor	Guatemala	C.F. Hill
Cy. australiense	CBS 112954; CPC 4714T	DQ190596	DQ190699	Ficus pleurocarpa	Australia	C. Pearce & B. Paulus
Cy. clavatum (synonym of Cy. gracile)	CBS 111776; ATCC 22833; CPC 2479T	AF232850	DQ190700	Pinus caribaea	Brazil	C.S. Hodges
Cy. curvisporum	CPC 763T	AF333394	DQ190701	Soil	Madagascar	P.W. Crous
	CPC 765	AF333395	AY725664	Soil	Madagascar	P.W. Crous
Cy. ecuadoriae	CBS 111383; CPC 1587	DQ190597	DQ190702	Soil	Brazil	P.W. Crous
	CBS 111393; CPC 1627	DQ190598	DQ190703	Soil	Ecuador	M.J. Wingfield
	CBS 111394; CPC 1628	DQ190599	DQ190704	Soil	Ecuador	M.J. Wingfield
	CBS 111406; CPC 1635T	DQ190600	DQ190705	Soil	Ecuador	M.J. Wingfield
	CBS 111412; CPC 1648	DQ190601	DQ190706	Soil	Ecuador	M.J. Wingfield
	CBS 111425; CPC 1657	DQ190602	DQ190707	Soil	Ecuador	M.J. Wingfield
Cy. gordoniae	CBS 112142; ATCC 201837; CPC 3136T	AF449449	DQ190708	Gordonia lasianthus	U.S.A.	D. Chiappini
Cy. gracile	CBS 110676; CPC 623	AF333405	DQ190709	Soil	Brazil	M.J. Wingfield
	CBS 111129; CPC 921	DQ190603	DQ190710	Soil	Brazil	M.J. Wingfield
	CBS 111382; CPC 1586	AF232863	—	Soil	Brazil	P.W. Crous
	CBS 111390; CPC 1616	DQ190604	DQ190711	Soil	Ecuador	M.J. Wingfield
	CBS 111456; CPC 1918	DQ190605	DQ190712	Soil	Brazil	A.C. Alfenas
	CBS 111458; CPC 1910	DQ190606	DQ190713	Soil	Brazil	A.C. Alfenas
	CBS 111465;CPC 1902	DQ190607	DQ190714	Soil	Brazil	A.C. Alfenas
	CBS 111468; CPC 1905	DQ190608	DQ190715	Soil	Brazil	A.C. Alfenas
	CBS 111474; CPC 1908	DQ190609	DQ190716	Soil	Brazil	A.C. Alfenas
	CBS 111475; IMI 167580; CPC 1967	AF333404	DQ190717	Camellia sinensis	Mauritius	A. Peerally
	CBS 111476; CPC 1909	DQ190610	DQ190718	Soil	Brazil	A.C. Alfenas
	CBS 111478; CPC 1921	DQ190611	DQ190719	Soil	Brazil	A.C. Alfenas
	CBS 111869; PC 551197; CPC 2409T	AF232857	DQ190720	Argyreia sp.	South East Asia	—
	CBS 112673; Cam 14	DQ190612	DQ190721	Musa sp.	Cameroon	J.M. Abadie
	CBS 112694; CPC 617	DQ190613	DQ190722	Soil	Brazil	M.J. Wingfield
	CBS 114167; CPC 1912	DQ190614	DQ190723	Soil	Brazil	A.C. Alfenas
	CBS 114171; CPC 1891	DQ190615	DQ190724	Soil	Brazil	A.C. Alfenas
	CBS 115680; CPC 922	DQ190616	DQ190725	Soil	Brazil	M.J. Wingfield
	CBS 115769; CPC 920	DQ190617	DQ190726	Soil	Brazil	M.J. Wingfield
Cy. hurae	CBS 114182; UFV 216; CPC 1714	DQ190618	DQ190727	Rumohra adiantiformis	Brazil	A.C. Alfenas
	CBS 114551; CPC 2344	AF333408	DQ190728	R. adiantiformis	U.S.A.	N.E. El-Gholl
	FTCC 1002; CPC 2395	AF232866	DQ190729	R. adiantiformis	U.S.A.	N.E. El-Gholl
	FTCC 1003; CPC 2419	AF232867	DQ190730	R. adiantiformis	U.S.A.	N.E. El-Gholl
Cylindrocladium sp.	CBS 112957; CPC 4710	DQ190619	DQ190731	Eleaeocarpus angustifolius	Australia	I. Steer & B. Paulus

CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Pedro Crous working collection housed at CBS; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; ATCC: American Type Culture Collection, Virginia, U.S.A.; UFV: Univeridade Federal de Viçosa, Brazil; CMW: Mike Wingfield collection housed at FABI, Pretoria, South Africa; FTCC: Food Technology Culture Collection, Malaysian Agricultural Research and Development Institute, Food Technology Centre, MARDI, GPO Box 12301, Kuala Lumpur, 50774, Malaysia; MUCL: Mycotheque de l'Université Catholique de Louvain, Louvain-la-Neuve, Belgium; PC: Laboratoire de Cryptogamie, Paris, France.

All ex-type cultures are indicated with a superscript “T”.

GenBank accession numbers.

Taxonomy

Morphological examinations were made from cultures sporulating on CLA. Structures were mounted in lactic acid, and 30 measurements at × 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

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 %.

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.

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.

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.

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

Anamorph: 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 × 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 × 4–6 μm; secondary branches aseptate or 1-septate, 30–50 × 3–6 μm, tertiary and quaternary branches aseptate, 15–30 × 3–5 μm, each terminal branch producing 1–4 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 10–20 × 4–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, (55–)68–75(–95) × (5–)6(–7) μm (av. = 70 × 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) × (4–)5–6 μm (av. = 65 × 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.

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

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.

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) × (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 × 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 × 5–7 μm; secondary branches aseptate, 12–20 × 5–6 μm, tertiary and additional branches (–6), aseptate, 10–15 × 5–6 μm, each terminal branch producing 1–4 phialides; phialides cylindrical to allantoid, hyaline, aseptate, 10–15 × 3.5–4.5 μm; apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, (48–)57–68(–75) × (6–)6.5(–7) μm (av. = 63 × 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) × (6–)6.5(–7) μm than Cy. colhounii [(45–)60–70(–80) × (4–)5(–6) μm], and Cy. madagascariense Crous [(42–)52–58(–65) × (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) × 5–6(–7) μm, and also forms megaconidia and a Calonectria teleomorph with red perithecia in culture (Crous 2002).

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

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.

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) × (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 × 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 × 3–5 μm; secondary branches aseptate, 15–25 × 3–5 μm, tertiary branches aseptate, 12–17 × 3–5 μm, additional branches (–7), aseptate, 10–15 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–15 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, (45–)48–55(–65) × (4–)4.5(–5) μm (av. = 51 × 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) × (4–)4.5(–5) μm (av. = 51 × 4.5 μm), thus longer and wider than those of Cy. graciloideum Crous & G.R.A. Mchau [(35–)40–48(–60) × 4–5(–6) μm (av. = 45 × 4.5 μm)], narrower than those of Cy. gracile [(38–)40–55(–65) × (3.5–)4–5(–6) μm (av. = 53 × 4.5 μm)], and shorter than those of Cy. flexuosum Crous [(44–)50–70(–80) × (4–)5–6 μm (av. = 65 × 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) × (3.5–)4–5 μm (av. = 56 × 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

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 , 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.

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) × (4–)5–6 μm (av. 65 × 5 μm)] (Crous 2002), while those of the banana isolates are [(55–)68–75(–95) × (5–)6–7 μm (av. 70 × 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 ).

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 × 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.