Calonectria Species and Their Cylindrocladium Anamorphs: Species with Clavate Vesicles

STUDIES IN MYCOLOGY 55: 213–226. 2006.

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

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

*Correspondence: Pedro W. Crous, [email protected]

Abstract: 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. Key words: Ascomycetes, Calonectria, Cylindrocladium, Hypocreales, leaf spots, soil fungi, systematics.

INTRODUCTION MATERIALS AND METHODS

Members of the genus Calonectria De Not. (Ca.) Isolates (Nectriaceae, Hypocreales, Ascomycetes) and Isolates were obtained from plant hosts, or baited their Cylindrocladium Morgan (Cy.) anamorphs are from soil as explained in Crous (2002). Cultural commonly associated with a wide range of plant disease characteristics and morphology were determined on symptoms (Crous 2002). The current paper represents plates containing 2 % malt extract agar (MEA) (20 g/ the second in a series assessing the taxonomy of L), and carnation leaf agar (CLA) [1 % water agar (10 species of Cylindrocladium, by integrating morphology g/L) with autoclaved carnation leaves placed onto the with DNA sequence data and sexual compatibility medium] in the other (Gams et al. 1998). Plates were studies (Crous et al. 2004). incubated for 7 d at 25 °C under continuous near-UV Cylindrocladium species with clavate vesicles are light, to promote sporulation. well-known pathogens from a wide range of hosts in most subtropical to tropical countries (Crous & Wingfield DNA phylogeny 1992, Crous et al. 1995, 1997, 1999, 2000, Kang et al. Genomic DNA was isolated from fungal mycelia 2001, Crous 2002). In the current study, we obtained collected from the plates using the isolation protocol numerous isolates of Cylindrocladium from baited soils collected in tropical areas. Further Cylindrocladium of Lee & Taylor (1990). Two loci were amplified and isolates were obtained from a biotic complex sequenced as explained in Crous et al. (2004), namely, including root rot fungi and plant-parasitic nematodes part of the β-tubulin gene, amplified with primers T1 associated with toppling disease of banana (Risède (O’Donnell & Cigelnik 1997) and CYLTUB1R (Crous et & Simoneau 2001). Previous studies have shown al. 2004); and part of the histone H3 gene using primers that isolates resembling Cy. gracile (Bugn.) Boesew. CYLH3F and CYLH3R (Crous et al. 2004). were pathogenic to banana, and associated with stem The sequences generated in this study were added lesions, root breakage and toppling disease (Risède & to other sequences obtained from GenBank (http://www. Simoneau 2001, 2004). The aim of the present study ncbi.nlm.nih.gov) and TreeBASE (http://www.treebase. was to analyze all available Cylindrocladium strains with org) and the alignment was assembled using Sequence clavate vesicles using morphology and DNA sequence Alignment Editor v. 2.0a11 (Rambaut 2002) with manual analysis of their β-tubulin and histone H3 gene regions adjustments for improvement made visually where in order to resolve the status of Cylindrocladium species necessary. Sequences for Cylindrocladiella peruviana with clavate vesicles. A further aim was to identify the (Bat., J.L. Bezerra & M.M.P. Herrera) Boesew. and Cylindrocladium sp. associated with toppling disease Cylindrocladiella lageniformis Crous, M.J. Wingf. & of banana. Alfenas were added to the alignments as outgroups. 213 CROUS ET AL.

Cylindrocladiella peruviana AY725653 Cylindrocladiella lageniformis AY725652 89 Cy. penicilloides CBS 174.55 100 CPC 765 CPC 763 Cy. curvisporum 98 FTCC 1003 CBS 114551 Cy. hurae 98 CBS 114182 100 79 FTCC 1002 100 CBS 114529 99 UFV 215 Cy. rumohrae 54 CBS 111431 CBS 114544 100 CBS 112133 CBS 114767 Cy. angustatum 91 CBS 114766 Ca. leguminum CBS 728.68 CBS 115674 100 CBS 111141 Cy. graciloideum CBS 111040 CBS 114175 100 CBS 111399 67 CBS 111705 Cy. theae 66 CBS 114684 ATCC 48895 Cy. avesiculatum ATCC 38226 100 CBS 114454 CBS 111284 Cy. pseudogracile AR2677 CBS 111383 88 CBS 111425 CBS 111406 Cy. ecuadoriae 80 CBS 111412 CBS 111393 64 CBS 111394 CBS 114557 CBS 114666 100 CPC 11347 CPC 11352 CPC 11350 Cy. flexuosum CBS 112676 63 CBS 112675 CPC 11349 CPC 11351 CPC 11348 CBS 111382 62 CBS 112694 CBS 111475 64 CBS 110676 62 CBS 111869 63 CBS 112673 CBS 114167 CBS 111478 91 CBS 111458 CBS 111465 CBS 115769 Cy. gracile CBS 111456 CBS 111468 CBS 115680 63 CBS 111129 CBS 111474 Cy. clavatum CBS 111776 CBS 111476 CBS 111390 CBS 114171 Cy. hawksworthii CBS 111870 Cy. gordoniae CBS 112142 CBS 111793 CPC 12114 74 CPC 12072 CPC 11619 99 CPC 11618 CPC 11286 CPC 11284 CBS 111867 Cy. pteridis CBS 111871 CBS 111778 60 CPC 2869 CPC 2190 CPC 11285 CPC 11435 86 CPC 11569 CPC 2322 93 CBS 114572 CBS 114571 Cy. madagascariense 97 CBS 114539 100 CBS 114880 Cy. macroconidiale CPC 413 CBS 114660 99 CBS 114704 CBS 114695 96 CBS 112948 68 CBS 111367 CBS 112951 CBS 111205 Cy. colhounii CBS 293.79 CPC 705 CPC 681 68 CPC 1237 CBS 114036 64 CBS 112739 Cy. australiense CBS 112954 Cylindrocladium sp. CBS 112957 100 CBS 112682 Cy. multiseptatum 99 CPC 1602 CBS 114812 100 CPC 10898 Cy. acicola 77 CBS 114813 CBS 113925 CBS 112955 80 83 CBS 112634 CBS 112151 71 97 CBS 112155 Fig. 1. One of 657 most parsimonious trees CBS 112149 CBS 112146 obtained from a heuristic search with 100 CBS 112144 Ca. reteaudii CBS 112153 random taxon additions of the β-tubulin sequence 73 CBS 112147 alignment. The scale bar shows 10 changes and CBS 582.50 CPC 3217 bootstrap support values from 1000 replicates CBS 113582 10 changes CBS 113583 are shown at the nodes. Thickened lines indicate CBS 112143 branches present in the strict consensus tree. The tree was rooted to two Cylindrocladiella species. 214 CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS

Cylindrocladiella peruviana AY725700 Cylindrocladiella lageniformis AY725699 Cy. avesiculatum ATCC 38226 92 CBS 114529 100 UFV 215 Cy. rumohrae CBS 111431 CBS 114551 93 100 CBS 114182 FTCC 1002 Cy. hurae 53 FTCC 1003 Ca. leguminum CBS 728.68 65 CBS 114544 100 CBS 112133 CBS 114767 Cy. angustatum CBS 114766 Cy. hawksworthii CBS 111870 CBS 115674 100 CBS 111141 Cy. graciloideum CBS 111040 Cy. gordoniae CBS 112142 88 CBS 111793 CPC 11286 97 CPC 11284 CPC 11285 CPC 2190 54 CPC 11569 CPC 11435 CBS 111778 Cy. pteridis 56 CBS 111867 CBS 111871 CPC 12114 CPC 12072 62 CPC 11619 CPC 11618 CBS 111383 98 CBS 111425 CBS 111393 92 CBS 111394 Cy. ecuadoriae 57 CBS 111412 62 CBS 111406 CBS 114454 100 CBS 111284 Cy. pseudogracile AR2677 CBS 114557 CBS 114666 99 CPC 11347 CPC 11352 CPC 11350 CBS 112676 Cy. flexuosum 54 CBS 112675 CPC 11349 60 CPC 11351 CPC 11348 Cy. clavatum CBS 111776 CBS 111475 CBS 111478 CBS 111458 98 CBS 111465 CBS 114167 CBS 111456 CBS 111468 CBS 115680 CBS 111129 Cy. gracile 53 CBS 111474 CBS 111476 CBS 111390 CBS 114171 CBS 115769 CBS 112673 CBS 111869 CBS 112694 CBS 110676

CBS 111399 95 CBS 111705 CBS 114684 Cy. theae 98 CBS 114175 100 CBS 114880 Cy. macroconidiale CPC 413 100 CPC 765 CPC 763 Cy. curvisporum CBS 114036 72 CPC 705 CPC 1237 CBS 112739 100 CBS 112951 CPC 681 CBS 293.79 Cy. colhounii 52 CBS 112948 CBS 111367 81 CBS 114660 CBS 114704 CBS 114695 100 CBS 114572 Cy. madagascariense CBS 114571 Cy. australiense CBS 112954 Cylindrocladium sp. CBS 112957 Cy. multiseptatum CBS 112682 100 CBS 114812 Cy. acicola 88 CPC 10898 70 CBS 114813 CBS 112955 CBS 112634 85 CBS 112149 94 CBS 112146 CBS 112155 72 CBS 112151 CBS 113925 CPC 3217 Ca. reteaudii CBS 582.50 100 CBS 113582 CBS 113583 CBS 112143 CBS 112147 CBS 112153 10 changes CBS 112144 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. 215 CROUS ET AL. . Crous . Crous M.J. Wingfield M.J. Wingfield Africa Brazil P.W Guadeloupe J.M. Risède MartiniqueMartinique J.M. Risède Martinique J.M. Risède Saint Lucia J.M. Risède Saint Lucia J.M. Risède Martinique J.M. Risède J.M. Risède AustraliaU.S.A. C. Pearce & B. Paulus AustraliaAustralia C.S. Hodges D. Hutton Australia D. Hutton Indonesia D. Hutton Thailand – South M.J. Wingfield Brazil P.W Ecuador M.J. Wingfield Country Collector Africa South U.S.A. – U.S.A.Alfieri S.A. U.S.A.U.S.A. N.E. El-Gholl N.E. El-Gholl Colombia M.J. Wingfield Brazil – Mauritius A. Peerally Colombia M.J. Wingfield IndonesiaU.S.A. M.J. Wingfield U.S.A. M.J. Wingfield – Colombia M.J. Wingfield Indonesia M.J. Wingfield Saint Lucia J.M. Risède sp. cv. Kingfisher cv. sp. sp. sp. sp. Musa sp. Musa sp. Musa sp. Musa sp. Musa sp. Musa sp. Musa sp. Eleaeocarpus angustifolius Rhododendron Arachis pintoi A. pintoi A. pintoi Host Eucalyptus Ilex vomitoria Callistemon viminalis Eucalyptus Manilkara Eucalyptus Eucalyptus grandis – – Rhododendron Musa sp. 3 Histone H3 DQ190622 DQ190625 DQ190626 DQ190628 DQ190629 DQ190630 DQ190634 DQ190635 DQ190636 DQ190637 DQ190638 DQ190632 DQ190633 – DQ190621 3 AF232864 DQ190648 Soil DQ190548 DQ190551 DQ190552DQ190553 DQ190627 DQ190554 DQ190555 DQ190560 DQ190561 DQ190562 DQ190563 DQ190564AF232852 DQ190639AF232854 DQ190640 – DQ190641 Soil Soil DQ190567 DQ190647 – DQ190568 DQ190650 – AF232853 DQ190642 DQ190550 AF232861 β-tubulin AF333392 DQ190620 AF333396DQ190549 DQ190623 DQ190624 – DQ190566 DQ190644 AF232858 DQ190646 AF333407 DQ190649 – DQ190565 DQ190643 – AF232851DQ190557 DQ190631 DQ190558 DQ190559 AF333406 DQ190645 Soil DQ190547 DQ190556 – T T T T T 153 1 1,2 T 1284; CPC 1483 1399; CPC 1620 1040; CPC 1159 1367; CPC 1339 CPC 1211 1141; 1205; CPC 1330 1 ) species studied. CBS 111870; MUCL 30866; CPC 2405 MUCL CBS 111870; CBS 112675; SLU2 CBS 112675; CPC 1588 CBS 114454; CBS 114557; CPC 2536 CBS 114557; Mar23 CPC 11348; Mar1 CPC 11349; SLU5 CPC 11350; Gua12 CPC 11351; CPC 11352 CBS 1 CPC 10718 CBS 114036; CPC 2407 CBS 114660; CPC 2424 CBS 114695; CPC 2422 CBS 114704; CBS 293.79 CPC 681 CPC 705 CPC 1237 CBS 11 Isolate number CBS 114666; CPC 2537 CBS 114666; Mar8 CPC 11347; CPC 1 CBS 115674; CBS 11 48895; CPC 2383 ATCC CBS 11 CBS 112739; CPC 4082 CBS 112739; CPC 4669 CBS 112948; CPC 4717 CBS 112951; AR2677 CBS 11 CBS 11 CBS 112676; Gua9 CBS 112676; ( Calonectria ) ) ) Cy. avesiculatum ) ( Cy. 38226; CPC 2373 ATCC Cy. graciloideum ( Cy. Cy. theae ) ( Cy. Cy. colhounii ( Cy. Cy. flexuosum ) ( Cy. Cy. pseudogracile ( Cy. Isolates of Cylindrocladium Ca. clavata Cy. hawksworthii Cy. Ca. colhounii Ca. avesiculata Species Table 1. Table Ca. indusiata Ca. gracilis Ca. gracilipes

216 CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS aylor Thu & K.M. Old . Crous . Crous . Crous P.W. Crous P.W. Thu M.J. Dudzinski & P.Q. Thu M.J. Dudzinski & P.Q. Thu M.J. Dudzinski & P.Q. Thu M.J. Dudzinski & P.Q. M.B. Figueiredo – P.W. Crous P.W. Thu M.J. Dudzinski & P.Q. ietnam U.S.A. Uchida J.Y. Madagascar P.W Australia Baigent T. Netherlands R. Pieters PanamaAlfenas A.C. U.S.A. N.E. El-Gholl Congo J. Roux U.S.A. Uchida J.Y. Vietnam AustraliaVietnam C. Hanwood Thailand M.J. Wingfield Vietnam Country Collector Africa South Australia B. Brown Australia M. Ramsden Australia P.Q. – Africa South Madagascar P.W IndonesiaIndonesia M.J. Wingfield M.J. Wingfield Vietnam PanamaAlfenas A.C. Japan Tubaki Australia C. Pearce & B. Paulus Madagascar P.W – IndonesiaV K.B. Boedijn & J. Reitsma Brazil J.C. Dianese MadagascarT J.E. sp. sp. Hibiscus sabdariffa sp. sp. sp. sp. sp. Xanthorrhoea australis Rumohra adiantiformis Strelitzia nicolaii Rhododendron Adiantum Soil Scolopendrium Rumohra adiantiformis E. camaldulensis E. urophylla E. camaldulensis Eucalyptus sp. Eucalyptus Host Eucalyptus E. urophylla E. urophylla E. pellita Eucalyptus Eucalyptus grandis Eucalyptus E. camaldulensis Rumohra adiantiformis Prunus sp. Ficus pleurocarpa Eucalyptus sp. E. camaldulensis Soil 3 Histone H3 DQ190663 DQ190665 DQ190666 DQ190670 DQ190660 DQ190671 – 3 DQ190571 DQ190657 – DQ190576 DQ190668 AF232872 DQ190677 DQ190570AF232862 DQ190652 DQ190653 AF232873 DQ190676 AF333417 – AF389843 DQ190672 DQ190569 DQ190651 AF389831 AF232856 DQ190656 AF389835AF389830 DQ190662 AF389832DQ190575 DQ190664 AF389834 DQ190667 AF210866 – β-tubulin AF389837AF232855 DQ190654 DQ190655 – DQ190572 DQ190658DQ190573 – DQ190659 DQ190574 AF389833 DQ190661 AF232871 DQ190675 AF333414 – DQ190577AF389846 DQ190669 AF389836AF389829 DQ190673 DQ190674 Seedling of AF333416 AF232859 DQ190678 – AF389847 T T T T T T T 1,2 1705; CPC 1713 1431; UFV 218; CPC 1716 1778; UFV 43; CPC 2451 14175; CPC 1712 14684; UFV 16A; CPC 2446 14529; CPC 1603 12955; CPC 4716 12682; CPC 1589 CBS 1 CBS 112143; CPC 3200 CBS 112143; CBS 1 CPC 2253 CBS 114571; CPC 4233 CBS 112634; UFV 215; CPC 1831 CBS 174.55; CPC 2388 CBS 114572; CPC 2252 CBS 114572; CPC 2322 16550; IMI 114953; ATCC CBS 113925; CPC 2366 CBS 728.68; IMI 299578 CBS 1 CPC 3205 CBS 112153; CPC 759 CBS 113583; CBS 11 CPC 413 CPC 3213 CBS 112146; CPC 3214 CBS 112147; CPC 3216 CBS 112149; CPC 3202 CBS 112151; CPC 3210 CBS 112155; CBS 11 Isolate number CPC 1602; CMW 4054 CBS 1 CBS 113582; CPC 516 CBS 113582; CBS 582.50; CPC 3701 CPC 3217 CBS 11 CBS 114539; CPC 2321 CBS 114539; CPC 3201 CBS 112144; ) CPC 307 CBS 114880; ) ) Cy. ( Cy. Cy. macroconidiale ( Cy. ) Cy. multiseptatum ) ( Cy. CBS 1 Cy. leguminum ( Cy. Cy. rumohrae ) ( Cy. Cy. reteaudii ( Cy. Cy. pteridis ) Cy. ( (Continued). . penicilloides Cy Ca. leguminum Ca. multiseptata Ca. rumohrae Table 1. Table Species Ca. macroconidialis Ca. reteaudii Ca. pteridis Ca. madagascariensis madagascariense

217 CROUS ET AL. . Crous . Crous . Crous . Crous . Crous C.F. Hill C.F. R.M. Leahy N.E. El-Gholl Hill C.F. Hill C.F. C.F. Hill C.F. H. Pearson M. Dick New Zealand New U.S.A. Guatemala Brazil P.W BrazilBrazilBrazil P.W BrazilAlfenas A.C. BrazilAlfenas A.C. BrazilAlfenas A.C. Netherlands Alfenas A.C. Alfenas A.C. Ecuador M.J. Wingfield New ZealandNew H. Pearson Zealand New Spain Krugner T.L. U.S.A. Guatemala Brazil M.J. Wingfield Country Collector Netherlands J. Dijksterhuis U.S.A. – AustraliaBrazil C. Pearce & B. Paulus Madagascar P.W Madagascar C.S. Hodges P.W Ecuador M.J. Wingfield U.S.A. D. Chiappini SpainBrazil Krugner T.L. P.W Netherlands Hill C.F. Zealand New Ecuador M.J. Wingfield EcuadorEcuador M.J. Wingfield M.J. Wingfield

Phoenix P. canariensis P. sp. sp. sp. sp. fuse leaf lesions of canariensis Soil Eucalyptus E. grandis E. grandis E. grandis Eucalyptus sp. Eucalyptus wagneriana Tillandsia Pinus sp. Pinus caribaea capitata Tillandsia tricolor T. Host Drosera Arachnoides adiantiformis capitata T. Ficus pleurocarpa Pinus caribaea Gordonia lasianthus Pinus sp. Eucalyptus Guzmania wittmackii tricolor T. 3 Histone H3 DQ190693 Dif DQ190697 DQ190682 DQ190690 – 3 DQ190597 DQ190702 Soil AF333415DQ190581 – DQ190582 DQ190683 DQ190583 DQ190684 DQ190584 DQ190685 DQ190585 DQ190686 DQ190586 DQ190687 DQ190688 AF333395 AY725664 DQ190598 DQ190703 Soil DQ190592 DQ190694 DQ190594 DQ190579 DQ190680 DQ190590 DQ190692 leaf lesions of Diffuse DQ190593 DQ190695 AF333405 DQ190709 Soil DQ190589 DQ190691 DQ190591 β-tubulin DQ190578 DQ190679 AF207543 DQ190696 DQ190596AF232850 DQ190699 AF333394 DQ190700 DQ190701 Soil DQ190600 DQ190705 Soil AF449449 DQ190708 DQ190580AF232860 DQ190681 DQ190587DQ190588 DQ190689 DQ190595 DQ190698 DQ190599 DQ190704DQ190601 Soil DQ190602 DQ190706 DQ190707 Soil Soil T T T T T T 1,2 T 1793; ATCC 34395; CPC 2372 ATCC 1793; 1867; CPC 2447 1776; ATCC 22833; CPC 2479 ATCC 1776; 1394; CPC 1628 1406; CPC 1635 1412; CPC 1648 1425; CPC 1657 1285 1286 1435 1569 1618 1619 14812 12133; P99-1321; CPC 3152 10676; CPC 623 11871; UFV 10-A; CPC 2443 11871; 14767; CPC 4523 201837; CPC 3136 ATCC 12142; CBS 111383; CPC 1587 CBS 111383; CPC 2869 CPC 11284 CPC 765 CPC 1627 CBS 111393; CPC 1 CPC 1 CPC 1 CPC 1 CPC 1 CBS 11 CBS 1 CPC 10898T P99-0454; CPC 2347 CBS 114544; CPC 4522 CBS 114766; CBS 1 CBS 11 CPC 12114 CBS 1 CBS 114813 Isolate number CBS 11 CPC 763 CBS 11 CBS 1 CBS 112954; CPC 4714 CBS 112954; CPC 2190 CPC 12072 CBS 11 CBS 11 CBS 1 CPC 1 CBS 1 Cy. gracile ) (synonym of Cy. CBS 11 (Continued). . australiense Cy. ecuadoriae Cy. Cy. acicola Cy. Table 1. Table Species Cy. clavatum Cy. curvisporum Cy. gracile Cy. Cy. gordoniae Cy. Cy. angustatum Cy. Cy

218 CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS . Crous – BrazilBrazilBrazilAlfenas A.C. BrazilAlfenas A.C. MauritiusAlfenas A.C. Alfenas A.C. A. Peerally BrazilAlfenas A.C. BrazilBrazil M.J. Wingfield BrazilAlfenas A.C. M.J. Wingfield U.S.A.U.S.A.U.S.A. N.E. El-Gholl N.E. El-Gholl N.E. El-Gholl Brazil M.J. Wingfield BrazilAlfenas A.C. CameroonAbadie J.M. Country Collector Ecuador M.J. Wingfield South East Asia BrazilBrazil P.W Alfenas A.C. BrazilAlfenas A.C. BrazilBrazil M.J. Wingfield Alfenas A.C. Australia I. Steer & B. Paulus sp. Soil R. adiantiformis R. adiantiformis Camellia sinensis R. adiantiformis Musa sp. Host Argyreia Soil Rumohra adiantiformis Eleaeocarpus angustifolius 3 Histone H3 DQ190721 DQ190723 Soil DQ190726 Soil DQ190731 3 AF232866AF232867 DQ190729 DQ190730 DQ190606DQ190607 DQ190713DQ190608 DQ190714 Soil DQ190609 DQ190715 Soil DQ190716 Soil Soil DQ190611 DQ190719 Soil DQ190613 DQ190722DQ190615 Soil DQ190616 DQ190724 DQ190725 Soil Soil AF333408 DQ190728 DQ190604 DQ190711 DQ190603 DQ190710 Soil DQ190610 DQ190718 Soil DQ190612 β-tubulin AF232857 DQ190720 AF232863 – DQ190605 DQ190712 Soil DQ190614 DQ190617 DQ190618 DQ190727 DQ190619 T 1,2 1129; CPC 921 1129; 1476; CPC 1909 1382; CPC 1586 1456; CPC 1918 11458; CPC 1910 11458; 1902 11465;CPC CPC 1905 11468; CPC 1908 11474; IMI 167580; CPC 196711475; CPC 1921 11478; AF333404 CPC 2409 PC 551197; 11869; DQ190717 12694; CPC 617 14171; CPC 1891 15680; CPC 922 14551; CPC 2344 12957; CPC 4710 14182; UFV 216; CPC 1714 CBS 1 CBS 1 CBS 1 CBS 1 CBS 1 CBS 11 CBS 1 CPC 1912 CBS 114167; CBS 1 CBS 1 CPC 920 CBS 115769; CBS 1 FTCC 1002; CPC 2395 FTCC 1003; CPC 2419 CBS 1 CBS 111390; CPC 1616 CBS 111390; CBS 11 CBS 1 CBS 1 CBS 11 Isolate number CBS 11 Cam14 CBS 112673; CBS 1 sp. (Continued). CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CPC: Pedro Crous working collection housed at CBS; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham CBS: Centraalbureau voor Schimmelcultures, Utrecht, All ex-type cultures are indicated with a superscript “T”. GenBank accession numbers. Cylindrocladium 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 Pretoria, South Mike Wingfield collection housed at FABI, Brazil; CMW: Univeridade Federal de Viçosa, U.S.A.; UFV: Culture Collection, Virginia, Type American ATCC: Lane, U.K.; 50774, Malaysia; MUCL: Mycotheque de Centre, MARDI, GPO Box 12301, Kuala Lumpur, Technology Agricultural Research and Development Institute, Food Culture Collection, Malaysian Technology l’Université Catholique de Louvain, Louvain-la-Neuve, Belgium; PC: Laboratoire Cryptogamie, Paris, France. Table 1. Table Species 1 2 3 Cy. hurae Cy.

219 CROUS ET AL.

The phylogenetic analyses of sequence data single taxon were placed as unsupported sister taxa were done using PAUP (Phylogenetic Analysis Using to the other clades in the tree. The only exception was Parsimony) v. 4.0b10 (Swofford 2002). Phylogenetic Cy. penicilloides (Tubaki) Tubaki, which grouped with analysis of both datasets in PAUP consisted of the Cy. curvisporum clade with a bootstrap support distance (using the uncorrected “p”, Jukes-Cantor and value of 89 %. Association with support values were HKY85 substitution models) and parsimony analysis also observed between some clades, for example the as described in Crous et al. (2004). Sequences were clades containing Cy. hurae, Cy. rumohrae and Cy. deposited in GenBank (Table 1) and the alignments in angustatum grouped with a bootstrap support value of TreeBASE (S1508, M2711). 79 %. Approximately 480 bases of the histone gene were Taxonomy determined for the isolates in Table 1. The manually Morphological examinations were made from cultures adjusted alignment contained 115 isolates (including sporulating on CLA. Structures were mounted in lactic the two outgroups), and for each taxon 425 characters acid, and 30 measurements at × 1000 magnification including alignment gaps were analysed. Of these were made of each structure. The 95 % confidence characters, 168 were parsimony-informative, 9 were levels were determined, and the extremes of spore variable and parsimony-uninformative, and 248 were measurements given in parentheses. Colony reverse constant. Neighbour-joining analysis using the three colours were noted after 6 d on MEA at 25 °C in the substitution models, as well as parsimony analysis, dark, using the colour charts of Rayner (1970) for yielded trees in which the same clades were supported. comparison. All cultures studied are maintained in For distance analysis, the Jukes-Cantor and HKY85 the culture collection of the Centraalbureau voor substitution models yielded trees with identical Schimmelcultures (CBS), Utrecht, the Netherlands topologies, but the tree obtained from the uncorrected (Table 1). “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 RESULTS 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 DNA phylogeny Cy. colhounii clade. As with the β-tubulin tree, taxa from Approximately 550 bases of the β-tubulin gene were the same species clustered together in well-supported determined for the isolates indicated in Table 1. The clades (Fig. 2). Clade order was not supported at the manually adjusted alignment contained 123 isolates deeper nodes. (including the two outgroups) and 533 characters including alignment gaps. Of these characters, 220 Taxonomy were parsimony-informative, 60 were variable and Calonectria clavata Alfieri, El-Gholl & E.L. Barnard, parsimony-uninformative, and 253 were constant. Mycotaxon 48: 206. 1993. Neighbour-joining analysis using the three substitution Anamorph: Cylindrocladium flexuosum Crous, Syst. models, as well as parsimony analysis, yielded trees Appl. Microbiol.18: 248. 1995. in which the same clades were supported. In some analyses, for example between the uncorrected "p" and Macroconidiophores consisting of a stipe, a penicillate HKY85 substitution models, the basal order of the clades arrangement of fertile branches, a stipe extension, and were different (data not shown). Parsimony analysis of a terminal vesicle; stipe septate, pale brown at base, the alignment yielded 657 most parsimonious trees (TL hyaline, smooth, septate, 60–260 × 5–7 µm; stipe = 881 steps; CI = 0.529; RI = 0.853; RC = 0.451), one extensions septate, straight to flexuous, 120–450 µm of which is shown in Fig. 1. Most of these trees resulted long, 3–4 µm wide at apical septum, terminating in a from the reordering of taxa within the Cy. colhounii narrowly clavate vesicle, 4–5 µm diam. Conidiogenous Peerally and Ca. reteaudii (Bugn.) C. Booth clades. All apparatus 70–120 µm long, 25–60 µm wide; primary taxa from the same species clustered in well-supported branches aseptate or 1-septate, 30–65 × 4–6 µm; clades, namely Cy. curvisporum Crous & D. Victor (100 secondary branches aseptate or 1-septate, 30–50 × % bootstrap support), Cy. hurae (Linder & Whetzel) 3–6 µm, tertiary and quaternary branches aseptate, Crous (98 %), Cy. rumohrae El-Gholl & Alfenas (100 15–30 × 3–5 µm, each terminal branch producing 1– %), Cy. angustatum Crous & El-Gholl (100 %), Cy. 4 phialides; phialides elongate doliiform to reniform, graciloideum Crous & M.R.A. Mchau (100 %), Cy. hyaline, aseptate, 10–20 × 4–5 µm, apex with minute theae (Petch.) Subram. (100 %), Cy. pseudogracile periclinal thickening and inconspicuous collarette. Crous (100 %), Cy. ecuadoriae Crous & M.J. Wingf. (88 Conidia cylindrical, rounded at both ends, straight, %), Cy. flexuosum Crous (100 %), Cy. gracile (Bugn.) (55–)68–75(–95) × (5–)6(–7) µm (av. = 70 × 6 µm), 1- Boesew. (91 %), Cy. pteridis F.A. Wolf (99 %), Cy. septate (but up to 5-septate at germination), lacking madagascariense Crous (93 %), Cy. macroconidiale a visible abscission scar, held in parallel cylindrical (Crous, M.J. Wingf. & Alfenas) Crous (100 %), Cy. clusters by colourless slime (description based on colhounii (96 %), Cy. multiseptatum Crous & M.J. isolates obtained from Musa). Wingf. (100 %), Cy. acicola Gadgil & M. Dick (100 %) Specimens examined: U.S.A. Florida, Lake Placid, roots and stems and Ca. reteaudii (80 %). All species represented by a of Callistemon viminalis, 5 Apr. 1978, C.P. Seymour & E.L. Barnard, 220 CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS

PREM 51721 holotype of Cy. flexuosum, P078-1543 = ATCC 66389 Cylindrocladium australiense Crous & K.D. Hyde, = STE-U 2536 = CBS 114557culture ex-type, heterothallic mating sp. nov. MycoBank MB500864. Figs 3–4. with P078-1261 = STE-U 2537 = CBS 114666, Florida, Lee County, root debris in non-sterilized peat, 4 Mar. 1978, D. Ferrin, Aug. 1989, Etymology: Named after the country from which it was N.E. El-Gholl, FLAS F55430, holotype of Ca. clavata. Guadeloupe, Musa sp., J.M. Risède & Ph. Simoneau, Gua12 = CPC 11351, CPC collected. 11352 = CBS 119338, Gua9 = CBS 112676. Martinique, Musa sp., Cylindrocladio colhounii simile sed conidiis latioribus, (48–)57–68(– J.M. Risède & Ph. Simoneau, Mar11 = CPC 11349 = CBS 119336, 75) × (6–)6.5(–7) µm, distinguendum. Mar23 = CPC 11348 = CBS 119335, Mar8 = CPC 11347 = CBS 119334. Saint Lucia, Musa sp., SLU2 = CBS 112675, SLU5 = CPC Teleomorph unknown. Conidiophores consisting of 11350 = CBS 119337. a stipe bearing a penicillate arrangement of fertile Cultural characteristics: See Crous (2002). branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, 60–150 × 6–7 µm; Substrates and distribution: Musa spp., Guadeloupe, stipe extensions septate, straight to flexuous, 300– Martinique, Saint Lucia; Callistemon viminalis, and root 450 µm long, 2.5–3 µm wide at the apical septum, debris in peat U.S.A. (Florida) (Crous 2002). terminating in a clavate vesicle, (3.5–)5(–6) µm diam. Notes: Cylindrocladium flexuosum is known to have Conidiogenous apparatus 40–80 µm long, and 40–60 conidia that are straight to curved, (44–)50–70(–80) × µm wide; primary branches aseptate or 1-septate, 15– (4–)5–6 µm (av. = 65 × 5 µm) and 1(–3)-septate. The 30 × 5–7 µm; secondary branches aseptate, 12–20 × isolates obtained from Musa differ from the ex-type 5–6 µm, tertiary and additional branches (–6), aseptate, strains by having conidia that are up to 7 µm wide. 10–15 × 5–6 µm, each terminal branch producing 1–4 Although we originally suspected the Musa isolates phialides; phialides cylindrical to allantoid, hyaline, to represent an undescribed taxon, they clustered in aseptate, 10–15 × 3.5–4.5 µm; apex with minute the same clade as those of Cy. flexuosum. None of periclinal thickening and inconspicuous collarette. the isolates were able to mate, and since its original Conidia cylindrical, rounded at both ends, straight, description, it has not proven possible to reproduce (48–)57–68(–75) × (6–)6.5(–7) µm (av. = 63 × 6.5 perithecia of Calonectria clavata in culture. µm), (1–)3-septate, lacking a visible abscission scar,

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. 221 CROUS ET AL.

Cylindrocladium ecuadoriae Crous & M.J. Wingf., sp. nov. MycoBank MB500865. Figs 5–6. 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, Fig. 4. Cylindrocladium australiense. Penicillate conidiophore, holotype CBS H-17871, culture ex-type CBS 111406 = CPC 1635; clavate vesicles and conidia. Scale bar = 10 µm. 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, held in parallel cylindrical clusters by colourless slime. dense, occurring throughout the medium, forming Megaconidia and microconidia unknown. microsclerotia, with moderate to extensive sporulation Specimen examined: Australia, Queensland, Topaz, Atherton on the aerial mycelium. Tablelands, Ficus pleurocarpa, 2 Apr. 2001, C. Pearce & B. Paulus, holotype CBS H-17872, culture ex-type CBS 112954 = CPC 4714. Substrate: Soil.

Cultural characteristics: Colonies fast growing Distribution: ?Brazil, Ecuador. with abundant white aerial mycelium; surface and Notes: When ﬁrst isolated, isolates of Cy. ecuadoriae reverse sienna (13i), with moderate numbers of were observed to also form a few conidia that were chlamydospores. 3-septate when studied on CLA. Presently, however, strains seem to have lost this ability and only form Substrate: Ficus pleurocarpa. 1-septate conidia. The same phenomenon was also Distribution: Australia. observed in the strain obtained from Brazil (CBS 111383). Although the Brazilian strain clusters close to Notes: This species can be confused with taxa in the those obtained from Ecuador, its conidia are somewhat Cylindrocladium colhounii Peerally species complex shorter (av. 44 µm) than those from Ecuador (av. 51 that form 3-septate conidia of similar dimensions, and µm), and it might very well end up representing a cryptic yellow Calonectria perithecia. It can be distinguished species closely related to Cy. ecuadoriae. by having wider conidia (48–)57–68(–75) × (6–)6.5(–7) Cylindrocladium ecuadoriae is morphologically µm than Cy. colhounii [(45–)60–70(–80) × (4–)5(–6) similar to others in the Cy. gracile (Bugn.) Boesew. µm], and Cy. madagascariense Crous [(42–)52–58(– species complex. Its conidia are (45–)48–55(–65) × 65) × (3.5–)4–5 µm]. Another species that needs to (4–)4.5(–5) µm (av. = 51 × 4.5 µm), thus longer and be compared to Cy. australiense is Cy. theae (Petch) wider than those of Cy. graciloideum Crous & G.R.A. Subram., which again has larger conidia (65–)70–88(– Mchau [(35–)40–48(–60) × 4–5(–6) µm (av. = 45 × 4.5 96) × 5–6(–7) µm, and also forms megaconidia and a µm)], narrower than those of Cy. gracile [(38–)40–55(– Calonectria teleomorph with red perithecia in culture 65) × (3.5–)4–5(–6) µm (av. = 53 × 4.5 µm)], and shorter (Crous 2002). than those of Cy. ﬂexuosum Crous [(44–)50–70(–80) × 222 CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS

(4–)5–6 µm (av. = 65 × 5 µm)]. In the past, isolates DISCUSSION of Cy. ecuadoriae were treated as representative of Cy. pseudogracile Crous, which has conidia of similar Several studies in recent years have focused on dimensions of [(40–)53–58(–65) × (3.5–)4–5 µm (av. = resolving the taxonomy of Cylindrocladium spp. with 56 × 4.5 µm)]and are 1(–3)-septate. Cylindrocladium clavate vesicles (Crous et al. 1995, 1997, 1999, 2000, ecuadoriae can be distinguished from Cy. pseudogacile Kang et al. 2001, Crous 2002). In a study focusing based on its lower average conidial length, and the on taxa with sphaeropedunculate vesicles, Crous et absence of a Calonectria state in culture (Crous al. (2004) described nine new species from the Cy. 2002). ﬂoridanum species complex. Contrary to what we

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. 223 CROUS ET AL.

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. 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 Fig. 6. Cylindrocladium ecuadoriae. Penicillate conidiophores, clavate vesicles and conidia. Scale bar = 10 µm. 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 × 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 Fig. 7. Cylindrocladium ilicicola (epitype). Conidiophore, vesicles may be yet more cryptic taxa within this complex that and conidia. Scale bar = 10 µm. need to be resolved. 224 CALONECTRIA SPECIES AND THEIR CYLINDROCLADIUM ANAMORPHS

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

Calonectria pyrochroa (Desm.) Sacc., Michelia 1: Curvicladiella cignea (Decock & Crous) Decock & 308. 1878. Crous, comb. nov. MycoBank MB500867. ≡ Nectria pyrochroa Desm., Pl. Crypt. France Ed. 2: 372. 1856. ≡ Curvicladium cigneum Decock & Crous, Mycologia 90: 277. = Calonectria daldiniana De Not., Comment. Soc. Crittogam. 1998. Ital. 2: 477. 1867. = Ophionectria puiggarii Speg., Bol. Acad. Nac. Ci. 11: 532. Specimens examined: French Guiana, Matoury, first part of the 1889. Lamirande trail, on decaying leaf of unknown angiosperm, 23 Jan. = Nectria abnormis Henn., Hedwigia 36: 219. 1897. 1997, C. Decock FG2240, MUCL 40269 = CPC 1595 = CBS 109167 Anamorph: Cylindrocladium ilicicola (Hawley) (ex-type culture); on decaying seed of unknown angiosperm, 20 Jan. 1997, C. Decock FG2158, MUCL 40268 = CPC 1594 = CBS Boedijn & Reitsma, Reinwardtia 1: 57. 1950. Fig. 7. 109168. ≡ Candelospora ilicicola Hawley, Proc. Roy. Irish Acad. 31: 11. 1912. Notes: It was recently brought to our attention (J. = Tetracytum lauri Vanderw., Parasitica 1: 145. 1945. (as “laurii”). Bischoff, NCBI), that the generic name “Curvicladium”, which was proposed by Decock & Crous (1998) for a Macroconidiophores consisting of a stipe, a penicillate anamorphic fungus collected from leaf litter in French arrangement of fertile branches, a stipe extension, and Guiana, was already occupied for a species of moss a terminal vesicle; stipe septate, hyaline, smooth, up (Enroth 1993). A new name is thus called for, and to 70 µm long, 5–6 µm wide; stipe extensions septate, herewith we propose Curvicladiella Decock & Crous, to straight to flexuous, 160–210 µm long, 3–4 µm wide at replace Curvicladium Decock & Crous (1998). apical septum, terminating in an obpyriform to broadly ellipsoidal vesicle, 5–8 µm diam. Conidiogenous apparatus with primary branches that are aseptate ACKNOWLEDGEMENTS or 1-septate, 15–20 × 3–5 µm; secondary branches aseptate, 10–20 × 3–5 µm, tertiary branches aseptate, The authors acknowledge collections made by various researchers, rarely observed, 8–15 × 3–5 µm, each terminal branch 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. producing 2–4 phialides; phialides doliiform to reniform, Alfenas and M.J. Wingfield, who placed numerous collections from hyaline, aseptate, 9–15 × 3–4 µm, apex with minute diverse locations at our disposal for study. Bart van Asten (CBS) is periclinal thickening and inconspicuous collarette. thanked for the sequencing of isolates. Conidia cylindrical, rounded at both ends, straight, (50–)63–68(–70) × 5(–6) µm, (1–)3-septate, lacking a visible abscission scar, held in parallel cylindrical REFERENCES clusters by colourless slime. Megaconidia and microconidia unknown. Brayford D, Chapman AU (1987). Cylindrocladium ilicicola on cuttings of evergreen ornamental shrubs in the U.K. Plant Pathology 36: Specimens examined: Ireland, Clare Island, Ilex aquifolium, Hawley, 413–414. K 61269!, holotype of Cy. ilicicola, IMI 76542 isotype. Netherlands, Crous PW (2002). Taxonomy and pathology of Cylindrocladium South-East Limburg, Vijlenerbos, Vijlen, Ilex aquifolium, Aug. 1970, (Calonectria) and allied genera. APS Press, St. Paul, MN, H.A van der Aa, epitype designated here CBS H-15110, ex-epitype U.S.A. culture CBS 749.70. Crous PW, Groenewald JZ, Risède J-M, Simoneau P, Hywel-Jones NL (2004). Calonectria species and their Cylindrocladium Cultural characteristics: Cultures sterile, white. anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50: 415–430. Substrate and distribution: See Crous (2002). Crous PW, Kang JC, Schoch CL, Mchau GRA (1999). Phylogenetic relationships of Cylindrocladium pseudogracile and Notes: The genus Calonectria is based upon Calonectria Cylindrocladium rumohrae with morphologically similar taxa, pyrochroa (= Ca. daldiniana), which is linked to a based on morphology and DNA sequences of internal transcribed Cylindrocladium ilicicola anamorph (Rossman 1979, spacers and β-tubulin. Canadian Journal of Botany 77: 1813– 1820. Brayford & Chapman 1987, Crous 2002). All cultures thus Crous PW, Korf A, Van Zyl WH (1995). Nuclear DNA polymorphisms far collected by us, and thought to be representative of of Cylindrocladium species with 1-septate conidia and clavate Cy. ilicicola, have turned out to represent other species, vesicles. Systematic and Applied Microbiology 18: 224–250. and hence no authentic cultures of Cy. ilicicola have as Crous PW, Theron L, Van Zyl WH (1997). Delineation of Cylindrocladium species with 1–3-septate conidia and clavate yet been obtained. A strain not previously studied by us vesicles based on morphology and rDNA RFLPs. Mycological was recently retrieved from the CBS collection (CBS Research 101: 210–214. 749.70). Although the isolate sporulated poorly, it was Crous PW, Schoch CL, El-Gholl N, Schubert TS, Leahy RM (2000). accompanied by a very good specimen, which proved Cylindrocladium angustatum sp. nov., a new leaf spot pathogen to be identical to the original holotype collection. We of Tillandsia capitata from Florida, U.S.A. Mycoscience 41: 521– 526. therefore designate this specimen as epitype, thereby Crous PW, Wingfield MJ (1992). Cylindrocladium leucothoes and obtaining an authentic strain of Cy. ilicicola for further C. hederae, synonyms of C. reteaudii. South African Journal of study. Botany 58: 397–400. Decock C, Crous PW (1998). Curvicladium gen. nov., a new hyphomycete genus from French Guiana. Mycologia 90: 276– Curvicladiella Decock & Crous, nom. nov. MycoBank 281. MB500866. Enroth J (1993). Notes on the Neckeraceae (Musci). 18. Description ≡ Curvicladium Decock & Crous, Mycologia 90: 276. 1998 [non of Curvicladium, a new genus from Southern and Southeastern Curvicladium Enroth, 1993]. Asia. Annales Botanici Fennici 30: 109–117. 225 CROUS ET AL.

Gams W, Hoekstra ES, Aptroot A (eds) (1998). CBS Course of Department of Zoology, University of Oxford, Oxford. Mycology. 4th ed. CBS, Baarn, Netherlands. Rayner RW (1970). A mycological colour chart. CMI and British Kang JC, Crous PW, Old KM, Dudzinski MJ (2001). Non-conspeciﬁcity Mycological Society. Kew, Surrey, England. of Cylindrocladium quinqueseptatum and Calonectria Risède J-M, Simoneau P (2001). Typing Cylindrocladium species by quinqueseptata based on beta-tubulin gene phylogeny and analysis of ribosomal DNA spacers polymorphism: application morphology. Canadian Journal of Botany 79: 1241–1247. to ﬁeld isolates from the banana rhizosphere. Mycologia 93: Lee SB, Taylor JW (1990). Isolation of DNA from fungal mycelia 494–504. and single spores. In: PCR Protocols: A guide to methods and Risède J-M, Simoneau P (2004). Pathogenic and genetic diversity applications (Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds). of soilborne isolates of Cylindrocladium from banana cropping Academic Press, San Diego, California: 282–287. systems. European Journal of Plant Pathology 110: 139–154. O’Donnell K, Cigelnik E (1997). Two divergent intragenomic rDNA Rossman AY (1979). Calonectria and its type species, C. daldiniana, ITS2 types within a monophyletic lineage of the fungus Fusarium a later synonym of C. pyrochroa. Mycotaxon 8: 321–328. are nonorthologous. Molecular Phylogenetics and Evolution 7: Swofford DL (2002). PAUP*. Phylogenetic Analysis Using Parsimony 103–116. (*and other methods). Version 4. Sinauer Associates, Sunderland, Rambaut A (2002). Sequence Alignment Editor. Version 2.0. MA, U.S.A.

226