Diversity and potential impact of Calonectria species in Eucalyptus plantations in Brazil.

Species in the genus Calonectria (Hypocreales) represent an important group of plant pathogenic fungi that cause serious losses to plant crops in tropical and subtropical climates. Calonectria leaf blight is currently one of the main impediments to Eucalyptus cultivation in Brazil, and various species of Calonectria have been associated with this disease. Since most previous identifications were solely based on morphological characters, much of the published literature needs to be re-evaluated. The aim of this study was thus to identify and determine the phylogenetic relationships among species that occur in the Eucalyptus growing regions of Brazil by using partial sequences of the β-tubulin, calmodulin, translation elongation factor 1-α and histone H3 gene regions. Based on extensive collections from soil and infected eucalypt leaf samples from plantations, phylogenetic inference revealed the Ca. pteridis complex to be the most common species complex present in Eucalyptus plantations in Brazil. By elucidating taxa in the Ca. pteridis, Ca. cylindrospora and Ca. candelabra species complexes, 20 novel Calonectria species were identified, and a new name in Calonectria provided for Cylindrocladium macrosporum as Ca. pseudopteridis.

Introduction

Calonectria species (asexual morph previously known as Cylindrocladium (Cy.)) are widely distributed around the world and cause diseases on a broad range of host plants in tropical and subtropical climates (Crous, 2002, Lombard et al., 2010a, Vitale et al., 2013). In Brazil, Calonectria species have been reported as pathogens of numerous important agronomic crops, such as potatoes (Solanum tuberosum; Dianese ), soybeans (Glycine max; Bolkan ), acerola (Malpighia glabra; Silva ), mango (Mangifera indica; Tozetto & Ribeiro 1996), Eugenia spp. (Poltronieri ), and several ornamentals (Reis ). Thus far however, the majority of the reports from Brazil focused on forestry crops, such as Pinus and Acacia (Hodges and May, 1972, Hodges et al., 1973, Alfenas, 1986, Dianese et al., 1986, Novaes et al., 2012, Alfenas et al., 2013a, Alfenas et al., 2013b) and in particular on the epidemiology and disease control of Calonectria spp. associated with diseases of Eucalyptus in commercial plantations and nurseries (Blum et al., 1992, Mafia et al., 2008, Mafia et al., 2009, Graça et al., 2009, Ferreira et al., 2012, Alfenas et al., 2013c).

Based on the increasing global market for paper and wood pulp, and renewable energy, commercial Eucalyptus plantations in Brazil have expanded towards the warm and humid regions of northern and north-eastern Brazil where Calonectria leaf blight (CLB) has become the primary fungal leaf disease of this crop (Alfenas et al., 2009, Alfenas et al., 2013c). Other prominent diseases associated with Calonectria species on Eucalyptus in Brazil include damping-off, cutting rot and root rot (Alfenas and Ferreira, 1979, Alfenas et al., 1979, Alfenas, 1986, Alfenas et al., 2009). Calonectria leaf blight was first observed in commercial plantation trees of E. grandis in 1970, with more than 80 % of the trees showing severe defoliation (Alfenas & Ferreira 1979). Three Calonectria species were identified as the causal agents, which included Calonectria cylindrospora (= Cylindrocladium scoparium; see Lombard et al., 2015a, Lombard et al., 2015b), Ca. ilicicola (= Cy. parasiticum) and Ca. pyrochroa (= Cy. ilicicola). Additional species also reported to cause CLB and damping-off of Eucalyptus in Brazil include Ca. ovata (= Cy. ovatum), Ca. candelabra (= Cy. candelabrum; see Lombard et al., 2015a, Lombard et al., 2015b), and Ca. brassicae (= Cy. gracile) (Alfenas et al., 1979, Almeida and Bolkan, 1981, Alfenas, 1986, El-Gholl et al., 1993, Crous et al., 1998). However, these Calonectria species have been identified based solely on morphological characters of the asexual morphs (conidial dimensions and vesicle shape; Alfenas 1986, Ferreira 1989, Alfenas ), which could have resulted in incorrect identifications.

In the 1990's, Eucalyptus leaf blight and defoliation caused by Ca. pteridis (= Cy. pteridis), in south-eastern Bahia and Pará provinces resulted in severe defoliation of E. grandis trees in these regions (Ferreira ). Since then, Ca. pteridis has become the most common species reported from commercial plantations, primarily on E. camaldulensis, E. cloeziana, E. grandis, E. saligna, E. tereticornis, E. urophylla and hybrid E. grandis × E. urophylla (Alfenas ). For most Eucalyptus species, the disease is characterised by spots that are initially small, circular or elongated and pale grey to pale brown, progressing and extending throughout the leaf blade, resulting in leaf drop and in some cases severe defoliation (Alfenas and Ferreira, 1979, Alfenas et al., 1979). It is believed that defoliation caused by CLB decreases timber volume due to the reduced photosynthetic area (Ferreira et al., 1995, Berger et al., 2007, Alfenas et al., 2009) and that weed growth is promoted due to light in the understory, which further subjects the trees to competition from weeds (Alfenas ).

Planting of resistant genotypes is the most effective and economical method to control this disease in the field (Alfenas et al., 2009, Fonseca et al., 2010). However, selecting resistant genotypes has proven difficult since several Calonectria species appear to be associated with CLB. Pathogenicity trials done by Rehn showed that several Calonectria species isolated from soil can be highly aggressive to Eucalyptus, but hardly any information is presently available on the diversity of Calonectria species occurring in soil in eucalypt plantations in Brazil.

Although morphological characters provide valuable information for species discrimination in Calonectria, incorporation of a polyphasic identification process with multi-gene DNA sequence data has elucidated various previously unknown species complexes (Crous et al., 2004b, Crous et al., 2006, Lombard et al., 2010b, Lombard et al., 2010c, Chen et al., 2011, Lombard et al., 2015a, Lombard et al., 2015b). For some of these species complexes, cryptic members can only be accurately identified on the basis of DNA sequence data. Except for a few recent studies (Alfenas et al., 2013a, Alfenas et al., 2013b), most previous reports of Calonectria species in Brazil need to be re-evaluated. Therefore, the aims of the present study were to conduct extensive surveys of soils and trees in various commercial Eucalyptus plantations in Brazil, cultivate as many isolates as possible, and subject them to DNA sequence analyses, to determine which morphological groups are dominant, and establish their distribution in Brazil.

Material and methods

Sampling and isolation

Samples of Eucalyptus leaves showing characteristic symptoms of CLB were collected in the main eucalypt growing regions of Brazil. Since the clonal plantations are established in different Management Operational Units (MOU), according to the characteristics of the soil and climatic conditions, a sample of 30 leaves per infected clone/species was collected. A random soil sample (400 g in the 0–20 cm layer) was also collected for each MOU and another from the surrounding native vegetation (Table 1). Additionally, diseased Azadirachta indica and Eucalyptus cuttings were collected from nurseries in the states of Minas Gerais (Viçosa) and Pará (Santana). The symptomatic plant material were kept in paper bags and the soil samples in plastic bags and transported to the Forest Pathology Laboratory/Bioagro of the Universidade Federal de Viçosa. All the collected plant materials were incubated in moist chambers at room temperature (25 °C ± 3 °C) for up to 14 d and inspected daily for fungal sporulation. The collected soil samples were baited with mature leaf discs of castor bean (Ricinus communis) and eucalypt twig segments as described by Gonçalves . Direct isolations were made onto malt extract agar (2 % w/v; MEA; Vetec, Brazil) and incubated for 7 d at 25 °C under continuous near-ultraviolet light. From these primary isolations, single conidial cultures were prepared on MEA and deposited in the culture collection of the CBS-KNAW Fungal Biodiversity Centre (CBS), Utrecht, The Netherlands, the working collections of Pedro W. Crous (CPC) maintained at CBS, and Acelino C. Alfenas (LPF) maintained at the Forest Pathology Laboratory/Bioagro, Universidade Federal de Viçosa, Brazil.

Table 1

Collection details and GenBank assessions of Calonectria isolates included in this study.

Species	Isolate nr.1	Substrate	Locality	Collector	GenBank assession2
tub2	cmdA	his3	tef1
Calonectria brachiatica	CBS 111478; CMW 30981; CPC 1921	Soil	Brazil	A.C. Alfenas	DQ190611	GQ267383	DQ190719	FJ918568
	CBS 123699; CMW 25303	Pinus tecunumanii	Buga, Colombia	M.J. Wingfield	FJ716708	GQ267365	FJ716712	GQ267295
	CBS 123700; CMW 25298	Pinus maximinoi	Buga, Colombia	M.J. Wingfield	FJ696388	GQ267366	FJ696396	GQ267296
	CBS134665; LPF305	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395933	KM396020	KM396103	KM395846
	CBS134666; LPF298	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395934	KM396021	KM396104	KM395847
Ca. brasiliensis	CBS 230.51; IMI 299576; CPC 2390	Eucalyptus sp.	Brazil	R. Ciferri	GQ267241	GQ267421	GQ267259	GQ267328
	CBS 114257; CMW 32949; CPC 1944	Eucalyptus sp.	Brazil	A.C. Alfenas	GQ267242	GQ267422	GQ267260	GQ267329
Ca. brassiana	CBS 134855; LPF378	Soil (E. brassiana plantation)	Teresina, Piauí, Brazil	R.F. Alfenas	KM395969	KM396056	KM396139	KM395882
	CBS 134856; LPF379	Soil (E. brassiana plantation)	Teresina, Piauí, Brazil	R.F. Alfenas	KM395970	KM396057	KM396140	KM395883
	CBS 134857; LPF380	Soil (E. brassiana plantation)	Teresina, Piauí, Brazil	R.F. Alfenas	KM395971	KM396058	KM396141	KM395884
Ca. brassicae (= Cy. clavatum)	CBS 111869; CPC 2409; PC 551197	Argyreia splendens	Indonesia	F. Bugnicourt	AF232857	GQ267382	DQ190720	FJ918567
	CBS 143.72; ATCC 22833; IMI 164057	Pinus caribaea	Itabira, Minas Gerais, Brazil	C.S. Hodges	KM395988	KM396075	–	KM395901
	CBS 134657; LPF236	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395918	KM396005	KM396088	KM395831
	CBS 134658; LPF234	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395919	KM396006	KM396089	KM395832
	CBS 134659; LPF216	Soil	Salinas, Minas Gerais, Brazil	D.B. Pinho	KM395920	KM396007	KM396090	KM395833
	CBS 134660; LPF493	Soil	Salinas, Minas Gerais, Brazil	D.B. Pinho	KM395921	KM396008	KM396091	KM395834
	LPF235	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395922	KM396009	KM396092	KM395835
	LPF237	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395923	KM396010	KM396093	KM395836
Ca. candelabra	CMW 31000; CPC 1675	Eucalyptus sp.	Amazonas, Brazil	A.C. Alfenas	FJ972426	GQ267367	FJ972476	FJ972525
	CMW 31001; CPC 1679	Eucalyptus sp.	Amazonas, Brazil	A.C. Alfenas	GQ421779	GQ267368	GQ267246	GQ267298
Ca. cerciana	CBS 123693; CMW 25309	Eucalyptus hybrid	Zhanjiang Prov., CERC nursery, China	M.J. Wingfield & X.D. Zhou	FJ918510	GQ267369	FJ918528	FJ918559
	CBS 123695; CMW 25290	Eucalyptus hybrid	Zhanjiang Prov., CERC nursery, China	M.J. Wingfield & X.D. Zhou	FJ918511	GQ267370	FJ918529	FJ918560
Ca. clavata	CBS 114557; ATCC 66389; CPC 2536	Callistemon viminalis	USA	C.P. Seymour & E.L. Barnard	AF333396	GQ267377	DQ190623	GQ267305
	CBS 114666; CMW 30994; CPC 2537	Root debris in peat	USA	D. Ferrin & N.E. El-Gholl	DQ190549	GQ267378	DQ190624	GQ267306
Ca. colombiana	CBS 115127; CPC 1160	Soil	La Selva, Colombia	M.J. Wingfield	FJ972423	GQ267455	FJ972442	FJ972492
	CBS 115638; CPC 1161	Soil	La Selva, Colombia	M.J. Wingfield	FJ972422	GQ267456	FJ972441	FJ972491
Ca. colombiensis	CBS 112220; CPC 723	Soil	La Selva, Colombia	M.J. Wingfield	GQ267207	AY725748	AY725662	AY725711
	CBS 112221; CPC 724	Soil	La Selva, Colombia	M.J. Wingfield	AY725620	AY725749	AY725663	AY725712
Ca. cylindrospora	CBS 110666	Rosa sp.	USA	N.E. El-Gholl	FJ918509	GQ267423	FJ918527	FJ918557
Ca. densa	CBS 125249; CMW 31184	Soil	Las Golondrinas, Pichincha, Ecuador	M.J. Wingfield	GQ267230	GQ267442	GQ267279	GQ267350
	CBS 125261; CMW 31182	Soil	Las Golondrinas, Pichincha, Ecuador	M.J. Wingfield	GQ267232	GQ267444	GQ267281	GQ267352
Ca. duoramosa	CBS 134656; LPF434	Soil (tropical rainforest)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395940	KM396027	KM396110	KM395853
	LPF453	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395941	KM396028	KM396111	KM395854
Ca. ecuadoriae	CBS 111394; CPC 1628	Soil	Ecuador	M.J. Wingfield	DQ190599	GQ267376	DQ190704	GQ267304
	CBS 111406; CPC 1635	Soil	Ecuador	M.J. Wingfield	DQ190600	GQ267375	DQ190705	GQ267303
Ca. eucalypticola	CBS 134846; LPF121	Eucalyptus sp. (leaf)	Eunápolis, Bahia, Brazil	A.C. Alfenas	KM395963	KM396050	KM396133	KM395876
	CBS 134847; LPF124	Eucalyptus sp. (seeding)	Santa Bárbara, Minas Gerais, Brazil	A.C. Alfenas	KM395964	KM396051	KM396134	KM395877
	CBS 134848; LPF451	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395965	KM396052	KM396135	KM395878
Ca. glaebicola	CBS 134852; LPF406	Soil (Eucalyptus plantation)	Martinho Campos, Minas Gerais, Brazil	A.C. Alfenas	KM395966	KM396053	KM396136	KM395879
	CBS 134853; LPF407	Eucalyptus sp. (leaf)	Bico do Papagaio, Tocantins, Brazil	R.F. Alfenas	KM395967	KM396054	KM396137	KM395880
	CBS 134854; LPF408	Eucalyptus sp. (leaf)	Bico do Papagaio, Tocantins, Brazil	R.F. Alfenas	KM395968	KM396055	KM396138	KM395881
Ca. gordoniae	CBS 112142; CPC 3136; ATCC 201837	Gordonia liasanthus	USA	D. Chiappini	AF449449	GQ267381	DQ190708	GQ267309
Ca. gracilipes	CBS 111141	Soil	La Selva, Colombia	M.J. Wingfield	DQ190566	GQ267385	DQ190644	GQ267311
	CBS 115674	Soil	La Selva, Colombia	M.J. Wingfield	AF333406	GQ267384	DQ190645	GQ267310
Ca. gracilis	CBS 111284	Soil	Brazil	P.W. Crous	DQ190567	GQ267408	DQ190647	GQ267324
	CBS 111807	Manilkara zapota	Belém, Pará, Brazil	M. Aragaki	AF232858	GQ267407	DQ190646	GQ267323
Ca. hodgesii	CBS 133608; LPF244	Piptadenia gonoacantha	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KC491227	KC491221	–	KC491224
	CBS 133609; LPF245	Anadenanthera peregrina	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KC491228	KC491222	–	KC491225
	CBS 133610; LPF261	Azadirachta indica	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KC491229	KC491223	–	KC491226
Ca. humicola	CBS 125251	Soil	Las Golondrinas, Pichincha, Ecuador	M.J. Wingfield	GQ267233	GQ267445	GQ267282	GQ267353
	CBS 125269	Soil	Las Golondrinas, Pichincha, Ecuador	L. Lombard	GQ267235	GQ267447	GQ267284	GQ267355
Ca. insularis	CBS 114558; CPC 768	Soil	Tamatave, Madagascar	P.W. Crous	AF210861	GQ267389	FJ918526	FJ918556
	CBS 114559; CPC 954	Soil	Tamatave, Madagascar	P.W. Crous	AF210862	GQ267390	FJ918525	FJ918555
Ca. leucothoës	CBS 109166; ATCC 64824; CPC 2385	Leucothoë axillaris	USA	N.E. El-Gholl	FJ918508	GQ267392	FJ918523	FJ918553
Ca. pseudopteridis	CBS 163.28	Washingtonia robusta	USA	C.D. Sherbakoff	–	KM396076	–	KM395902
Ca. maranhensis	CBS 134811; LPF142	Eucalyptus sp. (leaf)	Açailândia, Maranhão, Brazil	A.C. Alfenas	KM395948	KM396035	KM396118	KM395861
	CBS 134812; LPF143	Eucalyptus sp. (leaf)	Açailândia, Maranhão, Brazil	A.C. Alfenas	KM395949	KM396036	KM396119	KM395862
	CBS 134825; LPF370	Soil (Eucalyptus plantation)	Imperatriz, Maranhão, Brazil	R.F. Alfenas	KM395950	KM396037	KM396120	KM395863
	CBS 134828; LPF441	Soil (Eucalyptus plantation)	Urbano Santos, Maranhão, Brazil	E. Zauza	KM395951	KM396038	KM396121	KM395864
	CBS 134829; LPF443	Soil (Eucalyptus plantation)	Urbano Santos, Maranhão, Brazil	E. Zauza	KM395952	KM396039	KM396122	KM395865
Ca. metrosideri	CBS 133603; LPF101	Metrosideros polymorpha	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KC294313	KC294304	KC294307	KC294310
	CBS 133604; LPF103	Metrosideros polymorpha	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KC294314	KC294305	KC294308	KC294311
	CBS 133605; LPF104	Metrosideros polymorpha	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KC294315	KC294306	KC294309	KC294312
Ca. multinaviculata	CBS 134858; LPF233	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395985	KM396072	KM396155	KM395898
	CBS 134859; LPF418	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395986	KM396073	KM396156	KM395899
	CBS 134862; LPF472	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395987	KM396074	KM396157	KM395900
Ca. multiphialidica	CBS 112678	Soil	Cameroon	Abadie	AY725628	AY725761	AY725673	AY725723
Ca. naviculata	CBS 101121	Leaf litter	João Pessoa, Brazil	R.F. Castañeda	GQ267211	GQ267399	GQ267252	GQ267317
	CBS 116080	Soil	Amazonas, Brazil	M.J. Wingfield	AF333409	GQ267398	GQ267251	GQ267316
Ca. nemuricola	CBS 134837; LPF085	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395979	KM396066	KM396149	KM395892
	CBS 134838; LPF090	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395980	KM396067	KM396150	KM395893
	CBS 134839; LPF094	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395981	KM396068	KM396151	KM395894
Ca. orientalis	CBS 125259	Soil	Teso East, Indonesia	M.J. Wingfield	GQ267237	GQ267449	GQ267286	GQ267357
	CBS 125260	Soil	Lagan, Indonesia	M.J. Wingfield	GQ267236	GQ267448	GQ267285	GQ267356
	LPF032	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395910	KM395996	–	KM395822
	LPF300	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395911	KM395997	–	KM395823
	LPF301	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395912	KM395998	–	KM395824
	LPF435	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395913	KM395999	–	KM395825
Ca. ovata	CBS 111299	E. tereticornis	Tucuruí, Pará, Brazil	P.W. Crous	GQ267212	GQ267400	GQ267253	GQ267318
	CBS 111307	E. tereticornis	Tucuruí, Pará, Brazil	P.W. Crous	AF210868	GQ267401	GQ267254	GQ267319
Ca. paraensis	CBS 134669; LPF430	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395924	KM396011	KM396094	KM395837
	LPF306	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395925	KM396012	KM396095	KM395838
	LPF308	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395926	KM396013	KM396096	KM395839
	LPF309	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395927	KM396014	KM396097	KM395840
	LPF429	Soil (tropical rainforest)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395928	KM396015	KM396098	KM395841
Ca. pauciramosa	CMW 5683	E. grandis	Knysna, South Africa	P.W. Crous	FJ918514	GQ267405	FJ918531	FJ918565
	CMW 30823	Soil	Tzaneen, South Africa	S. de Buisson	FJ918515	GQ267404	FJ918532	FJ918566
Ca. piauiensis	CBS 134849; LPF291	Soil (tropical rainforest)	Serra das Confusões, Piauí	O.L. Pereira	KM395972	KM396059	KM396142	KM395885
	CBS 134850; LPF377	Soil (Eucalyptus plantation)	Teresina, Piauí, Brazil	R.F. Alfenas	KM395973	KM396060	KM396143	KM395886
	CBS 134851; LPF381	Soil (tropical rainforest)	Teresina, Piauí, Brazil	R.F. Alfenas	KM395974	KM396061	KM396144	KM395887
Ca. pini	CBS 123698	Pinus patula	Buga, Colombia	C.A. Rodas	GQ267224	GQ267436	GQ267273	GQ267344
	CBS 125253	Pinus patula	Buga, Colombia	C.A. Rodas	GQ267225	GQ267437	GQ267274	GQ267345
Ca. polizzii	CBS 125270	Callistemon citrinus	Sicily, Messina, Italy	G. Polizzi	FJ972417	GQ267461	FJ972436	FJ972486
	CBS 125271	Arbustus unedo	Sicily, Messina, Italy	G. Polizzi	FJ972418	GQ267462	FJ972437	FJ972487
Ca. propaginicola	CBS 134815; LPF220	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395953	KM396040	KM396123	KM395866
	CBS 134816; LPF222	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395954	KM396041	KM396124	KM395867
	CBS 134817; LPF223	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395955	KM396042	KM396125	KM395868
	CBS 134820; LPF287	Used planting substrate	Santana, Pará, Brazil	A.C. Alfenas	KM395956	KM396043	KM396126	KM395869
	CBS 134821; LPF289	Used planting substrate	Santana, Pará, Brazil	A.C. Alfenas	KM395957	KM396044	KM396127	KM395870
	LPF218	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395958	KM396045	KM396128	KM395871
	LPF221	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395959	KM396046	KM396129	KM395872
Ca. pseudobrassicae	CBS 134661; LPF260	Soil (Eucalyptus plantation)	Santana, Pará, Brazil	A.C. Alfenas	KM395935	KM396022	KM396105	KM395848
	CBS 134662; LPF280	Soil (Eucalyptus plantation)	Santana, Pará, Brazil	A.C. Alfenas	KM395936	KM396023	KM396106	KM395849
Ca. pseudocerciana	CBS 134822; LPF365	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395960	KM396047	KM396130	KM395873
	CBS 134823; LPF366	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395961	KM396048	KM396131	KM395874
	CBS 134824; LPF367	Eucalyptus sp. (seeding)	Santana, Pará, Brazil	A.C. Alfenas	KM395962	KM396049	KM396132	KM395875
Ca. pseudohodgesii	CBS 134813; LPF205	Eucalyptus sp. (seeding)	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KM395903	KM395989	KM396077	KM395815
	CBS 134814; LPF206	Eucalyptus sp. (seeding)	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KM395904	KM395990	KM396078	KM395816
	CBS 134818; LPF262	Azadirachta indica (leaf)	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KM395905	KM395991	KM396079	KM395817
	CBS 134819; LPF265	Azadirachta indica (leaf)	Viçosa, Minas Gerais, Brazil	R.F. Alfenas	KM395906	KM395992	KM396080	KM395818
Ca. pseudometrosideri	CBS 134843; LPF100	Metrosideros polymorpha	Viçosa, Minas Gerais, Brazil	A.C. Alfenas	KM395907	KM395993	KM396081	KM395819
	CBS 134844; LPF147	Eucalyptus sp. (leaf)	Açailândia, Maranhão, Brazil	R.F. Alfenas	KM395908	KM395994	KM396082	KM395820
	CBS 134845; LPF210	Soil (Eucalyptus plantation)	Maceió, Alagoas, Brazil	M.M. Coutinho	KM395909	KM395995	KM396083	KM395821
Ca. pseudonaviculata	CBS 114417; CPC 10926	Buxus sempervirens	West Auckland, New Zealand	C. Crepel	GQ267214	GQ267409	GQ267258	GQ267325
	CBS 116251; CPC 3399	Buxus sempervirens	West Auckland, New Zealand	C.R. MacDiarmid	AF449455	KM396000	–	KM395826
Ca. pseudoscoparia	CBS 125255	E. grandis	Pichincha, Ecuador	M.J. Wingfield	GQ267227	GQ267439	GQ267276	GQ267347
	CBS 125257	E. grandis	Pichincha, Ecuador	M.J. Wingfield	GQ267229	GQ267441	GQ267278	GQ267349
Ca. pseudospathulata	CBS 134840; LPF066	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395982	KM396069	KM396152	KM395895
	CBS 134841; LPF072	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395983	KM396070	KM396153	KM395896
	CBS 134842; LPF087	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395984	KM396071	KM396154	KM395897
Ca. pseudovata	CBS 134674; LPF267	Soil (Eucalyptus plantation)	Santana, Pará, Brazil	A.C. Alfenas	KM395945	KM396032	KM396115	KM395858
	CBS 134675; LPF285	Soil (Eucalyptus plantation)	Santana, Pará, Brazil	A.C. Alfenas	KM395946	KM396033	KM396116	KM395859
	LPF286	Soil (Eucalyptus plantation)	Santana, Pará, Brazil	A.C. Alfenas	KM395947	KM396034	KM396117	KM395860
Ca. pseudospathiphylli	CBS 109165; CPC 1623	Soil	Ecuador	M.J. Wingfield	FJ918513	GQ267412	AF348241	FJ918562
Ca. pteridis	CBS 111793; ATCC 34395; CPC 2372	Arachnoides adiantiformis	USA	P.W. Crous	DQ190578	GQ267413	DQ190679	FJ918563
	CBS 111871; CPC 2443	Pinus sp.	Spain	T.L. Krugner	DQ190579	GQ267414	DQ190681	FJ918564
	CBS 134670; LPF410	Eucalyptus sp. (leaf)	Imperatriz, Maranhão, Brazil	R.F. Alfenas	KM395914	KM396001	KM396084	KM395827
	CBS 134671; LPF422	Eucalyptus sp. (leaf)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395915	KM396002	KM396085	KM395828
	CBS 134672; LPF201	Eucalyptus sp. (leaf)	Imperatriz, Maranhão, Brazil	R.F. Alfenas	KM395916	KM396003	KM396086	KM395829
	CBS 134673; LPF202	Eucalyptus sp. (leaf)	Imperatriz, Maranhão, Brazil	R.F. Alfenas	KM395917	KM396004	KM396087	KM395830
Ca. quinqueramosa	CBS 134654; LPF065	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395942	KM396029	KM396112	KM395855
	CBS 134655; LPF281	Soil (Eucalyptus plantation)	Santana, Pará, Brazil	A.C. Alfenas	KM395943	KM396030	KM396113	KM395856
	CBS 134863; LPF302	Soil (Eucalyptus plantation)	Monte Dourado, Pará, Brazil	R.F. Alfenas	KM395944	KM396031	KM396114	KM395857
Ca. robigophila	CBS 134652; LPF192	Eucalyptus sp. (leaf)	Açailândia, Maranhão, Brazil	R.F. Alfenas	KM395937	KM396024	KM396107	KM395850
	CBS 134653; LPF193	Eucalyptus sp. (leaf)	Açailândia, Maranhão, Brazil	R.F. Alfenas	KM395938	KM396025	KM396108	KM395851
	LPF190	Eucalyptus sp. (leaf)	Açailândia, Maranhão, Brazil	R.F. Alfenas	KM395939	KM396026	KM396109	KM395852
Ca. silvicola	CBS 134836; LPF079	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395975	KM396062	KM396145	KM395888
	CBS 135237; LPF081	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395978	KM396065	KM396148	KM395891
	CPC 18741; LPF071	Soil (tropical rainforest)	Araponga, Minas Gerais, Brazil	A.C. Alfenas & P.W. Crous	KM395976	KM396063	KM396146	KM395889
	CPC 18766; LPF096	Soil (tropical rainforest)	Mucuri, Bahia, Brazil	E. Zauza	KM395977	KM396064	KM396147	KM395890
Ca. spathiphylli	CBS 114540; ATCC 44730; CPC 2378	Spathiphyllum sp.	USA	S. A. Alfieri	AF348214	GQ267424	AF348230	GQ267330
	CBS 116168; CPC 789	Spathiphyllum sp.	Switzerland	L. Petrini	FJ918512	GQ267425	FJ918530	FJ918561
Ca. spathulata	CBS 555.92	Eucalyptus viminalis	Brazil	N.E. El-Gholl	AF308463	GQ267426	FJ918524	FJ918554
	CBS 112689	Araucaria angustifolia	São Paulo, Brazil	C.S. Hodges	GQ267215	GQ267427	GQ267261	GQ267331
Ca. sulawesiensis	CBS 125248	Eucalyptus sp.	Sulawesi, Indonesia	M.J. Wingfield	GQ267223	GQ267435	GQ267272	GQ267343
	CBS 125277	Eucalyptus sp.	Sulawesi, Indonesia	M.J. Wingfield	GQ267222	GQ267434	GQ267271	GQ267342
Ca. telluricola	CBS 134663; LPF214	Soil (tropical rainforest)	Salinas, Minas Gerais, Brazil	D.B. Pinho	KM395929	KM396016	KM396099	KM395842
	CBS 134664; LPF217	Soil (tropical rainforest)	Mucuri, Bahia, Brazil	E. Zauza	KM395930	KM396017	KM396100	KM395843
	CBS 134667; LPF263	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395931	KM396018	KM396101	KM395844
	CBS 134668; LPF254	Soil (Eucalyptus plantation)	Mucuri, Bahia, Brazil	E. Zauza	KM395932	KM396019	KM396102	KM395845
Ca. variabilis	CBS 112691; CPC 2506	Theobroma grandiflorum	Brazil	F. Carneiro	GQ267240	GQ267458	GQ267264	GQ267335
	CBS 114677; CPC 2436	Schefflera morototoni	Brazil	F. C. de Albuquerque	AF333424	GQ267457	GQ267263	GQ267334
Ca. zuluensis	CBS 125268	E. grandis	Kwa-Zulu Natal, Kwambonambi, South Africa	L. Lombard	FJ972414	GQ267459	FJ972433	FJ972483
	CBS 125272	E. grandis	Kwa-Zulu Natal, Kwambonambi, South Africa	L. Lombard	FJ972415	GQ267460	FJ972434	FJ972484

ATCC: American Type Culture Collection, Virginia, U.S.A., CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, CMW: collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa, CPC: Pedro W. Crous working collection housed at CBS, IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham lane, U.K., LPF: Laboratório de Patologia Florestal, Universidade Federal de Viçosa, Viçosa, Brazil. Ex-type strains indicated in bold.

tub2 = β-tubulin, cmdA = calmodulin, his3 = histone H3, tef1 = translation elongation factor-1α; sequences generated in this study indicated in italics.

DNA sequencing and phylogenetic analyses

Genomic DNA was isolated from 7-d-old fungal mycelium grown on MEA following the protocol of the Wizard® Genomic DNA Purification (Promega Corporation, WI, USA) kit. For amplification of gene regions, the DreamTaq™ Master Mix (MBI Fermentas, Vilnius, Lithuania) was used, following the manufacturer's protocol. Initially, partial gene sequences of the translation elongation factor 1-α (tef1) were determined for all isolates collected using the primers EF1-728F (O'Donnell ) and EF-2 (Carbone & Kohn 1999) following the protocol and conditions outlined by Crous . Subsequently, partial fragments of β-tubulin (tub2), calmodulin (cmdA) and histone 3 (his3), were determined following the protocols and primers outlined by Crous and Groenewald . DNA sequencing reactions were performed using the BigDye® Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems Life Technologies, Carlsbad, CA, USA) following the protocol provided by the manufacturer. To ensure the integrity of the sequences, amplicons were sequenced in both directions using the same primers used for amplification. Purified sequence reactions were run on an ABI Prism 3730xl DNA Sequencer (Life Technologies, Carlsbad, CA, USA). The quality of the electropherograms generated were evaluated using Sequence Scanner Software v. 1.0 (Applied Biosystems) and PHPH (http://www.biomol.unb.br/phph/). Consensus sequences were determined using Seqman (DNAStar Inc., Madison, Wisconsin, USA). All sequences were manually corrected and the arrangement of nucleotides in ambiguous positions was corrected using comparisons of the sequences generated from both the forward and reverse primers. In addition to the sequences generated in this study, other sequences were obtained from NCBI's GenBank nucleotide database (www.ncbi.nlm.nih.gov) and added to the DNA sequence datasets generated in this study (Table 1).

Sequence datasets for the four loci were aligned in MAFFT v. 7.0 (Katoh & Standley 2013), and manually corrected where necessary using MEGA v. 5 (Tamura ). Single nucleotide polymorphisms (SNP's) were determined for each gene region with the aid of DnaSP v. 5.00.06 (Librado & Rozas 2009). The best evolutionary model of nucleotide substitution for each gene region was selected according to Akaike Information Criterion (AIC) using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses.

An initial phylogenetic analysis was done for the aligned tef1 data set which included 1019 taxa, including outgroup, using MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003) to identify the possible groups present in our samples. Based on this, 85 Calonectria isolates were selected for further study and divided into four separate datasets representing the (1) Ca. brassicae and Ca. pteridis complex, (2) Ca. cylindrospora complex, (3) Ca. candelabra complex and (4) Ca. naviculata complex, to reduce the number of gaps in the alignments and consequently improve the resolution of the analyses. To determine whether the four gene regions determined were congruent, congruence index trees (De Vienne ) and a 70 % reciprocal bootstrap method (Gueidan ) were applied to each gene region used.

Phylogenetic analyses were based on both Bayesian Inference (BI) and Maximum Parsimony (MP). For BI, the best evolutionary model of nucleotide substitution for each gene region was incorporated into the analyses. Analyses in MrBayes v. 3.2.1 (Ronquist & Huelsenbeck 2003) used the Markov Chain Monte Carlo (MCMC) algorithm and employed two sets of four chains started in parallel from a random tree topology with the heating parameter set at 0.3. The MCMC analysis ran until the average standard deviation of split frequencies came 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) determined from the remaining trees.

MP analyses were performed in PAUP (Phylogenetic Analysis Using Parsimony, v. 4.0b10; Swofford 2003) with phylogenetic relationships estimated by heuristic searches with 1 000 random addition sequences. The tree-bisection-reconnection option was used, with the branch swapping option set to “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 resulting trees were illustrated using Geneious v. 5.5.4 (Drummond ). Sequences derived in this study were deposited in GenBank (Table 1) and the alignments in TreeBASE (www.treebase.org/treebase/index.html).

Taxonomy

Single conidial isolates were grown on synthetic nutrient-poor agar (SNA; Nirenburg 1981) at 24 °C, following the protocol of Lombard . After 7 d of incubation, the morphological characteristics of the asexual morphs were determined by mounting fungal structures in clear lactic acid and 30 measurements at ×1 000 magnification were determined for each isolate using a Zeiss Axioscope 2 microscope with interference contrast (DIC) optics. Additionally, crosses were made as described by Lombard et al., 2010b, Lombard et al., 2010c in all possible combinations based on the identities determined by DNA sequence analysis of the tef1 gene region. Isolates were crossed with themselves as controls, thus making it possible to distinguish between heterothallic and homothallic mating systems of the isolates. The plates were stacked in plastic containers and incubated at room temperature (25°C ± 3 °C) for 6–8 wk. Crosses were regarded as successful when isolate combinations produced ascomata extruding viable ascospores. Morphological characteristics of the sexual morphs were determined by mounting ascomata in tissue freezing medium (Leica Biosystems, Nussloch, Germany) and cutting sections with a Leica CM1100 cryostate (Leica Biosystems, Nussloch, Germany). The 10 μm sections were mounted in 85 % lactic acid and 3 % KOH. The 95 % confidence levels were determined and extremes of conidial and ascospore measurements are given in parentheses. For other structures only extremes are presented. Colony morphology was assessed using 7-d-old cultures on MEA and oatmeal agar (OA) incubated at 24 °C and the colour charts of Rayner (1970). All descriptions, illustrations and nomenclatural data were deposited in MycoBank (Crous ).

Results

A total of 1 017 isolates were obtained of which 646 were from Eucalyptus leaves displaying symptoms of CLB in plantations, 320 isolates were baited from soils collected within the commercial Eucalyptus plantations, 13 isolates were obtained from Eucalyptus seedlings and two from A. indica seedlings in the nursery, and 36 isolates were obtained from the surrounding native vegetation. Eighty-five of these isolates were selected for further study (Table 1) based on preliminary phylogenetic analysis of the tef1 gene region (results not shown).

Phylogenetic analyses

Approximately 500–550 bases were determined for the his3, tef1 and tub2 gene regions and 650 bases for the cmdA gene region. The preliminary tef1 sequence analysis, which included 1 019 taxa as well as outgroup taxa (Ca. colombiensis CBS 112220 & CBS 112221), showed that the majority of the collected isolates from Eucalyptus with CLB symptoms belonged to the Ca. pteridis species complex (484 of 545 isolates; results not shown). The remaining isolates were divided among the Ca. brassicae, Ca. cylindrospora, Ca. naviculata and Ca. candelabra species complexes.

For the Bayesian analyses, the evolutionary model selected for each gene region for each dataset is presented in Table 2. The Bayesian consensus trees for each of the datasets confirmed the tree topologies obtained from the MP analyses, and therefore, only the Bayesian consensus trees are presented with bootstrap support values (BS) and posterior probabilities (PP) shown for well-supported nodes. Congruency tests revealed no conflicts in tree topologies for the four gene regions used in each of the four separate datasets and were therefore combined.

Table 2

Evolutionary substitution models determined for each gene region used in the Baysian phylogenetic inference.

Calonectria complex	Evolution model
tef1	tub2	cmdA	his3
Ca. brassicae and Ca. pteridis complexes	HKY + G	HKY + G	GTR + G	GTR + I + G
Ca. cylindrospora complex	HKY + G	HKY + I + G	HKY + I + G	GTR + I + G
Ca. naviculata complex	HKY + I	HKY + I	HKY + I	GTR + I
Ca. candelabra complex	GTR + G	GTR + G	GTR + I + G	HKY + G

The combined dataset for the Ca. brassicae and Ca. pteridis complexes included 61 ingroup taxa, with Ca. colombiensis (CBS 112220 & CBS 112221) as the outgroup taxon. The sequence dataset consisted of 1 958 characters, including alignment gaps. Of these, 1 289 were constant, 35 were parsimony-uninformative and 634 parsimony-informative. The MP analysis yielded 1 000 trees (TL = 1 304, CI = 0.685, RI = 0.902, RC = 0.618). The BI analysis lasted 1 625 000 generations and the consensus tree (Fig. 1) and posterior probabilities (PP) were calculated from 2 439 trees. In the tree, five main clades could be resolved. Clade 1 included four smaller clades, of which two of the clades represented Ca. orientalis (ex-type CBS 125260; BS = 77, PP = 0.97) and Ca. pini (ex-type CBS 123698; BS = 100, PP = 1.0), respectively. The remaining two clades (BS = 53, PP = 0.98 and BS = 81, PP = 0.98, respectively), which include CBS 134669, LPF 306, LPF308, LPF309, LPF429 in one clade, and CBS 134663, CBS 134664, CBS 134667, CBS 134668 in the other, appear to represent two distinct lineages. Clade 2 consisted of four smaller clades, of which three represented known Calonectria species. The clade representing Ca. brassicae, which included the ex-types of Cy. gracile (CBS 111869) and Cy. clavatum (CBS 134.71), could not be resolved in this study. Similarly, the clade representing Ca. brachiatica (ex-type CBS 123700) could not be resolved. Two isolates (CBS 134661, CBS 1346620) formed a basal sister clade (BS = 100, PP = 1.0) to the clades representing Ca. brassicae and Ca. brachiatica, possibly indicating a previously unrecognised lineage. Clade 3 included three well-supported sister clades, one of which (BS = 100, PP = 1.0) represents Ca. ecuadoriae (ex-type CBS 111406). The other two clades, one incorporating CBS 134652, CBS 134653 and LPF190 (BS = 100, PP = 1.0) and the other CBS 134656 and LPF453 (BS = 98, PP = 1.0), each represent novel lineages. Isolates CBS 134654, CBS 134655 and CBS 134863, in Clade 4, clustered together in a clade (BS = 91, PP < 0.95) closely related to but separate from the clade (BS = 100, PP = 1.0) representing Ca. gracilis (ex-type CBS 111807), representing a novel lineage. In Clade 5, three isolates obtained from Eucalyptus leaves (CBS 134674, CBS 134675, LPF286) formed a sister clade (BS = 93, PP = 1.0) closely related to but separate from the clade (BS = 100, PP = 1.0) representing Ca. ovata (CBS 111299 and CBS 111307). Four representative isolates (CBS 134670 – CBS 134673) collected during this study, clustered within the clade (BS = 100, PP = 1.0) representing Ca. pteridis (ex-type CBS 111793). The ex-type of Cy. macrosporum (CBS 163.28) formed a distinct basal lineage to the Ca. pteridis clade, indicating that this species was incorrectly synonymised under Ca. pteridis (Crous 2002).

Fig. 1

Consensus phylogram of 2 439 trees resulting from a Bayesian analysis of the combined four gene sequence alignment of the Calonectria brassicae and Ca. pteridis complexes. Accession numbers in bold represent ex-type strains. Bayesian posterior probabilities and Maximum Parsimony bootstrap support values are indicated at the nodes and the scale bar represents the number of expected changes per site. The tree was rooted to Ca. colombiensis (CBS 112220, CBS 112221).

The combined dataset for the Ca. cylindrospora species complex included 41 ingroup taxa, with Ca. colombiensis (CBS 112220 & CBS 112221) as the outgroup taxon. The sequence dataset consisted of 1 975 characters, including alignment gaps. Of these, 1 355 were constant, 85 were parsimony-uninformative and 535 parsimony-informative. The MP analysis yielded 1 000 trees (TL = 1 002, CI = 0.767, RI = 0.891, RC = 0.684). The BI analysis lasted 985 000 generations and the consensus tree (Fig. 2) and posterior probabilities (PP) were calculated from 1 480 trees. In the tree, four main clades could be resolved with the isolates collected in this study clustering in Clades 1 & 3. In Clade 1, isolates obtained during this study clustered in two smaller clades, one of which formed a distinct basal clade (BS = 98, PP = 1.0) to the clades representing Ca. hodgesii (ex-type CBS 133609), Ca. brasiliensis (ex-type CBS 230.51) and Ca. sulawesiensis (ex-type CBS 125277), representing a distinct lineage. The remaining isolates (CBS 134813, CBS 134814, CBS 134818, CBS 134819) formed a clade (BS = 79, PP = 0.99) closely related to, but distinct from, the Ca. hodgesii clade, also representing a previously unrecognised lineage. In Clade 3, the newly collected isolates also clustered in two well-supported but distinct clades (containing CBS 134815; BS = 69, PP = 0.95 & containing CBS 134824; BS = 73, PP < 0.95, respectively) with Ca. cerciana (ex-type CBS 123693) forming a basal clade to both these lineages.

Fig. 2

Consensus phylogram of 1 480 trees resulting from a Bayesian analysis of the combined four gene sequence alignment of the Calonectria cylindrospora complex. Accession numbers in bold represent ex-type strains. Bayesian posterior probabilities and Maximum Parsimony bootstrap support values are indicated at the nodes and the scale bar represents the number of expected changes per site. The tree was rooted to Ca. colombiensis (CBS 112220, CBS 112221).

The combined dataset for the Ca. naviculata species complex included eight ingroup taxa, with Ca. colombiensis (CBS 112220 & CBS 112221) as the outgroup taxon. The sequence dataset consisted of 1 994 characters, including alignment gaps. Of these, 1 431 were constant, 86 were parsimony-uninformative and 477 parsimony-informative. The MP analysis yielded 1 000 trees (TL = 764, CI = 0.919, RI = 0.931, RC = 0.855). The BI analysis lasted 275 000 generations and the consensus tree (Fig. 3) and posterior probabilities (PP) were calculated from 15 trees. Only three isolates (CBS 134858, CBS 134859, CBS 134862), collected in this study, grouped in this dataset. They formed a well-supported clade (BS = 91, PP < 0.95) closely related to, but distinct from, the clade representing Ca. naviculata (ex-type CBS 101121).

Fig. 3

Consensus phylogram of 15 trees resulting from a Bayesian analysis of the combined four gene sequence alignment of the Calonectria naviculata complex. Accession numbers in bold represent ex-type strains. Bayesian posterior probabilities and Maximum Parsimony bootstrap support values are indicated at the nodes and the scale bar represents the number of expected changes per site. The tree was rooted to Ca. colombiensis (CBS 112220, CBS 112221).

The combined dataset for the Ca. candelabra species complex included 42 ingroup taxa, with Ca. colombiensis (CBS 112220 & CBS 112221) as the outgroup taxon. The sequence dataset consisted of 1 930 characters, including alignment gaps. Of these, 1 448 were constant, six were parsimony-uninformative and 476 parsimony-informative. The MP analysis yielded 1 000 trees (TL = 690, CI = 0.813, RI = 0.922, RC = 0.750). The BI analysis lasted 1 565 000 generations and the consensus tree (Fig. 4) and posterior probabilities (PP) were calculated from 2 348 trees. In the tree, three main clades are resolved, with each clade incorporating isolates collected in this study. In Clade 1, the newly collected isolates clustered into five well-supported clades. The first of these clades (containing CBS 134845; BS = 61, PP = 0.99) is closely related but separate from the Ca. metrosideri clade (ex-type CBS 133603). The remaining four clades of newly collected isolates formed basal sister clades to Ca. candelabra (CMW31000 & CMW31001), Ca. pseudoscoparia (ex-type CBS 125257) and Ca. metrosideri. In Clade 2, the isolates from the current study clustered into two separate well-supported clades (containing CBS 135237; BS = 99, PP = 1.0, and containing CBS 134837; BS = 100, PP = 1.0) sister to the clades of Ca. pauciramosa (ex-type CMW5683), Ca. polizzii (CBS 125270 & CBS 125271) and Ca. zuluensis (ex-type CBS 125268). The newly collected isolates in Clade 3 clustered together in a well-supported clade (containing CBS 134841; BS = 100, PP = 0.99) closely related to, but distinct from, the Ca. spathulata clade (CBS 555.92 & CBS 112689).

Fig. 4

Consensus phylogram of 2 348 trees resulting from a Bayesian analysis of the combined four gene sequence alignment of the Calonectria candelabra complex. Accession numbers in bold represent ex-type strains. Bayesian posterior probabilities and Maximum Parsimony bootstrap support values are indicated at the nodes and the scale bar represents the number of expected changes per site. The tree was rooted to Ca. colombiensis (CBS 112220, CBS 112221).

Morphological observations (Table 3) supported by phylogenetic inference showed that the majority of the strains collected in this study belonged to Ca. pteridis. The remaining isolates are shown to represent several distinct taxa that are provided with names in Calonectria. Furthermore, Ca. metrosideri is invalid, as Alfenas did not include collection and specimen details and it is, therefore, validated here. Calonectria pseudopteridis (= Cylindrocladium macrosporum) is resurrected to species rank based on phylogenetic inference.

Table 3

Morphological characterisitics of Calonectria spp. included in this study.

Species	Perithecia		Asci	Ascospores		Conidiogenous apparatus		Stipe extention	Vesicle		Macroconidia			Reference
Size (μm)	Shape	Size (μm)	Size (μm)	Septation	Size (μm)	Branches	Size (μm)	Diam (μm)	Shape	Size (μm)	Septation	Length/Diam ratio
Calonectria brassicae species complex
Ca. brachiatica						40–81 × 35–84	5	134–318 × 4–5	5–7	clavate	(37–)40–48(–50) × 4–6	1(–2)	8.8	Lombard et al. (2009)
Ca. brassicae						35–75 × 15–60	5	140–350 × 2.5–3	2–6	clavate	(38–)40–55(–65) × 3.5–6	1	11.78	Crous (2002)
Ca. clavata	360–630 × 290–500	subglobose to ovoid	53–155 × 10–22.5	(30–)40–50(–54) × (4–)5–6(–6.5)	1(–3)	40–70 × 25–50	4	60–110 × 5–6	3–4	narrowly clavate	(44–)50–70(–80) × 4–6	1(–3)	13	Crous (2002)
Ca. duoramosa						20–60 × 30–50	2	175–310 × 3–5	4–6	acicular to clavate	(35–)44–48(–55) × 3–5	1	11.38	This study
Ca. ecuadoriae						30–100 × 30–100	7	200–300 × 2–3	3–5	clavate	(45–)48–55(–65) × 4–5	1(–3)	11.33	Crous et al. (2006)
Ca. gracilipes	350–400 × 300–380	subglobose to ovoid	80–120 × 12–18	(28–)33–40(–45) × (5–)6–7(–7.5)	1	30–70 × 25–35	3	150–260 × 2.5–3	3–4	clavate	(35–)40–48(–60) × 4–6	1	10	Crous (2002)
Ca. gracilis	350–400 × 330–380	subglobose to ovoid	75–100 × 8–15	(27–)33–45(–50) × (4–)4.5–5(–6)	1	60–100 × 30–70	4	160–350 × 2–3	2–11	clavate	(40–)53–58(–65) × 3.5–5	1(–3)	12.44	Crous (2002)
Ca. orientalis						54–174 × 67–92	5	90–218 × 5–10	5–10	clavate to broadly clavate	(43–)46–50(–53) × 4–5	1	12	Lombard et al. (2010c)
Ca. paraensis						45–55 × 60–75	2	120–195 × 3–5	4–6	clavate	(35–)40–43(–50) × 3–6	1	8.85	This study
Ca. pini						49–81 × 35–84	3	121–266 × 5–7	4–6	clavate	(37–)40–48(–50) × 4–6	1	8.8	Lombard et al. (2010c)
Ca. pseudobrassicae						50–115 × 60–100	3	190–300 × 3–5	3–5	clavate	(30–)39–42(–48) × 4–6	1	8.04	This study
Ca. quinqueramosa	160–400 × 115–250	pyriform to subglobose	50–105 × 10–25	(25–)39–42(–50) × 5–7	1	30–60 × 35–65	5	170–340 × 2–4	3–5	narrowly clavate to clavate	(45–)57–61(–70) × 4–6	1	11.57	This study
Ca. robigophila						15–60 × 30–70	6	125–225 × 3–4	4–5	acicular to clavate	(45–)49–52(–60) × 3–5	1	12.6	This study
Ca. telluricola						45–95 × 40–80	4	100–225 × 2–4	3–6	clavate	(35–)40–42(–50) × 3–6	1	9.13	This study
Calonectria candelabra species complex
Ca. brassiana						50–135 × 50–80	3	90–172 × 2–3	3–7	ellipsoid to narrowly obpyriform	(35–)50–56(–65) × 3–5	1	12.91	This study
Ca. candelabra	350–450 × 300–350	subglobose to ovoid	70–130 × 7–15	(40–)45–50(–60) × 5–6	1	30–70 × 50–80	5	100–220 × 3–3.5	5–8	ellipsoid to narrowly obpyriform	(45–)58–68(–80) × 4–5(–6)	1	13.33	Crous (2002)
Ca. colombiana	270–410 × 175–285	subglobose to ovoid	87–162 × 12–18	(28–)31–36(–40) × 3–5	1	38–115 × 35–91	4	143–173 × 5–7	8–12	obpyriform to ellipsoidal	(33–)35–39(–40) × 3–4	1	12.33	Lombard et al. (2010b)
Ca. eucalypticola						45–75 × 35–62	3	145–170 × 2–4	5–7	ellipsoid to obpyriform	(43–)49–52(–55) × 3–5	1	12.2	This study
Ca. glaebicola						25–40 × 27–45	2	100–165 × 2–4	3–5	ellipsoid to narrowly obpyriform	(45–)50–52(–55) × 3–5	1	12.06	This study
Ca. metrosideri						60–75 × 40–65	4	90–170 × 2–4	5–9	spathulate to obpyriform	(40–)44–46(–51) × 3–5	1	11.25	Alfenas et al. (2013a)
Ca. mossambicensis						37–87 × 19–59	3	91–203 × 2–6	2–8	obpyriform to ellipsoidal	(35–)38–46(–50) × 3–6	1	10.5	Crous et al. (2013)
Ca. nemuricola						50–80 × 40–60	4	150–205 × 6–12	7–13	obpyriform	(40–)44–46(–50) × 3–5	1	11.06	This study
Ca. pauciramosa	250–400 × 170–300	subglobose to ovoid	70–140 × 8–25	(30–)33–38(–40) × 6–7(–8)	1	20–50 × 35–85	3	120–230 × 2–3	5–11	obpyriform to ellipsoidal	(30–)45–55(–60) × (3.5–)4–5	1	12.5	Schoch et al. (1999)
Ca. piauiensis						35–80 × 20–60	2	95–130 × 2–3	3–7	ellipsoid to narrowly obpyriform	(38–)47–52(–60) × 3–5	1	11.27	This study
Ca. polizzii						28–51 × 27–57	3	111–167 × 5–6	6–9	obpyriform to ellipsoidal	(31–)32–42(–49) × 3–5	1	9.25	Lombard et al. (2010a)
Ca. pseudometrosideri						30–76 × 45–65	3	160–210 × 2–4	5–7	ellipsoid to obpyriform	(40–)49–52(–60) × (3–)4.5(–5)	1	11.34	This study
Ca. pseudoscoparia						52–74 × 34–87	4	124–201 × 4–6	6–10	obpyriform to ellipsoidal	(41–)45–51(–52) × 3–3	1	12	Lombard et al. (2010b)
Ca. pseudospathulata						60–100 × 30–70	3	145–190 × 2–4	7–10	obpyriform	(35–)41–44(–50) × 3–5	1	10.46	This study
Ca. silvicola						45–105 × 35–90	3	130–195 × 3–4	7–10	obpyriform	(30–)40–42(–50) × 3–5	1	9.17	This study
Ca. spathulata	300–500 × 200–350	subglobose to ovoid	90–150 × 13–17	(38–)45–55(–60) × (4.5–)5–6(–7)	(1)–3	60–100 × 30–70	3	150–300 × 3–4	6–10	ellipsoid to obpyriform to clavate	(48–)75–90(–100) × (4–)5–6	(1–)3(–6)	13.33	Crous 2002
Ca. zuluensis	292–394 × 170–285	subglobose to ovoid	92–140 × 10–16	(26–)29–34(–38) × 4–5	1	37–70 × 35–67	3	110–171 × 5–8	6–10	ellipsoid to obpyriform	(31–)34–38(–40) × 3–5	1	8	Lombard et al. (2010a)
Calonectria cylindrospora species complex
Ca. brasiliensis						81–103 × 58–90	3	204–266 × 6–7	7–11	ellipsoid to obpyriform	(35–)36–40(–41) × 3–5	1	10.86	Lombard et al. (2010a)
Ca. cerciana						62–113 × 70–98	4	148–222 × 5–6	8–13	fusiform to obpyriform	(37–)41–46(–49) × 5–6	1	8.8	Lombard et al. (2010d)
Ca. cylindrospora	280–520 × 280–400	globose to subglobose	75–100 × 8–15	(24–)30–40(–49) × (4–)5–6(–8)	1	60–100 × 60–110	6	150–200 × 3–4	6–8	ellipsoid to pyriform or clavate	(40–)42–50(–66) × 3–4(–5)	1	11.25	Crous (2002)
Ca. densa						49–78 × 63–123	4	149–192 × 5–6	10–12	ovoid to ellipsoid to sphaeropedunculate	(47–)50–58(–62) × (5–)6	1	9	Lombard et al. (2010d)
Ca. hodgesii						61–72 × 45–65	3	136–196 × 2–4	6–11	pyriform to ellipsoidal or ovoid to sphaeropedunculate	(44–)49–51(–55) × 3–5	1	12.5	Alfenas et al. (2013b)
Ca. humicola						43–71 × 42–49	3	126–157 × 4–5	10–12	globose to ovoid to sphaeropedunculate	(45–)48–54(–56) × 4–5	1	10.2	Alfenas et al. (2013b)
Ca. insularis	350–450 × 300–350	subglobose to ovoid	70–125 × 7–18	(27–)30–36(–42) × 5–6(–7)	1	45–90 × 45–80	6	110–250 × 4–5	4–13	obpyriform to broadly ellipsoidal	(33–)40–50(–60) × 3.5–4	1	11.25	Crous (2002)
Ca. leucothoës						25–50 × 50–80	6	160–250 × 3–6	6–11.5	ellipsoid to obpyriform	(45–)68–78(–97) × (4–)5–5.5(–6.5)	(1–)3(–6)	14.6	Crous (2002)
Ca. maranhensis						45–65 × 45–71	3	125–190 × 3–5	7–11	ellipsoid, obpyriform to sphaeropedunculate	(50–)56–58(–65) × (3–)5(–6)	1	11.85	This study
Ca. propaginicola						40–75 × 31–85	4	130–250 × 2–5	5–12	ellipsoid, obpyriform to sphaeropedunculate	(40–)48–51(–55) × 3–5	1	12.67	This study
Ca. pseudocerciana						50–90 × 50–95	3	160–250 × 2–5	4–10	clavate or ellipsoidal to obpyriform	(45–)53–55(–65) × (3–)4.5(–5)	1	11.95	This study
Ca. pseudohodgesii						50–90 × 40–95	3	130–190 × 2–5	7–12	obpyriform to sphaeropendunculate	(35–)43–46(–55) × 3–5	1	11.95	This study
Ca. pseudospatiphylli	350–550 × 300–500	globose to subglobose	90–150 × 7–25	(30–)38–45(–55) × 5–6	1(–3)	70–100 × 25–70	4	100–250 × 2.5–3.5	8–12	sphaeropendunculate to ellipsoidal	(40–)47–55(–60) × 4–5	1(–3)	13	Crous (2002)
Ca. spathiphylli	380–655 × 340–650	subglobose to ovoid	120–230 × 7–25	(22–)40–52(–65) × (3–)4.5–5.5(–7)	1(–3)	60–150 × 40–90	4	170–260 × 3–4	8–15	globoid or ellipsoid to obpyriform	(45–)46–80(–120) × (5–)6(–7)	1(–3)	11.67	Crous (2002)
Ca. sulawesiensis						43–81 × 41–79	5	113–262 × 5–7	5–7	broadly clavate to ellipsoid	(41–)45–51(–54) × (3–)4(–6)	1	12	Lombard et al. (2010b)
Ca. variabilis	260–450 × 220–350	globose to ovoid	90–120 × 10–20	(34–)38–50(–60) × 4–5(–6)	1(–3)	40–70 × 20–100	3	130–250 × 2–3	6–11	sphaeropendunculate to ovoid or ellipsoid to clavate	(48–)68–77(–85) × 4–5(–7)	(1–)3(–4)	14.6	Crous (2002)
Calonectria pteridis species complex
Ca. gordoniae							4		3–6	narrowly clavate	(45–)62(–81) × 4–6	1	12.6	Leahy et al. (2000)
Ca. ovata	350–550 × 350–450	globose to ovoid	70–120 × 10–25	(35–)55–70(–90) × (4–)5–6	1	30–55 × 20–45	3	185–230 × 2.5–4	8–14	ovate	(50–)65–80(–110) × 4–6	1(–3)	10.9	Crous 2002
Ca. pseudovata						55–121 × 75–105	3	140–280 × 3–6	8–12	ovate to ellipsoidal	(55–)67–70(–80) × (4–)5(–7)	1	13.73	This study
Ca. pteridis	300–500 × 280–350	subglobose to ovoid	70–120 × 10–25	(30–)45–60(–75) × (4–)5–6(–7)	1(–3)	75–150 × 45–170	5	150–300 × 2.5–4	4–6	clavate to narrowly ellipsoidal	(50–)70–100(–130) × (4–)5–6	1(–3)	14.91	Crous 2002
Calonectria naviculata species complex
Ca. multinaviculata						30–65 × 40–70	3	75–140 × 2–5	4–7	naviculate	(40–)44–49(–52) × (2–)3.5(–4)	1	13.72	This study
Ca. multiphialidica						70–150 × 70–150	8	170–300 × 4–5	8–16	sphaeropedunculate to clavate	(45–)48–55(–65) × (4–)4.5(–5)	1	11.78	Crous et al., 2004a, Crous et al., 2004b
Ca. naviculata	350–450 × 350–400	globose to ovoid	70–100 × 8–12	(20–)40–48(–52) × (3–)5–6(–6.5)	3	45–90 × 25–100	4	150–200 × 3–4.5	5–11	naviculate to ellipsoidal	(40–)42–50 × 3(–4)	1	15	Crous 2002
Ca. pseudonaviculata						30–60 × 30–45	4	120–180 × 3–4	4–8	naviculate	(50–)55–65(–80) × 4–5(–6)	1(–3)	18	Crous 2002

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810001. Fig. 5.

Fig. 5

Calonectria brassiana (ex-type CBS 134855). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F–G. Ellipsoidal to narrowly obpyriform vesicles. H–I. Macroconidia. Scale bars: A = 50 μm (apply to B); C = 20 μm; D = 10 μm (apply to F, H); E = 10 μm (apply to G, I).

Etymology: Name refers to Eucalyptus brassiana, the plantation tree species associated with the soil from which this fungus was isolated.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 55–155 × 5–8 μm; stipe extensions septate, straight to flexuous, 90–172 μm long, 2–3 μm wide at the apical septum, terminating in ellipsoidal to narrowly obpyriform vesicles, 3–7 μm diam. Conidiogenous apparatus 50–80 μm long, 50–135 μm wide; primary branches aseptate, 20–30 × 4–6 μm, secondary branches aseptate, 15–25 × 3–6 μm, and tertiary branches aseptate, 10–17 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 9–15 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (35–)50–56(–65) × 3–5 μm (av. = 53 × 4 μm), L/W ratio = 12.91, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff on the surface and sepia in reverse; extensive white aerial mycelium with moderate sporulation on the aerial mycelium; chlamydospores sparse, occurring throughout the medium and forming microsclerotia. Colonies moderately fast growing (40–60 mm diam) on MEA and OA, after 7 d at 25 °C.

Material examined: Brazil, Piauí state, Teresina, from soil collected in Eucalyptus brassiana plantation, Jul. 2011, R.F. Alfenas (holotype CBS H-21376, culture ex-type CBS 134855 = LPF378), CBS 134856 = LPF379, CBS 134857 = LPF380.

Note: The macroconidia of Ca. brassiana are larger than those of Ca. eucalypticola, Ca. glaebicola, Ca. metrosideri, Ca. piauiensis, Ca. pseudoscoparia and Ca. pseudometrosideri, but smaller than those of Ca. candelabra (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810002. Fig. 6.

Fig. 6

Calonectria duoramosa (ex-type CBS 134656). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–I. Clavate vesicles. J–K. Macroconidia. Scale bars: A = 50 μm; B = 50 μm (apply to C); D = 10 μm (apply to E–K).

Etymology: Name refers to the two levels of fertile branches formed in the conidiogenous apparatus of this fungus.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 45–95 × 4–7 μm; stipe extensions septate, straight to flexuous, 175–310 μm long, 3–5 μm wide at the apical septum, terminating in acicular to clavate vesicles, 4–6 μm diam. Conidiogenous apparatus 20–60 μm long, 30–50 μm wide; primary branches aseptate, 20–30 × 4–6 μm and secondary branches aseptate, 10–20 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–15 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (35–)44–48(–55) × 3–5 μm (av. = 46 × 4 μm), L/W ratio = 11.38, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies umber to fawn on the surface and dark brick in reverse; sparse aerial mycelium; chlamydospores sparse, occurring throughout the medium, with moderate to extensive sporulation on the aerial mycelium. Colonies slow growing (33–43 mm diam) on MEA, and fast growing (79–83 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Pará state, Monte Dourado, from soil collected in tropical rainforest, Aug. 2010, R.F. Alfenas (holotype CBS H-21380, culture ex-type CBS 134656 = LPF434); from soil collected in Eucalyptus plantation, Aug. 2010, R.F. Alfenas, culture LPF453.

Notes: Calonectria duoramosa characteristically forms only two levels of branching in its conidiogenous apparatus distinguishing it from Ca. ecuadoriae and Ca. robigophila. The macroconidia of Ca. duoramosa are also slightly smaller than those of Ca. ecuadoriae and Ca. robigophila (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810003. Fig. 7.

Fig. 7

Calonectria eucalypticola (ex-type CBS 134847). A–C. Macroconidiophores. D. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. E–F. Ellipsoidal to obpyriform vesicles. G. Macroconidia. Scale bars: A = 50 μm (apply to B–C); D = 10 μm; E = 10 μm (apply to F); G = 10 μm.

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

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 50–242 × 5–10 μm; stipe extensions septate, straight to flexuous, 145–170 μm long, 2–4 μm wide at the apical septum, terminating in ellipsoidal to obpyriform vesicles, 5–7 μm diam. Conidiogenous apparatus 35–62 μm long, 45–75 μm wide; primary branches aseptate, 20–25 × 4–6 μm; secondary branches aseptate, 16–19 × 3–5 μm, tertiary branches aseptate, 9–16 × 2–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–12 × 2–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (43–)49–52(–55) × 3–5 μm (av. = 50 × 4 μm), L/W ratio = 12.20, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies cinnamon to dark brick on the surface and sepia in reverse; moderate to extensive sporulation on the aerial mycelium, especially at the margins; chlamydospores moderate occurring throughout the medium forming microsclerotia. Colonies slow growing (40–45 mm diam) on MEA, and moderate growing (50–55 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Minas Gerais state, Santa Bárbara, from stem of Eucalyptus seedling, Dec. 2010, A.C. Alfenas (holotype CBS H-21359, culture ex-type CBS 134847 = LPF124); Bahia state, Eunápolis, from Eucalyptus leaf, Mar. 2012, A.C. Alfenas, CBS 134846 = LPF121; Pará state, Monte Dourado, from soil collected in Eucalyptus plantation, July 2012, R.F. Alfenas, CBS 134848 = LPF451.

Note: Calonectria eucalypticola can be distinguished from its closest relatives (Fig. 4) based on macroconidial dimensions and the number of fertile branches produced in the conidiogenous apparatus (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810004. Fig. 8.

Fig. 8

Calonectria glaebicola (ex-type CBS 134852). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F–G. Ellipsoidal to narrowly obpyriform vesicles. H. Macroconidia. I. Chlamydospores. Scale bars: A = 50 μm (apply to B–C); D = 10 μm (apply to E–I).

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

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 50–130 × 5–7 μm; stipe extensions septate, straight to flexuous, 100–165 μm long, 2–4 μm wide at the apical septum, terminating in ellipsoidal to narrowly obpyriform vesicles, 3–5 μm diam. Conidiogenous apparatus 27–45 μm long, 25–40 μm wide; primary branches aseptate, 14–22 × 3–5 μm, secondary branches aseptate, 11–15 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 5–13 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (45–)50–52(–55) × 3–5 μm (av. = 50 × 4 μm), L/W ratio = 12.06, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff on the surface and sepia to umber in reverse; extensive aerial mycelium with moderate sporulation on the aerial mycelium; chlamydospores sparse, occurring throughout the medium forming microsclerotia. Colonies moderate growing (45–60 mm diam) on MEA and OA, after 7 d at 25 °C.

Materials examined: Brazil, Minas Gerais state, Martinho Campos, from soil collected in Eucalyptus plantation, Jul. 2010; A.C. Alfenas (holotype CBS H-21378, culture ex-type CBS 134852 = LPF406); Tocantins, Bico do Papagaio, from Eucalyptus leaf, Aug. 2012, R.F. Alfenas, CBS 134853 = LPF407, CBS 134854 = LPF408.

Note: Calonectria glaebicola is morphologically similar to its closest relatives (Fig. 4), from which it can be distinguished only by phylogenetic inference.

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810005. Fig. 9.

Fig. 9

Calonectria maranhensis (ex-type CBS 134811). A–C. Macroconidiophores. D–F. Ellipsoidal, obpyriform to sphaeropedunculate vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 20 μm (apply to B–C); D = 10 μm (apply to E–F); G = 10 μm (apply to H–I).

Etymology: Name refers to Maranhão state, Brazil, the region where this fungus was first collected.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 55–105 × 6–9 μm; stipe extensions septate, straight to flexuous, 125–190 μm long, 3–5 μm wide at the apical septum, terminating in ellipsoidal, obpyriform to sphaeropedunculate vesicles, 7–11 μm diam. Conidiogenous apparatus 45–71 μm long, 45–65 μm wide; primary branches aseptate, 20–45 × 3–6 μm; secondary branches aseptate, 15–20 × 3–5 μm, tertiary branches aseptate, 11–16 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 8–15 × 3–5 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (50–)56–58(–65) × (3–)5(–6) μm (av. = 57 × 5 μm), L/W ratio = 11.85, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies greyish sepia to dark brick on the surface and sepia to umber in reverse; extensive white aerial mycelium with moderate sporulation on the aerial mycelium; chlamydospores moderate to extensive occurring throughout the medium. Colonies moderately slow growing (50–55 mm diam) on MEA, and fast growing (80–85 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Maranhão state, Açailândia, from Eucalyptus leaf, May 2011, A.C. Alfenas (holotype CBS H-21360, culture ex-type CBS 134811 = LPF142), CBS 134812 = LPF143; Imperatriz, from soil in Eucalyptus plantation, May 2011, R.F. Alfenas, CBS 134825 = LPF370.

Note: Calonectria maranhensis can be distinguished from Ca. brasiliensis, Ca. hodgesii, Ca. sulawesiensis and Ca. variabilis by the dimensions of their macroconidia (Table 3).

R.F. Alfenas, O.L. Pereira, Crous & A.C. Alfenas, sp. nov. MycoBank MB810023.

≡ Calonectria metrosideri R.F. Alfenas, O.L. Pereira, Crous & A.C. Alfenas, Forest Pathology 43: 262. 2013. Nom. inval., Art 37.7.

See Alfenas for description and illustrations.

Material examined: Brazil, Minas Gerais state, Viçosa, Universidade Federal de Viçosa, forest nursery, isolated from leaf of Metrosideros polymorpha, Apr 2010, R.F. Alfenas (holotype CBS H-21146, culture ex-type CBS 133603).

Note: The original description of Ca. metrosideri is invalid, as no type specimen was designated. This issue is now addressed, and the name validly published.

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810006. Fig. 10.

Fig. 10

Calonectria multinaviculata (ex-type CBS 134858). A–C. Macroconidiophores. D–F. Naviculate vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 50 μm (apply to B–C); D = 10 μm (apply to E–I).

Entymology: Name refers to the multiple naviculate terminal vesicles formed by this fungus.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 45–90 × 5–7 μm; stipe extensions septate, straight to flexuous, 75–140 μm long, 2–5 μm wide at the apical septum, terminating in naviculate vesicles, 4–7 μm diam, abundant lateral stipe extension also present. Conidiogenous apparatus 30–65 μm long, 40–70 μm wide; primary branches aseptate, 19–22 × 3–6 μm, secondary branches aseptate, 9–18 × 3–6 μm, tertiary branches aseptate, 9–12 × 2–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–12 × 2–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (40–)44–49(–52) × (2–)3.5(–4) μm (av. = 46 × 3.5 μm), L/W ratio = 13.72, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff on the surface and sepia to umber in reverse; extensive white aerial mycelium with sparse to moderate sporulation on the aerial mycelium; chlamydospores not seen. Colonies moderately fast to fast growing (50–70 mm diam) on MEA and OA, after 7 d at 25 °C.

Materials examined: Brazil, Bahia state, Mucuri, from soil collected in Eucalyptus plantation, Aug. 2010; E. Zauza (holotype CBS 134858, preserved as metabolically inactive culture; culture ex-type CBS 134858 = LPF233), CBS 134862 = LPF472; Pará state, Monte Dourado, from soil collected in Eucalyptus plantation, July 2012, R.F. Alfenas, CBS 134859 = LPF418.

Note: Calonectria multinaviculata can be distinguished from Ca. naviculata by having fewer fertile branches in the conidiogenous apparatus, and having abundant lateral stipe extensions, which are absent in Ca. naviculata (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810007. Fig. 11.

Fig. 11

Calonectria nemuricola (ex-type CBS 134837). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–H. Obpyriform vesicles. I. Macroconidia. Scale bars: A = 100 μm; B = 50 μm (apply to C); D = 10 μm (apply to E–F); G = 10 μm (apply to H–I).

Etymology: Name refers to a forest, the habitat this fungus was collected from.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 50–105 × 6–12 μm; stipe extensions septate, straight to flexuous, 150–205 μm long, 2–4 μm wide at the apical septum, terminating in obpyriform vesicles, 7–13 μm diam. Conidiogenous apparatus 40–60 μm long, 50–80 μm wide; primary branches aseptate, 19–25 × 3–7 μm, secondary branches aseptate, 11–18 × 3–5 μm, tertiary branches aseptate, 9–12 × 3–5 μm, additional branches rare, (–4), aseptate, 7–10 × 3–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 5–11 × 2–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (40–)44–46(–50) × 3–5 μm (av. = 45 × 4 μm), L/W ratio = 11.06, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff on the surface and sepia to umber in reverse; extensive white aerial mycelium with sparse to moderate sporulation on the aerial mycelium; chlamydospores sparse, occurring throughout the medium, forming microsclerotia; Colonies fast growing (55–80 mm diam) on MEA and OA, after 7 d at 25 °C.

Material examined: Brazil, Minas Gerais state, Araponga (Serra do Brigadeiro), from soil collected in tropical rainforest, Aug. 2010, A.C. Alfenas & P.W. Crous (holotype CBS H-21358, culture ex-type CBS 134837 = LPF085), CBS 134838 = LPF090, CBS 134839 = LPF094.

Note: The macroconidia of Ca. nemuricola are larger than those of Ca. mossambicensis, Ca. polizzii, Ca. silvicola and Ca. zuluensis, but smaller than those of Ca. pauciramosa (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810008. Fig. 12.

Fig. 12

Calonectria paraensis (ex-type CBS 134669). A–C. Macroconidiophores. D–E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F. Macroconidia. G–I. Clavate vesicles. Scale bars: A = 50 μm (apply to B–C); D = 10 μm (apply to E–I).

Etymology: Name refers to the Pará state in Brazil where the fungus was collected.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate suites of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 52–110 × 5–7 μm; stipe extensions septate, straight to flexuous, 120–195 μm long, 3–5 μm wide at the apical septum, terminating in a clavate vesicle, 4–6 μm diam. Conidiogenous apparatus 45–55 μm long, 60–75 μm wide; primary branches aseptate, 18–24 × 4–6 μm, secondary branches aseptate, 14–23 × 3–5 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, (35–)40–43(–50) × 3–6 μm (av. = 42 × 5 μm), L/W ratio = 8.85, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff on the surface and ochraceous to umber in reverse; extensive aerial mycelium; chlamydospores not seen; sparse sporulation on aerial mycelium. Colonies moderate growing (40–60 mm diam) on MEA, and fast growing (75–85 mm diam) on OA, after 7 d at 25 °C.

Material examined: Brazil, Pará state, Monte Dourado, from soil in Eucalyptus plantation, Aug 2011, R.F. Alfenas (holotype CBS H–21379, culture ex-type CBS 134669 = LPF430), LPF429.

Note: Calonectria paraensis is a new member of the Ca. brassicae complex, distinguished from Ca. pini and Ca. orientalis by the size and length/diam ratio of its macroconidia, numbers of branches per conidiophores, and vesicle diameter (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810009. Fig. 13.

Fig. 13

Calonectria piauiensis (ex-type CBS 134850). A–C. Macroconidiophores. D–F. Ellipsoidal to narrowly obpyriform vesicles. G. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. H. Macroconidia. I. Chlamydospores. Scale bars: A = 100 μm; B = 50 μm (apply to C, I); D = 10 μm (apply to E–F); G = 10 μm (apply to H).

Etymology: Name refers to Piauí state, Brazil, the region this fungus was isolated from.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 50–110 × 4–6 μm; stipe extensions septate, straight to flexuous, 95–130 μm long, 2–3 μm wide at the apical septum, terminating in ellipsoidal to narrowly obpyriform vesicles, 3–7 μm diam. Abundant lateral stipe extensions also present. Conidiogenous apparatus 20–60 μm long, 35–80 μm wide; primary branches aseptate, 12–20 × 3–5 μm, secondary branches aseptate, 8–10 × 3–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–12 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (38–)47–52(–60) × 3–5 μm (av. = 49 × 4.5 μm), L/W ratio = 11.27, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff on the surface and sepia in reverse; extensive white aerial mycelium with moderate sporulation on the aerial mycelium; chlamydospores sparse, occurring throughout the medium, forming microsclerotia. Colonies moderately fast growing (50–75 mm diam) after 7 d at 25 °C on MEA and OA.

Materials examined: Brazil, Piauí state, Teresina, from soil collected in Eucalyptus brassiana plantation, Jul. 2011, R.F. Alfenas (holotype CBS H-21375, culture ex-type CBS 134850 = LPF377), CBS 134851 = LPF381; Serra das Confusões, from soil and leaf litter collected in semi-arid vegetation, Jul. 2012, D.B. Pinho & O.L. Pereira, CBS 134849 = LPF291.

Note: Calonectria piauiensis can be distinguished from other closely related species in the Ca. candelabra complex by the abundant lateral stipe extensions produced on the conidiogenous apparatus not reported for other member species in the Ca. candelabra complex (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810018. Fig. 14.

Fig. 14

Calonectria propaginicola (ex-type CBS 134815). A–C. Macroconidiophores. D–F. Ellipsoidal, obpyriform to sphaeropedunculate vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 50 μm (apply to B–C); D = 10 μm (apply to E–I).

Etymology: Name refers to cuttings from which this fungus was first isolated.

Sexual morph not observed. Conidiophores containing a stipe bearing penicillate suites of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 52–180 × 6–8 μm; stipe extensions septate, straight to flexuous, 130–250 μm long, 2–5 μm wide at the apical septum, terminating in ellipsoidal, obpyriform to sphaeropedunculate vesicles, 5–12 μm diam. Conidiogenous apparatus 31–85 μm long, 40–75 μm wide; primary branches 0–1-septate, 18–30 × 3–7 μm; secondary branches aseptate, 10–22 × 3–6 μm, tertiary branches aseptate, 11–20 × 3–5 μm, and additional branches (–4), aseptate, 9–15 × 3–4 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 5–12 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (40–)48–51(–55) × 3–5 μm (av. = 49 × 4 μm), L/W ratio = 12.67, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff to pale umber on the surface and sepia in reverse; moderate to extensive aerial mycelium with extensive sporulation on the aerial mycelium, especially in the centre of the colony; chlamydospores moderate, occurring throughout the medium, forming microsclerotia. Colonies fast growing (65–70 mm diam) on MEA, and (80–85 mm diam) on OA, after 7 d at 25 °C.

Material examined: Brazil, Pará state, Santana, from Eucalyptus seedling, Apr. 2011, A.C. Alfenas (holotype CBS H-21366, culture ex-type CBS 134815 = LPF220), CBS 134816 = LPF222.

Note: Calonectria propaginicola can be distinguished from Ca. cerciana and Ca. pseudocerciana based on its terminal vesicle morphology, and length/diam ratio of the macroconidia.

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810010. Fig. 15.

Fig. 15

Calonectria pseudobrassicae (ex-type CBS 134662). A–C. Macroconidiophores. D–F. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. G–H. Clavate vesicles. I. Macroconidia. Scale bars: A = 100 μm (apply to B); C = 50 μm; D = 10 μm (apply to E–I).

Etymology: Name refers to the fact that this species closely resembles Calonectria brassicae.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 50–125 × 5–8 μm; stipe extensions septate, straight to flexuous, 190–300 μm long, 3–5 μm wide at the apical septum, terminating in clavate vesicles, 5–6 μm diam. Conidiogenous apparatus 50–115 μm long, 60–100 μm wide; primary branches aseptate, 15–30 × 5–7 μm; secondary branches aseptate, 15–25 × 4–6 μm, tertiary branches aseptate, 10–20 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–15 × 3–5 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (30–)39–42(–48) × 4–6 μm (av. = 41 × 5 μm), L/W ratio = 8.04, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies light amber, forming a rosy buff concentric ring on the surface, and ochraceous to umber in reverse; extensive aerial mycelium; sparse sporulation on the aerial mycelium; chlamydospores not seen. Colonies moderately fast growing (58–61 mm diam) on MEA, and fast growing (80–84 mm diam) on OA, after 7 d at 25 °C.

Material examined: Brazil, Pará state, Santana, Apr. 2011, A.C. Alfenas (holotype CBS H-21371, culture ex-type CBS 134662 = LPF280), CBS 134661 = LPF260.

Note: Calonectria pseudobrassicae is morphologically distinguished from Ca. brassicae and Ca. brachiatica by the number of conidiophore branches and slightly smaller macroconidia (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810011. Fig. 16.

Fig. 16

Calonectria pseudocerciana (ex-type CBS 134824). A–C. Macroconidiophores. D–F. Obpyriform to sphaeropedunculate vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 50 μm (apply to B–C); D = 10 μm (apply to E–I).

Etymology: Name refers to the fact that this fungus closely resembles Calonectria cerciana.

Sexual morph not observed. Conidiophores consists of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 40–110 × 5–7 μm; stipe extensions septate, straight to flexuous, 130–190 μm long, 2–5 μm wide at the apical septum, terminating in obpyriform to sphaeropedunculate vesicles, 7–12 μm diam. Conidiogenous apparatus 40–95 μm long, 50–90 μm wide; primary branches 0–1-septate, 20–45 × 4–7 μm; secondary branches aseptate, 13–30 × 3–6 μm, tertiary branches aseptate, 8–18 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 5–15 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (35–)43–46(–55) × 3–5 μm (av. = 45 × 4 μm), L/W ratio = 10.6, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies pale buff on the surface and cinnamon to sepia in reverse; extensive white aerial mycelium; sparse sporulation on the aerial mycelium; chlamydospores sparse, occurring throughout the medium, forming microsclerotia. Colonies fast growing (65–70 mm diam) on MEA, and (70–80 mm diam) on OA, after 7 d at 25 °C.

Material examined: Brazil, Pará state, Santana, from stem of Eucalyptus seedling, Apr. 2011, A.C. Alfenas (holotype CBS H-21366, culture ex-type CBS 134824 = LPF367), CBS 134822 = LPF365.

Note: Calonectria pseudocerciana can be distinguished from Ca. cerciana and Ca. propaginicola based on the morphology of their terminal vesicle and length/diam ratio of the macroconidia.

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810012. Fig. 17.

Fig. 17

Calonectria pseudohodgesii (ex-type CBS 134818). A–C. Macroconidiophores. D–F. Ellipsoidal to obpyriform vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 100 μm; B = 50 μm (apply to C); D = 10 μm (apply to E–F, H–I); G = 20 μm.

Etymology: Name refers to the fact that this fungus closely resembles Calonectria hodgesii.

Sexual morph not observed. Conidiophores containing a stipe bearing penicillate suites of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 35–160 × 5–8 μm; stipe extensions septate, straight to flexuous, 160–250 μm long, 2–5 μm wide at the apical septum, terminating in clavate (rarely), ellipsoidal to obpyriform vesicles, 4–10 μm diam (av. = 8 μm). Conidiogenous apparatus 50–90 μm long, 50–95 μm wide; primary branches aseptate, 20–35 × 4–7 μm; secondary branches aseptate, 15–30 × 4.5–6 μm, tertiary branches aseptate, 10–20 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 7–15 × 3–5 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (45–)53–55(–65) × (3–)4.5(–5) μm (av. = 54 × 4.5 μm), L/W ratio = 11.95, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia were not observed.

Culture characteristics: Colonies fawn to cinnamon, rosy buff at the margin on the surface, and sepia in reverse; extensive white aerial mycelium with extensive sporulation on the aerial mycelium; chlamydospores moderate to extensive occurring throughout the medium. Colonies moderately fast growing (60–65 mm diam) on MEA, and fast growing (80–85 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Minas Gerais state, Viçosa, on leaf of rooted Azadirachta indica cutting, Mar. 2011, R.F. Alfenas (holotype CBS H-21368, culture ex-type CBS 134818 = LPF262), CBS 134819 = LPF265; from stem of Eucalyptus seedling, Mar. 2011, R.F. Alfenas, CBS 134813 = LPF205, CBS 134814 = LPF206.

Note: The macroconidia of Ca. pseudohodgesii are larger than those of Ca. hodgesii and the stipe extensions of Ca. pseudohodgesii are also longer than those of Ca. hodgesii (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810013. Fig. 18.

Fig. 18

Calonectria pseudometrosideri (ex-type CBS 134845). A–C. Macroconidiophores. D–F. Ellipsoidal to obpyriform vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 50 μm (apply to B, G); D = 10 μm (apply to E–F, H–I).

Etymology: Name refers to the fact that this fungus closely resembles Calonectria metrosideri.

Sexual morph not observed. Conidiophores containing a stipe bearing penicillate suites of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 62–220 × 6–8 μm; stipe extensions septate, straight to flexuous, 160–210 μm long, 2–4 μm wide at the apical septum, terminating in ellipsoidal to obpyriform vesicles, 5–7 μm diam. Conidiogenous apparatus 30–76 μm long, 45–65 μm wide; primary branches 0(–1)-septate, 21–30 × 5–7 μm; secondary branches aseptate, 16–22 × 4–7 μm, tertiary branches aseptate, 10–17 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides elongated 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 to slightly curved, (40–)49–52(–60) × (3–)4.5(–5) μm (av. = 51 × 4.5 μm), L/W ratio = 11.34, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies cinnamon to dark brick on the surface, and sepia in reverse; moderate aerial white mycelium with moderate to extensive sporulation on the aerial mycelium, especially at the margins; chlamydospores moderate to extensive, occurring throughout the medium forming microsclerotia. Colonies slow growing (35–40 mm diam) on MEA, and moderately slow growing (45–50 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Alagoas state, Maceió, from soil collected in Eucalyptus plantation, Apr. 2011, M.M. Coutinho (holotype CBS 134845, preserved as metabolically inactive culture, culture ex-type CBS 134845 = LPF210); Maranhão, Açailândia, from leaf of Eucalyptus sp., Aug 2012, A.C. Alfenas CBS 134844 = LPF147; Minas Gerais, Viçosa, from leaf of Metrosideros polymorpha, Mar. 2012, R.F. Alfenas, CBS 134843 = LPF100.

Note: Calonectria pseudometrosideri can be distinguished from Ca. metrosideri by their larger macroconidia and longer stipe extensions (Table 3).

(Sherb.) R.F. Alfenas, L. Lombard & Crous, nom. nov. MycoBank MB810024.

Basionym: Cylindrocladium macrosporum Sheb., Phytopathology 18: 219. 1928.

Notes: Sobers (1968) synonymised Cy. macrosporum under Ca. pteridis based on morphological similarities of the asexual morphs. This was further validated in the monographic studies of Crous & Wingfeld (1994) and Crous (2002). However, to our knowledge, the ex-type strain (CBS 163.28) of Cy. macrosporum has never been subjected to DNA sequence analysis. Phylogenetic inference in this study showed that the ex-type strain of Cy. macrosporum is closely related to, but distinct from, the ex-type strain (CBS 111793) of Ca. pteridis, and is therefore reinstated here as a separate species of Calonectria. As the name Ca. macrospora is already occupied, we provide a new name for this species.

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810014. Fig. 19.

Fig. 19

Calonectria pseudospathulata (ex-type CBS 134841). A–C. Macroconidiophores. D–F. Obpyriform vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 50 μm (apply to B–C); D = 10 μm (apply to G–I); E = 10 μm (apply to F).

Etymology: Name refers to the fact that this species closely resembles Calonectria spathulata.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 45–95 × 5–8 μm; stipe extensions septate, straight to flexuous, 145–190 μm long, 2–4 μm wide at the apical septum, terminating in obpyriform vesicles, 7–10 μm diam. Conidiogenous apparatus 30–70 μm long, 65–100 μm wide; primary branches aseptate, 15–25 × 4–7 μm, secondary branches aseptate, 12–20 × 4–5 μm, tertiary branches aseptate, 10–12 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 5–10 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (35–)41–44(–50) × 3–5 μm (av. = 43 × 4 μm), L/W ratio = 10.46, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies cinnamon to dark brick on the surface and sepia to umber in reverse; extensive aerial white mycelium with moderate sporulation on the aerial mycelium; chlamydospores moderately abundant, occurring throughout the medium, forming microsclerotia. Colonies slow to moderately slow growing (40–60 mm diam) on MEA and OA, after 7 d at 25 °C.

Material examined: Brazil, Minas Gerais state, Araponga (Serra do Brigadeiro), from soil collected in tropical rainforest, Aug. 2010, A.C. Alfenas & P.W. Crous (holotype CBS H-21356, living ex-type CBS 134841 = LPF072), CBS 134840 = LPF066, CBS 134842 = LPF087.

Note: The macroconidia of Ca. pseudospathulata are smaller than those of Ca. spathulata (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810015. Fig. 20.

Fig. 20

Calonectria pseudovata (ex-type CBS 134674). A–C. Macroconidiophores. D–F. Ovate to ellipsoidal vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. J–K. Microconidiophores. L. Microconidia. Scale bars: A = 50 μm (apply to B); C = 50 μm; D = 10 μm (apply to E–F, H–I); G = 10 μm (apply to J–L).

Etymology: Name refers to the fact that the species closely resembles Calonectria ovata.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 35–105 × 5–7 μm; stipe extensions septate, straight to flexuous, 140–280 μm long, 3–6 μm wide at the apical septum, terminating in fusiform, ovate to ellipsoidal vesicles, 8–12 μm diam. Conidiogenous apparatus 55–121 μm long, 75–105 μm wide; primary branches 0–1-septate, 25–75 × 5–8 μm; secondary branches aseptate, 15–35 × 4–7 μm, tertiary branches aseptate, 15–30 × 4–6 μm, each terminal branch producing 2–6 phialides; phialides elongate doliiform to reniform, hyaline, aseptate, 10–25 × 3–5 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (55–)67–70(–80) × (4–)5(–7) μm (av. = 69 × 5 μm), L/W ratio = 13.73, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Microconidiophores comprise a stipe, a stipe extension and a penicillate or subverticillate arrangement of fertile branches. Stipe extension septate, thin-walled, terminating in an ellipsoidal to ovoid vesicle, 3–5 μm diam. Primary branches aseptate, 8–15 × 2–4 μm, secondary branches aseptate, 5–10 × 2–4 μm, terminating in 1–3 phialides; phialides elongate doliiform to reniform, straight to slightly curved, hyaline, aseptate, 7–15 × 2–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Microconidia cylindrical, straight to curved, rounded at apex, (10–)20–23(–30) × (3–)4(–6) μm (av. = 22 × 4 μm), L/W ratio = 5.38, 1-septate, held in fascicles by colourless slime. Megaconidia not observed.

Culture characteristics: Colonies ochraceous to rosy buff on the surface and umber in reverse; moderate to extensive aerial mycelium; sparse sporulation on the aerial mycelium; chlamydospores not seen. Colonies moderately fast growing (55–64 mm diam) on MEA and on OA, after 7 d at 25 °C.

Material examined: Brazil, Pará state, Santana, from soil in Eucalyptus plantation, Apr. 2011, A.C. Alfenas (holotype CBS H-21370, culture ex-type CBS 134674 = LPF267), CBS 134675 = LPF285, LPF286.

Notes: Calonectria pseudovata can be distinguished from Ca. ovata by the shape of the terminal vesicle and smaller macroconidia produced by Ca. pseudovata. The microconidia of Ca. pseudovata are also slightly smaller than those of Ca. ovata.

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810016. Fig. 21.

Fig. 21

Calonectria quinqueramosa (ex-type CBS 134654). A. Ascoma. B–C. Vertical section through ascomata, showing wall structure. D–E. Asci. F. Ascospores. G–I. Macroconidiophores. J–L. Narrowly clavate to clavate vesicles. M–N. Conidiogenous apparatus with conidiophore branches, doliiform to reniform phialides. O. Macroconidia. Scale bars: A = 500 μm; B = 100 μm (apply to C); D = 50 μm (apply to I); E = 20 μm (apply to F, J–O); G = 50 μm (apply to H).

Etymology: Name refers to the characteristic five branches formed in the conidiogenous apparatus of this fungus.

Ascomata perithecial, solitary or in groups, orange to red, becoming brown with age; in section apex and body orange to red, base red-brown, pyriform to sub-globose, 160–400 μm high, 115–250 μm diam, body turning dark red, and base dark red-brown (KOH+). Perithecial walls rough, consisting of two thick-walled layers: outside layer of textura globulosa, 25–85 μm wide; becoming more compressed towards inner layer of textura angularis, 10–30 μm wide; becoming thin-walled and hyaline towards the centre, outer layer cells 10–20 × 10–30 μm; inner cells 4–6 × 8–15 μm: perithecial base up to 135 μm wide; consisting of dark red, angular cells; merging with an erumpent stroma, cells of the outer wall layer continuing into the pseudoparenchymatous cells of the erumpent stroma. Asci 8-spored, clavate, 50–105 × 10–25 μm, tapering to a long thin stalk. Ascospores aggregated in the upper third of the ascus, hyaline, guttulate, fusoid with rounded ends, straight to curved, (1–)3-septate, slightly constricted at the septum, (25–)39–42(–50) × 5–7 μm (av. = 40 × 6 μm). Cultures were homothallic. Conidiophores consists of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 50–145 × 5–7 μm; stipe extensions septate, straight to flexuous, 170–340 μm long, 2–4 μm wide at the apical septum, terminating in narrowly clavate to clavate vesicles, 3–5 μm diam. Conidiogenous apparatus 30–60 μm long, 35–65 μm wide; primary branches aseptate, 10–35 × 3–6 μm; secondary branches aseptate, 10–30 × 3–5 μm; tertiary branches aseptate, 10–20 × 2–4 μm and additional branches (–5), aseptate, 10–15 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–18 × 2–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (45–)57–61(–70) × 4–6 μm (av. = 59 × 5 μm), L/W ratio = 11.57, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia were not observed.

Culture characteristics: Colonies umber to fawn on the surface and dark brick in reverse; sparse aerial mycelium; chlamydospores sparse, occurring throughout the medium, with moderate to extensive sporulation on the aerial mycelium. Colonies moderately fast growing (57–70 mm diam) on MEA, and fast growing (78–81 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Pará state, Monte Dourado, from soil in Eucalyptus plantation, May 2011, R.F. Alfenas (holotype CBS H-21355, culture ex-type CBS 134654 = LPF065), LPF302; Santana, from soil in Eucalyptus plantation, Apr. 2011, A.C. Alfenas, CBS 134655 = LPF281.

Note: Calonectria quinqueramosa can be distinguished from Ca. gracilis and Ca. gracilipes by the size of its ascospores and macroconidia, and by the number of fertile branches formed in the conidiogenous apparatus (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810017. Fig. 22.

Fig. 22

Calonectria robigophila (ex-type CBS 134652). A–C. Macroconidiophores. D–F. Clavate vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars: A = 50 μm; B = 50 μm (apply to C); D = 10 μm (apply to I); E = 10 μm (apply to F–H).

Etymology: Name refers to Calonectria leaf blight, the disease this fungus is associated with.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 65–120 × 5–8 μm; stipe extensions septate, straight to flexuous, 125–225 μm long, 3–4 μm wide at the apical septum, terminating in acicular to clavate vesicles, 4–5 μm diam. Conidiogenous apparatus 15–60 μm long, 30–70 μm wide; primary branches aseptate, 18–35 × 4–7 μm; secondary branches aseptate, 10–20 × 3–5 μm; tertiary branches aseptate, 10–20 × 3–5 μm and additional branches (–6), aseptate, 10–15 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 5–10 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight, (45–)49–52(–60) × 3–5 μm (av. = 50 × 4 μm), L/W ratio = 12.6, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies umber to sienna on the surface, and umber to sepia in reverse; sparse to moderate aerial mycelium with moderate to extensive sporulation on the aerial mycelium; forming sparse chlamydospores occurring throughout the medium. Colonies slow growing (34–40 mm diam) on MEA, and fast growing (70–80 mm diam) OA, after 7 d at 25 °C.

Material examined: Brazil, Maranhão state, Açailândia, on leaves of Eucalyptus sp., May 2011, R.F. Alfenas (holotype CBS H-21361, living ex-type CBS 134652 = LPF192), LPF190, CBS 134653 = LPF193.

Notes: Calonectria robigophila can be distinguished from Ca. ecuadoriae by the dimensions and septation of its macroconidia. Furthermore, Ca. robigophila formed fewer fertile branches than reported for Ca. ecuadoriae (Crous , Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810019. Fig. 23.

Fig. 23

Calonectria silvicola (ex-type CBS 135237). A–C. Macroconidiophores. D–F. Obpyriform vesicles. G–H. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. I. Macroconidia. Scale bars A = 100 μm; B = 50 μm (apply to C); D = 10 μm (apply to E–I).

Etymology: Name refers to a forest, the habitat this fungus was isolated from.

Sexual morph not observed. Conidiophores consist of a stipe bearing a penicillate arrangement of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 50–220 × 7–9 μm; stipe extensions septate, straight to flexuous, 130–195 μm long, 3–4 μm wide at the apical septum, terminating in obpyriform vesicles, 7–10 μm diam. Conidiogenous apparatus 35–90 μm long, 45–105 μm wide; primary branches aseptate, 20–30 × 3–6 μm, secondary branches aseptate, 13–26 × 3–6 μm, tertiary branches aseptate, 8–15 × 3–5 μm, each terminal branch producing 2–6 phialides; phialides doliiform to reniform, hyaline, aseptate, 6–10 × 3–4 μm; apex with minute periclinal thickening and inconspicuous collarette. Macroconidia cylindrical, rounded at both ends, straight to slightly curved, (30–)40–42(–50) × 3–5 μm (av. = 41 × 4.5 μm), L/W ratio = 9.17, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies cinnamon to dark brick on the surface and sepia in reverse; moderate aerial mycelium with extensive sporulation on the aerial mycelium, especially at the centre; chlamydospores moderate to extensive, occurring throughout the medium, forming microsclerotia. Colonies slow growing (30–40 mm diam) on MEA, and moderately slow growing (45–50 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Bahia state, Mucuri, form soil collected in tropical rainforest, Aug. 2011, E. Zauza (holotype CBS H-21357, living ex-type CBS 135237 = LPF081); Minas Gerais state, Araponga, from soil collected in tropical rainforest, Aug. 2010, A.C. Alfenas & P.W. Crous, CBS 134836 = LPF079, CPC 18741 = LPF071, CPC 18766 = LPF096.

Note: The macroconidia of Ca. silvicola are larger than those of Ca. polizzii and Ca. zuluensis, but smaller than those of Ca. mossambicensis, Ca. nemuricola and Ca. pauciramosa (Table 3).

R.F. Alfenas, L. Lombard & Crous, sp. nov. MycoBank MB810020. Fig. 24.

Fig. 24

Calonectria telluricola (ex-type CBS 134664). A–C. Macroconidiophores. D. Clavate vesicle. E. Conidiogenous apparatus with conidiophore branches and doliiform to reniform phialides. F. Macroconidia. Scale bars: A = 100 μm (apply to B); C = 20 μm; D = 10 μm (apply to E–F).

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

Sexual morph not observed. Conidiophores consist of a stipe bearing penicillate suites of fertile branches, stipe extension, and terminal vesicle; stipe septate, hyaline, smooth, 55–125 × 5–7 μm; stipe extensions septate, straight to flexuous, 100–225 μm long, 2–4 μm wide at the apical septum, terminating in clavate vesicles, 3–6 μm diam. Conidiogenous apparatus 45–95 μm long, 40–80 μm wide; primary branches aseptate, 20–30 × 5–8 μm; secondary branches aseptate, 15–30 × 4–5 μm; tertiary branches aseptate, 10–20 × 5–6 μm, and additional branches (–4), aseptate, 10–15 × 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–)40–42(–50) × 3–6 μm (av. = 41 × 5 μm), L/W ratio = 9.13, 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colourless slime. Mega- and microconidia not observed.

Culture characteristics: Colonies buff on the surface and ochraceous to umber in reverse; extensive aerial mycelium; chlamydospores not seen; sparse sporulation on the aerial mycelium. Colonies moderately fast growing (45–60 mm diam) at 25 °C on MEA, and fast growing (76–83 mm diam) on OA, after 7 d at 25 °C.

Materials examined: Brazil, Bahia state, Mucuri, from soil collected in tropical rainforest, Oct. 2011, E. Zauza (holotype CBS H–21365, culture ex-type CBS 134664 = LPF217); from soil collected in Eucalyptus plantations, Apr. 2011, E. Zauza, CBS 134667 = LPF263.

Note: Calonectria telluricola can be morphologically distinguished from other members of the Ca. brassicae complex by the length/diam ratio of its macroconidia, and number of conidiophore branches (Table 3).

Discussion

The present study represents the largest number of Calonectria isolates and species from Brazil ever subjected to DNA sequence analyses. Phylogenetic studies published on the genus Calonectria in recent years have substantially influenced its taxonomy (Lombard ). In the past five years alone, the phylogenetic species recognition concept (Taylor ) has led to the description of at least 20 additional new species of Calonectria (Lombard et al., 2010b, Lombard et al., 2010c, Chen et al., 2011, Xu et al., 2012, Alfenas et al., 2013a, Alfenas et al., 2013b). In this study, a further 20 new Calonectria species are introduced from Brazil, with the name Ca. pseudopteridis being introduced for Cy. macrosporum, which is restored to species rank based on morphological characteristics and phylogenetic inference. The introduction of these novel Calonectria species supports the view that there are many more species in this genus to be discovered, particularly from the tropics and Southern Hemisphere (Crous et al., 2006, Lombard et al., 2010c).

Mendes & Urben (2014) list 23 Calonectria and 24 Cylindrocladium species in their list of fungi known from Brazil, although this is based on the old nomenclature and associated publications. However, recent literature (Crous, 2002, Lombard et al., 2009, Lombard et al., 2010c, Alfenas et al., 2013a, Alfenas et al., 2013b) provide DNA proof for at least 25 Calonectria species from Brazil, which include Ca. avesiculata, Ca, brasiliensis, Ca. brassicae, Ca. canadensis, Ca. cylindrospora, Ca. gracilis, Ca. hederae, Ca. hodgesii, Ca. hurae, Ca. ilicicola, Ca. indusiata, Ca. insularis, Ca. kyotensis, Ca. lauri, Ca. leguminum, Ca. metrosideri, Ca. naviculata, Ca. ovata, Ca. pauciramosa, Ca. penicilloides, Ca. pteridis, Ca. pyrochroa, Ca. spathiphylli, Ca. spathulata and Ca. variabilis. Additionally, Ca. rubropunctata (Silva & Minter 1995) and Ca. meliolae (Herbário Virtual da Flora e dos Fungos – http://inct.splink.org.br) have also been reported from Brazil, but we regard these taxa as dubious, pending their re-examination.

Most of the isolates obtained from Eucalyptus leaves displaying symptoms of CLB were identified as Ca. pteridis in this study. Calonectria pteridis is regarded as the most important causal agent of CLB of Eucalyptus throughout Brazil (Alfenas et al., 2004, Graça et al., 2009, Alfenas et al., 2013c) and was first reported in Brazil by Hodges & May (1972), causing needle blight of Pinus caribaea var. hondurensis. Thereafter, this fungal pathogen has been reported on various plant hosts in Brazil, mostly associated with leaf spot diseases of these respective hosts (Silva and Souza, 1981, Dianese et al., 1986, Silva, 1996, Trindade et al., 1998, Crous, 2002). Past taxonomic studies that included Ca. pteridis (Crous and Wingfeld, 1994, Crous et al., 1997, Crous, 2002, Crous et al., 2006) concluded that this species should be regarded as a species complex based on the morphological variation observed during these studies. However, phylogenetic inference in this study failed to identify any cryptic species among the hundreds of Ca. pteridis isolates obtained during this study. Only one new taxon, Ca. pseudovata, could be identified in the Ca. pteridis species complex, which in the past also included Ca. gordoniae and Ca. ovata (Crous et al., 1997, Crous et al., 2006, Lombard et al., 2010c). Calonectria pseudovata appears to have a limited distribution, as all isolates were obtained from soil collected in a commercial Eucalyptus plantation in the state of Pará, and its pathogenicity needs to be tested experimentally. Additionally, Ca. pseudopteridis is introduced for Cy. macrosporum, based on the phylogenetic inference in this study. This species was considered synonymous with Ca. pteridis (Sobers, 1968, Crous and Wingfeld, 1994, Crous, 2002) based on morphology only, but DNA sequence analysis revealed it to be distinct from that species. However, more isolates need to be (re)collected and strains from previous studies (Renard and Viennot-Bourgin, 1973, Renard and Quillec, 1979, Ahmad and Ahmad, 1982) need to be re-evaluated to determine the distribution and host range of Ca. pseudopteridis.

Species in the Ca. brassicae complex are characterised by having clavate vesicles and small (<60 μm), 1-septate macroconidia. Species known to belong to this complex include: Ca. brachiatica, Ca. brassicae, Ca. clavata, Ca. ecuadoriae, Ca. gracilipes, Ca. gracilis, Ca. orientalis and Ca. pini (Crous et al., 2006, Lombard et al., 2009, Lombard et al., 2010c). In the study by Crous re-evaluating Calonectria species with clavate vesicles, only two new species could be resolved at that time. However, in this study, six new Calonectria species were delineated within this complex, with only Calonectria robigophila associated with CLB. The remaining species (Ca. duoramosa, Ca. paraensis, Ca. pseudobrassicae, Ca. quinqueramosa and Ca. telluricola) were all isolated from soil, and the extent of their pathogenicity to Eucalyptus still needs to be assessed. Only Ca. paraensis and Ca. telluricola were isolated from soils collected in tropical rainforests surrounding established Eucalyptus plantations. However, whether these species originated from these natural environments and were introduced into plantations through the movement of soil, still needs to be determined.

The Ca. cylindrospora complex is characterised by having 1-septate macroconidia and vesicles varying from pyriform to obpyriform or ovoid to ellipsoidal, and includes Ca. brasiliensis, C. cerciana, Ca. cylindrospora, Ca. hawksworthii, Ca. hodgesii, Ca. insularis, Ca. leucothöes, Ca. sulawesiensis and Ca. variabilis (Crous, 2002, Lombard et al., 2010c, Alfenas et al., 2013b). This complex has been extended in this study by the introduction of four new species (Ca. maranhensis, Ca. pseudocerciana, Ca. pseudohodgesii and Ca. propaginicola), based on phylogenetic inference and morphological features. Previous studies (Crous et al., 1993, Overmeyer et al., 1996, Schoch et al., 1999, Schoch et al., 2000) focussing on the taxonomy of the Ca. cylindrospora complex initially regarded these species as either Ca. cylindrospora (= Cy. scoparium) or Ca. candelabra (= Cy. candelabrum) based on their morphological similarities. However, Ca. cylindrospora has been circumscribed as having ellipsoidal to pyriform vesicles and Ca. candelabra having ellipsoidal to obpyriform vesicles (Crous ). Of the four species described in this complex, only Ca. maranhensis was isolated from soil and Eucalyptus leaves displaying CLB collected in commercial plantations. The remaining three species were all isolated from Eucalyptus seedlings displaying symptoms of damping-off, with Ca. pseudohodgesii also isolated from Azadirachta indica leaves showing CLB symptoms, collected at the same nursery in Viçosa.

The Ca. candelabra complex is characterised by having ellipsoidal to obpyriform vesicles and 1-septate macroconidia (Schoch et al., 1999, Crous, 2002, Lombard et al., 2010b). This complex includes Ca. candelabra, Ca. colombiana, Ca. metrosideri, Ca. mexicana, Ca. mossambicensis, Ca. pauciramosa, Ca. pseudoscoparia, Ca. polizzii, Ca. spathulata and Ca. zuluensis (Schoch et al., 1999, Lombard et al., 2010b Lombard et al., 2011, Crous et al., 2013, Alfenas et al., 2013a). Eight new species (Ca. brassiana, Ca. eucalypticola, Ca. glaebicola, Ca. nemuricola, Ca. paiuiensis, Ca. pseudospathulata and Ca. silvicola) are introduced in this complex from this study. Of these, Ca. eucalypticola was the only species isolated from Eucalyptus seedlings displaying symptoms of damping-off, and from soil and Eucalyptus leaves with CLB symptoms in commercial plantations. This suggests that this species could have been introduced into the plantation environment from infected seedlings supplied by the nursery. Calonectria nemuricola, Ca. pseudospathulata and Ca. silvicola were only isolated from soils collected in tropical rainforests neighbouring commercial Eucalyptus plantations, and therefore their pathogenicity on Eucalyptus still needs to be determined. Calonectria piauiensis was isolated from soils collected in both commercial plantations and neighbouring tropical rainforests, whereas Ca. brassiana was only isolated from soils collected in a commercial plantation of E. brassiana. Both Ca. glaebicola and Ca. pseudometrosideri were found in soils and on Eucalyptus leaves, with the latter also isolated from Metrosideros polymorpha. The Ca. candelabra complex represents an important pathogen complex, having been reported worldwide on numerous plant hosts, and being regarded as dominant in commercial forest nurseries (Schoch et al., 1999, Crous, 2002, Lombard et al., 2010b, Vitale et al., 2013). Members of this complex have been reported in regions where the climatic conditions differ significantly, supporting the view that these species can tolerate a wide range of environmental conditions (Crous, 2002, Lombard et al., 2010b, Lombard et al., 2010c). Calonectria multinaviculata is introduced here as a new species in the Ca. naviculata complex. This species was isolated from soil collected in commercial Eucalyptus plantations, and therefore nothing is known about its pathogenicity.

Considering the distribution and substrates from which the new species described here were collected, it is apparent that the majority of species were isolated from soils collected in commercial Eucalyptus plantations. It is still uncertain whether these soil-borne species were originally present in the soil or were introduced during the establishment of these plantations. Extensive pathogenicity trials are, however, needed to determine if these species present a potential risk to Eucalyptus plantations in Brazil. The highest species diversity found in Eucalyptus plantations appears to be in the Brazilian states of Pará, Minas Gerais and Bahia, with fewer species recorded from the states of Maranhão and Piauí, and only single species from the states of Alagoas and Tocantins. More extensive surveys are required for the remaining areas in Brazil to obtain a clearer view on the species diversity of Calonectria in Brazil.

Recently, Lombard divided the genus Calonectria into two groups, the Prolate- and Sphaero-Naviculate groups, which corresponded well with terminal vesicle morphology of the respective species. The Prolate Group includes the majority of plant pathogenic Calonectria species, which appear to have distinct biogeographic distributions. For example, the C. reteaudii complex has only been reported from Australia, China, Indonesia and New Zealand, while the C. brassicae complex has only been reported from South and Central America, now including C. orientalis which is newly reported from Brazil here. The remaining members of the Prolate Group appear to have broad geographic distributions (Schoch et al., 2001, Lombard et al., 2010c). All novel taxa treated in this study, belonged to the Prolate Group. In the Sphaero-Naviculate Group there were no obvious patterns of distribution and pathogenicity, and only vesicle morphology appeared consistent. However, the highest species diversity in this group appears to be in the Northern Hemisphere and Asia (Crous et al., 2004b, Lombard et al., 2015a, Lombard et al., 2015b).

Other than clear global distribution patterns in the Prolate- and Sphaero-Naviculate groups of Calonectria species, there also appears to be a movement of taxa from natural forests to commercial forest nurseries, and again from nurseries to commercial plantations. The next logical question would be to establish if these species are also exported along with plant material in international trade. While this could be feasible in South America, no evidence yet has been found supporting such movement of any of the new species described here to other continents. There are however ample examples of the global movement of other pathogen groups along with this host, such as Ceratocystis fimbriata (Ferreira ), Teratosphaeria nubilosa (Teratosphaeria leaf Blight; Hunter et al., 2009, Quaedvlieg et al., 2014), and Chrysoporthe cubensis (Van der Merwe ). Global trade in forest products and plant material remains a serious concern for biosecurity, as Calonectria species could represent a more serious threat when introduced into favourable climate zones, and hence could pose a serious problem for the establisment of Eucalyptus plantations elsewhere in the world.