The forgotten Calonectria collection: Pouring old wine into new bags.

The genus Calonectria with its Cylindrocladium asexual morphs has been subject to several taxonomic revisions in the past. These have resulted in the recognition of 116 species, of which all but two species (C. hederae and C. pyrochroa) are supported by ex-type cultures and supplemented with DNA barcodes. The present study is based on a large collection of unidentified Calonectria isolates that have been collected over a period of 20 years from various substrates worldwide, which has remained unstudied in the basement of the CBS-KNAW Fungal Biodiversity Centre. Employing a polyphasic approach, the identities of these isolates were resolved and shown to represent many new phylogenetic species. Of these, 24 are newly described, while C. uniseptata is reinstated at species level. We now recognise 141 species that include some of the most important plant pathogens globally.

Introduction

The genus Calonectria, first introduced in 1867 (Rossman 1979), has been the subject of numerous taxonomic studies since the 1990s (Crous & Wingfield 1994, Crous, 2002, Lombard et al., 2010b, Lombard et al., 2015a, Alfenas et al., 2015). These studies have resulted in the recognition of 116 species, of which all but two (C. hederae and C. pyrochroa) are supported by ex-type cultures and supplemented by DNA barcodes (Crous, 2002, Lechat et al., 2010, Lombard et al., 2010b). This large number of species has arisen mainly due to the introduction of DNA sequence data and subsequent phylogenetic inference enabling delimitation of numerous previously unrecognised cryptic taxa. These species often share the same plant hosts, informing knowledge of the epidemiology and fungicide resistance (Graça et al., 2009, Vitale et al., 2013, Gehesquière et al., 2016).

Calonectria spp. are characterised by sexual morphs that have yellow to dark red perithecia, with scaly to warty ascocarp walls, and Cylindrocladium asexual morphs in which the cylindrical and septate conidia are produced from phialides clustered below and surrounding a stipe extention terminating in variously shaped vesicles (Rossman, 1993, Crous, 2002, Lombard et al., 2010b, Lombard et al., 2010c). For many years these fungi were best known by their Cylindrocladium names associated with important plant diseases (Crous and Wingfield, 1994, Crous, 2002, Lombard et al., 2010c). Following convention that only one scientific name should be used for a fungal species (Hawksworth, 2011, Hawksworth, 2012, Hawksworth et al., 2011, McNeill et al., 2012), Calonectria has been chosen (Rossman ). This newly adopted convention should resolve confusion regarding their names (Wingfield ). However, it is important to recognise that the asexual Cylindrocladium morph represents the life phase most commonly found in nature and many species are known only in this form, which also plays a major role in the dissemination of Calonectria spp.

Calonectria spp. cause important diseases in numerous plant hosts worldwide. This includes leaf blight, cutting rot, damping-off and root rot (Crous, 2002, Lombard et al., 2010c, Lombard et al., 2015a, Vitale et al., 2013, Alfenas et al., 2015). The majority of the diseases caused by Calonectria spp. are associated with forestry-related plants (see Lombard ), where Calonectria leaf blight (CLB) is an important constraint to plantation productivity in South America (Rodas et al., 2005, Alfenas et al., 2015) and Southeast Asia (Crous and Kang, 2001, Old et al., 2003, Chen et al., 2011, Lombard et al., 2015a). In other regions, such as southern Africa and Australia, Calonectria spp. appear mostly to be limited to forestry nurseries (Crous, 2002, Lombard et al., 2009, Lombard et al., 2010a, Lombard et al., 2010b, Lombard et al., 2010c). In agricultural and horticultural crops, Calonectria spp. have chiefly been reported only from South America and the Northern Hemisphere, where they are mostly associated with nursery diseases (Lombard et al., 2010c, Vitale et al., 2013), Cylindrocladium black rot of peanut (Bell and Sobers, 1966, Beute and Rowe, 1973, Hollowell et al., 1998) and box blight of Buxus spp. (Henricot et al., 2000, Crepel and Inghelbrecht, 2003, Brand, 2005, Saracchi et al., 2008, Saurat et al., 2012, Mirabolfathy et al., 2013, Gehesquière et al., 2016).

The present study is based on a large collection of unidentified Calonectria isolates that were collected over a period of 20 years from various substrates worldwide. This collection of isolates, deposited in the CBS-KNAW culture collection in 2002 has remained unstudied in the basement of the institute and hence, the title of this study “the forgotten basement collection”. The large majority of these isolates were initially identified based solely on morphology and at a time when robust and multigene DNA sequence data were not commonly available. This implied that cryptic species could not be resolved (Lombard et al., 2010b, Lombard et al., 2015a, Alfenas et al., 2015). The aim of the present study was to employ a polyphasic approach to identify these isolates.

Materials and methods

Isolates

Calonectria strains were obtained from the culture collection of the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands and the working collection of the senior author (CPC) housed at the CBS (Table 1).

Table 1

Calonectria spp. used in phylogenetic analyses.

Species	Isolate nr.1	Substrate	Locality	GenBank accession no.2
				tub2	cmdA	tef1
Calonectria acicola	CBS 114812	Phoenix canariensis	New Zealand	DQ190590	GQ267359	GQ267291
	CBS 114813	P. canariensis	New Zealand	DQ190591	GQ267360	GQ267292
C. aconidialis	CBS 136086; CMW 35174; CERC 1850	Soil in Eucalyptus plantation	Hainan, China	–	KJ463017	KJ462785
	CBS 136091; CMW 35384; CERC1886	Soil in Eucalyptus plantation	Hainan, China	–	–	KJ462786
C. amazonica	CBS 115486; CPC 3894	E. tereticornis	Brazil	KX784611	KX784554	KX784681
	CBS 116250; CPC 3534	E. tereticornis	Brazil	KX784612	KX784555	KX784682
C. amazoniensis	CBS 115438; CPC 3890	E. tereticornis	Brazil	KX784613	KX784556	KX784683
	CBS 115439; CPC 3889	E. tereticornis	Brazil	KX784614	KX784557	KX784684
	CBS 115440; CPC 3885	E. tereticornis	Brazil	KX784615	KX784558	KX784685
C. angustata	CBS 109065; CPC 2347	Tillandsia capitata	USA	AF207543	GQ267361	FJ918551
	CBS 112133; CPC 3152	T. capitata	USA	DQ190593	GQ267362	FJ918552
C. arbusta	CBS 136079; CMW 31370; CERC1705	Soil in Eucalyptus plantation	Guangxi, China	KJ462904	KJ463018	KJ462787
	CBS 136098; CPC 23519; CMW37981; CERC 1944	Soil in Eucalyptus plantation	Guangxi, China	–	KJ463019	KJ462788
C. asiatica	CBS 112711; CPC 3898	Leaf litter	Thailand	AY725613	AY725738	AY725702
	CBS 114073; CPC 3900	Leaf litter	Thailand	AY725616	AY725741	AY725705
C. australiensis	CBS 112954	Ficus pleurocarpa	Australia	DQ190596	GQ267363	GQ267293
C. blephiliae	CBS 136425; CPC 21859	Blephilia cliata	USA	KF777246	–	KF777243
C. brachiatica	CBS 123700; CMW 25298	Pinus maximinoi	Buga, Colombia	FJ696388	GQ267366	GQ267296
	CBS134665; LPF305	Soil in Eucalyptus plantation	Monte Dourado, Pará, Brazil	KM395933	KM396020	KM395846
C. brasiliana	CBS 111484; CPC 1924	Soil	Brazil	KX784616	KX784559	KX784686
	CBS 111485; CPC 1929	Soil	Brazil	KX784617	KX784560	KX784687
C. brasiliensis	CBS 230.51; CPC 2390	Anacardium sp.	Brazil	GQ267241	GQ267421	GQ267328
	CBS 114257; CPC 1944	Eucalyptus leaf	Brazil	GQ267242	GQ267422	GQ267329
C. brassiana	CBS 134855	Soil	Teresina, Piauí, Brazil	KM395969	KM396056	KM395882
	CBS 134856	Soil	Teresina, Piauí, Brazil	KM395970	KM396057	KM395883
C. brassicae	CBS 111478; CPC 1921	Soil	Brazil	DQ190611	GQ267383	FJ918568
	CBS 111869; CPC 2409	Argyeia splendens	Indonesia	AF232857	GQ267382	FJ918567
C. brassicicola	CBS 112756; CPC 4502	Brassica sp.	Indonesia	KX784618	–	KX784688
	CBS 112841; CPC 4552	Brassica sp.	Indonesia	KX784619	KX784561	KX784689
	CBS 112947; CPC 4668		New Zealand	KX784620	KX784562	KX784690
C. brevistipitata	CBS 110837; CPC 913	Soil	Mexico	KX784621	KX784563	KX784691
	CBS 110928; CPC 951	Soil	Mexico	KX784622	KX784564	KX784692
	CBS 115671; CPC 949	Soil	Mexico	KX784623	KX784565	KX784693
C. canadania	CBS 110817; CPC 499		Canada	AF348212	AY725743	GQ267297
C. candelabrum	CPC 1675	Eucalyptus sp.	Amazonas, Brazil	FJ972426	GQ267367	FJ972525
	CMW 31001	Eucalyptus sp.	Amazonas, Brazil	GQ421779	GQ267368	GQ267298
C. cerciana	CBS 123693; CMW 25309	Eucalyptus cutting	Zhanjiang, China	FJ918510	GQ267369	FJ918559
	CBS 123695; CMW 25290	Eucalyptus cutting	Zhanjiang, China	FJ918511	GQ267370	FJ918560
C. chinensis	CBS 112744; CPC 4104	Soil	Hong Kong, China	AY725618	AY725746	AY725709
	CBS 114827; CPC 4101	Soil	Hong Kong, China	AY725619	AY725747	AY725710
C. clavata	CBS 114557; ATCC 66389; CPC 2536	Callistemon viminalis	USA	AF333396	GQ267377	GQ267305
	CBS 114666; CMW 30994; CPC 2537	Root debris in peat	USA	DQ190549	GQ267378	GQ267306
C. cliffordiicola	CBS 111812; CPC 2631	Cliffordia feruginea	South Africa	KX784624	KX784566	KX784694
	CBS 111814; CPC 2617	Prunus avium	South Africa	KX784625	KX784567	KX784695
	CBS 111819; CPC 2604	P. avium	South Africa	KX784626	KX784568	KX784696
C. colhounii	CBS 293.79	Camellia sinensis	Bandung, Indonesia	DQ190564	GQ267373	GQ267301
	CBS 114704	Arachis pintoi	Australia	DQ190563	GQ267372	GQ267300
Ca. colombiana	CBS 115127; CPC 1160	Soil	La Selva, Colombia	FJ972423	GQ267455	FJ972492
	CBS 115638; CPC 1161	Soil	La Selva, Colombia	FJ972422	GQ267456	FJ972491
C. colombiensis	CBS 112220; CPC 723	Soil	La Selva, Colombia	GQ267207	AY725748	AY725711
	CBS 112221; CPC 724	Eucalyptus grandis	La Selva, Colombia	AY725620	AY725749	AY725712
C. crousiana	CBS 127198; CMW 27249	E. grandis	Fujian, China	HQ285794	–	HQ285822
	CBS 127199; CMW 27253	E. grandis	Fujian, China	HQ285795	–	HQ285823
C. cylindrospora	CBS 110666; CPC 496		USA	FJ918509	GQ267423	FJ918557
	CBS 119670; CPC 12766	Pistacia lentiscus	Italy	DQ521600	–	GQ421797
C. densa	CBS 125249; CMW 31184	Soil	Las Golondrinas, Pichincha, Ecuador	GQ267230	GQ267442	GQ267350
	CBS 125261; CMW 31182	Soil	Las Golondrinas, Pichincha, Ecuador	GQ267232	GQ267444	GQ267352
C. duoramosa	CBS 134656; LPF434	Soil	Monte Dourado, Pará, Brazil	KM395940	KM396027	KM395853
	LPF453	Soil in Eucalyptus plantation	Monte Dourado, Pará, Brazil	KM395941	KM396028	KM395854
C. ecuadoriae	CBS 111406; CPC 1635	Soil	Ecuador	DQ190600	GQ267375	GQ267303
	CBS 111394; CPC 1628	Soil	Ecuador	DQ190599	GQ267376	GQ267304
C. ericae	CBS 114456; CPC 1984	Erica sp.	USA	KX784627	KX784569	KX784697
	CBS 114457; CPC 1985	Erica sp.	USA	KX784628	KX784570	KX784698
	CBS 114458; CPC 2019	Erica sp.	USA	KX784629	KX784571	KX784699
C. eucalypti	CBS 125273; CMW 14890	E. grandis	Indonesia	GQ267217	GQ267429	GQ267337
	CBS 125275; CMW 18444	E. grandis	Indonesia	GQ267218	GQ267430	GQ267338
C. eucalypticola	CBS 134846	Eucalyptus leaf	Eunápolis, Bahia, Brazil	KM395963	KM396050	KM395876
	CBS 134847	Eucalyptus seedling	Santa Bárbara, Minas Gerais, Brazil	KM395964	KM396051	KM395877
C. expansa	CBS 136078; CMW 31441; CERC 1776	Soil in Eucalyptus plantation	Guangdong, China	KJ462913	KJ463028	KJ462797
	CBS 136247; CMW 31392; CERC 1727	Soil in Eucalyptus plantation	Guangxi, China	KJ462914	KJ463029	KJ462798
C. foliicola	CBS 136641; CMW 31393; CERC 1728	E. urophylla × E. grandis clone leaf	Guangxi, China	KJ462916	KJ463031	KJ462800
	CMW 31394; CERC 1729	E. urophylla × E. grandis clone leaf	Guangxi, China	KJ462917	KJ463032	KJ462801
C. fujianensis	CBS 127200; CMW 27254	E. grandis	Fujian, China	HQ285791	–	HQ285819
	CBS 127201; CMW 27257	E. grandis	Fujian, China	HQ285792	–	HQ285820
C. glaeboicola	CBS 134852	Soil	Martinho Campos, Minas Gerais, Brazil	KM395966	KM396053	KM395879
	CBS 134853	Soil	Bico do Papagaio, Tocantins, Brazil	KM395967	KM396054	KM395880
C. gordoniae	CBS 112142; CPC 3136; ATCC 201837	Gordonia liasanthus	USA	AF449449	GQ267381	GQ267309
C. gracilipes	CBS 111141	Soil	La Selva, Colombia	DQ190566	GQ267385	GQ267311
	CBS 115674	Soil	La Selva, Colombia	AF333406	GQ267384	GQ267310
C. gracilis	CBS 111284	Soil	Brazil	DQ190567	GQ267408	GQ267324
	CBS 111807	Manilkara zapota	Belém, Pará, Brazil	AF232858	GQ267407	GQ267323
C. guangxiensis	CBS 136092; CMW 35409; CERC 1900	Soil in Eucalyptus plantation	Guangxi, China	KJ462919	KJ463034	KJ462803
	CBS 136094; CMW 35411; CERC 1902	Soil in Eucalyptus plantation	Guangxi, China	KJ462920	KJ463035	KJ462804
C. hainanensis	CBS 136248; CMW 35187; CERC 1863	Soil in Eucalyptus plantation	Hainan, China	–	KJ463036	KJ462805
C. hawksworthii	CBS 111870; CPC 2405; MUCL 30866	Nelumbo nucifera	Mauritius	AF333407	GQ267386	FJ918558
C. henricotiae	CB041	Buxus sempervirens	Belgium	KF815129	KF815156	–
	CBS 138102; CB045	B. sempervirens	Belgium	JX535308	KF815157	–
C. hodgesii	CBS 133609; LPF 245	Anadenanthera peregrina	Viçosa, Brazil	KC491228	KC491222	KC491225
	CBS 133610; LPF 261	Azadirachta indica	Viçosa, Brazil	KC491229	KC491223	KC491226
C. hongkongensis	CBS 114711; CPC 686	Soil	Hong Kong, China	AY725621	AY725754	AY725716
	CBS 114828; CPC 4670	Soil	Hong Kong, China	AY725622	AY725755	AY725717
C. humicola	CBS 125251	Soil	Las Golondrinas, Pichincha, Ecuador	GQ267233	GQ267445	GQ267353
	CBS 125269	Soil	Las Golondrinas, Pichincha, Ecuador	GQ267235	GQ267447	GQ267355
C. hurae	CBS 114182; CPC 1714	Rumohra adiantiformis	Brazil	DQ190618	–	–
	CBS 114551; CPC 2344	R. adiantiformis	USA	AF333408	GQ267387	FJ918548
C. ilicicola	CBS 190.50; CMW 30998; IMI 299389	Solanum tuberosum	Bogor, Indonesia	AY725631	AY725764	AY725726
	CBS 115897; CPC 493; UFV 108	Anacardium sp.	Brazil	AY725647	GQ267403	AY725729
C. indonesiae	CBS 112823; CPC 4508	Soil	Warambunga, Indonesia	AY725623	AY725756	AY725718
	CBS 112840; CPC 4554	Syzygium aromaticum	Indonesia	AY725625	AY725758	AY725720
C. indonesiana	CBS 112826; CPC 4519		Indonesia	KX784630	KX784572	KX784700
	CBS 112936; CPC 4504		Indonesia	KX784631	KX784573	KX784701
C. indusiata	CBS 144.36	Camellia sinensis	Sri lanka	GQ267239	GQ267453	GQ267332
	CBS 114684	Rhododendron sp.	USA	AF232862	GQ267454	GQ267333
C. insularis	CBS 114558; CPC 768	Soil	Tamatave, Madagascar	AF210861	GQ267389	FJ918556
	CBS 114559; CPC 954	Soil	Tamatave, Madagascar	AF210862	GQ267390	FJ918555
C. kyotensis	CBS 114525; CPC 2367; ATCC 18834	Acacia dealbata	Japan
	CBS 114542; CPC 2352	Soil	China	KX784649	–	KX784720
	CBS 114550; CPC 2351	Soil	China	KX784650	KX784587	KX784721
	CBS 114692; CPC 2478; ATCC 18882	Prunus sp.	USA	KX784651	KX784588	KX784722
C. lageniformis	CBS 111324; CPC 1473	Eucalyptus sp.	Mauritius	KX784632	KX784574	KX784702
	CBS 112685; CPC 3418	Eucalyptus sp.	Brazil	KX784633	KX784575	KX784703
C. lateralis	CBS 136629; CMW 31412; CERC 1747	Soil in Eucalyptus plantation	Fangchenggang, Guangxi, China	KJ462955	KJ463070	KJ462840
C. lauri	CBS 749.70	Ilex aquifolium	Netherlands	GQ267210	GQ267388	GQ267312
C. leucothoes	CBS 109166; CPC 2385; ATCC 64824	Leucothoe axillaris	Gainsville, Florida, USA	FJ918508	GQ267392	FJ918553
C. machaerinae	CBS 123183; CPC 15378	Machaerina sinclairii	New Zealand	KX784636	–	KX784706
C. madagascariensis	CBS 114571; CPC 2253	Soil	Madagascar	DQ190571	GQ267395	GQ267315
	CBS 114572; CPC 2252	Soil	Madagascar	DQ190572	GQ267394	GQ267314
C. macroconidialis	CBS 114880; CPC 307	E. grandis	South Africa	AF232855	GQ267393	GQ267313
C. magnispora	CBS 136249; CMW 35184; CERC 1860	Soil in Eucalyptus plantation	Guangxi, China	KJ462956	KJ463071	KJ462841
C. malesiana	CBS 112710; CPC 3899	Leaf litter	Thailand	AY725626	AY725759	AY725721
	CBS 112752; CPC 4223	Soil	Sumatra, Indonesia	AY725627	AY725760	AY725722
C. maranhensis	CBS 134811	Eucalyptus sp.	Açailândia, Maranhão, Brazil	KM395948	KM396035	KM395861
	CBS 134812	Eucalyptus sp.	Açailândia, Maranhão, Brazil	KM395949	KM396036	KM395862
C. metrosideri	CBS 133603; LPF101	Metrosideros polymorpha	Viçosa, Brazil	KC294313	KC294304	KC294310
	CBS 133604; LPF 103	M. polymorpha	Viçosa, Brazil	KC294314	KC294305	KC294311
C. mexicana	CBS 110918; CPC 927	Soil	Mexico	AF210863	GQ267396	FJ972526
C. microconidialis	CBS 136636; CMW 31475; CERC 1810	E. urophylla × E. grandis clone seedling leaf	CERC Nursery, Zhanjiang, Guangdong, China	KJ462959	KJ463074	KJ462844
	CBS 136638; CMW 31487; CERC 1822	E. urophylla × E. grandis clone seedling leaf	CERC Nursery, Zhanjiang, Guangdong, China	KJ462960	KJ463075	KJ462845
C. monticola	CBS 140645; CPC 28835	Soil	Thailand	KT964769	KT964771	KT964773
	CPC 28836	Soil	Thailand	KT964770	KT964772	KT964774
C. mossambicensis	CBS 137243; CMW 36327	E. grandis × E. camaldulensis cutting	Mozambique	–	JX570722	JX570718
C. multilateralis	CBS 110926: CPC 947	Soil	Mexico	KX784639	KX784578	KX784709
	CBS 110927; CPC 948	Soil	Mexico	KX784640	KX784579	KX784710
	CBS 110931; CPC 956	Soil	Mexico	KX784641	–	KX784711
	CBS 110932; CPC 957	Soil	Mexico	KX784642	KX784580	KX784712
	CBS 115606
	CBS 115615; CPC 915	Soil	Mexico	KX784643	KX784581	KX784713
C. multinaviculata	CBS 134858; LPF233	Soil in Eucalyptus plantation	Mucuri, Bahia, Brazil	KM395985	KM396072	KM395898
	CBS 134859; LPF418	Soil in Eucalyptus plantation	Monte Dourado, Pará, Brazil	KM395986	KM396073	KM395899
C. multiphialidica	CBS 112678	Soil	Cameroon	AY725628	AY725761	AY725723
C. multiseptata	CBS 112682	Eucalyptus sp.	Indonesia	DQ190573	GQ267397	FJ918535
C. naviculata	CBS 101121	Leaf litter	João Pessoa, Brazil	GQ267211	GQ267399	GQ267317
	CBS 116080	Soil	Amazonas, Brazil	AF333409	GQ267398	GQ267316
C. nemicola	CBS 134837	Soil	Araponga, Minas Gerais, Brazil	KM395979	KM396066	KM395892
	CBS 134838	Soil	Araponga, Minas Gerais, Brazil	KM395980	KM396067	KM395893
C. orientalis	CBS 125259	Soil	Teso East, Indonesia	GQ267237	GQ267449	GQ267357
	CBS 125260	Soil	Lagan, Indonesia	GQ267236	GQ267448	GQ267356
C. ovata	CBS 111299	E. tereticornis	Tucuruí, Pará, Brazil	GQ267212	GQ267400	GQ267318
	CBS 111307	E. tereticornis	Tucuruí, Pará, Brazil	AF210868	GQ267401	GQ267319
C. pacifica	CBS 109063; CPC 2534; IMI 354528	Araucaria heterophylla	Hawaii, USA	GQ267213	AY725762	AY725724
	CBS 114038; CPC 10717	Ipomoea aquatica	Auckland, New Zealand	AY725630	GQ267402	GQ267320
C. papillata	CBS 136096; CMW 37972; CERC 1935	Soil in Eucalyptus plantation	Guangdong, China	KJ462963	KJ463078	KJ462848
	CBS 136097; CMW 37976; CERC 1939	Soil in Eucalyptus plantation	Guangdong, China	KJ462964	KJ463079	KJ462849
C. paracolhounii	CBS 114679; CPC 2445		USA	KX784644	KX784582	KX784714
	CBS 114705; CPC 2423		USA	KX784645	–	KX784715
C. paraensis	CBS 134669; LPF430	Soil in Eucalyptus plantation	Monte Dourado, Pará, Brazil	KM395924	KM396011	KM395837
	LPF306	Soil in Eucalyptus plantation	Monte Dourado, Pará, Brazil	KM395925	KM396012	KM395838
C. parakyotensis	CBS 136085; CMW 35169; CERC 1845	Soil in Eucalyptus plantation	Guangdong, China	–	KJ463081	KJ462851
	CBS 136095; CMW 35413; CERC 1904	Soil in Eucalyptus plantation	Guangxi, China	–	KJ463082	KJ462852
C. parva	CBS 110798; CPC 410	Soil	South Africa	KX784646	KX784583	KX784716
C. pauciramosa	CBS 138824; CMW 5683	E. grandis	South Africa	FJ918514	GQ267405	FJ918565
	CMW 30823	E. grandis	South Africa	FJ918515	GQ280404	FJ918566
C. penicilloides	CBS 174.55; IMI 299375	Prunus sp.	Japan	AF333414	GQ267406	GQ267322
C. pentaseptata	CBS 136087; CMW 35177; CERC 1853	Eucalyptus leaf	Hainan, China	KJ462966	KJ463083	KJ462853
	CBS 136089; CMW 35377; CERC 1879	Eucalyptus leaf	Hainan, China	KJ462967	KJ463084	KJ462854
C. piauiensis	CBS 134849	Soil	Serra das Confusões, Piauí	KM395972	KM396059	KM395885
	CBS 134850	Soil	Teresina, Piauí, Brazil	KM395973	KM396060	KM395886
C. pini	CBS 123698	Pinus patula	Buga, Colombia	GQ267224	GQ267436	GQ267344
	CBS 125253	P. patula	Buga, Colombia	GQ267225	GQ267437	GQ267345
C. polizzi	CBS 125270; CMW 7804	Callistemon citrinus	Messina, Sicily, Italy	FJ972417	GQ267461	FJ972486
	CBS 125271; CMW 10151	Arbustus unedo	Catania, Sicily, Italy	FJ972418	GQ267462	FJ972487
C. plurilateralis	CBS 111401; CPC 1637		Ecuador	KX784648	KX784586	KX784719
C. pluriramosa	CBS 136976; CMW 31440; CERC 1774	Soil in Eucalyptus plantation	Fangchenggang, Guangxi, China	KJ462995	KJ463112	KJ462882
C. propaginicola	CBS 134815; LPF220	Eucalyptus sp.	Santana, Pará, Brazil	KM395953	KM396040	KM395866
	CBS 134816; LPF222	Eucalyptus sp.	Santana, Pará, Brazil	KM395954	KM396041	KM395867
C. pseudobrassicae	CBS 134661; LPF260	Soil in Eucalyptus plantation	Santana, Pará, Brazil	KM395935	KM396022	KM395848
	CBS 134662; LPF280	Soil in Eucalyptus plantation	Santana, Pará, Brazil	KM395936	KM396023	KM395849
C. pseudocerciana	CBS 134823	Eucalyptus sp.	Santana, Pará, Brazil	KM395961	KM396048	KM395874
	CBS 134824	Eucalyptus seedling	Santana, Pará, Brazil	KM395962	KM396049	KM395875
C. pseudocolhounii	CBS 127195; CMW 27209	E. dunnii	Fujian, China	HQ285788	–	HQ285816
	CBS 127196; CMW 27213	E. dunnii	Fujian, China	HQ285789	–	HQ285817
C. pseudoecuadoriae	CBS 111402; CPC 1639		Ecuador	KX784652	KX784589	KX784723
	CBS 111412; CPC 1648	Soil	Ecuador	DQ190601	KX784590	KX784724
C. pseudohodgesii	CBS 134818	Azadirachta indica	Viçosa, Minas Gerais, Brazil	KM395905	KM395991	KM395817
	CBS 134819	A. indica	Viçosa, Minas Gerais, Brazil	KM395906	KM395992	KM395818
C. pseudokyotensis	CBS 137332; CMW 31439; CERC 1774	Soil in Eucalyptus plantation	Fangchenggang, Guangxi, China	KJ462994	KJ463111	KJ462881
C. pseudometrosideri	CBS 134844	Eucalyptus sp.	Açailândia, Maranhão, Brazil	KM395908	KM395994	KM395820
	CBS 134845	Soil	Maceió, Alagoas, Brazil	KM395909	KM395995	KM395821
C. pseudomexicana	CBS 130354	Callistemon sp.	Tunisia	JN607281	–	JN607496
	CBS 130355	Callistemon sp.	Tunisia	JN607282	–	JN607497
Ca. pseudonaviculata	CBS 114417; CPC 10926	Buxus sempervirens	West Auckland, New Zealand	GQ267214	GQ267409	GQ267325
	CBS 116251; CPC 3399	B. sempervirens	New Zealand	AF449455	KM396000	KM395826
C. pseudopteridis	CBS 163.28; IMI 299579	Washingtonia robusta	USA	–	KM396076	KM395902
C. pseudoreteaudii	CBS 123694; CMW 25310	Eucalyptus hybrid cutting	Guangdong, China	FJ918504	GQ267411	FJ918541
	CBS 123696; CMW 25292	Eucalyptus hybrid cutting	Guangdong, China	FJ918505	GQ267410	FJ918542
C. pseudoscoparia	CBS 125255; CMW 15216	E. grandis	Pichincha, Ecuador	GQ267227	GQ267439	GQ267347
	CBS 125256; CMW 15216	E. grandis	Pichincha, Ecuador	GQ267228	GQ267440	GQ267348
C. pseudospathiphylli	CBS 109165; CPC 1623	Soil	Ecuador	FJ918513	GQ267412	FJ918562
C. pseudospathulata	CBS 134840	Soil	Araponga, Minas Gerais, Brazil	KM395982	KM396069	KM395895
	CBS 134841	Soil	Araponga, Minas Gerais, Brazil	KM395983	KM396070	KM395896
C. pseudouxmalensis	CBS 110923; CPC 941	Soil	Mexico	KX784653	–	KX784725
	CBS 110924; CPC 942	Soil	Mexico	KX784654	–	KX784726
	CBS 115677; CPC 943	Soil	Mexico	KX784655	–	KX784727
C. pseudovata	CBS 134674; LPF267	Soil in Eucalyptus plantation	Santana, Pará, Brazil	KM395945	KM396032	KM395858
	CBS 134675; LPF285	Soil in Eucalyptus plantation	Santana, Pará, Brazil	KM395946	KM396033	KM395859
C. pteridis	CBS 111793; ATCC 34395; CPC 2372	Arachnoides adiantiformis	USA	DQ190578	GQ267413	FJ918563
	CBS 111871; CPC 2443	Pinus sp.	Spain	DQ190579	GQ267414	FJ918564
C. putriramosa	CBS 111449; CPC 1951	Eucalyptus cutting	Brazil	KX784656	KX784591	KX784728
	CBS 111470; CPC 1940	Soil	Brazil	KX784657	KX784592	KX784729
	CBS 111477; CPC 1928	Soil	Brazil	KX784658	KX784593	KX784730
	CBS 116076; CPC 604	Eucalyptus cutting	Brazil	GQ421776	–	GQ421792
C. queenslandica	CBS 112146; CPC 3213	E. urophylla	Australia	AF389835	GQ267415	FJ918543
	CBS 112155; CPC 3210	E. pellita	Australia	AF389834	GQ267416	FJ918544
C. quinqueramosa	CBS 134654; LPF065	Soil in Eucalyptus plantation	Monte Dourado, Pará, Brazil	KM395942	KM396029	KM395855
	CBS 134655; LPF281	Soil in Eucalyptus plantation	Santana, Pará, Brazil	KM395943	KM396030	KM395856
C. reteaudii	CBS 112143; CPC 3200	E. camaldulensis	Vietnam	GQ240642	GQ267418	FJ918536
	CBS 112144; CPC 3201	E. camaldulensis	Vietnam	AF389833	GQ267417	FJ918537
C. robigophila	CBS 134652	Eucalyptus sp.	Açailândia, Maranhão, Brazil	KM395937	KM396024	KM395850
	CBS 134653	Eucalyptus sp.	Açailândia, Maranhão, Brazil	KM395938	KM396025	KM395851
C. rumohrae	CBS 109062; CPC 1603	Adianthum sp.	Netherlands	AF232873	GQ267420	FJ918550
	CBS 111431; CPC 1716	R. adiantiformis	Brazil	AF232871	GQ267419	FJ918549
C. seminaria	CBS 136631; CMW 31449; CERC 1784	E. urophylla × E. grandis clone seedling leaf	CERC Nursery, Zhanjiang, Guangdong, China	KJ462997	KJ463114	KJ462884
	CBS 136632; CMW 31450; CERC 1785	E. urophylla × E. grandis clone seedling leaf	CERC Nursery, Zhanjiang, Guangdong, China	KJ462998	KJ463115	KJ462885
C. silvicola	CBS 134836	Soil	Araponga, Minas Gerais, Brazil	KM395975	KM396062	KM395888
	CBS 135237	Soil	Araponga, Minas Gerais, Brazil	KM395978	KM396065	KM395891
C. spathulata	CBS 555.92	E. viminalis	Brazil	AF308463	GQ267426	FJ918554
	CBS 115639; CPC 1148		Colombia	KX784659	KX784594	KX784732
	CBS 115644; CPC 1071	E. grandis	Colombia	KX784660	KX784595	KX784733
C. spathiphylli	CBS 114540; ATCC 44730; CPC 2378	Spathiphyllum sp.	USA	AF348214	GQ267424	GQ267330
	CBS 116168; CPC 789	Spathiphyllum sp.	Switzerland	FJ918512	GQ267425	FJ918561
C. sphaeropendunculata	CBS 136081; CMW 31390; CERC 1725	Soil in Eucalyptus plantation	Guangxi, China	KJ463003	KJ463120	KJ462890
C. stipitata	CBS 112513; CPC 3851	Eucalyptus sp.	Colombia	KX784661	KX784596	KX784734
C. sulawesiensis	CBS 125253; CMW 14879	Eucalyptus sp.	Sulawesi, Indonesia	GQ267220	GQ267432	GQ267340
	CBS 125277	Eucalyptus sp.	Sulawesi, Indonesia	GQ267222	GQ267434	GQ267342
C. sumatrensis	CBS 112829; CPC 4518	Soil	Sumatra, Indonesia	AY725649	AY725771	AY725733
	CBS 112934; CPC 4516	Soil	Indonesia	AY725651	AY725773	AY725735
C. syzygiicola	CBS 112827; CPC 4512	S. aromaticum	Indonesia	KX784662	KX784597	KX784735
	CBS 112831; CPC 4511	S. aromaticum	Indonesia	KX784663	–	KX784736
C. telluricola	CBS 134663; LPF214	Soil	Salinas, Minas Gerais, Brazil	KM395929	KM396016	KM395842
	CBS 134664; LPF217	Soil	Mucuri, Bahia, Brazil	KM395930	KM396017	KM395843
C. tereticornis	CBS 111301; CPC 1429	E. tereticornis	Brazil	KX784664	–	KX784737
C. terrae-reginae	CBS 112151; CPC 3202	E. urophylla	Queensland, Australia	FJ918506	GQ267451	FJ918545
	CBS 112634; CPC 4233	Xanthorrhoea australis	Victoria, Australia	FJ918507	GQ267452	FJ918546
C. terrestris	CBS 136642; CMW 35180; CERC 1856	Soil in Eucalyptus plantation	Guangdong, China	KJ463004	KJ463121	KJ462891
	CBS 136643; CMW 35364; CERC 1868	Soil in Eucalyptus plantation	Guangdong, China	KJ463005	KJ463122	KJ462892
C. terricola	CBS 116247; CPC 3583	Soil in Eucalyptus plantation	Brazil	KX784665	–	KX784738
	CBS 116248; CPC 3536	Soil in Eucalyptus plantation	Brazil	KX784666	–	KX784739
C. tetraramosa	CBS 136635; CMW 31474; CERC 1809	E. urophylla × E. grandis clone seedling leaf	CERC Nursery, Zhanjiang, Guangdong, China	KJ463011	KJ463128	KJ462898
	CBS 136637; CMW 31476; CERC 1811	E. urophylla × E. grandis clone seedling leaf	CERC Nursery, Zhanjiang, Guangdong, China	KJ463012	KJ463129	KJ462899
C. tropicalis	CBS 116242; CPC 3543	Eucalyptus sp.	Brazil	KX784668	–	KX784741
	CBS 116271; CPC 3559	Eucalyptus sp.	Brazil	KX784669	KX784599	KX784742
C. turangicola	CBS 136077; CMW 31411; CERC 1746	Soil in Eucalyptus plantation	Fangchenggang, Guangxi, China	KJ463013	–	KJ462900
	CBS 136093; CMW 35410; CERC 1901	Soil in Eucalyptus plantation	Guangxi, China	KJ463014	KJ463130	KJ462901
C. tunisiana	CBS 130356	Callistemon sp.	Tunisia	JN607277	–	JN607292
	CBS 130357	C. laevis	Tunisia	JN607276	–	JN607291
C. uniseptata	CBS 413.67; CPC 2391; IMI 299577	Paphiopedilum callosum	Celle, Germany	GQ267208	GQ267379	GQ267307
	CBS 170.77; IMI 299388	Idesia polycarpa	Auckland, New Zealand	GQ267209	GQ267380	GQ267308
C. uxmalensis	CBS 110919; CPC 928	Soil	Mexico	KX784637	–	KX784707
	CBS 110925; CPC 945	Soil	Mexico	KX784638	–	KX784708
C. variabilis	CBS 112691; CPC 2506	Theobroma grandiflorum	Brazil	GQ267240	GQ267458	GQ267335
	CBS 114677; CPC 2436	Schefflera morotoni	Brazil	AF333424	GQ267457	GQ267334
C. venezuelana	CBS 111052; CPC 1183		Venezuela	KX784671	KX784601	KX784744
Ca. zuluensis	CBS 125268	E. grandis	South Africa	FJ972414	GQ267459	FJ972483
	CBS 125272	E. grandis	South Africa	FJ972415	GQ267460	FJ972484
Calonectria sp.	CBS 111423; CPC 1650		Ecuador	KX784673	KX784603	KX784746
	CBS 111465; CPC 1902	Soil	Brazil	DQ190607	KX784584	KX784717
	CBS 111706; CPC 1636		Ecuador	KX784674	KX784604	KX784747
	CBS 112152; CPC 3203	E. camaldulensis	Vietnam	KX784672	KX784602	KX784745
	CBS 112753; CPC 4225		Indonesia	KX784667	KX784598	KX784740
	CBS 113496; CPC 3155			KX784675	KX784605	KX784748
	CBS 113627; CPC 3232			KX784676	KX784606	KX784749
	CBS 114164; CPC 1634		Ecuador	KX784677	KX784607	KX784750
	CBS 114691; CPC 2472; AR 2574		Canada	KX784678	KX784608	KX784751
	CBS 114755; CPC 1403	E. tereticornis	Brazil	KX784670	KX784600	KX784743
	CBS 116108; CPC 726	Soil	Colombia	KX784647	KX784585	KX784718
	CBS 116249; CPC 3533	Eucalyptus sp.	Brazil	KX784679	KX784609	KX784752
	CBS 116265; CPC 3552	Eucalyptus sp.	Brazil	KX784680	KX784610	KX784753
	CBS 116305; CPC 3890	Eucalytus sp.	Brazil	KX784634	KX784576	KX784704
	CBS 116319; CPC 3761	Eucalytus sp.	Brazil	KX784635	KX784577	KX784705
Curvicladiella cignea	CBS 109167; CPC 1595; MUCL 40269	Leaf litter	French Guiana	KM232002	KM231287	KM231867

AR: Amy Y. Rossman working collection; ATCC: American Type Culture Collection, Virginia, USA; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CERC: China Eucalypt Research Centre, Zhanjiang, Guangdong Province, China; CMW: culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; CPC: Pedro Crous working collection housed at CBS; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, UK; LPF: Laboratório de Patologia Florestal, Universidade Federal de Viçosa, Viçosa, Brazil; MUCL: Mycothèque, Laboratoire de Mycologie Systématique st Appliqée, l’Université, Louvian-la-Neuve, Belgium; UFV: Universidade Federal de Viçosa, Viçosa, Brazil. Isolates obtained during the survey indicated in grey blocks.

tub2 = β-tubulin, cmdA = calmodulin, tef1 = translation elongation factor 1-alpha. Ex-type isolates indicated in bold. Sequences generated in this study indicated in italics.

Phylogeny

Total genomic DNA was extracted from 7-d-old axenic cultures, grown on MEA at room temperature, using the UltraClean™ Microbial DNA isolation kit (Mo Bio Laboratories, Inc., California, USA) following the protocols provided by the manufacturer. Based on previous studies (Lombard et al., 2010b, Lombard et al., 2015b, Alfenas et al., 2015), partial gene sequences were determined for β-tubulin (tub2), calmodulin (cmdA), and the translation elongation factor 1-alpha (tef1) regions as these regions provided the best phylogenetic signal at species level for the genus Calonectria. Therefore, the primers and protocols described by Lombard were used to determine these regions.

To ensure the integrity of the sequences, the amplicons were sequenced in both directions using the same primers used for amplification. Consensus sequences for each locus were assembled in MEGA v. 7 (Kumar ) and compared with representative sequences from Alfenas et al., 2013a, Alfenas et al., 2013b, Alfenas et al., 2015, Chen et al., 2011 and Lombard et al., 2010a, Lombard et al., 2010b, Lombard et al., 2011, Lombard et al., 2015a. Subsequent alignments for each locus were generated in MAFFT v. 7.110 (Katoh & Standley 2013) and the ambiguously aligned regions of both ends were truncated. Congruency of the three loci was tested using the 70 % reciprocal bootstrap criterion (Mason-Gamer & Kellogg 1996) following the protocols of Lombard .

Phylogenetic analyses of the individual gene regions and the combined dataset were based on Bayesian inference (BI), Maximum Likelihood (ML) and Maximum Parsimony (MP). For BI and ML, the best evolutionary models for each locus were determined using MrModeltest (Nylander 2004) and incorporated into the analyses. MrBayes v. 3.2.1 (Ronquist & Huelsenbeck 2003) was used for BI to generate phylogenetic trees under optimal criteria for each locus. A Markov Chain Monte Carlo (MCMC) algorithm of four chains was initiated in parallel from a random tree topology with the heating parameter set at 0.3. The MCMC analysis lasted until the average standard deviation of split frequencies was below 0.01 with trees saved every 1 000 generations. The first 25 % of saved trees were discarded as the “burn-in” phase and posterior probabilities (PP) were determined from the remaining trees.

The ML analyses were preformed using RAxML v. 8.0.9 (randomised accelerated (sic) maximum likelihood for high performance computing; Stamatakis 2014) through the CIPRES website (http://www.phylo.org) to obtain another measure of branch support. The robustness of the analysis was evaluated by bootstrap support (BS) with the number of bootstrap replicates automatically determined by the software.

For MP, analyses were done using PAUP (Phylogenetic Analysis Using Parsimony, v. 4.0b10; Swofford 2003) with phylogenetic relationships estimated by heuristic searches with 1 000 random addition sequences. Tree-bisection-reconnection was used, with branch swapping option set on “best trees” only. All characters were weighted equally and alignment gaps treated as fifth state. Measures calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistence index (RC). Bootstrap analyses (Hillis & Bull 1993) were based on 1 000 replications. All new sequences generated in this study were deposited in GenBank (Table 1) and alignments and trees in TreeBASE.

Taxonomy

Axenic cultures were transferred to synthetic nutrient-poor agar (SNA; Nirenburg 1981) and incubated at room temperature for 7 d. Gross morphological characteristics were studied by mounting the fungal structures in 85 % lactic acid and 30 measurements were made at ×1 000 magnification for all taxonomically informative characters using a Zeiss Axioscope 2 microscope with differential interference contrast (DIC) illumination. The 95 % confidence levels were determined for the conidial measurements with extremes given in parentheses. For all other fungal structures measured, only the extremes are provided. Colony colour was assessed using 7-d-old cultures on MEA incubated at room temperature and the colour charts of Rayner (1970). All descriptions, illustrations and nomenclatural data were deposited in MycoBank (Crous ).

Results

Phylogenetic analyses

Approximately 500−550 bases were determined for the three gene regions included in this study. The congruency analyses revealed no conflicts in tree topologies, with only minor differences in branch support. Therefore, the sequences of the three loci determined here were combined in a single dataset for analyses. For the BI and ML analyses, a HKY+I+G model was selected for all three gene regions and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI and MP analyses, and therefore, only the ML tree is presented.

The combined cmdA, tef1 and tub2 sequences dataset included 278 ingroup taxa and Curvicladiella cignea (CBS 109167) as outgroup taxon. This dataset consisted of 1 680 characters, of which 507 were constant, 198 parsimony-uninformative and 975 parsimony-informative. The MP analysis yielded 1 000 trees (TL = 6 998; CI = 0.344; RI = 0.867; RC = 0.298) and a single best ML tree with −InL = −32198.651254 which is presented in Fig. 1. The BI lasted for 10 M generations, and the consensus tree, with posterior probabilities, was calculated from 15 002 trees left after 5 000 trees were discarded as the ‘burn-in’ phase. In the phylogenetic tree (Fig. 1) the previously unnamed Calonectria species resolved in 21 distinct clades that were either well or strongly supported and 17 single lineages, each representing probable novel phylogenetic taxa.

Fig. 1

The ML consensus tree inferred from the combined cmdA, tef1 and tub2 sequence alignments. Thickened lines indicate branches present in the ML, MP and Bayesian consensus trees. Branches with ML-BS & MP-BS = 100 % and PP = 1.00 are in blue. Branches with ML-BS & MP-BS ≥ 75 % and PP ≥ 0.95 are in red. Dashed lines indicate branches shortened ×10. The scale bar indicates 0.09 expected changes per site. The tree is rooted to Curvicladiella cignea (CBS 109167). Epi- and ex-type strains are indicated in bold.

Based on phylogenetic inference supported by morphological observations, numerous Calonectria isolates included in this study represent novel species. No sexual morphs were observed for any of the novel taxa described below, even after 6 wk of incubation at room temperature. Fifteen of the lineages (CBS 111423, CBS 111468, CBS 111706, CBS 112152, CBS 112753, CBS 113496, CBS 113627, CBS 114164, CBS 114691, CBS 114755, CBS 116108, CBS 116249, CBS 116265, CBS 116305, CBS 116319) identified based on phylogenetic inference are not provided with names because they form part of a separate study (Crous et al. in prep.) or more taxa are required to resolve their phylogenetic position.

L. Lombard & Crous, sp. nov. MycoBank MB818698. Fig. 2.

Fig. 2

Calonectria amazonica (ex-type CBS 116250). A. Macroconidiophore. B–C. Conidiogenous apparatus with conidiophore branches and allantoid to elongate doliiform to reniform phialides. D–E. Clavate vesicles. F–G. Macroconidia. Scale bars: A = 50 μm; B−G = 10 μm.

Etymology: Name refers to the Amazonian region of Brazil where this fungus was collected.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 75–190 × 6–8 μm; stipe extension septate, straight to flexuous, 180–270 μm long, 4–5 μm wide at the apical septum, terminating in a clavate vesicle, 5–6 μm diam. Conidiogenous apparatus 45–55 μm wide, and 60–80 μm long; primary branches aseptate, 22–32 × 4–6 μm; secondary branches aseptate, 14–24 × 3–5 μm; tertiary branches aseptate, 10–18 × 2–4 μm; quaternary branches aseptate, 10−15 × 3 μm, each terminal branch producing 2–4 phialides; phialides allantoid to elongate doliiform to reniform, hyaline, aseptate, 9–20 × 3–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (68–)74–84(–88) × (4−)4.5−5.5(−6) μm (av. 79 × 5 μm), 1(−3)-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (40−65 mm diam) on MEA after 7 d at room temperature; surface sienna to sepia with moderate white, wooly aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse sienna to sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, Amazon, from foliar lesion of Eucalyptus tereticornis, 1993, P.W. Crous & A.C. Alfenas (holotype CBS-H22750, culture ex-type CBS 116250 = CPC 3534); ibid., cultures CBS 115486 = CPC 3894.

Notes: Calonectria amazonica resides in the C. pteridis complex. The macroconidia of C. amazonica [(68–)74–84(–88) × (4−)4.5−5.5(−6) μm (av. 79 × 5 μm)] are slightly smaller than those of C. pteridis and C. pseudopteridis [(50–)70–100(–130) × (4−)5−6 μm (av. 82 × 5.5 μm); Crous, 2002, Alfenas et al., 2015], but larger than those of C. amazoniensis, C. lageniformis and C. tropicalis (see below).

L. Lombard & Crous, sp. nov. MycoBank MB818699. Fig. 3.

Fig. 3

Calonectria amazoniensis (ex-type CBS 115440). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and elongate doliiform to reniform phialides. F. Conidiogenous apparatus with lateral stipe extension. G–J. Clavate vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the Amazonian region of Brazil where this fungus was collected.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 45–240 × 6–9 μm; stipe extension septate, straight to flexuous, 140–280 μm long, 4–5 μm wide at the apical septum, terminating in a clavate vesicle, 5–7 μm diam; lateral stipe extensions (90° to main axis) few, 80–95 μm long, 2–4 μm wide at the apical septum, terminating in clavate vesicles, 2–3 μm diam. Conidiogenous apparatus 30–110 μm wide, and 30–100 μm long; primary branches aseptate, 15–31 × 4–6 μm; secondary branches aseptate, 10–26 × 3–5 μm; tertiary branches aseptate, 9–31 × 3–5 μm; quaternary branches and additional branches (−5) aseptate, 9−18 × 3–5 μm each terminal branch producing 2–4 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 7–17 × 3–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (56–)64–74(–75) × (4−)4.5−5.5(−6) μm (av. 69 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (40−65 mm diam) on MEA after 7 d at room temperature; surface sienna to amber with moderate white, wooly aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse sienna with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, Amazon, from foliar lesion of Eucalyptus tereticornis, 1993, P.W. Crous & A.C. Alfenas (holotype CBS-H22751 culture ex-type CBS 115440 = CPC 3885); ibid., cultures CBS 115438 = CPC 3890, CBS 115439 = CPC 3889.

Notes: Calonectria amazoniensis resides in the C. pteridis complex. This species can be distinguished from other species in the C. pteridis complex by its greater number (−5) of branches in the conidiogenous apparatus and the presence of lateral stipe extensions (Crous, 2002, Alfenas et al., 2015).

L. Lombard & Crous, sp. nov. MycoBank MB818700. Fig. 4.

Fig. 4

Calonectria brasiliana (ex-type CBS 111484). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–J. Ellipsoid to obpyrifom vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to Brazil, the country where this fungus was collected.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 40–240 × 5–10 μm; stipe extension septate, straight to flexuous, 117–172 μm long, 4–6 μm wide at the apical septum, terminating in an ellipsoid to obpyriform vesicle, 6–9 μm diam. Conidiogenous apparatus 45–100 μm wide, and 40–70 μm long; primary branches aseptate, 16–23 × 4–6 μm; secondary branches aseptate, 10–17 × 3–6 μm; tertiary branches aseptate, 7–13 × 3–5 μm; quaternary branches and additional branches (–5) aseptate, 7–14 × 3–4 μm each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–12 × 3–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (36–)38–42(–46) × (3−)3.5−4.5(−5) μm (av. 40 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (30−60 mm diam) on MEA after 7 d at room temperature; surface cinnamon to brick with sparse, felty, white aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse cinnamon to sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, from soil, Jun. 1998, A.C. Alfenas (holotype CBS-H22752, culture ex-type CBS 111484 = CPC 1924); ibid., culture CBS 111485 = CPC 1929.

Notes: Calonectria brasiliana is a new species in the C. candelabrum complex (Schoch et al., 1999, Lombard et al., 2010a, Lombard et al., 2010b, Lombard et al., 2015a). The macroconidia of C. brasiliana [(36–)38–42(–46) × (3−)3.5−4.5(−5) μm (av. 40 × 4 μm)] are smaller than those of its closest phylogenetic neighbours (Fig. 1): C. candelabrum [(45–)58–68(–80) × 4−5(−6) μm (av. 60 × 4.5 μm); Crous 2002], C. eucalypticola [(43–)49–52(–55) × 3−5 μm (av. 50 × 4 μm); Alfenas ], C. glaebicola [(45–)50–52(–55) × 3−5 μm (av. 50 × 4 μm); Alfenas ], C. metrosideri [(40–)44–46(–51) × 3−5 μm (av. 45 × 4 μm); Alfenas et al., 2013a, Alfenas et al., 2015], C. pseudometrosideri [(40–)49–52(–60) × (3−)4.5(−5) μm (av. 51 × 4.5 μm); Alfenas ] and C. pseudoscoparia [(41–)45–51(–52) × 3−5 μm (av. 48 × 4 μm); Lombard ].

L. Lombard & Crous, sp. nov. MycoBank MB818701. Fig. 5.

Fig. 5

Calonectria brassicicola (ex-type CBS 112841). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with lateral stipe extensions and doliiform to reniform phialides. G–J. Sphaeropedunculate vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the host plant, Brassica, from which this fungus was isolated.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicles; stipe septate, hyaline, smooth, 30–90 × 6–9 μm; stipe extension septate, straight to flexuous, 90–140 μm long, 4–5 μm wide at the apical septum, terminating in a sphaeropedunculate vesicle, 6–10 μm diam; lateral stipe extensions (90° to main axis) sparse, 30–50 μm long, 2–4 μm wide at the apical septum, terminating in sphaeropedunculate vesicles, 3−5 μm. Conidiogenous apparatus 45–80 μm wide, and 35–50 μm long; primary branches aseptate, 12–20 × 4–6 μm; secondary branches aseptate, 8–13 × 3–5 μm; tertiary branches aseptate, 8–12 × 3–6 μm; quaternary branches aseptate, 8–11 × 2–5 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. Macroconidia cylindrical, rounded at both ends, straight, (36−)39–45(–48) × (4−)4.5–5.5(−6) μm (av. 42 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (50−65 mm diam) on MEA after 7 d at room temperature; surface buff with abundant white to buff, wooly aerial mycelium, and moderate sporulation on the colony surface; reverse sienna, chlamydospores not observed.

Specimens examined: Indonesia, from soil at Brassica sp., 1990s, M.J. Wingfield (holotype CBS-H22753, culture ex-type CBS 112841 = CPC 4552); ibid., culture CBS 112756 = CPC 4502. New Zealand, substrate unknown, 2001, C.F. Hill, Lynfield 484, culture CBS 112947 = CPC 4668.

Notes: Calonectria brassicicola is similar to C. sumatrensis in having few lateral stipe extensions (Crous ). The macroconidia of C. brassicicola [(36−)39–45(–48) × (4−)4.5–5.5(−6) μm (av. 42 × 5 μm)] are smaller than those of C. sumatrensis [(45−)55–65(–70) × (4.5–)5(−6) μm (av. 58 × 5 μm); Crous ].

L. Lombard & Crous, sp. nov. MycoBank MB818702. Fig. 6.

Fig. 6

Calonectria brevistipitata (ex-type CBS 115671). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and elongate doliiform to reniform phialides. F. Conidiogenous apparatus with lateral stipe extension. G–J. Fusiform to ellipsoid vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the short stipe extensions of the macroconidiophores in this fungus.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 50–210 × 5–12 μm; stipe extension septate, straight to flexuous, 90–135 μm long, 2–5 μm wide at the apical septum, terminating in an fusiform to obpyriform vesicle, 5–8 μm diam; lateral stipe extensions (90° to main axis) abundant, 60–80 μm long, 2–3 μm wide at the apical septum, terminating in broadly clavate vesicles, 2–3 μm diam. Conidiogenous apparatus 45–75 μm wide, and 45–70 μm long; primary branches aseptate, 13–25 × 4–6 μm; secondary branches aseptate, 10–19 × 3–5 μm; tertiary branches aseptate, 8–16 × 3–5 μm; quaternary branches aseptate, 7–11 × 3–4 μm each terminal branch producing 2–6 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 6–11 × 2–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, 29–33(–35) × 3−4 μm (av. 31 × 3.5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (40−70 mm diam) on MEA after 7 d at room temperature; surface cinnamon to brick to sienna with abundant, wooly, white to buff aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse cinnamon to sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Mexico, from soil, Apr. 1994, P.W. Crous (holotype CBS-H22754, culture ex-type CBS 115671 = CPC 949); ibid., cultures CBS 110837 = CPC 913, CBS 110928 = CPC 951.

Notes: Calonectria brevistipitata is a new species in the C. candelabrum complex. The lateral stipe extensions (up to 80 μm long) and macroconidia [29–33(–35) × 3−4 μm (av. 31 × 3.5 μm) of C. brevistipitata are shorter than the lateral stipe extensions (up to 125 μm long) and macroconidia [(35–)36–40(–43) × (3−)3.5−4.5(−5) μm (av. 38 × 4 μm)] of C. machaerinae, the only other species in the C. candelabrum complex to produce lateral stipe extensions.

L. Lombard & Crous, sp. nov. MycoBank MB818703. Fig. 7.

Fig. 7

Calonectria cliffordiicola (ex-type CBS 111812). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–J. Ellipsoid to obpyrifom vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to plant host plant genus, Cliffordia, from which this fungus was isolated.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 65–130 × 7–10 μm; stipe extension septate, straight to flexuous, 127–180 μm long, 4–6 μm wide at the apical septum, terminating in an ellipsoid to obpyriform vesicle, 7–9 μm diam. Conidiogenous apparatus 57–100 μm wide, and 40–85 μm long; primary branches aseptate, 15–32 × 4–6 μm; secondary branches aseptate, 11–23 × 3–6 μm; tertiary branches aseptate, 7–13 × 3–5 μm; quaternary branches aseptate, 8–13 × 3–4 μm each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–11 × 3–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (35–)38–42(–44) × (3−)3.5−4.5(−6) μm (av. 40 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (35−65 mm diam) on MEA after 7 d at room temperature; surface cinnamon to brick with sparse, felty, white to buff aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse cinnamon to sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: South Africa, Western Cape Province, George, from Cliffordia feruginea, 14 Apr. 1998, P.W. Crous (holotype CBS-H22755, culture ex-type CBS 111812 = CPC 2631); Stellenbosch, from Prunus avium saplings, 1 May 1999, C. Linde, cultures CBS 111814 = CPC 2617, CBS 111819 = CPC 2604.

Notes: Calonectria cliffordiicola is a new species in the C. candelabrum complex (Schoch et al., 1999, Lombard et al., 2010a, Lombard et al., 2010b, Lombard et al., 2015a). Morphologically, this species shows some overlap with C. brasiliana, but can be distinguished by its shorter stipe extensions (up to 180 μm) compared to C. brasiliana (up to 240 μm).

L. Lombard & Crous, sp. nov. MycoBank MB818704. Fig. 8.

Fig. 8

Calonectria ericae (ex-type CBS 114458). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–J. Ellipsoid to obpyriform vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to host plant genus, Erica, from which this species was isolated.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 40–100 × 6–9 μm; stipe extension septate, straight to flexuous, 105–160 μm long, 3–7 μm wide at the apical septum, terminating in an ellipsoid to obpyriform vesicle, 6–10 μm diam. Conidiogenous apparatus 40–75 μm wide, and 35–70 μm long; primary branches aseptate, 15–23 × 3–5 μm; secondary branches aseptate, 10–19 × 2–6 μm; tertiary branches aseptate, 6–16 × 2–5 μm; quaternary branches aseptate, 6–13 × 2–5 μm each terminal branch producing 2–6 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 6–11 × 2–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (29–)34–40(–42) × (3−)3.5−4.5(−5) μm (av. 37 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (40−65 mm diam) on MEA after 7 days at room temperature; surface cinnamon to brick with sparse, felty, white aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse cinnamon to umber with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: USA, California, from Erica capensis, Sep. 1998, S.T. Koike (holotype CBS-H22756, culture ex-type CBS 114458 = CPC 2019); ibid., cultures CBS 114456 = CPC 1984, CBS 114457 = CPC 1985.

Notes: Calonectria ericae is a new species in the C. candelabrum complex. This species produces the smallest macroconidia in the C. candelabrum complex. Kioke initially identified these isolates as C. pauciramosa based on morphology and mating studies using the C. pauciramosa mating tester strains (Schoch et al., 1999, Lombard et al., 2010a).

L. Lombard & Crous, sp. nov. MycoBank MB818705. Fig. 9.

Fig. 9

Calonectria indonesiana (ex-type CBS 112936). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F. Conidiogenous apparatus with lateral stipe extension. G–J. Sphaeropedunculate vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to Indonesia, the country where this fungus was collected.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicles; stipe septate, hyaline, smooth, 35–115 × 6–9 μm; stipe extension septate, straight to flexuous, 110–130 μm long, 3–5 μm wide at the apical septum, terminating in a sphaeropedunculate vesicle, 8–10 μm diam; lateral stipe extensions (90° to main axis) sparse, 30–50 μm long, 3–4 μm wide at the apical septum, terminating in sphaeropedunculate vesicles, 4−5 μm. Conidiogenous apparatus 40–100 μm wide, and 40–70 μm long; primary branches aseptate, 11–20 × 4–6 μm; secondary branches aseptate, 8–17 × 4–7 μm; tertiary branches aseptate, 9–14 × 3–6 μm; quaternary branches and additional branches (−6) aseptate, 7–12 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–14 × 3–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (38−)40–46(–48) × (3−)4.5–5.5(−6) μm (av. 43 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (50−65 mm diam) on MEA after 7 d at room temperature; surface buff with abundant white to buff, wooly aerial mycelium, and moderate sporulation on the colony surface; reverse sienna, chlamydospores not observed.

Specimens examined: Indonesia, north Sumatera, from soil, 1998, M.J. Wingfield (holotype CBS-H22757, culture ex-type CBS 112936 = CPC 4504); ibid., culture CBS 112826 = CPC 4519.

Notes: Calonectria indonesiana is similar to C. brassicicola and C. sumatrensis in having few lateral stipe extensions (Crous ). Calonectria indonesiana (−6) can be distinguished from C. brassicicola (−4) and C. sumatrensis (−3) by the number of branches of the conidiogenous apparatus (Crous ).

L. Lombard & Crous, sp. nov. MycoBank MB818706. Fig. 10.

Fig. 10

Calonectria lageniformis (ex-type CBS 111324). A–B. Macroconidiophores. C–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F–I. Lageniformis to ellipsoid vesicles. J. Macroconidia. Scale bars: A–B = 50 μm; C–J = 10 μm.

Etymology: Name refers to the characteristic lageniform vesicles in this fungus.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 65–220 × 4–9 μm; stipe extension septate, straight to flexuous, 135–185 μm long, 4–6 μm wide at the apical septum, terminating in a lageniform to ellipsoid vesicle, 6–10 μm diam. Conidiogenous apparatus 20–80 μm wide, and 35–60 μm long; primary branches aseptate, 16–28 × 4–6 μm; secondary branches aseptate, 10–18 × 3–6 μm; tertiary branches aseptate, 8–13 × 3–6 μm, each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–11 × 3–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (35–)37–43(–45) × (3−)4.5−5.5(−6) μm (av. 40 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (60−90 mm diam) on MEA after 7 d at room temperature; surface sepia with sparse buff, felty aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, from leaf lesion on Eucalyptus sp., 1993, P.W. Crous & A.C. Alfenas, culture CBS 112685 = CPC 3418. Mauritius, Rivière Noire, from foliar lesion on Eucalyptus sp., 10 Apr. 1996, H. Smith (holotype CBS-H22758 culture ex-type CBS 111324 = CPC 1473).

Note: Calonectria lageniformis is the only species that has lageniform vesicles (Crous, 2002, Lombard et al., 2010b, Lombard et al., 2015a, Alfenas et al., 2015).

L. Lombard & Crous, sp. nov. MycoBank MB818707. Fig. 11.

Fig. 11

Calonectria machaerinae (ex-type CBS 123183). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F. Conidiogenous apparatus with lateral stipe extension. G–J. Ellipsoid to obpyriform vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to plant host genus, Machaerina, from which this species was isolated.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 40–115 × 5–10 μm; stipe extension septate, straight to flexuous, 105–170 μm long, 3–5 μm wide at the apical septum, terminating in an ellipsoid to obpyriform vesicle, 6–9 μm diam; lateral stipe extensions (90° to main axis) few, 80–125 μm long, 3–5 μm wide at the apical septum, terminating in broadly clavate vesicles, 5–6 μm diam. Conidiogenous apparatus 40–80 μm wide, and 55–90 μm long; primary branches aseptate, 18–28 × 4–6 μm; secondary branches aseptate, 13–23 × 3–6 μm; tertiary branches aseptate, 8–19 × 3–5 μm; quaternary branches and additional branches (–6) aseptate, 7–15 × 3–5 μm each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–11 × 2–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (35–)36–40(–43) × (3−)3.5−4.5(−5) μm (av. 38 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (60−85 mm diam) on MEA after 7 d at room temperature; surface cinnamon to brick with sparse, wooly, white aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse cinnamon to umber with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimen examined: New Zealand, Auckland, Auckland University Campus, from foliar lesion of Machaerina sinclairii, 27 Jan. 2008, C.F. Hill (holotype CBS-H22760, culture ex-type CBS 123183 = CPC 15378).

Notes: Calonectria machaerinae is a new species in the C. candelabrum complex. This species, along with C. brevistipitata, are the only two species to produce lateral stipe extensions in the C. candelabrum complex (Schoch et al., 1999, Lombard et al., 2010a, Lombard et al., 2010b, Lombard et al., 2015a). See note under C. brevistipitata for additional distinguishing characters.

L. Lombard & Crous, sp. nov. MycoBank MB818708. Fig. 12.

Fig. 12

Calonectria multilateralis (ex-type CBS 110932). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform to elongate reniform phialides. F. Conidiogenous apparatus with lateral stipe extension. G–J. Naviculate vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the multiple lateral stipe extensions on the macroconidiophores of this species.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicles; stipe septate, hyaline, smooth, 25–130 × 4–8 μm; stipe extension septate, straight to flexuous, 135–375 μm long, 5–6 μm wide at the apical septum, terminating in a naviculate vesicle, 6–11 μm diam; lateral stipe extensions (90° to main axis) numerous, 55–100 μm long, 3–5 μm wide at the apical septum, terminating in naviculate vesicles, 4−8 μm. Conidiogenous apparatus 45–95 μm wide, and 30–70 μm long; primary branches aseptate, 10–25 × 3–6 μm; secondary branches aseptate, 6–20 × 3–5 μm; tertiary branches aseptate, 7–15 × 3–5 μm; quaternary branches and additional branches (–7) aseptate, 6–13 × 2–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform to elongate reniform, hyaline, aseptate, 6–12 × 2–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (27−)31–35(–38) × 3–4 μm (av. 33 × 3 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (55−85 mm diam) on MEA after 7 d at room temperature; surface buff with abundant white, wooly aerial mycelium and abundant sporulation on the colony surface; reverse buff to sienna, chlamydospores not observed.

Specimens examined: Mexico, Uxmal, from soil, Apr. 1994, P.W. Crous (holotype CBS-H22762, culture ex-type CBS 110932 = CPC 957); ibid., cultures CBS 110926 = CPC 947, CBS 110927 = CPC 948, CBS 110931 = CPC 956, CBS 115615 = CPC 915.

Notes: Calonectria multilateralis is a new species in the C. naviculata complex (Alfenas ). The macroconidia of C. multilateralis [31–35(–38) × 3–4 μm (av. 33 × 3 μm)] are smaller than those of C. naviculata [(40−)42–50 × 3(–4) μm (av. 45 × 3 μm); Crous 2002] and C. multinaviculata [(40−)44–49(–52) × (2.5−)3.5(–4) μm (av. 46 × 3.5 μm); Alfenas ].

L. Lombard & Crous, sp. nov. MycoBank MB818709. Fig. 13.

Fig. 13

Calonectria paracolhounii (ex-type CBS 114679). A–B. Macroconidiophores. C–D. Clavate vesicles. E–F. Conidiogenous apparatus with conidiophore branches and elongate doliiform to doliiform to reniform phialides. G. Macroconidia. Scale bars: A–B = 50 μm; C–G = 10 μm.

Etymology: Name refers to the fact that this species has an asexual morph that is very similar to that of C. colhounii.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 21–75 × 5–9 μm; stipe extension septate, straight to flexuous, 82–178 μm long, 3–5 μm wide at the apical septum, terminating in a narrowly clavate vesicle, 3–5 μm diam. Conidiogenous apparatus 31–77 μm wide, and 25–54 μm long; primary branches aseptate, 11–23 × 3–6 μm; secondary branches aseptate, 7–13 × 3–6 μm; tertiary branches aseptate, 7–12 × 2–4 μm, each terminal branch producing 2–6 phialides; phialides elongate doliiform to doliiform to reniform, hyaline, aseptate, 6–12 × 2–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (37–)39–43(–45) × 4−5 μm (av. 41 × 5 μm), 3-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (25−55 mm diam) on MEA after 7 d at room temperature; surface buff to sienna with abundant buff to white, felty to wooly aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse buff to sienna to umber with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: USA, substrate unknown, 1990s, A.Y. Rossman (holotype CBS-H22763 culture ex-type CBS 114679 = CPC 2445). Australia, fruit of Annona reticulata, 1988, D. Hutton, culture CBS 114705 = CPC 2423.

Notes: Calonectria paracolhounii is a new species in the C. colhounii complex (Lombard et al., 2010b, Chen et al., 2011). The macroconidia of C. paracolhounii [(37–)39–43(–45) × 4−5 μm (av. 41 × 5 μm)] are smaller than those of C. colhounii [(45–)60–70(–80) × (4−)5(−6) μm (av. 65 × 5 μm); Crous 2002], C. eucalypti [(66–)69–75(–80) × (5−)−6 μm (av. 72 × 6 μm); Lombard ], C. fujianensis [(48–)50–55(–60) × (2.5−)3.5−4.5(−5) μm (av. 52.5 × 4 μm); Chen ], C. monticola 46–51(–56) × 4−5 μm (av. 49 × 5 μm); Crous ] and C. pseudocolhounii [(49–)55–65(–74) × (3.5−)4−5(−5.5) μm (av. 60 × 4.5 μm); Chen ]. Hutton & Sanewski (1989) initially identified isolate CBS 114705 as C. colhounii, associated with leaf and fruit spots of custard apple (Annona reticulata). Their identification was based on morphological comparisons, as no DNA sequence data was available for the genus Calonectria at that time.

L. Lombard & Crous, sp. nov. MycoBank MB818710. Fig. 14.

Fig. 14

Calonectria parva (ex-type CBS 110798). A. Macroconidiophore. B–C. Conidiogenous apparatus with conidiophore branches and cylindrical to allantoid phialides. D–E. Narrowly clavate vesicles. F. Macroconidia. Scale bars = 10 μm.

Etymology: Name refers to the small macroconidiophores in this fungus.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and rarely a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 43–149 × 5–7 μm; stipe extension septate, straight to flexuous, 65–95 μm long, 2–4 μm wide at the apical septum, terminating in a narrowly clavate vesicle, 3–5 μm diam. Conidiogenous apparatus 18–33 μm wide, and 24–43 μm long; primary branches aseptate, 11–21 × 3–5 μm; secondary branches aseptate, 11–15 × 3–4 μm, each terminal branch producing 2–4 phialides; phialides cylindrical to allantoid, hyaline, aseptate, 9–19 × 3–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (60–)66–78(–83) × 5−7 μm (av. 72 × 6 μm), (1−)3-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (55−85 mm diam) on MEA after 7 d at room temperature; surface buff with abundant buff to white, felty aerial mycelium and sparse to moderate sporulation on the aerial mycelium and colony surface; reverse buff; chlamydospores not observed.

Specimen examined: South Africa, Mpumalanga, Sabie, D.R. de Wet nursery, from Eucalyptus grandis ramets (roots), 11 May 1990, P.W. Crous (holotype CBS-H22764, culture ex-type CBS 110798 = CPC 410 = PPRI 4001).

Note: Calonectria parva can be distinguished from other species in the genus by its relatively small macroconidiophores, which rarely bear a stipe extension.

L. Lombard & Crous, sp. nov. MycoBank MB818711. Fig. 15.

Fig. 15

Calonectria plurilateralis (ex-type CBS 111401). A–C. Macroconidiophores with lateral stipe extensions. D. Conidiogenous apparatus with lateral stipe extensions. E–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–H. Obpyriform to ellipsoidal vesicles. I. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the multiple lateral stipe extensions on the macroconidiophores of this fungus.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and numerous lateral stipe extensions terminating in vesicles, lacking a central stipe extension; stipe septate, hyaline, smooth, 50–130 × 4–7 μm; stipe extension septate, straight to flexuous, 110–180 μm long, 4–7 μm wide at the apical septum, terminating in obpyriform to ellipsoid vesicles, 7–11 μm diam; lateral stipe extensions (90° to main axis) abundant, 75–105 μm long, 3–6 μm wide at the apical septum, terminating in obpyriform to ellipsoid vesicles, 5−7 μm diam. Conidiogenous apparatus 25–80 μm wide, and 25–85 μm long; primary branches aseptate, 11–39 × 2–9 μm; secondary branches aseptate, 7–17 × 3–5 μm; tertiary branches aseptate, 6–12 × 3–5 μm; quaternary branches aseptate, 8 × 4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 4–11 × 3–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (27−)30–38(–41) × (3−)3.5−4.5(−5) μm (av. 34 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (60−85 mm diam) on MEA after 7 d at room temperature; surface sienna to sepia with moderate white, wooly aerial mycelium and abundant sporulation on the colony surface; reverse sienna to sepia, chlamydospores throughout the medium, forming microsclerotia.

Specimen examined: Ecuador, from soil, 20 Jun. 1997, M.J. Wingfield (holotype CBS-H22766, culture ex-type CBS 111401 = CPC 1637).

Note: Calonectria plurilateralis can be distinguished from other members of the C. cylindrospora complex by its numerous lateral stipe extensions.

L. Lombard & Crous, sp. nov. MycoBank MB818712. Fig. 16.

Fig. 16

Calonectria pseudoecuadoriae (ex-type CBS 111402). A–B. Macroconidiophores. C–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F–I. Clavate vesicles. J. Macroconidia. Scale bars: A–B = 50 μm; C–J = 10 μm.

Etymology: Name refers to the fact that this species has an asexual morph that is very similar to that of C. ecuadoriae.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 40–210 × 7–10 μm; stipe extension septate, straight to flexuous, 160–250 μm long, 4–5 μm wide at the apical septum, terminating in a clavate vesicle, 4–7 μm diam. Conidiogenous apparatus 70–105 μm wide, and 50–90 μm long; primary branches aseptate, 18–30 × 5–7 μm; secondary branches aseptate, 9–22 × 3–7 μm; tertiary branches aseptate, 7–17 × 3–5 μm; quaternary branches and additional branches (–6) aseptate, 7–12 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 8–12 × 3–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (34–)36–40(–43) × 3−4 (−5) μm (av. 38 × 3.5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (30−60 mm diam) on MEA after 7 d at room temperature; surface cinnamon to brick with sparse white, wooly aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse buff to cinnamon with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Ecuador, soil, 20 Jun. 1997, M.J. Wingfield (holotype CBS-H22768, culture ex-type CBS 111402 = CPC 1639); ibid., culture CBS 111412 = CPC 1648.

Notes: Calonectria pseudoecuadoriae is morphologically similar to C. ecuadoriae. The macroconidia of C. pseudoecuadoriae [(34–)36–40(–43) × 3−4 (−5) μm (av. 38 × 3.5 μm)] are smaller than those of C. ecuadoriae [(45–)48–55(–65) × (4−)4.5(−5) μm (av. 51 × 4.5 μm); Crous ]. Furthermore, C. pseudoecuadoriae has six tiers of branches in its conidiogenous apparatus in comparison to the seven in C. ecuadoriae (Crous ), although these differences are relatively minor.

L. Lombard & Crous, sp. nov. MycoBank MB818713. Fig. 17.

Fig. 17

Calonectria pseudouxmalensis (ex-type CBS 110924). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–J. Obpyriform to ellipsoidal vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the fact that this species has an asexual morph that is very similar to that of C. uxmalensis.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 30–60 × 6–8 μm; stipe extension septate, straight to flexuous, 100–140 μm long, 4–6 μm wide at the apical septum, terminating in a obpyriform to ellipsoidal vesicle sometimes with a papillate apex, 5–9 μm diam. Conidiogenous apparatus 25–65 μm wide, and 30–60 μm long; primary branches aseptate, 14–21 × 4–6 μm; secondary branches aseptate, 8–16 × 2–5 μm; tertiary branches aseptate, 5–13 × 2–5 μm; quaternary branches and additional branches (–6) aseptate, 5–9 × 2–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–9 × 3–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (26–)28–30(–32) × 3−4 μm (av. 29 × 3 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (75−90 mm diam) on MEA after 7 d at room temperature; surface sienna with abundant white, felty to wooly aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse sienna with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Mexico, from soil, Apr. 1994, P.W. Crous (holotype CBS-H22769, culture ex-type CBS 110924 = CPC 942); ibid., cultures CBS 110923 = CPC 941, CBS 115677 = CPC 943.

Notes: Calonectria pseudouxmalensis can be distinguished from C. uxmalensis by its lack of lateral stipe extensions. The macroconidia of C. pseudouxmalensis [(26–)28–30(–32) × 3−4 μm (av. 29 × 3 μm)] are smaller than those of C. mexicana [(35–)40–48(–52) × 3−4(−4.5) μm (av. 45 × 3 μm); Schoch et al., 1999, Crous, 2002], C. pseudomexicana [(40–)43–48(–49) × (4−)5−6 μm (av. 45 × 5 μm); Lombard ] and C. tunisiana [(43–)47–51(–53) × 4−6 μm (av. 49 × 5 μm); Lombard ]. Schoch was able to induce the sexual morph of C. mexicana through the heterothallic mating of CBS 110918 (= CPC 927) with CBS 110923 (= CPC 941), which was deposited as the holotype (PREM 55763) of C. mexicana. However, phylogenetic inference in this study showed that the one mating tester strain CBS 110923 (Schoch ) is distinct from the other mating tester strain (CBS 110918; ex-type of Cylindrocladium mexicanum). This phenomenon is not new to the genus Calonectria, as Neubauer & Zinkernagel (1995) and Overmeyer have shown that fertile perithecia can be induced in some Calonectria species when they are cultured in the presence of other species, but where sexual outcrossing has not occurred.

L. Lombard & Crous, sp. nov. MycoBank MB818714. Fig. 18.

Fig. 18

Calonectria putriramosa (ex-type CBS 111449). A–B. Macroconidiophores. C–E. Conidiogenous apparatus with conidiophore branches and elongate reniform to allantoid to cylindrical phialides. F–I. Ellipsoid to obpyrifom vesicles. J. Macroconidia. Scale bars: A–B = 50 μm; C–J = 10 μm.

Etymology: Name refers to cutting rot, the disease symptoms that are associated with infection by this fungus.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 40–170 × 5–10 μm; stipe extension septate, straight to flexuous, 145–185 μm long, 4–7 μm wide at the apical septum, terminating in an ellipsoid to obpyriform vesicle, 7–9 μm diam. Conidiogenous apparatus 45–60 μm wide, and 30–90 μm long; primary branches aseptate, 12–34 × 3–6 μm; secondary branches aseptate, 9–21 × 3–6 μm; tertiary branches aseptate, 9–17 × 3–5 μm; quaternary branches aseptate, 4–13 × 3–5 μm each terminal branch producing 2–6 phialides; phialides elongate reniform to allantoid to cylindrical, hyaline, aseptate, 6–15 × 3–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (35–)40–46(–49) × (4−)4.5−5.5(−6) μm (av. 43 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (35−75 mm diam) on MEA after 7 d at room temperature; surface cinnamon to brick with sparse, wooly, white to buff aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse cinnamon to sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, from Eucalyptus cuttings, Jun. 1998, A.C. Alfenas (holotype CBS-H22770, culture ex-type CBS 111449 = CPC 1951); Bahia do Sol, from Eucalyptus cuttings, Apr. 1993, P.W. Crous, culture CBS 116076 = CPC 604; from soil, Jun. 1998, A.C. Alfenas, cultures CBS 111470 = CPC 1940, CBS 111477 = CPC 1928.

Notes: Calonectria putriramosa is a new species in the C. candelabrum complex (Schoch et al., 1999, Lombard et al., 2010a, Lombard et al., 2010b, Lombard et al., 2015a). The macroconidia of C. putriramosa [(35–)40–46(–49) × (4−)4.5−5.5(−6) μm (av. 43 × 5 μm)] are smaller than those of its closest phylogenetic neighbours (see notes under C. brasiliana), but slightly larger than those of C. brasiliana [(36–)38–42(–46) × (3−)3.5−4.5(−5) μm (av. 40 × 4 μm)].

L. Lombard & Crous, sp. nov. MycoBank MB818715. Fig. 19.

Fig. 19

Calonectria stipitata (ex-type CBS 112513). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F. Conidiogenous apparatus with lateral stipe extension. G–J. Ellipsoid to obpyriform vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the lateral stipe extensions produced by this fungus.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 35–85 × 6–9 μm; stipe extension septate, straight to flexuous, 105–195 μm long, 4–6 μm wide at the apical septum, terminating in an ellipsoid to obpyriform vesicle, 7–11 μm diam; lateral stipe extensions (90° to main axis) abundant, 70–135 μm long, 3–6 μm wide at the apical septum, terminating in broadly clavate vesicles, 3–6 μm diam. Conidiogenous apparatus 50–120 μm wide, and 40–75 μm long; primary branches aseptate, 15–29 × 4–5 μm; secondary branches aseptate, 9–18 × 3–6 μm; tertiary branches aseptate, 8–19 × 2–5 μm; quaternary branches and additional branches (–6) aseptate, 6–14 × 2–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–13 × 2–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (27–)29–35(–37) × (3−)3.5−4.5(−6) μm (av. 32 × 4 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (60−85 mm diam) on MEA after 7 d at room temperature; surface sienna to sepia with abundant wooly, white aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse sienna to sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimen examined: Colombia, from Eucalyptus sp., 1990s, M.J. Wingfield (holotype CBS-H22771, culture ex-type CBS 112513 = CPC 3851).

Notes: Calonectria stipitata, like C. brevistipitata and C. machaerinae, produce lateral stipe extensions, a characteristic not usually associated with members of the C. candelabrum complex (Schoch et al., 1999, Lombard et al., 2010a, Lombard et al., 2010b, Lombard et al., 2015a). The lateral stipe extensions of C. stipitata (up to 135 μm) are longer than those of C. brevistipitata (up to 80 μm) and C. machaerinae (up to 125 μm).

L. Lombard & Crous, sp. nov. MycoBank MB818716. Fig. 20.

Fig. 20

Calonectria syzygiicola (ex-type CBS 112831). A–B. Macroconidiophores. C–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F–H. Sphaeropedunculate vesicles. I. Macroconidia. Scale bars: A–B = 50 μm; C–I = 10 μm.

Etymology: Name refers to the host plant, Syzygium aromaticum from which this fungus was isolated.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 30–170 × 4–8 μm; stipe extension septate, straight to flexuous, 65–105 μm long, 3–4 μm wide at the apical septum, terminating in a sphaeropedunculate vesicle, 4–7 μm diam; lateral stipe extensions (90° to main axis) sparse, 40–50 μm long, 2–3 μm wide at the apical septum, terminating in sphaeropedunculate vesicles, 3–6 μm diam. Conidiogenous apparatus 30–70 μm wide, and 30–45 μm long; primary branches aseptate, 12–21 × 4–6 μm; secondary branches aseptate, 8–14 × 3–5 μm; tertiary branches aseptate, 9–12 × 3–5 μm; quaternary branches aseptate, 8–10 × 2–3 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–11 × 2–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (39–)41–49(–56) × (4−)4.5−5.5(−7) μm (av. 45 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (45−65 mm diam) on MEA after 7 d at room temperature; surface amber to sienna with abundant wooly, white to buff aerial mycelium, and abundant sporulation on the aerial mycelium and colony surface; reverse sienna with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Indonesia, Sumatra, from soil under Syzygium aromaticum, 1998, M.J. Wingfield (holotype CBS-H22772, culture ex-type CBS 112831 = CPC 4511), culture CBS 112827 = CPC 4512.

Notes: Calonectria syzygiicola is closely related to C. asiatica (Fig. 1). However, the macroconidia of C. syzygiicola [(39–)41–49(–56) × (4−)4.5−5.5(−7) μm (av. 45 × 5 μm)] are smaller than those of C. asiatica [(42–)48–55(–65) × (4−)5(−5.5) μm (av. 53 × 5 μm); Crous ].

L. Lombard & Crous, sp. nov. MycoBank MB818717. Fig. 21.

Fig. 21

Calonectria tereticornis (ex-type CBS 111301). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and elongate doliiform to reniform phialides. G–J. Fusiform to ovoid vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the host plant, Eucalyptus tereticornis, from which this fungus was isolated.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 70–270 × 6–11 μm; stipe extension septate, straight to flexuous, 140–245 μm long, 3–7 μm wide at the apical septum, terminating in a fusiform to ovoid vesicle, 8–14 μm diam. Conidiogenous apparatus 35–65 μm wide, and 45–75 μm long; primary branches aseptate, 18–34 × 4–10 μm; secondary branches aseptate, 11–26 × 3–7 μm, each terminal branch producing 2–4 phialides; phialides elongate doliiform to allantoid, hyaline, aseptate, 9–15 × 3–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (51–)55–63(–71) × (3−)4.5−5.5(−6) μm (av. 59 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (55−75 mm diam) on MEA after 7 d at room temperature; surface cinnamon to sienna with sparse buff to white, wooly aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse sienna to umber with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, Tucurui, from leaves of Eucalyptus tereticornis, 20 Sep. 1996, P.W. Crous (holotype CBS-H22773 culture ex-type CBS 111301 = CPC 1429).

Notes: Calonectria tereticornis is closely related to C. gordoniae and C. ovata (Fig. 1). The macroconidia of C. tereticornis [(51–)55–63(–71) × (3−)4.5−5.5(−6) μm (av. 59 × 5 μm)] are smaller than those of C. gordoniae [(44–)50–70(–80) × (4−)5−6 μm (av. 65 × 5 μm); Crous 2002] and C. ovata [(50–)65–80(–110) × 4−5 (−6) μm (av. 70 × 5 μm); Crous 2002].

L. Lombard & Crous, sp. nov. MycoBank MB818718. Fig. 22.

Fig. 22

Calonectria terricola (ex-type CBS 116247). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and elongate doliiform to reniform phialides. G–J. Fusiform to ovoid vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to soil, the substrate from which this fungus was isolated.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 30–100 × 5–9 μm; stipe extension septate, straight to flexuous, 135–175 μm long, 4–5 μm wide at the apical septum, terminating in a fusiform to ovoid vesicle, 8–12 μm diam. Conidiogenous apparatus 30–100 μm wide, and 45–65 μm long; primary branches aseptate, 14–26 × 3–6 μm; secondary branches aseptate, 13–22 × 2–5 μm; tertiary branches aseptate, 15–18 × 4–5 μm, each terminal branch producing 2–4 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 9–17 × 3–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (40–)43–49(–53) × (3−)4−5(−6) μm (av. 46 × 4.5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (45−65 mm diam) on MEA after 7 d at room temperature; surface brick to sienna with sparse, buff to white, wooly aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse sienna with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, from soil in Eucalyptus plantation, 1996, P.W. Crous (holotype CBS-H22774; culture ex-type CBS 116247 = CPC 3583); ibid., culture CBS 116248 = CPC 3536.

Notes: Calonectria terricola is a new species in the C. pteridis complex. The macroconidia of C. terricola [(40–)43–49(–53) × (3−)4−5(−6) μm (av. 46 × 4.5 μm)] are smaller than those of C. ovata [(50–)65–80(–110) × 4−5 (−6) μm (av. 70 × 5 μm); Crous 2002], C. pseudovata [(55–)67–70(–80) × (4−)5 (−7) μm (av. 69 × 5 μm); Alfenas ] and C. tereticornis [(51–)55–63(–71) × (3−)4.5−5.5(−6) μm (av. 59 × 5 μm)].

L. Lombard & Crous, sp. nov. MycoBank MB818719. Fig. 23.

Fig. 23

Calonectria tropicalis (ex-type CBS 116271). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and elongate doliiform to reniform phialides. F–I. Clavate vesicles. J. Macroconidia. Scale bars: A–C = 50 μm; D–J = 10 μm.

Etymology: Name refers to the tropical region in Brazil where this fungus was collected.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 120–210 × 7–8 μm; stipe extension septate, straight to flexuous, 190–270 μm long, 4–6 μm wide at the apical septum, terminating in a clavate vesicle, 5–6 μm diam. Conidiogenous apparatus 50–70 μm wide, and 60–90 μm long; primary branches aseptate, 20–32 × 4–6 μm; secondary branches aseptate, 12–29 × 3–6 μm; tertiary branches aseptate, 12–20 × 2–4 μm, each terminal branch producing 2–4 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 10–16 × 2–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (69–)74–86(–89) × (4−)4.5−5.5(−6) μm (av. 80 × 5 μm), 1(−3)-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies moderately fast growing (45−65 mm diam) on MEA after 7 days at room temperature; surface sienna to sepia with moderate white, wooly aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse sienna to sepia with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Brazil, Amazon, from foliar lesion of Eucalyptus sp., 1993, P.W. Crous & A.C. Alfenas (holotype CBS-H22776 culture ex-type CBS 116271 = CPC 3559); ibid., cultures CBS 116242 = CPC 3543.

Notes: Calonectria tropicalis resides in the C. pteridis complex. This species can be distinguished from other species in the complex by the smaller numbers of fertile branches in its conidiogenous apparatus.

Gerlach, Phytopathol. Z. 61: 379. 1968. MycoBank MB327268.

Specimen examined: Germany, Celle, from root of Paphiopedilum callosum, May 1967, W. Gerlach, culture ex-type CBS 413.67 = IMI 299577.

Notes: Sobers (1972) reduced C. floridana and C. uniseptata to synonymy with C. kyotensis based on their similarities in morphology and pathogenicity. Phylogenetic inference in this study showed that the ex-type of C. uniseptata (CBS 413.67; Gerlach 1968) is distinct from C. kyotensis. Therefore, C. uniseptata is reinstated here as a distinct species of Calonectria.

L. Lombard & Crous, sp. nov. MycoBank MB818720. Fig. 24.

Fig. 24

Calonectria uxmalensis (ex-type CBS 110925). A–C. Macroconidiophores with lateral stipe extensions. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–J. Obpyriform to ellipsoidal vesicles. K. Macroconidia. Scale bars: A–C = 50 μm; D–K = 10 μm.

Etymology: Name refers to the ancient Maya city Uxmal, Mexico, the locality where this fungus was collected.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 35–155 × 6–8 μm; stipe extension septate, straight to flexuous, 60–140 μm long, 3–6 μm wide at the apical septum, terminating in a obpyriform to ellipsoidal vesicle sometimes with a papillate apex, 5–8 μm diam; lateral stipe extensions (90° to main axis) few, 88–100 μm long, 3–4 μm wide at the apical septum, terminating in broadly clavate to obpyriform to ellipsoid vesicles, 5–6 μm diam. Conidiogenous apparatus 30–90 μm wide, and 35–60 μm long; primary branches aseptate, 14–19 × 3–6 μm; secondary branches aseptate, 10–16 × 3–6 μm; tertiary branches aseptate, 7–11 × 3–5 μm; quaternary branches and additional branches (–6) aseptate, 7–11 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 8–11 × 2–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (26–)27–33(–35) × 3−4 μm (av. 30 × 3 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies fast growing (65−85 mm diam) on MEA after 7 d at room temperature; surface buff to sienna with abundant buff to white, felty to wooly aerial mycelium and moderate sporulation on the aerial mycelium and colony surface; reverse sienna to umber with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimens examined: Mexico, Uxmal, from soil, Apr. 1994, P.W. Crous (holotype CBS-H22761, culture ex-type CBS 110925 = CPC 945); ibid., culture CBS 110919 = CPC 928.

Notes: Calonectria uxmalensis can be distinguished from C. mexicana, C. pseudomexicana and C. tunisiana by its lateral stipe extensions, a characteristic not known for the latter three species (Schoch et al., 1999, Crous, 2002, Lombard et al., 2011).

L. Lombard & Crous, sp. nov. MycoBank MB818721. Fig. 25.

Fig. 25

Calonectria venezuelana (ex-type CBS 111052). A–B. Macroconidiophores. C–D. Conidiogenous apparatus with conidiophore branches and elongate doliiform to reniform phialides. E–F. Fusiform to ovoid to ellipsoid vesicles. G. Macroconidia. H. Microconidia. I. Micro- and macroconidia. Scale bars: A–B = 50 μm; C–I = 10 μm.

Etymology: Name refers to Venezuela, the country from which this fungus was collected.

Macroconidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, and a stipe extension terminating in a vesicle; stipe septate, hyaline, smooth, 35–100 × 4–8 μm; stipe extension septate, straight to flexuous, 85–190 μm long, 3–6 μm wide at the apical septum, terminating in a fusiform to ovoid to ellipsoid vesicle, 5–9 μm diam. Conidiogenous apparatus 25–60 μm wide, and 25–65 μm long; primary branches aseptate, 15–30 × 4–8 μm; secondary branches aseptate, 11–24 × 3–5 μm; tertiary branches aseptate, 8–14 × 3–6 μm, each terminal branch producing 2–4 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 8–17 × 2–5 μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (48–)54–62(–65) × (4−)4.5−5.5(−7) μm (av. 58 × 5 μm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Microconidiophores consists of a stipe and a penicillate or subverticillate arrangement of fertile branches; stipe septate, hyaline, smooth, 25–40 × 3–4 μm; primary branches aseptate, 8–12 × 2–4 μm, terminating in 1–4 phialides that are cylindrical, straight to slightly curved, 7–15 × 2–4 μm, apex with minute periclinal thickening and inconspicuous collarette. Microconidia cylindrical, straight to slightly curved, rounded at the apex and flattened at the base, 16–20(–22) × (2−)2.5−3.5(−4) μm (av. 18 × 3 μm), (0−)1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Megaconidia not observed.

Culture characteristics: Colonies fast growing (50−75 mm diam) on MEA after 7 d at room temperature; surface cinnamon to amber with sparse, buff to white, wooly aerial mycelium and abundant sporulation on the aerial mycelium and colony surface; reverse sienna to amber with abundant chlamydospores throughout the medium, forming microsclerotia.

Specimen examined: Venezuela, Acarigua, from soil, 27 Jun. 1995, M.J. Wingfield (holotype CBS-H22778 culture ex-type CBS 111052 = CPC 1183).

Notes: Calonectria venezuelana forms a single lineage closely related to C. eucalypticola (Fig. 1). The macroconidia of C. venezuelana [(48–)54–62(–65) × (4−)4.5−5.5(−7) μm (av. 58 × 5 μm)] are larger than those of C. eucalypticola [(43–)49–52(–55) × 3−5 μm (av. 50 × 4 μm); Alfenas ].

Discussion

A collection of isolates stored for many years and tentatively identified as species of Calonectria based on morphology, were shown to represent 24 new species. At the time that they were collected, it would not have been possible to recognise them as novel taxa and this vividly illustrates the power of the DNA-based sequencing tools that are now available to facilitate accurate species recognition. These species emerging from this study were isolated from various substrates collected globally over a 20 year period, and this study therefore highlights the value of the careful storage and maintenance of cultures for further study when appropriate opportunities arise to do so. This paper also highlights the fact that many undescribed species most likely remain hidden in culture collections, requiring a re-evaluation based on DNA sequence comparisons.

Most of the isolates collected in Brazil formed part of the C. pteridis species complex. This is regarded as one of the most prominent species complexes associated with CLB on Eucalyptus in that country (Alfenas et al., 2004, Alfenas et al., 2013c, Alfenas et al., 2015, Graça et al., 2009). Calonectria amazonica, C. amazoniensis, C. lageniformis, C. tereticornis and C. tropicalis were all isolated from CLB leaf lesions on Eucalyptus spp. propagated commercially as non-natives in plantations. Results of this study have raised the number of species known from Brazil to 55 (Alfenas et al., 2013a, Alfenas et al., 2013b, Alfenas et al., 2015). Calonectria terricola, isolated from soil collected in a Eucalyptus plantation in Brazil, also formed part of the C. pteridis complex in this study.

Calonectria parva, isolated from soil collected in South Africa, formed a basal lineage to the C. colhounii species complex. This species can be readily distinguished from other species in the C. colhounii species complex by its relatively small macroconidiophores, which rarely bear stipe extensions.

Both C. uxmalensis and C. pseudouxmalensis, isolated from soil collected in Mexico, are new additions to the C. mexicana species complex, which now include five species (Lombard ). This complex is characterised by the papillate apices of the vesicles terminating the stipe extensions (Lombard ). Calonectria uxmalensis can be distinguished from the other species in this complex by the formation of lateral stipe extensions, whereas macroconidial dimensions can distinguish C. pseudouxmalensis from the species in this complex.

Calonectria paracolhounii, collected in the USA and Australia, is a new addition to the C. colhounii complex (Crous, 2002, Crous et al., 2006, Chen et al., 2011). This species complex now includes seven species (Crous, 2002, Crous et al., 2006, Crous et al., 2015b, Chen et al., 2011, Xu et al., 2012), and is characterised by the formation of unique bright yellow perithecia. Although no perithecia were observed for C. paracolhounii in this study, the macroconidia of C. paracolhounii were smaller than those of the other species known in this complex.

The C. candelabrum species complex (Schoch ) accommodates the greatest number of species in the genus and includes 27 species (Schoch et al., 1999, Crous, 2002, Lombard et al., 2010a, Lombard et al., 2011, Lombard et al., 2015a, Crous et al., 2013, Alfenas et al., 2015) after the addition of C. brasiliana (Brazil), C. brevistipitata (Mexico), C. cliffordiicola (South Africa), C. ericae (USA), C. machaerinae (New Zealand), C. putriramosa (Brazil), C. stipitata (Colombia) and C. venezuelana (Venezuela) recognised in this study. Although some unique morphological characters could be identified to distinguish these eight new species, DNA sequence comparisons are required to provide accurate species identification.

Calonectria pseudoecuadoriae and C. plurilateralis (Ecuador) are both new additions to the C. brassicae and C. cylindrospora species complexes, respectively (Crous, 2002, Lombard et al., 2009, Alfenas et al., 2015). Calonectria pseudoecuadoriae is morphologically similar to C. ecuadoriae (Crous ) except for the additional branches in the conidiogenous apparatus and smaller macroconidia. Calonectria plurilateralis is the only species in the C. cylindrospora complex known to produce lateral stipe extensions, distinguishing it from other species in this complex.

Calonectria brassicicola (Indonesia and New Zealand), C. indonesiana (Indonesia), and C. syzygiicola (Indonesia) are new additions to the C. kyotensis species complex (Crous 2002, Crous , Lombard et al., 2010b, Lombard et al., 2015a). Species in this complex are characterised by their sphaeropedunculate vesicles and the formation of lateral stipe extensions on the conidiogenous apparatus (Crous , Lombard et al., 2010b, Lombard et al., 2015a). The three new species introduced in this study can be distinguished from their closest phylogenetic neighbours as well as from each other by the number of branches in the conidiogenous apparatus and their macroconidial dimensions.

Calonectria spp. are soil-borne fungi that are able to exist in this substrate for long periods of time due to their abundant production of sclerotia (Crous 2002). This also implies that they can be and most likely have been extensively moved between countries and continents. Given their importance as plant pathogens, it is ironical that very little is known regarding their genetic diversity or pathways of movement globally. This study has shown that there are many more species of Calonectria than has been recognised and it likely that many more species have yet to be discovered. Genomes have yet to be sequenced for Calonectria spp. and as these emerge, tools will become available to answer questions regarding the global movement of these fungi (Crous ). They will also contribute to reducing the impact of, for example, tree pathogens that are resulting in serious losses to planted forests (Wingfield ).

When the 24 species newly described in this study were collected, the genus Calonectria had only been peripherally studied. At that time, most species had been described based on their morphological characteristics, which included vesicle shape and macroconidial dimensions and septation (Crous and Wingfield, 1994, Crous, 2002). However, with a large number of DNA sequences now available from recent taxonomic studies of the genus Calonectria (Lombard et al., 2010b, Lombard et al., 2015a, Alfenas et al., 2015), the initial identifications could be either confirmed or corrected. This study, vividly highlights the impact that DNA sequence data have had in providing more accurate identifications of filamentous fungi (Crous et al., 2015a, Crous et al., 2016). Identifications at this level are already impacting substantially on agricultural and forestry practices as well as in the trade in food and fibre products (Crous ).