Multi-locus phylogeny and taxonomy of Exserohilum

Abstract

Exserohilum includes a number of plant pathogenic, saprobic and clinically relevant fungi. Some of these species are of great importance in human activities, but the genus has never been revised in a phylogenetic framework. In this study, we revise Exserohilum based on available ex-type cultures from worldwide collections, observation of the holotypes and/or protologues, and additional isolates from diverse substrates and geographical origins. Based on nine nuclear loci, i.e., ITS, LSU, act, tub2, cam, gapdh, his, tef1 and rpb2, as well as phenotypic data, the genus and species boundaries are assessed for Exserohilum. Three species, i.e., E. novae-zelandiae, E. paspali and E. sorghicola, are excluded from the genus and reallocated in Sporidesmiella and Curvularia, respectively, whereas E. heteropogonicola and E. inaequale are confirmed as members of Curvularia. Exserohilum rostratum is revealed as conspecific with species previously described in Exserohilum such as E. antillanum, E. gedarefense, E. leptochloae, E. longirostratum, E. macginnisii and E. prolatum. Additionally, E. curvatum is revealed as synonym of E. holmii, and E. fusiforme of E. oryzicola. A total of 11 Exserohilum phylogenetic species are described, illustrated and discussed, including one novel taxon, E. corniculatum. The placements of 15 other doubtful species are discussed, and E. elongatum is validated.

INTRODUCTION

The genus Helminthosporium was erected by Link (1809). The type species, H. velutinum, is a saprobic dematiaceous fungus occurring on dead stems of Alnus, Cornus, Salix and numerous other plants (Voglmayr & Jaklitsch 2017). It has macronematous, rather straight conidiophores arising from stromata. Conidia are elongate, distoseptate, brown and are produced laterally from small, inconspicuous pores while the conidiophore is elongating. The production of terminal conidia usually determines the end of conidiophore growth (Luttrell 1963b, Hughes 1978, Alcorn 1988a). Originally, Helminthosporium was defined very vaguely and over the years it became a repository for numerous taxa of which only a few were congeneric with the type species. MycoBank (http://www.mycobank.org/, consulted in September 2017) lists over 760 names in Helminthosporium, but currently less than 50 are considered to represent true Helminthosporium species (Seifert et al. 2011, Tanaka et al. 2015, Voglmayr & Jaklitsch 2017). Refinements in the taxonomy of this genus resulted in the reallocation of many of its members to genera such as Alternaria, Corynespora, Dendryphion, Septonema and others (Bolle 1924, Wei 1950, Subramanian & Jain 1966, Ellis 1971, 1976, Simmons 1971, 2007, Sivanesan 1984, Seifert et al. 2011, Voglmayr & Jaklitsch 2017). Several grass parasites originally described in Helminthosporium differed from the type species in producing conidia from sympodial, often strongly geniculate conidiophores (Drechsler 1923). The conidiogenous cells of these fungi had pores which were surrounded by dark scars, in contrast to the inconspicuous small pores of H. velutinum (Alcorn 1988a). Nisikado ( 1928) classified the graminicolous Helminthosporium species into two subgenera, Cylindro-Helminthosporium and Eu-Helminthosporium. Cylindro-Helminthosporium included species with straight, cylindrical conidia that germinate from any cell, which later were accommodated in the segregate genus Drechslera by Ito ( 1930) and were often associated with the dictyosporous sexual morph Pyrenophora (Shoemaker 1961, Paul & Parbery 1968, Paul 1972). Eu-Helminthosporium grouped species with fusiform, often curved conidia which germinate from end cells. Later, Shoemaker ( 1959) erected Bipolaris for taxa previously accommodated in Eu-Helminthosporium. Bipolaris was a heterogeneous entity which included two subgroups based on differences of the hilum morphology and associated with two different sexual morphs. The group that has conidia with non- or slightly protruding hila was often associated with the sexual morph Cochliobolus, characterised by filiform ascospores that often appear more or less coiled in a helix within the ascus (Drechsler 1934, Nelson 1964, Alcorn 1983, 1996, Manamgoda et al. 2011). On the other hand, the group that has conidia with a protruding hilum had sexual morphs with fusoid ascospores enveloped in gelatinous sheaths, which was originally described in Trichometasphaeria (Luttrell 1958, 1963a, Nelson 1965). Later Leonard & Suggs ( 1974) erected Exserohilum to accommodate those Bipolaris s.lat. species with a distinctly protruding hilum, and Setosphaeria for the sexual morph. Setosphaeria differs from Trichometasphaeria by the production of non-clypeate ascomata which can be erumpent or superficial and produce larger ascospores (Leonard & Suggs 1974). Recently, Rossman et al. ( 2015) recommend to use the name Exserohilum over Setosphaeria according to Article 57.2 of the International Code of Nomenclature for algae, fungi and plants (McNeill et al. 2012). MycoBank currently lists 38 taxa in Exserohilum, most of which are associated with diseases of grasses (Sivanesan 1984, 1987), although a few have been described from other substrates such as river sediments (Sivanesan et al. 1993), soil (Guiraud et al. 1997, Steiman et al. 2000), grains (El Shafie 1980), the palm tree Borassus flabellifer (Subramanian 1956), plant debris (Castañeda-Ruiz et al. 1995), and humans (McGinnis et al. 1986, Padhye et al. 1986). Members of this genus are distinguished mainly on the basis of morphological features such as conidial shape and size, number of distosepta and the presence or absence of thick, dark distosepta (McGinnis et al. 1986, Sivanesan 1987). So far, eight Setosphaeria species have been described, most of which were obtained by mating of compatible isolates (Luttrell 1958, 1963a, Leonard & Suggs 1974, Leonard 1976, Alcorn 1978). However, homothallism has also been described in this genus (El Shafie & Webster 1981, Alcorn 1986). Successful mating is achieved by inoculating compatible strains onto culture media with sterilized fragments of natural substrates such as barley grains, maize leaf or wheat straw (Leonard & Suggs 1974, Alcorn 1978). A pre-incubation step near 5 °C, for a few months may be required before performing the mating tests in some species (Leonard 1976). Some Exserohilum s.lat. species show an atypical morphology and have been excluded from the genus by some authors. For instance, E. heteropogonicola and E. inaequale were reallocated to Curvularia as C. heteropogonicola and C. crassiseptum, respectively (Alcorn 1991, Zhang et al. 2004), and E. paspali was considered a synonym of Bipolaris micropus (Alcorn 1991). Although species of other genera might belong to Exserohilum, as previously noticed by other authors, like in the case of Helminthosporium leptochloae, which was considered similar to E. rostratum by Alcorn ( 1991), no synonymy or new combination in Exserohilum was proposed.

The type species of Exserohilum, E. turcicum, was originally described from Italy as Helminthosporium turcicum (Passerini 1876). This fungus causes northern leaf blight of corn, a widespread foliar disease characterised by oblong, straw-coloured to greyish necrotic lesions which can coalesce and cause significant death of foliar tissue. The reduction of photosynthetic leaf area can lead, in severe cases, to grain yield losses of 20–25 % (Smith et al. 1988). Exserohilum turcicum is also an important blight agent in Sorghum spp. (Bunker & Mathur 2006). Other Exserohilum species attacking economically relevant crops include E. pedicellatum (causing root rot of maize and brown lesions on wheat roots), E. prolatum (producing leaf spots on maize), and E. rostratum (associated with leaf spot and foot rot of wheat, damping off of sugarcane seedlings, leaf spot of banana, and blackening and seed germination failure in many cereals) (Sivanesan 1987, Lin et al. 2011). Many Exserohilum species attack weeds and some of them have been proposed as potential biocontrol agents, e.g., E. monoceras against Echinochloa spp. (Zhang & Watson 1997, Tosiah et al. 2011) and E. prolatum against Rottboellia cochinchinensis (Alloub et al. 2009). Presently, Exserohilum species have been reported from over 30 plant genera (Sivanesan 1987, 1992, Pachkhede 1989, Wu 1990, Sun et al. 1997, Chen et al. 2002, Lin et al. 2011, Sakoda & Tsukiboshi 2011).

Exserohilum spp. are emerging agents of opportunistic, sometimes life-threatening infections in humans. The most commonly reported species is E. rostratum, but some cases are attributed to E. longirostratum and E. macginnisii (McGinnis et al. 1986, De Hoog et al. 2000, Al-Attar et al. 2006). They have a broad clinical spectrum that includes skin infections (Hsu & Lee 1993, Lin et al. 2009), keratitis (Bouchon et al. 1994, Mathews & Maharajan 1999, Joseph et al. 2012), non-invasive allergies (Friedman et al. 1991, Torres et al. 1996) and invasive sinusitis (Lasala et al. 2005, Togitani et al. 2007, Derber et al. 2010) and disseminated infections (Bhigjee et al. 1993, Aquino et al. 1995, Levy et al. 2003). Predisposing factors include traumatisms, especially with plant material for keratitis, atopy for non-invasive sinusitis and immunosuppression for skin infections, invasive sinusitis and disseminated disease (Adler et al. 2006, Joseph et al. 2012). Recently, E. rostratum was reported as the main etiological agent in a dramatic outbreak of infections associated with contaminated glucocorticoid injections in the USA. The outbreak involved several states and there were over 749 reported cases of infection of which 31 % presented meningitis. Six percent of the infections were fatal (Kainer et al. 2012, Smith et al. 2013). A case of dermal granulomas caused by Exserohilum sp. in a bovine was reported by Whitford et al. ( 1989). Some authors have suggested that the three mentioned clinically-relevant Exserohilum species are conspecific based on analyses of sequences of the internal transcribed spacer region (ITS), the large subunit ribosomal rDNA (LSU), the actin (act) and the translation elongation factor 1-alpha (tef1) genes (Lin et al. 2011, Da Cunha et al. 2012).

Phylogenetic studies based on different loci indicated that Exserohilum belongs to the Pleosporaceae, Pleosporales (Berbee et al. 1999, Olivier et al. 2000, Zhang & Berbee 2001, Rossman et al. 2002, Kodsueb et al. 2006, Zhang et al. 2009, 2012, Amaradasa et al. 2014). However, none of these studies included more than four species of the genus, and so a reassessment of the genus is necessary to determine the evolutionary relationships of the remaining species. A revision of these fungi is also necessary to clarify the phylogenetic placement of species with atypical morphology, and to assess whether previously suggested synonymies (Alcorn 1991, Zhang et al. 2004) are correct. In this paper we present a monograph of the genus Exserohilum based on the analysis of multi-locus sequence data and the morphological study of numerous isolates and herbarium collections. A robust phylogenetic tree based on seven loci is provided, representing the main plant-pathogens and clinically-relevant species.

MATERIAL AND METHODS

Fungal isolates

The Exserohilum/Setosphaeria isolates included in this study were obtained from various substrates and countries and acquired from public culture collections, including the Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands), the Faculty of Medicine of the Universitat Rovira i Virgili (FMR; Reus, Spain) and the Queensland Plant Pathology Herbarium (BRIP; Brisbane, Australia) as listed in Table 1. Herbarium specimens were loaned from the US National Fungus Collections (BPI; Maryland, USA), BRIP, Canadian National Mycological Herbarium (DAOM; Ottawa, Canada) and the Kew Royal Botanical Gardens (IMI; Kew, England).

Table 1.

Details of isolates included in phylogenetic analyses. GenBank accession numbers in bold were newly generated in this study. New species and new combinations are indicated in bold italic.

Taxon	Old name/identified as1	Strain no.2	Other collections2	Status of the strain1 , 3	Geographical origin (country, province, locality)	Substrate	GenBank accession numbers4
ITS	LSU	act	cam	tef1	gapdh	his	tub2	rpb2
Bipolaris chloridis		CBS 242.77B	ATCC 34706; IMI 208338		Australia	Chloris gayana	HF934928	HF934869	–	–	–	HG779083	–	–	HF934830
B. cynodontis		CBS 285.51			Kenya	Cynodon transvaalensis	HF934929	HF934874	–	–	–	HG779081	–	–	HF934831
		CBS 305.64			USA	Cynodon dactylon	HF934930	HF934883	–	–	–	HG779082	–	–	HF934832
B. maydis		CBS 130.26	ATCC 22246		Unknown	Unknown	HF934923	HF934873	–	–	–	HG779084	–	–	HF934825
		CBS 136.29		PT	Japan	Zea mays	HF934926	HF934879	–	–	–	HG779086	–	–	HF934828
B. microlaenae		CBS 280.91	BRIP 15613; IT: IMI 335218	T	Australia	Microlaena stipoides	HF934933	HF934877	–	–	–	HG779092	–	–	HF934835
B. oryzae		CBS 157.50			Indonesia	Oryza sativa	HF934931	HF934870	–	–	–	HG779090	–	–	HF934833
		CBS 199.54			New Guinea	Oryza sativa	HF934932	HF934884	–	–	–	HG779091	–	–	HF934834
B. sorghicola		CBS 249.49	MUCL 9689		Unknown	Sorghum vulgare var. sudanense	HF934927	HF934868	–	–	–	HG779087	–	–	HF934829
Curvularia aeria		CBS 294.61		T	Brazil	Air	HF934910	HF934902	–	–	–	HF565450	–	–	HF934812
C. akaii		CBS 318.86			Japan	Unknown	HF934921	HF934897	–	–	–	HG779118	–	–	HF934823
		CBS 127728	IMI 309517		Japan	Themeda triandra	HF9349 20	HF934898	–	–	–	HG7791 19	–	–	HF934822
C. akaiiensis		CBS 127726		T	India	Unknown	KJ415539	KJ415494	–	–	–	KJ415407	–	–	LT852469
C. andropogonis		CBS 186.49			Indonesia	Andropogon nardus	LT631354	LT715570	–	–	–	LT715835	–	–	LT852470
C. borreriae		CBS 859.73			Chile	Volcanic ash soil	HE861848	LT715573	–	–	–	HF565455	–	–	LT852471
C. caricapapayae		CBS 135941		T	India	Carica papaya	HG778984	HG779031	–	–	–	HG779146	–	–	HG779162
C. comoriensis		CBS 110673			Unknown	Unknown	LT631357	LT715576	–	–	–	LT715841	–	–	LT852472
C. crassiseptum	E. inaequale	CBS 503.90	IT: BRIP 14583	T	Nigeria	Plant material	LT631310	LT715613	–	–	–	LT715882	–	–	LT852473
C. cymbopogonis		CBS 4 19.78			Netherlands	Yuccasp.	HG778985	HG779032	–	–	–	HG779129	–	–	HG779163
C. deightonii		CBS 537.70			Denmark	Sorghum vulgare	LT631356	LT715574	–	–	–	LT715839	–	–	LT852474
C. gladioli		CBS 210.79			Romania	Gladiolus sp.	HG778987	HG779034	–	–	–	HG779123	–	–	HG779165
C. hawaiiensis		CBS 173.57		T	Hawaii	Oryza sativa	HG778988	HG779035	–	–	–	HG779140	–	–	HG779166
C. heteropogonicola	E. heteropogonicola	CBS 128052	BRIP 14579	T	India	Heteropogon contortus	KJ415548	KJ415503	–	–	–	KJ415398	–	–	LT715769
C. heteropogonis		CBS 284.91		T	Australia	Heteropogon contortus	HF9349 19	HF934893	–	–	–	HF9349 19	–	–	HF934821
		CBS 511.91			Australia	Heteropogon contortus	HF934918	HF934894	–	–	–	HF934918	–	–	HF9348 20
C. kusanoi		CBS 137.29		ET	Japan	Eragrostis major	JN 192381	JN600993	–	–	–	LT715862	–	–	LT715733
C. lunata		CBS 730.96		T	USA	Homo sapiens	HF934911	HF934900	–	–	–	JX256429	–	–	HF934813
C. micropus	B. micropus	BRIP 6516	CBS 127234, IMI 31 2021		USA	Paspalum notatum	HE792933	LT715598	–	–	–	LT715858	–	–	LT715730
	B. micropus	CBS 127235	BRIP 65 20, IMI 31 2022	ET	USA	Paspalum notatum	HE792934	LT715599	–	–	–	LT715859	–	–	LT715731
	B. micropus	BRIP 15689a	CBS 127236		USA	Paspalum notatum	HE792935	LT715600	–	–	–	LT715860	–	–	LT715732
	E. paspali	BRIP 16070	CBS 128057	A of E. paspali	Brazil	Paspalum conjugatum	LT837854	LT715597	–	–	–	LT715857	–	–	LT715729
C. nicotiae		CBS 655.74		IT	Algeria	Desert soil	KJ909772	KM243291	–	–	–	KM083614	–	–	–
C. nodulosa		CBS 161.58		A	Unknown	Eleusine indica	–	LT715603	–	–	–	LT715863	–	–	LT715734
C. portulacae		CBS 239.48		IT	USA	Portulaca oleracea	KJ909775	LT715594	–	–	–	LT715903	–	–	–
		CBS 127241			Unknown	Unknown	LT715593	–	–	–	–	LT715855	–	–	–
C. prasadii		CBS 143.64		T	India	Jasminum sambac	HG778996	HG779043	–	–	–	HG779147	–	–	HG779174
C. spicifera		CBS 198.31			Cyprus	Capsicum annuum	HF934916	HF934905	–	–	–	HG779136	–	–	HF934818
		CBS 199.31			Cyprus	Cucurbita maxima	HF934915	HF934903	–	–	–	HG779137	–	–	HF934817
C. trifolii		CBS 173.55			USA	Trifolium repens	HG779023	HG779077	–	–	–	HG779124	–	–	HG779 208
Exserohilum corniculatum	S. rostrata	BRIP 11426	IT: IMI 167611	T	Australia	Oryza sativa	LT837453	LT883391	LT837589	LT838283	LT883558	LT883533	LT860104	LT896678	LT852480
E. holmii	S. holmii	BRIP 12679			Australia	Dactyloctenium radulnas	LT837846	LT883453	LT837678	LT852460	LT896667	LT882542	LT860 190	LT899370	LT882525
		CBS 318.64	ATCC 58 199		Unknown	Dactyloctenium aegyptium	LT837457	LT883395	LT837596	LT838290	LT883565	LT883537	LT860111	LT896686	LT852487
		CBS 3 19.64			Unknown	Dactyloctenium aegyptium	LT837458	LT715622	LT837597	LT838291	LT883566	LT715891	LT860112	LT896685	LT852488
	H. holmii	CBS 413.65	ATCC 15226	IST of H. holmii	USA	Dactyloctenium aegyptium	LT837459	LT715621	LT837598	LT838292	LT883567	LT715890	LT860113	LT896687	LT852489
		CBS 414.65	ATCC 15225, IMI 103140	A of T. holmii	USA	Dactyloctenium aegyptium	LT837460	LT883396	LT837599	LT838293	LT883568	LT883538	–	–	–
	E. curvatum	CBS 505.90	IT: IMI 281326, CBS 132712	T of E. curvatum	Venezuela	Sorghum vulgare	KT265252	LT7156 20	LT837591	LT838285	LT883560	LT715889	LT860106	LT896680	LT852482
		CBS 128053	BRIP 12792		Thailand	Dactyloctenium aegyptium	KT265253	LT883441	LT837663	LT852447	LT896652	LT882555	LT860175	LT899383	LT882513
E. khartoumensis	S. khartoumensis	IMI 249 194	CBS 132708	IT	Sudan	Sorghum bicolor var. mayo	LT837461	LT7156 19	LT837600	LT838294	LT883569	LT715888	LT882489	LT896688	LT852490
E. minor	S. minor	BRIP 14612			Australia	Ascocarps formed by BRIP 13597	LT837467	LT715615	LT837609	LT838303	LT883577	LT715884	LT860121	LT896696	LT852499
	S. minor	BRIP 14614			Australia	Dactyloctenium aegyptium	LT837468	LT715616	LT837610	LT838304	LT883578	LT715885	LT860122	LT896697	LT852500
	S. minor	BRIP 14615	IT: IMI 294530b, DAR 51591, ATCC 62323	T of S. minor	Australia	Dactyloctenium aegyptium	LT837469	LT883402	LT837611	LT838305	LT883579	LT883544	LT860123	LT896698	LT852501
	E. minor	BRIP 14616	IT: IMI 294530a, DAR 51590	T of E. minor	Australia	Dactyloctenium aegyptium	LT837470	LT883403	LT837612	LT838306	LT883580	LT883545	LT860124	LT896699	LT852502
E. monoceras	S. monoceras	BRIP 11542			Australia	Setaria italica	LT837473	LT883404	LT837615	LT838309	LT896604	LT883546	LT860127	LT896702	LT852505
	S. monoceras	BRIP 12236			Australia	Echinochloa colona	LT837472	LT715637	LT837614	LT838308	LT896603	LT715876	LT860126	LT896701	LT852504
	S. monoceras	BRIP 12271	ATCC 36561, ATCC 36562	A of S. monoceras	Australia	Echinochloa colona	LT837475	LT883406	LT837617	LT838311	LT896606	LT883548	LT860129	LT898523	LT852507
	H. monoceras; D. monocera; H. crusgalli	CBS 198.29			Japan	Echinochloa crusgalli	LT837853	LT883460	LT837686	LT852468	LT896674	LT882534	LT860 198	LT899362	LT882533
	S. monoceras	CBS 239.77			Australia	Echinochloa colona	LT837474	LT883405	LT837616	LT838310	LT896605	LT883547	LT860128	LT898522	LT852506
	S. monoceras	CBS 209.78	77163-1		Australia	Echinochloa colona	LT837471	LT715638	LT837613	LT838307	LT883581	LT715875	LT860125	LT896700	LT852503
E. neoregeliae		CBS 132832	IM 201-D	T	Japan	Neoregelia carolinae	LT837476	LT715617	LT837618	LT838312	LT896607	LT715886	LT860130	LT898517	LT852508
		CBS 132833		IT	Japan	Neoregelia carolinae	LT837477	LT715618	LT8376 19	LT838313	LT896608	LT715887	LT860131	LT898518	LT715765
E. oryzicola		CBS 502.90	T: IMI 273 194	IT	Colombia	Oryza sativa	HF934949	HF934886	LT837640	LT838323	LT896629	LT715878	LT860152	LT899345	HF934851
	E. fusiforme	CBS 376.76			Turkey	Oryza sativa	LT837456	LT883393	LT837593	LT838287	LT883562	LT883535	LT860108	LT896682	LT852484
	E. fusiforme	BRIP 16229	IT: CBS 132709, IMI 354683	T of E. fusiforme	Australia	Echinochloa crusgalli	LT837455	LT715636	LT837592	LT838286	LT883561	LT715877	LT860107	LT896681	LT852483
E. pedicellatum	H. pedicellatum; D. pedicellata	CBS 322.64	MUCL 9617	ET	USA	Triticum aestivum	KT265258	HF934889	LT837641	LT838324	LT896630	LT715902	LT860153	LT899382	HF934854
	D. pedicellata	CBS 375.76			Turkey	Oryza sativa	KT265259	HF934890	LT837642	–	LT896631	LT715879	LT860154	LT899381	HF934855
	S. pedicellata	BRIP 1 2040			Australia	Oryza sativa	LT837452	LT883390	LT837588	LT838282	LT883557	LT883532	LT860103	LT896677	LT852479
E. protrudens		BRIP 14814	IT: CBS 132710, IMI 316693	T	Australia	Dactyloctenium aegyptium	LT631308	LT715611	LT837662	LT852446	LT896651	LT715880	LT860174	LT899384	LT715741
		BRIP 14816			Australia	Dactyloctenium aegyptium	LT631309	LT715612	LT837661	LT852445	LT896650	LT715881	LT860173	LT899385	LT715728
E. rostratum	S. holmii	BRIP 10724			Australia	Eragrostis tenella	LT837828	LT883436	LT837654	–	LT896643	LT882560	LT860166	–	LT882508
	S. rostrata	BRIP 10995	IMI 170 197		Australia	Zea mays	LT837823	LT883430	LT837648	LT852437	LT896637	LT882566	LT860160	LT899375	LT882502
	S. rostrata	BRIP 11416	IMI 170188		Australia	Zea mays	LT837466	LT883401	LT837608	LT838302	LT883576	LT883543	LT8601 20	LT896695	LT852498
	S. rostrata	BRIP 11417	IMI 167610		Australia	Zea mays	LT837836	LT883443	LT837667	LT852451	LT896656	LT882553	LT860179	LT899355	LT882516
	S. rostrata	BRIP 11422			Australia	Zea mays	LT837464	LT883399	LT837606	LT838300	LT883574	LT883541	LT860118	LT896693	LT852496
	S. rostrata	BRIP 11432			Australia	Poaceae	LT837824	LT883431	LT837649	LT852438	LT896638	LT882565	LT860161	LT899374	LT882503
	S. rostrata	BRIP 1 2090			Australia	Eragrostis brownii	LT837847	LT883454	LT837679	LT852461	LT896668	LT882539	LT860 191	LT899369	LT882526
	S. rostrata	BRIP 12147			Australia	Dinebra retroflexa	LT837499	LT883426	LT837644	LT838326	LT896633	LT882570	LT860156	LT899379	LT882498
	S. rostrata	BRIP 12270			Australia	Eragrostis pilosa	LT837851	LT883458	LT837684	LT852466	LT896672	LT882536	LT860 196	LT899364	LT882531
	S. rostrata	BRIP 13560			Australia	Paspalidium distans	LT837822	LT883429	LT837647	LT838329	LT896636	LT882567	LT860159	LT899376	LT882501
	S. rostrata	BRIP 13592			Australia	Ischaemum villosum	LT837849	LT883456	LT837681	LT852463	LT896670	LT882541	LT860 193	LT899367	LT882528
	S. holmii	BRIP 13599			Australia	Dactyloctenium aegyptium	LT837844	LT883451	LT837676	LT852458	LT896665	LT882544	LT860188	LT899348	LT882523
	E. longirostratum	BRIP 14916			Australia	Zea mays	LT837835	LT883442	LT837666	LT852450	LT896655	LT882552	LT860178	LT899356	LT882515
	E. longirostratum	BRIP 15274	DNAP 1390		Australia	Areca catechu	LT837840	LT883447	LT837672	LT852454	LT896661	LT882548	LT860184	LT899352	LT8825 19
	S. rostrata	BRIP 15403			Australia	Chrysalidocarpus lutescens	LT837845	LT883452	LT837677	LT852459	LT896666	LT882543	LT860189	LT899371	LT882524
	S. rostrata	BRIP 15489			Australia	Triticum aestivum	LT837821	LT883428	LT837646	LT838328	LT896635	LT882568	LT860158	LT899377	LT882500
	E. longirostratum	BRIP 16078			Australia	Spinifex hirsutus	LT837826	LT883433	LT837651	LT852440	LT896640	LT882563	LT860163	LT899372	LT882505
	E. longirostratum	BRIP 16114			Australia	Cymbopogon citratus	LT837843	LT883450	LT837675	LT852457	LT896664	LT882545	LT860187	LT899349	LT882522
	E. longirostratum	BRIP 20144b			Australia	Megathyrsus maximus	LT837500	LT883427	LT837645	LT838327	LT896634	LT882569	LT860157	LT899378	LT882499
	S. rostrata	BRIP 28001			Australia	Sorghum sp.	LT837479	LT883408	LT837621	LT838315	LT896610	LT883550	LT860133	LT8985 20	LT852510
	S. rostrata	BRIP 29236c			Australia	Hordeum vulgare	LT837492	LT8834 19	LT837634	LT838317	LT896623	LT882577	LT860146	LT899344	LT882491
	Exserohilum sp.	BRIP 46107			Australia	Vitis vinifera	LT837827	LT883434	LT837652	LT852441	LT896641	LT882562	LT860164	LT899393	LT882506
	S. rostrata	BRIP 52639			Australia	Hordeum vulgare	LT837825	LT883432	LT837650	LT852439	LT896639	LT882564	LT860162	LT899373	LT882504
	Exserohilum sp.	BRIP 53634b			Australia	Croton sp.	LT837498	LT883425	LT837643	LT838325	LT896632	LT882571	LT860155	LT899380	LT882497
		CBS 188.68			South Africa	Unknown	LT837839	LT883446	LT837671	LT852453	LT896660	LT882549	LT860183	LT899354	LT882518
	H. leptochloae	CBS 196.29	ATCC 6700, MUCL 18 207, MUCL 9609	ST of H. leptochloae	Japan	Leptochloa chinensis	LT837462	LT715627	LT837601	LT838295	LT883570	LT715896	LT882488	LT896689	LT852491
	H. halodes; D. halodes	CBS 229.39	MUCL 18 205		South Africa	Triticum aestivum	LT837829	LT883437	LT837655	LT852443	LT896644	LT882559	LT860167	LT899391	LT882509
	H. rostratum; D. rostrata	CBS 230.39	MUCL 18214, MUCL 9692		South Africa	Triticum aestivum	LT837852	LT883459	LT837685	LT852467	LT896673	LT882535	LT860 197	LT899363	LT882532
		CBS 273.52	IMI 048842, MUCL 18221, MUCL 96 19		Zambia	Pennisetum spicatum	LT837830	LT883438	LT837656	LT852444	LT896645	LT882558	LT860168	LT899390	LT882510
	E. gedarefense	CBS 297.80		T of E. gedarefense	Sudan	Sorghum bicolor	KT265244	LT715626	LT837594	LT838288	LT883563	LT715895	LT860109	LT896683	LT852485
	S. rostrata; H. halodes; D. halodes	CBS 3 20.64			USA	Bromus inermis	LT837490	LT883417	LT837632	LT838316	LT896621	LT882579	LT860144	LT899343	LT852517
		CBS 323.64			USA	Zea mays	LT837833	LT715632	LT837664	LT852448	LT896653	LT715901	LT860176	LT899358	LT715751
	E. macginnisii	CBS 325.87	ATCC 60408, CDC B-4030, NCMH 2445	T of E. macginnisii	USA	Homo sapiens	KT265237	LT715629	LT837602	LT838296	HE664082	LT715898	LT860114	–	LT852492
	E. antillanum	CBS 412.93	FMR 4455, IMI 358615	IT of E. antillanum	Cuba	Plant debris from forest soil	KT265246	LT715625	LT837587	LT838281	LT883556	LT715894	LT860102	LT896676	LT852478
	S. rostrata	CBS 467.75	ATCC 32 198, IMI 197560, SrA3		Unknown	Unknown	LT837850	HE664026	LT837682	LT852464	HE664081	LT882538	LT860 194	LT899366	LT882529
	S. prolata	CBS 571.73	ATCC 24775, IMI 175436	A of S. prolata	USA	Zea mays	LT837831	LT715623	LT837657	–	LT896646	LT715892	LT860169	LT899389	LT715760
		CBS 572.73	ATCC 24774, IMI 175435	A of S. prolata	Guatemala	Zea mays	LT837832	LT715624	LT837658	–	LT896647	LT715893	LT860170	LT899388	LT715759
		CBS 504.90	IMI 276558		Sudan	Sorghum bicolor	KT265243	LT883394	LT837595	LT838289	LT883564	LT883536	LT860110	LT896684	LT852486
	B. australiensis; D. australiensis	CBS 705.71			India	Soil	LT837841	LT883448	LT837673	LT852455	LT896662	LT882547	LT860185	LT899351	LT8825 20
	E. rostratum	CBS 706.71			India	Soil	LT837842	LT883449	LT837674	LT852456	LT896663	LT882546	LT860186	LT899350	LT882521
	S. rostrata	CBS 732.96	AMMRL 106.9, PPCC 19686		Unknown	Zea mays	KT265240	LT715631	LT837669	LT852452	LT896658	LT715900	LT860181	LT899353	LT715752
	E. macginnisii	CBS 1 20308			Unknown	Homo sapiens	KT265236	LT883435	LT837653	LT852442	LT896642	LT882561	LT860165	LT899392	LT882507
	D. micropus	CBS 127233	DAOM 71176		USA	Leptochloa filiformis	LT837454	LT883392	LT837590	LT838284	LT883559	LT883534	LT860105	LT896679	LT852481
	E. longirostratum	CBS 128054	BRIP 21343 R.11,1,OB40		Namibia	Acacia mellifera subsp. detinens	LT837451	LT715628	LT837586	LT838280	LT883555	LT715897	LT860101	LT896675	LT852477
	E. longirostratum	CBS 128055	BRIP 21347		Namibia	Acacia mellifera subsp. detinens	LT837478	LT883407	LT8376 20	LT838314	LT896609	LT883549	LT860132	LT8985 19	LT852509
	S. rostrata, mating type A	CBS 128060	BRIP 12214, Lutt. 8686		USA	Zea mays	KT265245	LT883397	LT837604	LT838298	LT883572	LT883539	LT860116	LT896691	LT852494
	S. rostrata, mating type A	CBS 128061	BRIP 12218, Lutt. 8868		USA	Zea mays	KT265240	LT715631	LT837669	LT852452	LT896658	LT715900	LT860181	LT899353	LT715752
	S. rostrata	CBS 128062	BRIP 12224		Australia	On Barley seed on Sach's agar	KT265247	LT883457	LT837683	LT852465	LT896671	LT882537	LT860 195	LT899365	LT882530
	S. rostrata	CBS 128063	BRIP 12223, SrA10		USA	Ascospore isolate from Hay 3 × IMI 76563	KT265239	LT883398	LT837605	LT838299	LT883573	LT883540	LT860117	LT896692	LT852495
		FMR 11028	UTHSC 08-655		USA	Homo sapiens	LT837837	LT883444	LT837668	–	LT896657	LT882551	LT860180	–	–
		FMR 11271	UTHSC 05-3456		USA	Homo sapiens	LT837496	LT883423	LT837638	LT838321	LT896627	LT882573	LT860150	–	LT882495
		FMR 11278	UTHSC 06-2113		USA	Homo sapiens	LT837493	LT8834 20	LT837635	LT838318	LT896624	LT882576	LT860147	–	LT882492
		FMR 11280	UTHSC 06-3237		USA	Homo sapiens	LT837494	LT883421	LT837636	LT8383 19	LT896625	LT882575	LT860148	–	LT882493
		FMR 11286	UTHSC 07-1292		USA	Homo sapiens	LT837848	LT883455	LT837680	LT852462	LT896669	LT882540	LT860 192	LT899368	LT882527
		FMR 11372	UTHSC 07-1310		USA	Homo sapiens	LT837497	LT883424	LT837639	LT838322	LT896628	LT882572	LT860151	–	LT882496
		FMR 11390	UTHSC 08-2940		USA	Homo sapiens	LT837495	LT883422	LT837637	LT8383 20	LT896626	LT882574	LT860149	–	LT882494
		FMR 11392	UTHSC 08-3638		USA	Homo sapiens	LT837489	LT883416	LT837631	–	LT8966 20	LT882580	LT860143	–	–
		FMR 11395	UTHSC 09-131		USA	Homo sapiens	LT837491	LT883418	LT837633	–	LT896622	LT882578	LT860145	–	LT852518
		FMR 11399	UTHSC 09-1259		USA	Homo sapiens	LT837838	LT883445	LT837670	–	LT896659	LT882550	LT860182	–	LT882517
	D. longirostrata	FMR 11773	IP 1229.80		Martinique	Homo sapiens	LT837834	HE664025	LT837665	LT852449	LT896654	LT882554	LT860177	LT899357	LT882514
E. turcicum	S. turcica	BRIP 12267			Australia	Sorghum bicolor	LT837482	LT883411	LT837624	–	LT896613	LT883553	LT860136	LT899337	LT852513
	S. turcica	BRIP 13326			Australia	Sorghum sudanense	LT837480	LT883409	LT837622	–	LT896611	LT883551	LT860134	LT898521	LT852511
	S. turcica	CBS 195.26			Indonesia	Zea mays	LT837485	LT883413	LT837627	–	LT896616	LT882583	LT860139	LT899340	–
	S. turcica	CBS 384.58			USA	Single ascospore isolate from holotype of S. turcica	LT837481	LT883410	LT837623	–	LT896612	LT883552	LT860135	LT899336	LT852512
	S. turcica	CBS 330.64			USA	Zea mays	LT837484	LT715639	LT837626	–	LT896615	LT715874	LT860138	LT899339	LT852515
	S. turcica	CBS 385.58			USA	Single ascospore isolate from holotype of S. turcica	LT837488	LT715640	LT837630	–	LT8966 19	LT715873	LT860142	–	LT852516
	S. turcica	CBS 386.58	ATCC 13068, NRRL 5239		USA	Sorghum halepense	LT837486	LT883414	LT837628	–	LT896617	LT882582	LT860140	LT899341	–
	S. turcica	CBS 387.58	NRRL 5240		USA	Zea mays	LT837483	LT883412	LT837625	–	LT896614	LT883554	LT860137	LT899338	LT852514
	S. turcica	CBS 690.71		ET	Germany	Zea mays	LT837487	LT883415	LT837629	–	LT896618	LT882581	LT860141	LT899342	–
Porocercospora seminalis	Ce. seminalis	CBS 134906	CPC 21305	ET	USA	Bouteloua dactyloides	HF934942	HF934865	–	–	–	–	–	–	HF934847
	Ce. seminalis	CPC 21330			USA	Bouteloua dactyloides	HF934948	HF934863	–	–	–	–	–	–	HF934849
	Ce. seminalis	CPC 21333			USA	Bouteloua dactyloides	HF934946	HF934859	–	–	–	–	–	–	HF934850
	D. graminea; H. gramineum	CBS 280.31				Hordeum vulgare	HF934954	HF934857	–	–	–	LT715872	–	–	HF934856

¹ B: Bipolaris; C: Curvularia; Ce: Cercospora; D: Drechslera; E: Exserohilum; H: Helminthosporium; S: Setosphaeria; T: Trichometasphaeria.

² ATCC: American Type Culture Collection, Bethesda, Maryland, USA; BRIP: Queensland Plant Pathology Herbarium, Brisbane, Australia; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; FMR: Faculty of Medicine collection, Reus, Spain; DAOM: Canadian National Mycological Herbarium, Ottawa, Canada; IMI: Kew Royal Botanical Gardens, Kew, England; MUCL: Mycothèque de L'Université Catholique de Louvain, Louvain-la-Neuve, Belgium; UTHSC: Fungus Testing Laboratory, University of Texas Health Science Center, San Antonio Texas, USA.

³ ET: ex-epitype; IT: ex-isotype; IST: ex-isosyntype; NT: ex-neotype; PT: ex-paratype; ST: ex-syntype; T: ex-holotype; A: Authentic strain.

⁴ ITS: internal transcribed spacer region; LSU: large subunit ribosomal RNA gene; act: partial actin gene; tub2: partial β-tubulin gene; cam: partial calmodulin gene; gapdh: partial glyceraldehyde-3-phosphate dehydrogenase gene; his: partial histone H3 gene; tef1: partial translation elongation factor-1 alpha gene; rpb2: RNA polymerase II second largest subunit gene.

Phenotypic study and species descriptions

Colony morphology was studied mainly on synthetic nutrient-poor agar (SNA, Nirenberg 1976) supplemented with fragments of sterilized maize or banana leaves, after 7 d of incubation at 24 °C in the dark. Microscopic features were studied in clear lactic acid from colonies growing on the culture media mentioned above after 7–14 d of incubation at 24 °C under near UV light with a 12 h photoperiod. Fungal structures from herbarium material were also mounted in lactic acid, but these were gently heated with the flame of a Bunsen burner before observation if they appeared dehydrated. Size ranges of each structure in the species descriptions are derived from at least 30 measurements.

Molecular study

DNA extraction was carried out from colonies growing on MEA with the UltraClean® Microbial DNA Isolation Kit (Mo Bio Laboratories, Inc., Solana Beach, CA, USA). Amplification and sequencing of nine nuclear loci, i.e., the internal transcribed spacer (ITS) region, large subunit ribosomal RNA gene (LSU), actin (act), β-tubulin (tub2), calmodulin (cam), glyceraldehyde-3-phosphate dehydrogenase (gapdh), histone H3 (his), translation elongation factor-1 alpha (tef1) and RNA polymerase II second largest subunit (rpb2) were performed with primers V9G (De Hoog & Gerrits van den Ende 1998) + ITS4 (White et al. 1990), LR0R + LR5 (Vilgalys & Hester 1990), Act1 + Act4 (Voigt & Wöstemeyer 2000), T1 (O’Donnell & Cigelnik 1997) + Bt2b (Glass & Donaldson 1995), CAL228F + CAL737R (Carbone & Kohn 1999), gpd1 + gpd2 (Berbee et al. 1999), CYLH3F + CYLH3R (Crous et al. 2004), 983F + 2218R (Rehner & Buckley 2005) and 5F2 + 7cR (O’Donnell et al. 2007), respectively. Sequencing was performed with the BigDye terminator sequencing kit v. 3.1 (Applied Biosystems) and an ABI Prism^TM 3100 DNA sequencer (Applied Biosystems). The program SeqMan Pro (Lasergene, Madison, Wisconsin) was used to obtain consensus sequences from the complementary sequences of each isolate. BLAST queries (Altschul et al. 1990) were performed to compare sequences of the studied isolates with those of other fungi deposited in GenBank. Alignments were produced with MAFFT v. 7 (Katoh & Standley 2013), checked and refined using MEGA v. 6 (Tamura et al. 2013) and SequenceMatrix (Vaidya et al. 2011).

Two multi-locus phylogenies were analysed in order to evaluate the generic placement of the strains and to establish the phylogenetic relationship among species of Exserohilum s.str. The generic placement is based on a concatenated ITS, LSU, gapdh and rpb2 dataset including species of Exserohilum s.lat. and representatives of other helminthosporoid genera (i.e., Bipolaris, Curvularia, Johnalcornia and Porocercospora), and Pyrenophora used as outgroup. This phylogeny was constructed to assess if Exserohilum is a well-delimited genus, and to corroborate if the previous reallocation of E. heteropogonicola and E. inaequalis to Curvularia, and the synonymy of E. paspali with Bipolaris micropus are correct.

The second multi-locus phylogeny was based on a concatenated alignment of ITS, act, tub2, gapdh, his, tef1 and rpb2 and included 98 isolates of Exserohilum/Setosphaeria, excluding E. paspali. This analysis was performed to evaluate species boundaries and species groupings within Exserohilum. Individual alignments of each locus and the concatenated four- and seven-locus datasets were analysed by maximum likelihood (ML) with gamma model of rate heterogeneity using the RAxML HPC BlackBox v. 8.2.8 (Stamatakis 2014) online server of the Cipres Science gateway portal (Miller et al. 2010). The maximum likelihood search option was used to search for the best-scoring tree after bootstrapping. By default, the RAxML BlackBox calculates statistical support for branches by rapid bootstrap analyses of 1 000 replicates (Stamatakis 2014). Bootstrap support (bs) values ≥ 70 % were considered significant. Incongruence among datasets was tested by a visual inspection of all groups with ≥ 70 % bs in partial trees of each locus to search for potentially conflicting groups. A Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities from the concatenated four-locus and seven-locus datasets using MrBayes v. 3.2.6 (Ronquist et al. 2012). The best models of nucleotide substitution for each locus were determined using MrModeltest v. 2.3 (Nylander 2004) and the critical value for the topological convergence diagnostic set to 0.01. Two analyses of four MCMC chains were run from random trees, trees were sampled every 100 generations and 25 % of them were discarded as the burn-in phase. Posterior probabilities (pp) were determined from the remaining trees. The sequences generated during this study and the alignments used in the phylogenetic analyses were deposited in GenBank (Table 1) and TreeBASE (Submission 21627), respectively.

RESULTS

Molecular and phylogenetic analysis

In Exserohilum, amplification success rate varied among the different loci tested, i.e., 100 % for ITS, LSU, act, gapdh and tef1, 99 % for his, 94 % for rpb2, 87 % for tub2 and 82 % for cam. BLAST searches with the ITS sequences revealed that E. heteropogonicola and E. inaequale are in fact members of Curvularia as suggested by Sivanesan ( 1984) and Zhang et al. ( 2004). The closest hit for the ITS sequence of CBS 128057, the authentic isolate of E. paspali, were three sequences of B. micropus, i.e., GenBank accession numbers HE792933, HE792934 and HE792935 (corresponding to CBS 127234, CBS 127235, CBS 127236, respectively), all of them 99 % identical. No close hits were found for the ITS sequence of E. novaezelandiae CBS 135842 (not type strain), but its LSU was 91 % identical to Conlarium duplumascospora (GenBank accession numbers JN936991, JN936992, JN936993), a member of Annulatascaceae, Sordariomycetes (Liu et al. 2012). This clearly indicates that E. novae-zelandiae is not a member of Exserohilum and should be excluded from this genus, thus a new combination is proposed in Sporidesmiella.

The first concatenated matrix contains 3 192 nucleotide characters, i.e., 616 from gapdh, 834 from ITS, 882 from LSU and 860 from rpb2. The second concatenated alignment contains 4 736 nucleotide characters, i.e., 653 from act, 597 from gapdh, 387 from his, 794 from ITS, 860 from rpb2, 896 from tef1 and 549 from tub2. For Bayesian analysis, MrModeltest proposed a GTR + I + G model for ITS, SYM + G for act, gapdh and rpb2, HKY + G for tub2, HKY + I + G for his and GTR + I + G for tef1. These models were incorporated in the analysis. During the generation of the Bayesian seven-locus tree, a total of 49 666 trees were sampled out of the 66 222 trees generated (75 %). The consensus tree obtained from the Bayesian analysis agreed with the topology of the best-scoring ML tree for the concatenated four-locus dataset (Fig. 1). Species of Exserohilum formed a well-supported clade (100 % bs / 1 pp) clearly separated from other graminicolous helminthosporoid genera, such as Bipolaris, Curvularia, Johnalcornia, Porocercospora and Pyrenophora. Interestingly, the authentic isolate of Exserohilum paspali CBS 128057 formed a clade with three isolates of Bipolaris micropus (CBS 127234, CBS 127235 and CBS 127236), which appeared more closely related to the genus Curvularia than to Exserohilum or Bipolaris. These results are in partial concordance with a previous proposal of E. paspali as a synonym of B. micropus by Sivanesan ( 1987) based on morphology. Nevertheless, they are phylogenetically closely related to Curvularia (Fig. 1) and a new combination is proposed in the taxonomy section.

Fig. 1.

Phylogenetic tree inferred from a RAxML analysis based on a concatenated alignment of ITS, LSU, gapdh and rpb2 sequences of Exserohilum and related genera in Pleosporaceae. The bootstrap support values and Bayesian posterior probabilities are given at the nodes (MLBS/BPP). Clades with 100 % MLBS and 1 BPP are indicated by thick lines. In red font are indicated taxa previously known as Exserohilum. Ex-type, ex-isotype, ex-epitype and ex-paratype and authentic strains are indicated as ^{T, IT, ET, PT, A}, respectively. Generic types are indicated in bold. The tree was rooted to Pyrenophora graminea CBS 280.31.

The combined phylogenetic tree inferred based on seven loci (Fig. 2) revealed the existence of 11 phylogenetic species in Exserohilum, including one novel taxon, E. corniculatum, which is described in the taxonomy section. Species in Exserohilum (Fig. 2) were distributed into two major fully supported subclades. The first clade includes isolates identified as E. corniculatum, E. holmii, E. khartoumensis, E. minor, E. neoregeliae and E. rostratum, whereas the second clade included isolates of E. monoceras, E. oryzicola, E. pedicellatum, E. protrudens and E. turcicum. Most species in the first subclade show one or more accentuated septa (dark and thick), especially in polar cells. The subclade comprising most isolates includes E. rostratum and E. corniculatum. Besides the numerous isolates of E. rostratum, it also includes several isolates identified as E. longirostratum and the ex-type strains of E. antillanum, E. gedarefense, E. macginnisii, E. prolatum and Helminthosporium leptochloae. Morphological and molecular analyses suggest that those species are conspecific with E. rostratum and are treated here as synonyms in the taxonomy section. All clinical isolates included in our study belong to E. rostratum. The second subclade includes isolates of E. curvatum, E. holmii, E. khartoumensis and E. neoregeliae. Morphological and molecular analyses suggest that E. curvatum is conspecific with E. holmii and is treated here as synonym in the taxonomy section. A clade formed by four isolates of E. minor revealed mostly fusiform conidia which lack accentuated septa, and they have a homothallic sexual behaviour.

Fig. 2.

Phylogenetic tree inferred from a RAxML analysis based on a concatenated alignment of ITS, act, gapdh, his, rpb2, tef1 and tub2 sequences of Exserohilum s.str. The bootstrap support values and Bayesian posterior probabilities are given at the nodes (MLBS/BPP). Clades with 100 % MLBS and 1 BPP are indicated by thickened lines. In the E. rostratum clade in green font are indicated taxa previously identified as E. macginnisii, in blue identified as E. longirostratum, in orange as E. holmii. Ex-type, ex-isotype, ex-neotype and ex-epitype strains are indicated in bold. The tree was rooted to Bipolaris maydis CBS 136.29, B. microlaenae CBS 280.91 and Curvularia heteropogonicola CBS 128052.

Another subclade included E. fusiforme, E. monoceras, E. oryzicola and E. turcicum. Morphological and molecular analyses suggest that E. fusiforme is conspecific with E. oryzicola and is treated here as synonym in the taxonomy section. All isolates in this subclade are characterised by mostly fusiform conidia which lack accentuated septa and show heterothallic sexuality. Two subclades grouped species showing conidia with a prominent subcylindrical basal extension in their conidia, at the base of which the hilum appears. One of these subclades includes two isolates of E. protrudens and the other one includes isolates of E. pedicellatum. In the former subclade, the basal extension is pale, while in the latter subclade it is strongly pigmented.

TAXONOMY

Dothideomycetes, Pleosporales, Pleosporaceae

Exserohilum K.J. Leonard & Suggs, Mycologia 66: 290. 1974

Synonyms. Setosphaeria K.J. Leonard & Suggs, Mycologia 66: 294. 1974.

Luttrellia Khokhr. & Gornostaĭ (as ‘Lutrellia’; non Luttrellia Shearer), Vodorosli, Griby i Mkhi Dal’nego Vostoka [Algae, Fungi and Mosses of the Soviet Far-East] (Vladivostok): 80. 1978.

Type species. Exserohilum turcicum (Pass.) K.J. Leonard & Suggs.

Adapted from Sivanesan ( 1987). Vegetative hyphae septate, branched, pale brown to dark brown, smooth to finely verruculose. Asexual morph. Conidiophores macronematous, mononematous, septate, cylindrical, olivaceous brown to brown, smooth to verruculose, often geniculate above. Conidiogenous cells integrated, terminal and intercalary, sympodial, mono- to polytretic, cicatrized; conidiogenous nodes smooth to rough. Conidia fusiform, cylindrical or obclavate, straight to curved, multi-distoseptate, with a protruding hilum. Sexual morph. Ascomata superficial, immersed or erumpent, globose to ellipsoid, unilocular, dark brown to black, with or without a beak, ostiolate, with simple rigid setae over the ostiolar apex and on the upper half of the ascoma where they are often mixed with hyaline, filiform, septate hyphae; peridium composed of pseudoparenchymatous cells, dark brown and thick-walled on the outside, but with more or less hyaline cells towards the inside forming a textura angularis. Pseudoparaphyses filiform, hyaline, septate, branched, anastomosing. Asci arising from a basal cushion of thin-walled pseudoparenchymatous cells, bitunicate, 1–8-spored, cylindrical to cylindrical-clavate, short or moderately long-stalked, thick-walled, with an apical nasse and fissitunicate dehiscence. Ascospores fusoid, hyaline to pale brown, smooth, 2–6 or rarely more transversely septate, constricted at the septa, surrounded by a hyaline mucilaginous sheath which often extends some distance beyond the ends of the spore.

Exserohilum corniculatum Madrid, Hern.-Restr., Y.P. Tan & Crous, sp. nov. — MycoBank MB821483; Fig. 3

Fig. 3.

Exserohilum corniculatum (BRIP 11426 ex-type). a. Conidiophores; b–d. conidiogenous cells and conidia; e–h. conidia. — Scale bars: 10 μm.

Etymology. From the Latin corniculatum - horn-like, referring to the narrow apical extensions observed in rostrate conidia of this fungus, which resemble a horn.

Type material. Australia, Queensland, Home Hill, on leaf spot of Oryza sativa, 4 May 1972, W. Pont (BRIP 11426 holotype; BRIP 11426 culture ex-type; CBS H-21815, IMI 167611 isotypes).

On SNA + maize leaves. Vegetative hyphae septate, branched, pale olivaceous brown to dark olivaceous brown, smooth to finely verruculose, 2–7 μm wide. Conidiophores macronematous, mononematous, straight to flexuous, sometimes geniculate towards the apex, septate, unbranched, subcylindrical, brown, smooth-walled, but sometimes becoming finely verruculose near the conidiogenous loci, with cell walls usually thicker than those of the vegetative hyphae, 158–458 × 5–8 μm, with occasional subnodulose to nodulose swellings up to 9.5 μm wide. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- to polytretic, proliferating sympodially, 11–56.5 μm long, with scars up to 5.5 μm wide. Conidia mostly subcylindrical to fusiform, straight to slightly curved, pale olivaceous brown to dark olivaceous brown, smooth to irregularly verruculose, 4–10(–12)-septate, sometimes with accentuated septa delimiting the basal cell or both the basal and apical cells, often becoming rostrate by means of a narrow apical extension, 41–94.5(–104.5) × (11.5–)15–24 μm, with a strongly protruding hilum. Sexual morph not observed.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 11 mm diam after 7 d, hairy, olivaceous black towards the periphery, greenish black on maize leaves, with a fimbriate margin; reverse concolorous with obverse.

Notes — Exserohilum corniculatum is unique in producing very narrow apical conidial extensions. The rostrate conidia of E. rostratum are usually much broader and do not resemble horns as in E. corniculatum.

Exserohilum holmii (Luttr.) K.J. Leonard & Suggs, Mycologia 66: 291. 1974 — Fig. 4

Fig. 4.

Exserohilum holmii (CBS 413.65 ex-isotype (a–d), CBS 128053 (e–h), CBS 505.90 (i–l), BRIP 12679 (m–p)). a–b, e–g, i–j, m–n, p. Conidiophores and conidia; c–d, h, k–l, o. conidia. — Scale bars: a = 50 μm; e, m–o = 20 μm; b–d, f–l, p = 10 μm.

Basionym. Trichometasphaeria holmii Luttr., Phytopathology 53: 285. 1963.

Synonyms. Helminthosporium holmii Luttr., Phytopathology 53: 285. 1963.

Drechslera holmii (Luttr.) Subram. & B.L. Jain, Curr. Sci. 35: 354. 1966.

Keissleriella holmii (Luttr.) Arx, Gen. Fungi Sporul. Cult. (Lehr): 126. 1970.

Setosphaeria holmii (Luttr.) K.J. Leonard & Suggs, Mycologia 66: 295. 1974.

Exserohilum curvatum Sivan. & Muthaiyan, Trans. Brit. Mycol. Soc. 83: 3 19. 1984.

Type material. usa, Georgia, Griffin, on Hordeum vulgare, 15 Aug. 1961, E.S. Luttrell No. 7607 (BPI 623928 lectotype designated here (of Trichometasphaeria holmii, MBT3798 20)); on Dactyloctenium aegyptium, 15 Aug. 1961, E.S. Luttrell No. 1607-7 (CBS H-7027 isosyntype (of Helminthosporium holmii); CBS 413.65 culture ex-isosyntype); on Dactyloctenium aegyptium, 15 Aug. 1961, E.S. Luttrell No. 1607-5 (CBS 414.65 culture ex-isosyntype).

On SNA + maize leaves. Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth-walled, 1–6.5 μm wide. Asexual morph. Conidiophores macronematous, mononematous, straight, curved or more or less flexuous, sometimes geniculate towards the apex, septate, unbranched, subcylindrical, pale to dark olivaceous brown, becoming paler at the apex, smooth-walled, but sometimes very finely verruculose around the conidiogenous loci, with cell walls usually thicker than those of the vegetative hyphae, 57–857.5 × 5–9 μm, occasionally with subnodulose to nodulose swellings up to 9.5 μm wide. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- to polytretic, proliferating sympodially, 11–36 μm long, with scars up to 4.5 μm wide. Conidia fusoid with obtuse ends, obovoid to clavate, obclavate rostrate, straight to moderately curved, mid olivaceous brown, with a small paler area at each pole, finely asperulate, but apical cell usually smooth, 3–9-distoseptate, 38.5–117.5 × 16.5–32 μm, with a strongly protruding hilum 2–4 μm wide. Sexual morph adapted from Luttrell ( 1963a) and Sivanesan ( 1987). Ascomata globose, 262–644 μm diam, unilocular, black, covered by rigid setae on the upper part, and a ostiole surrounded by short, rigid, dark brown setae; peridium composed of an outer layer of dark brown, thick-walled, pseudoparenchymatous polyhedral cells, which become thin-walled and hyaline towards the inner wall. Pseudoparaphyses numerous, hyaline, filiform, branched, sometimes anastomosing. Asci 1–8-spored, thick-walled when young, clavate, 174–232 × 28–36 μm. Ascospores fusoid, straight to curved, hyaline, (2–)3(–6)-septate, not or slightly constricted at the septa, surrounded by a mucilaginous sheath which may extend beyond either end after discharge, 47–78 × 12– 20 μm.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 48–70 mm diam after 7 d, flat, translucent toward the periphery, hairy to powdery and greenish black at the centre and on the maize leaves, with a fimbriate margin; reverse concolorous with obverse.

Additional materials examined. Australia, Queensland, Goondiwindi, on leaf spot of Dactyloctenium radulans, 25 Apr. 1979, Y. Brouwer No. 7795b2 (BRIP 12679). – Thailand, Nakhon Pathom, on Dactyloctenium aegyptium, 19 Sept. 1990, J.L. Alcorn No. 9084b (CBS 128053). – Unknown country, on Dactyloctenium aegyptium, unknown date, R.R. Nelson (CBS 318.64); on Dactyloctenium aegyptium, unknown date, R.R. Nelson (CBS 3 19.64). – Venezuela, on seed of Sorghum vulgare, 24 Oct. 1983, M.C. Muthaiyan (culture ex-type of E. curvatum CBS 505.90).

Notes — Exserohilum holmii, originally described as Helminthosporium, was the cause of leaf blight of Dactyloctenium aegyptium in Georgia, USA (Luttrell 1963a). Luttrell ( 1963a) obtained the sexual morph in culture by mating compatible conidial strains on Sach's agar supporting sterilized barley grains. In the protologue, conidial size were longer and with more number of septa than those observed in this study (56–134 × 14–31 μm, 5–11-distoseptate vs 38.5–117.5 × 16.5–32 μm, 3–9-distoseptate). Exserohilum holmii has also been isolated from other grasses and other hosts including Coffea, Cymbopogon, Gossypium, Musa, Oryza, Psidium, Triticum, Solanum, etc. Besides the USA, E. holmii has been reported from Australia, India and Nigeria (Sivanesan 1987, Farr & Rossman 2017). Later, E. curvatum was introduced for a fungus growing on Sorghum vulgaris in Venezuela (Sivanesan 1984). It was distinguished from other species by the distinctively curved conidia (Sivanesan 1984). Nevertheless, E. curvatum appears to be a morphological variant. With the culture media and growth conditions used in our study, the conidia of this fungus were predominantly asymmetrical to slightly curved. Based on a culture on tap water agar (TWA) + wheat straw, conidia in the protologue of E. curvatum are longer (up to 1 20 μm vs 92.5 μm) and distinctly curved (Sivanesan 1984) than those observed in our study. In culture, E. curvatum is morphologically similar to E. holmii which also produces elongated conidia with end cells usually delimited by an accentuated distoseptum. Based on these morphological similarities and supported by the multi-locus sequence data analysis (Fig. 2), we consider E. curvatum as a synonym of E. holmii.

Exserohilum khartoumensis (El Shafie & J. Webster) P.M. Kirk, Index Fungorum 269: 1. 2015 — Fig. 5

Fig. 5.

Exserohilum khartoumensis (CBS 132708 ex-isotype). a–e. Sexual morph: a. habit; b. ascoma; c, d. asci; e. ascospores; f–i. asexual morph:; f. conidiophore; g. conidiophore and conidia; h–i. conidia. — Scale bars: b = 50 μm; c–d = 20 μm; e–i = 10 μm.

Basionym. Setosphaeria khartoumensis El Shafie & J. Webster, Trans. Brit. Mycol. Soc. 77: 442. 1981.

Type material. Sudan, Khartoum, on seed of Sorghum bicolor var. mayo (HME 4006 holotype, not seen; IMI 249 194 (= CBS 132708) culture ex-isotype (of Setosphaeria khartoumensis)).

On maize meal agar with autoclaved Sorghum grains. Vegetative hyphae branched, septate, pale to mid-brown. Asexual morph. Conidiophores macronematous single or in small groups, straight to flexuous, geniculate above, septate, unbranched, brown to mid-brown, paler towards the apex, smooth, up to 240 × 5–7.5 μm thick. Conidia variable, broadly obclavate-rostrate, broadly ellipsoidal to cylindrical, straight sometimes slightly curved, end cells often rather pale and often cut off by a thick, dark septum, intermediate cells mid-dark golden brown, smooth, (6–)7–10(–12)-distoseptate, 55–160 × 15–25 μm, with a distinctly protuberant hilum. Sexual morph adapted from El Shafie & Webster ( 1981) and Sivanesan ( 1987). Ascomata unilocular, globose to ellipsoid, 200–300 × 190–300 μm, sometimes with a short cylindrical ostiolate beak, surrounded by rigid setae which also occur scattered over the upper surface of the ascomata. Setae dark brown, thick-walled, septate, bluntly rounded at the end, swollen at the base, 50–180 × 5–6 μm. Pseudoparaphyses filamentous, hyaline, septate, branched and anastomosing. Asci 1–8-spored, clavate to clavate-cylindrical, bitunicate, tapered at the base, thick-walled when young, 100–155 × 25–32.5 μm. Ascospores always 3-septate, constricted at the septa, fusoid, curved to straight, hyaline to pale brown, middle cells darker than the end cells, 42–44 × 10–15 μm, surrounded by hyaline, thin mucilaginous sheath which extends beyond the end of the spore after discharge.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 52 mm diam after 7 d, hairy, with scarce aerial mycelium, translucent at the periphery, except for sparse strands of dark brown hyphae, cottony and pale mouse grey on maize leaves, with a fimbriate margin; reverse concolorous with obverse.

Notes — Exserohilum khartoumensis is a homothallic species isolated from Sorghum, and only known from the type locality, Khartoum, Sudan, (El Shafie & Webster 1981). In the protologue, both the sexual and asexual morphs were described under the name Setosphaeria khartoumensis. Recently, Kirk ( 2015) proposed the new combination in Exserohilum. In our phylogenetic tree, this species is represented by the isotype strain IMI 249 194 which forms a basal clade of E. holmii (Fig. 2).

Exserohilum minor Alcorn, Trans. Brit. Mycol. Soc. 86: 313. 1986 — Fig. 6

Fig. 6.

Exserohilum minor (IMI 294530 isotype). a–f. Sexual morph: a. ascoma; b–d. asci; e. ascospore; f. setae of the ascoma; g–i. asexual morph: g. conidiophore; h–i. conidia. — Scale bars: a = 50 μm; b–i = 10 μm.

Synonym. Setosphaeria minor Alcorn, Trans. Brit. Mycol. Soc. 86: 313. 1986.

Type material. Australia, Queensland, Saibai Island, on leaf spot of Dactyloctenium aegyptium, 1 June 1981, J.L. Alcorn (BRIP 14616 holotype; IMI 294530a isotype; BRIP 14616 culture ex-type).

Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth to asperulate, 3–8.5 μm wide. Asexual morph based on IMI 294530b. Conidiophores macronematous, mononematous, rather straight, septate, unbranched, olivaceous brown, often paler at the apex, smooth to verruculose, length indeterminate, 3.5–7.5 μm wide, sometimes with a bulbous base up to 9.5 μm wide. Conidiogenous cells integrated, intercalary and terminal, mono- or polytretic, proliferating sympodially, mostly subcylindrical to slightly swollen, 13–48.5 × 3.5–7.5 μm, with scars up to 4.5 μm wide. Conidia fusiform, straight to slightly curved, pale olivaceous brown, smooth to verruculose, 6–8-distoseptate, 63.5–86 × 12– 19.5 μm, with a strongly protruding hilum 2–3 μm wide. Sexual morph based on IMI 294530a. Ascomata amphigenous, solitary to gregarious, erumpent, unilocular, subglobose to ovoid, black, often flattened at the base, ostiolate and sometimes papillate, 232.5–343 × 157.5–344 μm, covered by rigid setae on the upper part; peridium with outer wall layer of textura angularis. Setae dark brown, septate, unbranched, smooth to asperulate, thick-walled, 27.5–123.5 × 4.5–7.5 μm, with an obtuse, often paler apex, base sometimes swollen. Pseudoparaphyses filiform, hyaline, septate, branched, anastomosing, 2–4 μm wide. Asci 8-spored, subcylindrical to clavate, with a short stalk, 79.5–144.5 × 19.5–32.5 μm. Ascospores fusoid with obtuse ends, straight to slightly curved, pale olivaceous to pale olivaceous brown, 3(–5) septate, with central cells darker than the polar ones, constricted at the septa, 35.5–51.5 × 10–14.5 μm, enveloped by a mucilaginous sheath that extends from each end as a simple tubular appendage up to 47.5 μm long (in lactic acid mounts).

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 40–50 mm diam after 7 d, flat with scarce hairy aerial mycelium and whitish at the periphery, becoming cottony and pale olivaceous grey on the maize leaves, with a fimbriate to feathery margin; reverse concolorous with obverse.

Additional materials examined. Australia, Queensland, Saibai Island, ascocarps formed by BRIP 13597 in the laboratory (no culture), Mar. 1985, J.L. Alcorn (BRIP 14612); Queensland, Saibai Island, ascocarps formed by BRIP 13597 in the laboratory (no culture), Mar. 1985, J.L. Alcorn (BRIP 14614); Queensland, Saibai Island, on leaf spot of Dactyloctenium aegyptium, 1 June 1981, J.L. Alcorn (holotype of Setosphaeria minor BRIP 14615, isotype IMI 294530b, culture ex-type BRIP 14615).

Notes — Exserohilum minor is a homothallic species known only from Australia (Alcorn 1986). It is the cause of leaf spots on Dactyloctenium aegyptium. The isolates included in the present study appear to be degenerated since none of them produced the asexual morph in culture and the ascomata showed few short setae and ascospores with abnormal morphology. Therefore, in our study the description of microscopic features are based on the isotypes of E. minor IMI 294530a and S. minor IMI 294530b. However, in the protologue conidia were longer and with more distosepta (up to 135 μm long; 5–11-distoseptate) (Alcorn 1986). Exserohilum minor was phylogenetically placed in a separate basal clade to E. corniculatum, E. holmii, E. khartoumensis, E. neoregeliae and E. rostratum.

Exserohilum monoceras (Drechsler) K.J. Leonard & Suggs, Mycologia 66: 291. 1974 — Fig. 7, 8

Fig. 7.

Original drawing of Helminthosporium monoceras (reproduced from Drechsler 1923). a. From natural substrate Echinochloa crus-galli; b. from culture.

Fig. 8.

Exserohilum monoceras CBS 198.29 (a–h), CBS 239.77 (i, j, l, m) and BRIP 11542 (k, n, o)). a, c–g, k–m. Conidiogenous cells with conidia; b, i–j. conidiogenous cells; h, n–o. conidia. — Scale bars: 10 μm.

Basionym. Helminthosporium monoceras Drechsler, J. Agric. Res. 24: 706. 1923.

Synonyms. Bipolaris monoceras (Drechsler) Shoemaker, Canad. J. Bot. 37: 883. 1959.

Drechslera monoceras (Drechsler) Subram. & B.L. Jain, Curr. Sci. 35: 354. 1966.

Setosphaeria monoceras Alcorn, Mycotaxon 7: 411. 1978.

Luttrellia monoceras (Drechsler) Khokhr., as ‘Lutrellia’, Vodorosli, Griby i Mkhi Dal’nego Vostoka [Algae, Fungi and Mosses of the Soviet Far-East] (Vladivostok): 80. 1978.

Setomelanomma monoceras (Alcorn) S.A. Ahmed et al., Persoonia 33: 144. 2014 (nom. invalid Art. 41.1).

Helminthosporium crus-galli Y. Nisik. & C. Miyake, Ber. Ohara Inst. Landw. Forsch. Kurashiki 2: 597. 1925.

Type material. USA, New York, Long Island, Port Washington, on Echinochloa crus-galli, 20 Sept. 1922, C. Drechsler (BPI 429633 holotype, not seen (of Helminthosporium monoceras)).

Asexual morph adapted from Drechsler (1923). Conidiophores macronematous, single or in groups of 2–3, straight to flexuous, sometimes geniculate above, dark brown to olivaceous, paler at the apex, 1 20–325 × 6–9 μm. Conidiogenous cells integrated, terminal and intercalary, mono- to polytretic, proliferating sympodially, mostly subcylindrical. Conidia fusoid, mainly straight, yellowish when young, becoming dark olivaceous when fully matured, smooth, 3–10-septate, 40–150 × 15–22 μm, with a protruding hilum. On SNA + maize leaves (this study). Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth, 3–6.5 μm wide. Conidiophores macronematous, mononematous, straight to flexuous, occasionally geniculate towards the apex, septate, mostly unbranched, pale to dark olivaceous brown, smooth, with cell walls often thicker than those of the vegetative hyphae, 181–743 × 4.5–10.5 μm, with occasional subnodulose intercalary swellings up to 11.5 μm wide. Conidiogenous cells terminal and intercalary, mostly subcylindrical, mono- to polytretic, proliferating sympodially, 33.5–103.5 μm long, conidiogenous loci with scars up to 6 μm wide. Conidia fusiform, straight to slightly curved, pale to dark olivaceous brown, smooth slightly verruculose near the hilum, 3–9-distoseptate, 76–1 19(–139.5) × 16–31 μm, hilum strongly protruding, 2.5–4.5 μm wide. Sexual morph adapted from Sivanesan ( 1987). Ascomata immersed, erumpent or superficial on the substrate, dark brown to black, globose to ellipsoid or ovoid, 300–500 × 260–400 μm, ostiolate, sometimes with a short broad beak, setose, especially on the upper half. Setae dark brown, unbranched, straight, paler towards the apex, septate, up to 450 μm long, 6–15 μm wide at the base which is sometimes swollen. Pseudoparaphyses filiform, hyaline, septate, branched and anastomosing. Asci 1–8-spored, cylindrical to clavate, sometimes with a short pedicel, 135–245 × 21–35 μm. Ascospores hyaline, fusoid to oblong, straight to mostly slightly curved, 2–5-(usually 3-)septate, constricted at the septa, 45–75 × 11– 20 μm, surrounded by a thin, hyaline mucilaginous sheath.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 60–92 mm diam after 7 d, hairy to cottony or floccose, whitish to iron grey on maize leaves or olivaceous black, with a fimbriate margin; reverse concolorous with obverse.

Specimens examined. Australia, Queensland, Beerwah, on Echinochloa colona, Apr. 1972, J.L. Alcorn No. 19677 (BRIP 11418, CBS 239.77); Brisbane, on leaf spot of Setaria italica, 20 Mar. 1973, B. Campion No. 200 20a (BRIP 11542); from pairing single-spore cultures 77163-1 × 77163-5 on leaf sheaths of Triticum aestivum on modified Sach's agar, 23 Feb. 1978, J.L. Alcorn No. 7804b (holotype specimen of Setosphaeria monoceras BRIP 12567); Biloela, on leaf spot of Echinochloa colona, 13 Apr. 1977, M. Vincent No. 7792a (BRIP 12236); Biloela, on leaf spot of Echinochloa colona, 30 May 1977, J.L. Alcorn No. 77163 (paratype specimen of Setosphaeria monoceras BRIP 12271, culture ex-type BRIP 12271); Biloela, on leaf spot of Echinochloa colona, 30 May 1977, J.L. Alcorn No. 77163-1 (CBS 209.78). – Japan, on Echinochloa crus-galli, Nov. 1929, Y. Nisikado (CBS 198.29).

Notes — This species, formerly introduced as Helminthosporium monoceras, was isolated from a splotch in the grass Echinochloa crus-galli in Long Island, USA (Drechsler 1923) (Fig. 7). The holotype of H. monoceras (BPI 429633) is preserved in the US National Fungus Collection. The sexual morph, S. monoceras was obtained by Alcorn ( 1978) in Australia by pairing compatible single conidial isolates in modified Sach's agar media supporting sterilized wheat leaf sheaths. Unfortunately, none of the strains of E. monoceras serves as epitype, since they were collected in Australia and Japan, very distant geographically from the type locality in the USA. The type specimens of H. monoceras and S. monoceras are different and yet to be confirmed as the same using molecular phylogenetic studies. The correct phylogenetic position of E. monoceras is still unclear until molecular data from type material of H. monoceras becomes available.

It has been reported from Dichanthelium clandestinum, Echinochloa spp., Eragrostis spp., Panicum spp., Oryza sativa and Setaria viridis (Farr & Rossman 2017). In our phylogenetic tree, E. monoceras is represented by five Australian strains, which were isolated mainly from Echinochloa, but also from Setaria, and one strain isolated from Echinochloa crus-galli in Japan. Exserohilum monoceras formed a clade together with E. turcicum (Fig. 2). These two species are also similar in conidial morphology, dimensions and septation. Nevertheless, we consider them as different species based on substrate preferences; E. monoceras is mainly isolated from Echinochloa, Panicum and Setaria, while E. turcicum is mainly isolated from Zea mays and Sorghum.

Exserohilum neoregeliae Sakoda & Tsukib., Mycotaxon 118: 214. 2011 — Fig. 9

Fig. 9.

Exserohilum neoregeliae (CBS 132832 ex-type and CBS 132833). a–b. Conidiophores; c–g. conidiophores and conidia; h. upper part of conidium; i–k. conidia. — Scale bars: 10 μm.

Type material. Japan, Chiba, Narita, from living leaves of Neoregelia carolinae (imported from the Netherlands), 24 May 2006, T. Sakoda IM 201-D (NIAESH 20605 holotype, not seen; CBS 132832 culture ex-type; NIAESH 20606 isotype, not seen; CBS 132833 culture ex-isotype).

On SNA + maize leaves. Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth to verruculose, 2.5–6.5 μm wide. Asexual morph. Conidiophores macronematous, mononematous, straight to flexuous, often strongly geniculate towards the apex, septate, almost always unbranched, pale olivaceous brown to dark brown, paler at the apex, smooth, with cell walls often thicker than those of the vegetative hyphae, 14–596 × 5.5–10 μm, often with a bulbous basal cell up to 22.5 μm wide. Conidiogenous cells integrated, terminal and intercalary, mono- to polytretic, proliferating sympodially, mostly subcylindrical, 9.5–52 μm long, conidiogenous loci with scars 2–4.5 μm wide. Conidia ellipsoidal, clavate, subcylindrical or fusiform, often appearing strongly rostrate at maturity, straight to more or less curved, pale olivaceous to dark brown, smooth to verruculose, 2–11-distoseptate, 22–161(– 191.5) × (10.5–)12–24.5(–33.5) μm, with the basal (and sometimes also the apical) cell delimited by a dark septum; hilum usually strongly protruding, 2.5–4.5 μm wide. Sexual morph not reported.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 61–72 mm diam after 7 d, flat, with scarce aerial mycelium, hairy, becoming cottony on the maize leaves, grey olivaceous to olivaceous black, with a fimbriate margin; reverse concolorous with obverse.

Notes — Exserohilum neoregeliae caused leaf spots on Neoregelia carolinae plants imported from the Netherlands to Japan (Sakoda & Tsukiboshi 2011). It has not been reported from other countries or hosts since its original description. As in other members of Exserohilum, conidial size in this species can vary greatly depending on growth conditions. Based on colonies on V8 juice agar, the protologue describes conidia much longer (up to 285 μm in length) and with more septa (6–26-distosepta) than those obtained on SNA + maize leaves in our study.

Exserohilum oryzicola Sivan., Trans. Brit. Mycol. Soc. 83: 325. 1984 — Fig. 10

Fig. 10.

Exserohilum oryzicola CBS 502.90 ex-isotype (a–j) and BRIP 16229 (k–t)). a–h, k–o. Conidiophores, conidiogenous cells with conidia; i–j, q–t. conidia; p. chlamydospore. — Scale bars: a = 50 μm; b–t = 10 μm.

Synonym. Exserohilum fusiforme Alcorn, Mycotaxon 41: 337. 1991.

Type material. Colombia, Meta, Villavicencio, on leaves of Oryza sativa, 2 Nov. 1982, E.A. Urresta (IMI 273 194 holotype; CBS 502.90 culture ex-isotype).

On SNA + maize leaves. Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth to verruculose, 2.5–7.5 μm wide. Conidiophores macronematous, mononematous, straight to flexuous, geniculate at the fertile part, septate, unbranched, pale olivaceous brown to dark brown, often paler at the apex, smooth to finely verruculose, with cell walls often thicker than those of the vegetative hyphae, 180–1436 × 4.5–8.5 μm, with subnodulose and nodulose intercalary swellings up to 11 μm wide, sometimes with a swollen basal cell up to 15.5 μm wide. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- to polytretic, proliferating sympodially, 14.5–86.5 μm long, conidiogenous loci with scars up to 5.5 μm wide. Conidia fusiform, straight to slightly curved, pale to dark olivaceous brown, smooth to finely verruculose near the hilum, 4–10-distoseptate, (41.5–) 67–179(–221) × (11–)16.5–22(–30) μm; hilum strongly protruding, 2.5–5 μm wide. Chlamydospores (only produced by isolate CBS 376.76) terminal and intercalary, ellipsoidal to subglobose, pale olivaceous brown, smooth, 7.5–22.5 μm wide. Sexual morph not reported.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 44–94 mm diam after 7 d, hairy to cottony, grey olivaceous to olivaceous black, greenish black on maize leaves, with a fimbriate margin; reverse concolorous with obverse.

Additional materials examined. Australia, Queensland, Beaudesert, on leaf of Echinochloa crus-galli, 17 Mar. 1988, J.L. Alcorn (culture ex-holotype of E. fusiforme BRIP 16229 = CBS 132709). – Turkey, Ege Region, on Oryza sativa, July 1976, J. Oktar (CBS 376.76).

Notes — Exserohilum oryzicola was described growing on leaves of Oryza sativa from Colombia, characterized by long, tapered and fusiform conidia (Sivanesan 1984). Later, a morphologically similar fungus named E. fusiforme was introduced, but was distinguished from E. oryzicola by having smaller conidia (up to 141 μm) (Alcorn 1991). Exserohilum fusiforme is pathogenic to Echinochloa crus-galli causing numerous small leaf lesions and also can produce a few small linear spots on O. sativa (Alcorn 1991). In the phylogenetic tree, E. oryzicola represented by the ex-isotype strain CBS 502.90 and E. fusiforme represented by two isolates CBS 132709 (the ex-type strain) and CBS 376.76 were placed in the same clade, representing the same phylogenetic species (Fig. 2). Since both species are morphologically similar, they are known from the same substrate O. sativa, and their phylogenetic affinities, we consider E. fusiforme conspecific with E. oryzicola.

Exserohilum pedicellatum (A.W. Henry) K.J. Leonard & Suggs, Mycologia 66: 291. 1974 — Fig. 11

Fig. 11.

Exserohilum pedicellatum (CBS 375.76 (a–i) and BRIP 1 2040 (j–p)). a–c. Conidiophores; d–g, j–n. conidiophores and conidia; h–i, o–p. conidia. — Scale bars: 10 μm.

Basionym. Helminthosporium pedicellatum A.W. Henry, Tech. Bull. Minn. Agric. Exp. Stn. 22: 42. 1924.

Synonyms: Bipolaris pedicellata (A.W. Henry) Shoemaker, Canad. J. Bot. 37: 884. 1959.

Drechslera pedicellata (A.W. Henry) Subram. & B.L. Jain, Curr. Sci 35: 354. 1966.

Trichometasphaeria pedicellata R.R. Nelson, Mycologia 57: 665. 1965.

Setosphaeria pedicellata (R.R. Nelson) K.J. Leonard & Suggs, Mycologia 66: 295. 1974.

Type material. Usa, Minnesota, St. Paul, University Farm, substrate unknown, 23 Sept. 1925, A.W. Henry (BPI 429735 lectotype of Helminthosporium pedicellatum designated here (MBT379822)); on Triticum aestivum, Sept. 1964, R.R. Nelson (CBS H-12242 epitype designated here (MBT378850); CBS 322.64 culture ex-epitype).

On SNA + maize leaves. Vegetative hyphae septate, branched, subhyaline to pale olivaceous brown, smooth, 2.5–6 μm wide. Asexual morph. Conidiophores semi- to macronematous, mononematous, usually strongly geniculate towards the apex, unbranched to branched, septate, pale olivaceous brown to dark brown, smooth to asperulate, with cell walls usually thicker than those of the vegetative hyphae, 16– 196 × 4–8 μm, with subnodulose to nodulose intercalary swellings up to 9 μm wide, swellings with conidiogenous loci. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 10–43.5 μm long, with pores up to 1 μm wide, surrounded by scars 3.5–4.5 μm wide. Conidia mostly fusiform, straight to more or less curved, occasionally sigmoid, pale olivaceous brown to dark brown, paler in the area around the hilum, smooth, 4–8-distoseptate, (37–)44–88(–89) × (18–) 19.5–29(–32) μm, with a basal pedicel-like, subcylindrical extension, 6–15.5 × 4–6 μm, often delimited by a dark septum; hilum usually strongly protruding, 2.5–3.5 μm wide. Sexual morph adapted from Sivanesan ( 1987). Ascomata black, globose to ellipsoid, 250–625 × 210–600 μm, ostiolate, non-beaked, with rigid, dark brown setae surrounding the ostiole and also on the upper surface of the ascomata. Pseudoparaphyses filiform, hyaline, branched, septate and anastomosing. Asci 1–8-spored, cylindrical to cylindrical-clavate, short pedicellate, 125–210 × 21–32 μm. Ascospores fusoid, straight to curved, hyaline, (2–)3(–6)-septate, 40–60 × 11–18 μm, surrounded by a thin, hyaline mucilaginous sheath which may extend beyond the ends of the spore after discharge.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 80 mm diam after 7 d, hairy to powdery, grey olivaceous to olivaceous black, with a fimbriate margin; reverse concolorous with obverse.

Additional materials examined. Australia, Queensland, Clare, on root of Oryza sativa, 11 Oct. 1976, M. Finlay (BRIP 1 2040). – Turkey, Ege region, on Oryza sativa, July 1976, J. Oktar (CBS 375.76).

Notes — This species was originally described as Helminthosporium pedicellatum from wheat roots in the USA (Henry 1924). It has been reported from root rots of various hosts, including Echinochloa, Oryza, Paspalum, Setaria, Sorghum, Triticum and Zea in Egypt, India, Pakistan, South Africa and the USA (Henry 1924, Sivanesan 1987, Gilbert 2002). It causes brown lesions on wheat roots and root rot of maize (Sivanesan 1987). The isolate CBS 322.64 was chosen as the epitype since it was collected in the same country and substrate as stated in the protologue. Unfortunately, CBS 322.64 no longer sporulate, so the morphology was described from CBS 375.76, which shows good sporulation, fits well with the description given in the protologue, and it is conspecific with CBS 322.64 (Fig. 2). In this species, some morphological variation among strains was observed; conidia in the strain BRIP 1 2040 were mostly clavate with rounded apex, while those of CBS 375.76 were mostly fusiform with acute apex. Exserohilum pedicellatum is easy to identify on account of its mostly broadly fusiform conidia with a basal, cylindrical, pedicel-like extension.

Exserohilum protrudens Alcorn, Trans. Brit. Mycol. Soc. 90: 146. 1988 — Fig. 12

Fig. 12.

Exserohilum protrudens (BRIP 14816). a. Conidiophore and conidia; b–i. conidia. — Scale bars: 10 μm.

Type material. Australia, Queensland, Torres Strait, Yorke Island, on leaf spot of Dactyloctenium aegyptium, 30 May 1985, R.A. Peterson (BRIP 14814 holotype; BRIP 14814 culture ex-type; IMI 316693 isotype).

Asexual morph adapted from Alcorn ( 1988b). Conidiophores macronematous, mononematous, straight to flexuous, geniculate, septate, unbranched, cylindrical, mid to dark olivaceous brown, paler towards the apex, smooth, basal cell commonly swollen, 11–21 μm wide, 6–10 μm wide near the base, 5–7.5 μm wide at the apex, up to 2 100 μm long. Conidiogenous cells terminal and intercalary, mono- and polytretic, cicatrized, verruculose and slightly swollen at the conidiogenous nodes. Conidia fusoid to obclavate-fusoid, sometimes shortly and broadly rostrate, straight or curved, (5–)7–8(–9)-distoseptate, 55–105 × 14–27 μm, smooth, olivaceous brown, concolorous except for the hilar protrusion which is paler; hilum is borne on a distinct truncate conical protrusion 2.5–5 μm long, 2.5–4 μm wide proximal to the body of the spore, and 2–3 μm wide at the hilar extremity. Sexual morph not reported.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 40–61 mm diam after 7 d, mycelium mostly immersed, with scarce aerial mycelium, whitish, becoming pale mouse grey to mouse grey and cottony on the maize leaves, with a fimbriate margin; reverse concolorous with obverse.

Additional material examined. Australia, Queensland, Coconut Island, 1 June 1985, R.A. Peterson (BRIP 14816).

Notes — This species is only known from two specimens causing leaf spot on Dactyloctenium aegyptium from the Torres Strait Islands (Yorke Island and Coconut Island). Attempts to produce the sexual morph were not successful (Alcorn 1988b). Exserohilum protrudens is phylogenetically placed in a basal clade to E. monoceras, E. oryzicola, E. pedicellatum and E. turcicum.

Exserohilum rostratum (Drechsler) K.J. Leonard & Suggs, Mycologia 66: 290. 1974 — Fig. 13, 14, 15, 16, 17

Fig. 13.

Original drawing of Helminthosporium rostratum from dry leaves of Eragrostis major collected near Washington (lectotype) (reproduced from Drechsler 1923).

Fig. 14.

Exserohilum rostratum (BPI 429032 (holotype of Helminthosporium halodes) (a–f), CBS 128061 (g–j), CBS 1 20380 (k–n)). a. Conidiophore; b–f, j, n. conidia; g–i, k–m. conidiophores and conidia. — Scale bars: a, k = 20 μm; b–j, l–n= 10 μm.

Fig. 15.

Exserohilum rostratum (CBS 325.87 (ex-type of E. macginnisii) (a–d), BRIP 11416 (e–h), CBS 128060 (i–k), BRIP 11422 (l–n), CBS 128054 (o–q), CBS 412.93 (ex-type of E. antillanum) (r–t)). a–b, r–t. Conidiophores and conidia; c. chlamydospore; d–q. conidia. — Scale bars: a–n, p–t = 10 μm; o = 40 μm.

Fig. 16.

Exserohilum rostratum (CBS 572.73 (as E. prolatum) (a–f), CBS 127233 (g–j), CBS 196.29 (ex-syntype of H. leptochloae) (k–n), CBS 3 20.64 (o–r)). a–d, g–i, k–m, o–p. Conidiophores and conidia; e–f, j, n, q–r. conidia. — Scale bars: a–j, l–r = 10 μm; k = 20 μm.

Fig. 17.

Phylogenetic tree inferred from a RAxML analysis based on rpb2 sequences of Exserohilum rostratum. The bootstrap support values are given at the nodes. In green font are indicated taxa previously identified as E. macginnisii, in blue identified as E. longirostratum, in orange as E. holmii. Ex-type, strains are indicated in bold. The tree was rooted to E. minor BRIP 14612 and BRIP 14614.

Basionym. Helminthosporium rostratum Drechsler, J. Agric. Res. 24: 724. 1923.

Synonyms: Bipolaris rostrata (Drechsler) Shoemaker, Canad. J. Bot. 37: 883. 1959.

Drechslera rostrata (Drechsler) M.J. Richardson & E.M. Fraser, Trans. Brit. Mycol. Soc. 51: 148. 1968.

Luttrellia rostrata (Drechsler) Gornostaĭ, as ‘Lutrellia’, Vodorosli, Griby i Mkhi Dal’nego Vostoka [Algae, Fungi and Mosses of the Soviet Far-East] (Vladivostok): 81. 1978.

Helminthosporium halodes Drechsler, J. Agric. Res. 24 (8): 709. 1923.

Helminthosporium halodes Drechsler var. tritici Mitra, Trans. Brit. Mycol. Soc. 15 (3-4): 287. 1931.

Helminthosporium halodes Drechsler var. elaeicola Kovachich, Trans. Brit. Mycol. Soc. 37 (4): 423. 1954.

Bipolaris halodes (Drechsler) Shoemaker, Canad. J. Bot. 37: 883. 1959.

Drechslera halodes (Drechsler) Subram. & B.L. Jain, Curr. Sci. 35: 354. 1966.

Drechslera halodes (Drechsler) Subram. & B.L. Jain var. halodes (Drechsler) Subram. & B.L. Jain, Curr. Sci. 35: 354. 1966.

Drechslera halodes (Drechsler) Subram. & B.L. Jain var. elaeicola Kovachich, Trans. Brit. Mycol. Soc. 37: 423. 1954.

Exserohilum halodes (Drechsler) K.J. Leonard & Suggs, Mycologia 66: 290. 1974.

Helminthosporium leptochloae Y. Nisik. & C. Miyake, Ber. Ohara Inst. Landw. Forsch. Kurashiki 2: 483. 1924.

Helminthosporium longirostratum Subram., J. Indian Bot. Soc. 35: 463. 1957.

Exserohilum longirostratum (Subram.) Sivan., Trans. Brit. Mycol. Soc. 83 (2): 328. 1984.

Exserohilum prolatum K.J. Leonard & Suggs, Mycologia 66: 290. 1974.

Setosphaeria prolata K.J. Leonard & Suggs, Mycologia 66: 294. 1974.

Setosphaeria rostrata K.J. Leonard, Mycologia 68: 409. 1976.

Exserohilum gedarefense (El Shafie) Alcorn, as ‘gedarefensis’, Mycotaxon 17: 68. 1983.

Exserohilum macginnisii A.A. Padhye & Ajello, as ‘mcginnisii’, J. Clin. Microbiol. 24: 247. 1986.

Exserohilum antillanum R.F. Castañeda, Guarro & Cano, Mycol. Res. 99: 825. 1995.

Setomelanomma rostrata Green et al., Allergy, Asthma & Clinical Immunology 10: 3. 2014 (nom. invalid Art. 41.1).

Type material. USA, Washington DC, on dry leaves of Eragrostis major, Sept. 1921, C. Drechsler (BPI 430144 holotype (of Helminthosporium rostratum)).

Adapted from Leonard ( 1976). Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth to verruculose, 2.5–8 μm wide. Asexual morph. Conidiophores macronematous, mononematous, straight to flexuous, geniculate towards the apex, septate, unbranched, subcylindrical, pale olivaceous brown to dark olivaceous brown, with cell walls usually thicker than those of the vegetative hyphae, smooth, becoming finely verruculose around the conidiogenous loci, 65–395.5 × 4–7.5 μm, base up to 9 μm wide. Conidiogenous cells integrated, terminal and intercalary, mono- to polytretic, proliferating sympodially, irregularly pigmented, mostly subcylindrical, 5.5–27.5 μm long, pores surrounded by scars up to 3–4.5 μm wide. Conidia ellipsoid, clavate, obclavate or subcylindrical, rostrate or not, straight to moderately curved, pale olivaceous brown to dark olivaceous brown, basal and apical cells often delimited by a dark septum, smooth to verruculose, 1–15-distoseptate, 15– 190 × 7–29 μm, hilum 3–4 μm wide. Sexual morph. Ascomata superficial, globose to ellipsoid, black, 340–600 × 330–580 μm, ostiolate, sometimes with a short, cylindrical, beak, with rigid, dark brown, septate setae surrounding the ostiole, and the upper surface of the ascomata. Pseudoparaphyses filiform, hyaline, septate, branched and anastomosing. Asci 1–8-spored, clavate to cylindrical-clavate, short stalked, 105–260 × 26–42 μm. Ascospores hyaline to pale brown, fusoid, straight to curved, (2–)3(–5)-septate, constricted at the septa, 29–85 × 9–21 μm, surrounded by a thin, hyaline mucilaginous sheath which may extend beyond the ends of the spore after discharge.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 64–108 mm diam after 7 d, hairy, grey olivaceous to olivaceous black, with patches of cottony whitish to olivaceous grey mycelium on maize leaves and a fimbriate margin; reverse concolorous with obverse.

Additional materials examined. Australia, New South Wales, Broken Head, on leaf spot of Spinifex hirsutus, 6 Jan. 1988, J.L. Alcorn (BRIP 16078); Leadville, on Hordeum vulgare, 19 July 2002, G. Platz (BRIP 29236c); Sydney, on Sorghum sp., 1998, F. Benyon (BRIP 28001); Northern Territory, Howard Springs, on leaf spot of Areca catechu, 28 July 1986, M. Connelly (BRIP 15274); Queensland, Atherton, on stalk rot of Zea mays, 9 Apr. 1985, M.D. Ramsey (BRIP 14916); Bamaga, on leaf spot of Eragrostis tenella, 29 May 1981, J.L. Alcorn (BRIP 10724); Biloela, on leaf lesion of Zea mays, 12 Apr. 1972, N.T. Vock (BRIP 11416); Brisbane, on leaf blight of Cymbopogon citratus, 22 Feb. 1988, J.L. Alcorn (BRIP 16114); on leaf spot of Croton sp., 7 June 2010, L.I. Forsberg (BRIP 53634b); from pairing single-spore cultures BRIP 122 20 (USA, North Carolina, from Zea mays, 14 Jan. 1977, K.J. Leonard SrA3) × BRIP 11761 (Kingaroy, from stalk rot of Zea mays, 13 Apr. 1976, P.E. Mayers) on Barley seeds on Sach's agar, 22 Mar. 1997, J.L. Alcorn (BRIP 12224, culture CBS 128062); Gatton, on crown rot of Triticum aestivum, Oct. 1986, R.L. Dodman (BRIP 15489); Goomeri, on leaf spot of Zea mays, 26 Apr. 1972, C. Euler (BRIP 11422); Gympie, on leaf spot of Megathyrsus maximus, 6 July 1992, D.S. Loch (BRIP 20144b); Lawes, on leaf spot of Dinebra retroflexa, 4 Apr. 1977, J.L. Alcorn (BRIP 12147); Mundubbera, on leaf spot of Vitis vinifera, 11 Feb. 2005, C.M. Horlock and P. Jackson (BRIP 46107); Nebo, on undetermined Poaceae, 6 Sept. 1972, unknown collector (BRIP 11432); Norwin, on leaf spot of Zea mays, 28 Mar. 1972, K.M. Middleton (BRIP 10995); Parada, on ear rot of Zea mays, 13 Apr. 1972, W. Pont (BRIP 11417); Peregian Beach, on leaf spot of Paspalidium distans, 7 Mar. 1982, J.L. Alcorn No. 8230b (BRIP 13560); Rockhampton, on leaf spot of Chrysalidocarpus lutescens, 23 July 1986, unknown collector (BRIP 15403); Sabai Island, on leaf spot of Eragrostis brownii, 28 Feb. 1977, J.L. Alcorn No. 7728 (BRIP 1 2090); Sabai Island, on leaf spot of Ischaemum villosum, 1 June 1982, J.L. Alcorn No. 8 194a (BRIP 13592); Saibai Island, on leaf of Dactyloctenium aegyptium, 1 June 1981, J.L. Alcorn (BRIP 13599); Samford, on leaf spot of Eragrostis pilosa, 25 May 1977, J.L. Alcorn No. 77162 (BRIP 12270); Warwick, on Hordeum vulgare, 22 June 2009, K. Stephen (BRIP 52639). – Cuba, Ciudad de la Habana, Santiago de las Vegas, on plant debris from forest soil, 10 Feb. 1993, R.F. Castañeda-Ruiz (culture ex-isotype of E. antillanum CBS 412.93). – Guatemala, on seed of Zea mays, Feb. 1972, unknown collector, No. Ep1 (authentic mating type ‘minus’ of Setosphaeria prolata CBS 572.73). – India, Poona, from soil, unknown date, P.G. Patwardhan (CBS 705.71, CBS 706.71). – Japan, Okayama, Kurashiki, on Leptochloa chinensis, Oct. 19 19, Y. Nisikado (syntype of H. leptochloae CBS H-72 20; culture ex-syntype of H. leptochloae CBS 196.29). – Martinique, on heart valve prosthesis of Homo sapiens, 1980, unknown collector (FMR 11773). – Namibia, Windhoek, endophytic on 5-mo-old seedlings of Acacia mellifera subsp. detinens, Apr. 1993, G. Holz (CBS 128054); Windhoek, endophytic on 5-mo-old seedlings of Acacia mellifera subsp. detinens, Apr. 1993, G. Holz (CBS 128055). – South Africa, Gauteng Province, Johannesburg, unknown substrate, unknown date, P. Martin No. 1323 (CBS 188.68); Free State Province, Bethlehem, on Triticum aestivum, Apr. 1939, K. Putterill No 30456 (CBS 230.39); Gauteng Province, on Triticum aestivum, Apr. 1939, K. Putterill No. 30434 (CBS 229.39). – Sudan, on grains of Sorghum bicolor, Apr. 1979, A.E. El Shafie (culture ex-type of E. gedarefense CBS 297.80); 21 Mar. 1983, J.L. Alcorn (as E. gedarefense CBS 504.90). – Unknown country, on Zea mays, unknown date, J.L. Alcorn (CBS 732.96); from nasal mucosa of 45-yr-old male with acute myelogenous leukemia, unknown date, dep. I. Polacheck (as E. macginnisii CBS 1 20308); unknown substrate, unknown date, unknown collector (CBS 467.75 = ATCC 32 198). – Usa, Florida, Gainesville, on Zea mays, 27 May 1972, E.S. Luttrell 8868 ‘mating type A’ (CBS 128061); Georgia, Decatur County, on Zea mays, 8 June 1971, E.S. Luttrell 8686 ‘mating type A’ (CBS 128060); Georgia, from eye (wood splinter) of Homo sapiens, 2009, S.A. Sutton (FMR 11399); Mississippi, State College, on leaf of Zea mays, Oct. 1971, M.C. Futrell No. Ep3 (authentic mating type ‘plus’ of Setosphaeria prolata CBS 571.73); Montana, from maxillary sinus of Homo sapiens, 2009, S.A. Sutton (FMR 11395); New York, Douglaston, on Distichlis spicata, 26 Sept. 19 20, C. Drechsler (holotype of Helminthosporium halodes BPI 429032); North Carolina, ascospore isolate from Hay3 × IMI 76563, 14 Jan. 1977, K.J. Leonard (CBS 128063); North Carolina, on barley grains on Sach's agar by mating compatible isolates Ep1 × Ep3, 1974, K.J. Leonard (holotype specimen of Setosphaeria prolata BPI 622161); Oklahoma, Stillwater, on Leptochloa filiformis, 29 Aug. 1960, R.A. Shoemaker (CBS 127233); Texas, on elbow tissue of Homo sapiens, 2008, D.A. Sutton (FMR 11028); sinus, 2005, D.A. Sutton (FMR 11271); from cornea of Homo sapiens, 2006, D.A. Sutton (FMR 11278); great toe, 2006, D.A. Sutton (FMR 11280); shin skin of Homo sapiens, 2007, D.A. Sutton (FMR 11286); nasal inferior turbinate, 2008, D.A. Sutton (FMR 11390); unknown tissue of Homo sapiens, 2007, D.A. Sutton (FMR 11372); Tucson, Arizona, from nasal polyp from Homo sapiens, unknown date, A.A. Padhye (culture ex-type of E. macginnisii CBS 325.87); unknown state, on Bromus inermis, unknown date, R.R. Nelson (CBS 3 20.64); unknown state, on Zea mays, unknown date, R.R. Nelson No. 26 (CBS 323.64); Utah, maxillary sinus of Homo sapiens, 2008, D.A. Sutton (FMR 11392). – Zambia, on seed of Pennisetum spicatum, 1951, W.E. Kerr, No G. 32 (CBS 273.52).

Notes — Exserohilum rostratum is by far the most commonly recorded and known species of the genus. This cosmopolitan species has been recorded from numerous hosts, especially Poaceae and other monocots, causing leaf spot and foot rot of wheat and other grasses, blight, damping-off, rots including leaf spot of banana (Sivanesan 1987, Farr & Rossman 2017). Clinical human reports of this species have also been increasing (Aquino et al. 1995, Adler et al. 2006, Al-Attar et al. 2006, Derber et al. 2010, Da Cunha et al. 2012, Kainer et al. 2012, Smith et al. 2013).

Exserohilum rostratum was first described as Helminthosporium rostratum, isolated from Eragrostis major in the USA (Drechsler 1923). The holotype, BPI 430144, is preserved at the US National Fungus Collection. Unfortunately, no culture is linked to the holotype. Among the specimens examined in our study, none of them were suitable to serve as an epitype, since there are no isolates from E. major from the USA. In the protologue, Drechsler ( 1923) described ellipsoidal conidia with 3–9 septa and rostrate conidia with 8–15 septa, measuring 32–184 × 14–22 μm (Fig. 13). Leonard ( 1976) noticed the wide morphological variability of this species when he introduced the synonymy of E. halodes (conidial size after emendation 15– 190 × 7–29, 1–15-septate). He pointed out that isolates of E. rostratum that originally produce strongly rostrate conidia may lose that characteristic in culture. Another factor that influences the conidial morphology was the light. Isolates that were exposed to light formed strongly rostrate conidia, but in the dark they formed only ellipsoidal conidia (Leonard 1976). During our study, conidial morphology in the specimens examined was also highly variable (Fig. 14, 15, 16). Furthermore, phylogenetic analysis based on multi-locus data show that E. antillanum, E. gedarefense, E. longirostratum, E. macginnisii, E. prolatum and Helminthosporium leptochloae are conspecific to E. rostratum, and therefore they are listed here as synonyms (Fig. 1).

In E. rostratum, 14 lineages were discerned with the individual analysis of the most informative gene, rpb2 (Fig. 17). The clinical isolates were mainly distributed in three lineages, i.e., ‘rostratum’, ‘macginnisii’ and lineage 12, except for the isolate FMR 11286 which formed an independent lineage. The lineage ‘rostratum’ includes 26 strains isolated mainly from monocotyledon plants (Fig. 17, Table 1) but also include four clinical isolates (FMR 11773, FMR 11028, FMR 11399 and CBS 1 20308) from Australia, USA, South Africa, India and Zambia. The lineage ‘macginnisii’ includes CBS 325.87 (the ex-type of E. macginnisii) isolated from a clinical sample, CBS 128060 (the mating type A used by Luttrell to produce the sexual morph), CBS 128063 (ascospore isolate from crossing: Hay 3 (R.R. Nelson) × IMI 76563), and three isolates from Zea mays CBS 732.96, BRIP 11422 and BRIP 11416 from Australia and the USA. The lineage 12 includes mostly clinical isolates from the USA (FMR 11390, FMR 11287, FMR 11271, FMR 11280, FMR 11372, FMR 11395, FMR 11392) and two isolates from plants BRIP 29236 (Hordeum vulgare, Australia) and CBS 3 20.64 (Bromus inernis, USA). The lineage ‘prolatum’ includes three isolates, two of them CBS 571.73 and CBS 572.73, isolated from Zea mays in the USA and Guatemala, respectively, and CBS 127233 deposited as ‘Drechslera micropus’ isolated from Leptochloa filiformis in the USA. The lineage ‘gedarefense’ includes two isolates identified as E. gedarefense including the type strain CBS 297.80 and CBS 504.90, both isolated from Sorghum bicolor in Sudan. The lineage ‘antillanum’ includes CBS 412.93 (ex-type strain of E. antillanum) isolated from plant debris in Cuba. The clade ‘leptochloae’ includes CBS 196.29 (ex-type strain of H. leptochloae) isolated from Leptochloae chinensis in Japan. Other lineages were formed by individual strains CBS 230.39, BRIP 13592, BRIP 1 2090, CBS 128054, and BRIP 12270. Interestingly, the isolate CBS 128062 (= BRIP 12224) which was the offspring of a cross among BRIP 122 20 (USA, North Carolina, from Zea mays, 14 Jan. 1977, K.J. Leonard SrA3) × BRIP 11761 (Kingaroy, from stalk rot of Zea mays, 13 Apr. 1976, P.E. Mayers) formed a basal lineage together with CBS 467.75 (= ATCC 32 198 = IMI 197560), which is labelled at the ATCC database as SrA3. However, with the information available in the CBS and IMI database we cannot corroborate this data.

Exserohilum turcicum (Pass.) K.J. Leonard & Suggs, Mycologia 66: 291. 1974 — Fig. 18

Fig. 18.

Exserohilum turcicum (BPI 431157 holotype (a–j), CBS 690.71 ex-epitype (k–s)). a–e. Conidiophores and conidiogenous cells; f–j. conidia; k–s. conidiophores and conidia. — Scale bars: a–j = 10 μm; k–s = 20 μm.

Basionym. Helminthosporium turcicum Pass., Boln Comiz. Agr. Parmense 10: 3. 1876.

Synonyms. Bipolaris turcica (Pass.) Shoemaker, as ‘turcicum’, Canad. J. Bot. 37: 884. 1959.

Drechslera turcica (Pass.) Subram. & B.L. Jain, Curr. Sci. 35: 355. 1966.

Luttrellia turcica (Pass.) Khokhr., as ‘Lutrellia’, Vodorosli, Griby i Mkhi Dal’nego Vostoka [Algae, Fungi and Mosses of the Soviet Far-East] (Vladivostok): 81. 1978.

Trichometasphaeria turcica Luttr., Phytopathology 48: 282. 1958.

Keissleriella turcica (Luttr.) Arx, Gen. Fungi Sporul. Cult. (Lehr): 126. 1970.

Setosphaeria turcica (Luttr.) K.J. Leonard & Suggs, Mycologia 66: 295. 1974.

Helminthosporium inconspicuum Cooke & Ellis, Grevillea 6 (no. 39): 88. 1878.

Type material. Italy, Parma, on Zea mays, date unknown, G. Passerini (BPI 431157 lectotype designated here (of Helminthosporium turcicum MBT379823)). – Germany, Lower Saxony, Einbeck, on Zea mays, unknown date, D. Heitmann No. W64A (CBS H-23323 epitype designated here, MBT378854; CBS 690.71 culture ex-epitype).

On Zea mays leaves (BPI 431157). Vegetative hyphae mostly immersed, septate, branched, pale olivaceous to pale olivaceous brown, smooth, 3–7.5 μm wide. Asexual morph. Conidiophores macronematous, single to caespitose, usually emerging from stomata, straight to flexuous, often geniculate above, septate, mostly unbranched, subcylindrical, septate, mostly simple, olivaceous brown, becoming paler towards the apex, finely verruculose, with cell walls thicker than those of the vegetative hyphae, length indeterminate, 5.5–10.5 μm wide, often with a bulbous base up to 19.5 μm wide, rarely with subnodulose and nodulose intercalary swellings up to 15 μm wide. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 10–86.5 μm long; pores up to 1 μm wide, surrounded by scars 4–5.5 μm wide. Conidia ellipsoidal, obclavate to fusiform, straight to slightly curved, pale olivaceous brown, smooth, sometimes asperulate at the base, (1–)3–9-distoseptate, (51–)60.5–126(–140) × (16.5–) 19.5–31(–33) μm, with a slightly to strongly protruding hilum 2–4.5 μm wide.

On SNA + maize leaves. Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth to verruculose, with anastomoses, 2–9 μm wide. Asexual morph. Conidiophores semi-macronematous to macronematous, straight to flexuous, often geniculate at the fertile part, septate, often unbranched, subcylindrical, pale olivaceous brown to dark brown, paler at the apex, smooth to asperulate, with cell walls thicker than those of the vegetative hyphae, 169–1324.5 × 5.5–10 μm, often with a swollen, sometimes bulbous base up to 22 μm wide, with subnodulose to nodulose intercalary swellings up to 12.5 μm wide, swellings with conidiogenous loci. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- to polytretic, proliferating sympodially, 11–74 μm long, with scars up to 4.5 μm wide. Conidia fusoid, straight to more or less curved, pale to dark olivaceous brown, smooth to finely verruculose, (1–)4–7-distoseptate, (43–)76–135.5(–141) × (10–)16.5–22(–25) μm, with a strongly protruding hilum 2–4.5 μm wide. Microcyclic conidiation occasionally observed. Sexual morph (setosphaeria-like) adapted from Sivanesan ( 1987). Ascomata globose to ellipsoid, black, 350–725 × 345–500 μm, ostiolate, with rigid, dark brown, septate setae surrounding the ostiole, and the upper surface of the ascomata. Pseudoparaphyses filiform, hyaline, septate, branched and anastomosing. Asci 1–8-spored, cylindrical-clavate, short stalked, 175–250 × 24–31 μm. Ascospores hyaline, fusoid, straight to curved, (1–)3(–6)-septate, constricted at the septa, 40–78 × 12–18 μm, surrounded by a thin, hyaline mucilaginous sheath which may extend beyond the ends of the spore after discharge.

Culture characteristics — Colonies on SNA + sterilized maize leaves at 24 °C reaching 50–80 mm diam after 7 d, hairy to floccose, olivaceous grey to olivaceous black, cottony on maize leaves, with a fimbriate margin; reverse concolorous with obverse.

Additional materials examined. Australia, Queensland, Samford, on leaf blight of Sorghum bicolor, 25 May 1977, J.L. Alcorn No. 77159 (BRIP 12267); on leaf speckle of Sorghum sudanense, 6 Apr. 1981, R. Jones No. 22233 (BRIP 13326). – Indonesia, Lembang, on leaf of Zea mays, June 1926, M.B. Schwarz (CBS 195.26). – Usa, on leaf spot of Zea mays, Sept. 1964, R.R. Nelson (CBS 330.64); Georgia, dried culture of a crossing of compatible isolates on Sach's agar with Hordeum vulgare straw, 1954, E.S. Luttrell No. 6001 (holotype of Setosphaeria turcica BPI 623931); single ascospore isolate, Mar. 1958, E.S. Luttrell No. 1 198-9 (‘plus’ strain of S. turcica CBS 384.58); E.S. Luttrell No. 1 198-6 (‘minus’ strain of S. turcica CBS 385.58); on leaf of Sorghum halepense, Mar. 1958, E.S. Luttrell (CBS 386.58); Tifton, on leaf of Zea mays, Mar. 1958, E.S. Luttrell (CBS 387.58).

Notes — Helminthosporium turcicum was described on maize from Italy (Passerini 1876, Saccardo 1886). Luttrell ( 1958) obtained the ascomata by mating opposite compatible sexual strains, and introduce Trichometasphaeria turcica for the sexual morph. Later, Leonard & Suggs ( 1974) introduce Exserohilum with E. turcicum, based on H. turcicum, as the generic type and introduce the sexual morph as Setosphaeria. The herbarium material preserved at the US National Fungal Collection, BPI 431157 bears the label ‘Type?’. This material was collected by G. Passerini from the same locality and host as the type, and is therefore designated here as the lectotype. To stabilize the name, the isolate CBS 690.71 from Zea mays in Germany, is proposed as the ex-epitype, being from the same host and geographically close to the type locality, Italy, and its morphology fits well with the description of the species. This species causes the disease known as northern leaf blight of maize. It has also been reported on Euchlaena, Sorghum and other graminicolous plants. It is widespread in both tropical and subtropical areas. In the phylogenetic tree, E. turcicum is represented by eight strains isolated from Zea mays and Sorghum spp. collected from different geographical origins, i.e., Australia, Germany, Indonesia and the USA (Fig. 2).

DOUBTFUL OR EXCLUDED SPECIES

In this section are included species retained in Exserohilum based on morphology (without molecular data), and species transferred to other genera based on molecular and/or morphological data.

Exserohilum curvisporum Sivan., Abdullah & B.A. Abbas, Mycol. Res. 97: 1486. 1993 — Fig. 19

Fig. 19.

Exserohilum curvisporum (IMI 356632 holotype). a–e. Conidiophores and conidiogenous cells; f–j. conidia. — Scale bars: 10 μm.

Type material. Iraq, Basrah, isolated from sediments of Shatl-al-Arab River, 15 Dec. 1991, S.K. Abdullah & A. Abbas BSRA 10260 (IMI 356632 holotype).

Adapted from Sivanesan et al. ( 1993). Colonies effuse, pale brown. Vegetative hyphae pale brown, branched, septate, smooth, 4–5 μm wide. Conidiophores commonly unbranched, straight to flexuous, geniculate above, cicatrized, cylindrical, olivaceous brown, paler towards the apex, 125–450 × 6–8 μm. Conidia cylindrical to cylindrical-fusiform, mostly strongly curved, sometimes slightly curved or sigmoid, rarely straight, concolorous, pale brown, surface often granulose, 1–12-distoseptate, 65–165 × 12.5–22 μm, mostly 80–125 × 14–15 μm, with a basal distinctly protruding hilum up to 1 μm wide.

Notes — Exserohilum curvisporum is only known from the type locality, isolated from sediments of a river in Iraq. Although no molecular data exist for E. curvisporum, this species is retained in Exserohilum based on the characteristic hilum structure and conidial morphology.

Exserohilum echinochloae Sivan., Trans. Brit. Mycol. Soc. 83: 3 19. 1984 — Fig. 20

Fig. 20.

Exserohilum echinochloae (IMI 237838 holotype). a–d. Conidiogenous cells; e. lower part of conidium; f–j. conidia. — Scale bars: 10 μm.

Type material. Bangladesh, on leaves of Echinochloae colona, 10 Apr. 1979, M.A. Miah (IMI 237838 holotype).

Herbarium material. Colonies dark brown, effuse, sporulating abundantly. Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, 2.5–6 μm wide. Conidiophores macronematous, mononematous, straight, more or less bent or flexuous, septate, mostly unbranched, subcylindrical, brown, becoming paler and sometimes appearing geniculate toward the apex, smooth to asperulate, with cell walls thicker than those of the vegetative hyphae, 216–812.5 × 5.5–9.5 μm, and with a bulbous base up to 14.5 μm wide, often with subnodulose and nodulose intercalary swellings up to 13 μm wide, swellings with conidiogenous loci. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 26–88 μm long, pores 0.5–1 μm wide, surrounded by scars 4.5–5 μm wide. Conidia fusiform, straight to curved, olivaceous brown, smooth, asperulate at the base, 7–11-distoseptate, 111– 197.5 × 19–37 μm, basal cell often bulging a little, with a strongly protruding hilum 2.5–3.5 μm wide.

Notes — Exserohilum echinochloae is morphologically similar to E. monoceras and E. turcicum, but differs from them by having longer and wider conidia (Sivanesan 1984). Although no molecular data exist for E. echinochloae, this species is retained in Exserohilum based on the characteristic hilum structure and conidial morphology.

Exserohilum elongatum Hern.-Restr. & Crous, sp. nov. — MycoBank MB823162; Fig. 21

Fig. 21.

Exserohilum elongatum (IMI 321829 holotype). a–e. Conidiogenous cells; f–j. conidia. — Scale bars: 10 μm.

Synonym. Exserohilum elongatum Del Serrone et al., Phytopath. Mediterr. 30: 152. 1991 [nom. invalid Art. 40.1].

Type material. Italy, Piemonte, Cherasco (Cuneo), on leaves of Echinochloa crus-galli, 1979, Porta-Puhglia, IMI 321829 holotype designated here.

Herbarium material. Colonies brown, effuse, with abundant sporulation. Vegetative hyphae septate, branched, pale olivaceous to pale olivaceous brown, smooth to asperulate, 2–6 μm wide. Conidiophores macronematous, straight to flexuous, septate, mostly unbranched, subcylindrical, pale olivaceous brown to dark brown, often paler toward the apex, smooth to asperulate, with cell walls thicker than those of the vegetative hyphae, 168.5–1 190 × 7–9.5 μm, often with a bulbous base up to 18.5 μm wide, with subnodulose intercalary swellings up to 11.5 μm wide, swellings with conidiogenous loci. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 19.5–97 μm long, pores 0.5–1 μm wide, surrounded by scars 4–6 μm wide. Conidia clavate, cylindrical to fusiform, olivaceous brown, often somewhat paler at the apex or at the ends, smooth, asperulate at the base, 4–8-distoseptate, (42–)81–168 × 19–28.5 μm, with a strongly protruding hilum 2–3.5 μm wide.

Notes — Exserohilum elongatum is morphologically comparable with E. echinochloae and E. oryzicola. However, the protologue of the former (Del Serrone et al. 1991) describes longer conidia in respect to the other two species (i.e., 114–247 × 28–30 μm vs 150–210 × 25–35 μm and 170–210 × 20–28 μm, respectively). Nevertheless, we examined the material deposited at Kew Botanical Garden (IMI 321829) and conidia were shorter (42–168 × 19–28.5 μm) than those described in the original description (Del Serrone et al. 1991). The high morphological variation in species of Exserohilum, and the lack of molecular data in this species, make the correct placement of E. elongatum problematic.

Exserohilum frumentacei (Mitra) K.J. Leonard & Suggs, as ‘frumentaceum’, Mycologia 66: 291. 1974

Basionym. Helminthosporium frumentacei Mitra, Trans. Brit. Mycol. Soc. 15: 288. 1931.

Type material. India, Pusa, leaf-sheaths of Panicum frumentaceum, unknown date, Mitra.

Notes — This species was described from Panicum frumentaceum in India (Mitra 1931). Exserohilum frumentacei resembles E. monoceras in conidial size and morphology and probably represents the same species. However, culture of this species is not available for phylogenetic comparison.

Exserohilum glycinea (L.S. Srivast. et al.) P.M. Kirk, Index Fungorum 269: 1. 2015

Basionym. Setosphaeria glycinea L.S. Srivast. et al., Indian J. Mycol. Pl. Pathol. 12: 241. 1983.

Type material. India, Meghalaya, on leaves of Glycine max, 11 Aug. 1976 (IMI 209021 holotype, not seen).

Notes — The inclusion of this species in Exserohilum is doubtful. Originally, E. glycinea was described as the causal agent of leaf spot and blight of leaves of Glycine max (Srivastava et al. 1983), an uncommon substrate for Exserohilum species. It is only known by the sexual morph and was distinguished from other species of Setosphaeria by the presence of well-developed, long and septate setae on the ascomata, ascospores 5–6-septate, the enlargement of the third cell from the top of the ascospore and by the absence of asexual morph (Srivastava et al. 1983).

Exserohilum heteromorphum G.Y. Sun, Mycotaxon 92: 174. 2005

Type material. China, Shaanxi, on leaves of Echinochloa crus-galli var. mitis, unknown date, unknown collector (HMUABO 20579 holotype, not seen).

Notes — Exserohilum heteromorphum was differentiated from other species of the genus by the presence of strongly curved conidia (Sun et al. 2005). However, E. heteromorphum is morphologically similar to E. monoceras, which also has curved conidia (Drechsler 1923). Furthermore, E. heteromorphum was described from leaves of Echinochloa crus-galli, the same substrate as E. monoceras, and therefore likely represent the same species. Unfortunately, no cultures are available to confirm the phylogenetic relationship.

Exserohilum israeli Steiman et al., Antonie van Leeuwenhoek 78: 155. 2000

Type material. Israel, Timna Park, Negev desert, Arava valley, from soil, Aug. 1994 (CMPG 1339 holotype, not seen).

Notes — Exserohilum israeli was described from soil in Israel (Steiman et al. 2000). According to the protologue, this fungus is morphologically compatible with E. rostratum. No living culture of E. israeli is available for study.

Exserohilum lagenarioides Pachkhede, Geobios, New Rep. 8: 64. 1989

Notes — This name is currently considered as invalid in Index Fungorum following Art. 40.1, 40.4 and 8.4 of the International Code of Nomenclature for algae, fungi and plants (Melbourne Code).

Exserohilum longisporum G.Y. Sun, Mycol. Res. 101: 776. 1997

Type material. China, Hunan, Changsha, on Miscanthus sinensis, 12 Oct. 1992 (HMUABO 100133 holotype, not seen; HMAS 73782 isotype, not seen).

Notes — Exserohilum longisporum was described from Miscanthus sinensis in China (Sun et al. 1997). According to the protologue, this fungus is morphologically compatible with E. rostratum. No living culture of E. longisporum is available for study.

Exserohilum oryzae Sivan., Mycol. Pap. 158: 231. 1987 — Fig. 22

Fig. 22.

Exserohilum oryzae (IMI 214168 holotype). a–c. Conidiophores and conidiogenous cells; d–f. conidiogenous cells and conidia; g–i. conidia; j. lower part of conidium. — Scale bars: 10 μm.

Type material. Yugoslavia (currently Macedonia), Kočani, on Oryzae sativa, June 1977, Karov Ilija 5 (IMI 214168 holotype).

Herbarium material. Vegetative hyphae septate, branched, pale olivaceous to pale brown, smooth-walled, 3.5–5.5 μm wide, anastomosing. Conidiophores macronematous, mononematous, straight, curved or geniculate, mostly unbranched, subcylindrical, pale olivaceous brown to dark brown, becoming paler towards the apex, smooth to asperulate, with cell walls usually thicker than those of the vegetative hyphae, 21–7 20 × 3.5–7.5 μm, sometimes slightly swollen at the base up to 10 μm wide, often with subnodulose and nodulose intercalary swellings up to 10 μm wide, swellings with conidiogenous loci. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 10–54 μm long, with pores up to 1 μm wide, surrounded by scars 3–5 μm wide. Conidia fusiform with an obtuse apex and a truncate obconic base, rarely clavate, straight to more or less curved, pale olivaceous brown to dark brown, often paler at both poles, smooth to asperulate with the ornamentation more evident at the base, (3–)7–9-distoseptate, (34–)61–150 × 10–24 μm, with a protruding hilum, 2.5–5.5 μm wide. Germination unipolar or bipolar. Microcyclic conidiation frequent.

Notes — The protologue describes narrower hyphae (up to 4.5 μm wide), conidiophores slightly shorter and narrower (up to 600 μm long, 5–6 μm wide) and conidia slightly longer and wider, 96–160 × 18–25 μm (Sivanesan 1987) than those observed here (34–150 × 10–24 μm). Sivanesan possibly excluded short conidia from his description because he might have considered them immature. They were included here because even these short conidia were able to germinate. Exserohilum oryzae and E. oryzicola are very similar species isolated from the same substrate. According to Sivanesan ( 1987), they differ in conidial size. Nevertheless, the high morphological variation in species of Exserohilum, and the lack of molecular data in this species, makes the correct placement of E. oryzae difficult to assess.

Exserohilum oryzinum Sivan., Trans. Brit. Mycol. Soc. 83: 325. 1984 — Fig. 23

Fig. 23.

Exserohilum oryzinum (IMI 152682 holotype). a. Conidiophore with a swelling base; b–e. conidiogenous cells; f–j. conidia. — Scale bars: 10 μm.

Type material. Egypt, Alexandria, from leaves of Oryza sp., 8 Nov. 1970, M.K. El-Kazaz (IMI 152682 holotype).

Herbarium material. Vegetative hyphae septate, branched, pale olivaceous to medium brown, smooth to asperulate 3–7.5 μm wide, anastomosing. Conidiophores macronematous, straight to flexuous, septate, mostly unbranched, subcylindrical, pale olivaceous brown to dark brown, becoming paler towards the apex, smooth to asperulate, with cell walls thicker than the vegetative hyphae, up to 810 μm long, 4–8 μm wide, sometimes swollen at the base up to 9 μm wide, with subnodulose and nodulose intercalary swellings up to 11 μm wide, swellings with conidiogenous loci. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 9–61.5 μm long, pores up to 1 μm wide, surrounded by scars 3.5–5.5 μm wide. Conidia mostly falcate or sigmoid, rarely fusiform or clavate, pale to medium olivaceous brown, smooth to asperulate with the ornamentation more evident at the base, (4–)6–10-distoseptate, (56–)80–155 × 14–27 μm, with a protruding hilum, 2.5–4 μm wide. Germination uni- or bipolar. Microcyclic conidiation frequent.

Notes — Exserohilum oryzinum is only known from Egypt growing on Oryza sp. This species is distinguished from other Exserohilum spp. by the distinctively curved to sigmoid, pale to mid-brown conidia. Although no molecular data exist for E. oryzinum, this species is retained in Exserohilum based on the characteristic hilum structure and conidial morphology (Sivanesan 1984).

Exserohilum parlierense W.Q. Chen & Michailides, as ‘parlierensis’, Mycotaxon 83: 153. 2002

Type material. USA, California, on leaves of Pistacia vera, 16 Aug. 2001, Q.W. Chen (ATCC MYA-2456 holotype, not seen; CH-26 culture ex-type).

Notes — Exserohilum parlierense was described from Pistacia vera in the USA (Chen et al. 2002). According to the protologue, this fungus is morphologically compatible with E. rostratum.

Exserohilum phragmitis W.P. Wu, as ‘phragmatis’, J. Hebei Acad. Sci., Selected papers: 60. 1990

Type material. China, Hebei, on leaves of Phragmites (IBHAS 4150 holotype, not seen).

Notes — This name we considered here as invalid following Art. 8 and 40 of the International Code of Nomenclature for algae, fungi and plants (Melbourne Code). The data of the type material listed here is accordingly to information in Index Fungorum. However, the publication linked to the protologue did not describe a new species.

Exserohilum psidii Sivan., Mycol. Res. 96: 489. 1992 — Fig. 24

Fig. 24.

Exserohilum psidii (IMI 299549 holotype). a–d. Conidiogenous cells; e–j. conidia. — Scale bars: a–i = 10 μm; j = 20 μm.

Type material. India, Warangal, on Psidium sp., 15 Oct. 1985, Madhukar D2 (IMI 299549 holotype, not seen).

Herbarium material. Colonies on TWA + wheat straw effuse floccose, dark brown. Vegetative hyphae septate, branched, subhyaline to mid olivaceous brown, smooth to asperulate, 3–7 μm wide. Conidiophores macronematous, straight to flexuous, septate, unbranched, subcylindrical, pale olivaceous brown to dark brown, smooth to asperulate, with cell walls thicker than those of the vegetative hyphae, up to 1 086 μm long, 5.5–11 μm wide, often swollen at the base up to 18 μm wide, with subnodulose and nodulose intercalary swellings up to 12 μm wide, swellings with conidiogenous loci. Conidiogenous cells integrated, terminal and intercalary, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 16.5–118.5 μm long, with pores up to 1 μm wide, surrounded by scars up to 4.5–5.5 μm wide. Conidia mostly fusiform, rarely clavate, with an obtuse apex and a truncate obconic base, straight to slightly curved, pale to mid olivaceous brown, smooth, asperulate at the base, (5–)6(–7)-distoseptate, (53–)112.5–148 × 16–23 μm, with a protruding hilum 2.5–3.5 μm wide. Germination uni- or bi-polar.

Notes — The protologue describes narrower conidiophores (up to 6 μm wide) and conidia much longer and wider 150–175 (– 190) × 27–30(–35) μm (Sivanesan 1992) than those observed here (53–148 × 16–23 μm). The morphology of this species resembles E. monoceras and E. turcicum. Nevertheless, the high morphological variation in species of Exserohilum and the lack of molecular data in this species, make the correct placement of E. psidii difficult.

Exserohilum sodomii Guiraud et al., Antonie van Leeuwenhoek 72: 318. 1997

Type material. Israel, Neguev desert, Dead Sea (road of Sodom), from soil, Aug. 1994, unknown collector (CMPG1340 holotype, not seen).

Notes — Exserohilum sodomii was described from soil in Israel, as well as E. israeli by the same authors (Guiraud et al. 1997, Steiman et al. 2000). According to the protologue, this fungus is morphological compatible with E. rostratum. No living culture of E. sodomii is available for study.

Curvularia micropus (Drechsler) Hern.-Restr., Y.P. Tan & Crous, comb. nov. — MycoBank MB822994; Fig. 25

Fig. 25.

Curvularia micropus (CBS 127235 ex-epitype (a–d), CBS 127236 (e–g), CBS 128057 (h–k)). a–c, e, h. Conidiophores and conidia; d, f–g, i–k. conidia. — Scale bars: 10 μm.

Basionym. Helminthosporium micropus Drechsler, J. Agric. Res. 24: 722. 1923.

Synonyms. Bipolaris micropus (Drechsler) Shoemaker, Canad. J. Bot. 37: 884. 1959.

Drechslera micropus (Drechsler) Subram. & B.L. Jain, as ‘micropa’, Curr. Sci. 35: 354. 1966.

Exserohilum paspali J.J. Muchovej & Nesio, Trans. Brit. Mycol. Soc. 89: 126. 1987.

Type material. USA, Florida, Wauchula, on Paspalum boscianum (?), 2 May 1921, C. Drechsler (IMI 296605, not seen (holotype of Helminthosporium micropus); BPI 429621, syntype (of Helminthosporium micropus)); Georgia, Tifton, on Paspalum notatum, 17 July 1970, E.S. Luttrell No. Lutt. 8530 (BRIP 65 20 epitype designated here (MBT378847); CBS 127235 = BRIP 65 20 culture ex-epitype.

On SNA + banana leaf. Vegetative hyphae pale brown, septate. Conidiophores macronematous, single or in small groups, erect, septate, unbranched, cylindrical, brown, smooth, 80–335 × 4–8 μm. Conidiogenous cells terminal and intercalary, geniculate, mono- and polytretic, 8–37.5 × 3.5–6.5 μm. Conidia cylindrical (longer ones) to ellipsoid (shorter ones) or sigmoid, straight or slightly curved, subhyaline to pale brown, verruculose, 3–9-distoseptate, 30–70 × 10–18.5 μm, protruding hilum 1–3.5 μm long, 1.5–3 μm wide. Sexual morph not observed.

Culture characteristics — On SNA + sterilized maize leaf after 7 d at 24 °C in the dark, hairy to floccose, olivaceous grey to olivaceous black, cottony on maize leaves, with a fimbriate margin; reverse concolorous with obverse.

Additional materials examined. Brazil, Minas Gerais, Viçosa, on Paspalum conjugatum, 10 May 1986, J.J. Muchovej (authentic culture of E. paspali BRIP 16070 = CBS 128057). – Usa, Florida, Lakeland, on Paspalum notatum, 3 Apr. 1970, E.S. Luttrell, Lutt. 8452 (BRIP 6516 = CBS 127234); Georgia, Tifton, on Paspalum notatum, 3 Apr. 1987, A.Y. Rossman (BRIP 15689a = CBS 127236).

Notes — Curvularia micropus attacks leaf blades of young plants of Paspalum, killing the foliar tissues (Drechsler 1923, Muchovej & Ribeiro Nesio 1987, Sivanesan 1987). Curvularia micropus, originally described as Helminthosporium (Drechsler 1923), was transferred to Bipolaris (Shoemaker 1959) and later to Drechslera (Subramanian & Jain 1966). Other species listed as synonyms of B. micropus are Helminthosporium leptochloae (Sivanesan 1987) and E. paspali (Alcorn 1991). However, molecular data generated in this study revealed that H. leptochloae is conspecific with E. rostratum (Fig. 2), whereas E. paspali and B. micropus are conspecific (Fig. 1). Nevertheless, this species is better placed in Curvularia. In the multi-gene tree, three strains isolated from Paspalum in the USA and one from Brazil, are placed in a basal clade of Curvularia (Fig. 1). Type material of H. micropus is preserved at the IMI (holotype IMI 296605) and at the US National Fungal Collection (BPI 4296 20, BPI 429621, BPI 429615, as syntype), unfortunately there is no culture available. The strain CBS 127234 was collected from the same state as the type material (Florida) but this isolate was sterile under the culture conditions tested. It is, however, genetically identical to the strain CBS 127235. In order to stabilize the use of the name, we propose CBS 127235 as the ex-epitype strain of C. micropus, since it fits well with the description given in the protologue and it was collected in a neighbouring state.

Curvularia sorghicola (Sivan.) Madrid & Crous, comb. nov. — MycoBank MB822997; Fig. 26

Fig. 26.

Curvularia sorghicola (IMI 225559 holotype). a–f. Conidiophores; g–i. conidia. — Scale bars: 10 μm.

Basionym. Exserohilum sorghicola Sivan., Mycol. Pap. 158: 237. 1987.

Type material. Ethiopia, on leaves of Sorghum sp., 15 Sept. 1976, unknown collector (IMI 225559 holotype).

Adapted from Sivanesan ( 1987). Leaf spots irregularly elongate, running parallel to the midrib, surrounded by a thick dark purple border. Vegetative hyphae mostly immersed, septate, branched, pale olivaceous to pale olivaceous brown, sometimes with purplish tinges, smooth, 2.5–7.5 μm wide, giving rise to chains and clumps of swollen subcylindrical, globose to irregularly shaped cells up to 24 μm wide. Conidiophores macronematous, single to fasciculate, straight to flexuous, often geniculate above, septate, mostly unbranched, subcylindrical, olivaceous brown, becoming paler towards the apex and sometimes also toward the base, smooth to asperulate, with cell walls thicker than those of the vegetative hyphae, 28.5–112 × 3–7.5 μm, often with a bulbous base up to 14 μm wide. Conidiogenous cells integrated, intercalary and terminal, mostly subcylindrical, mono- and polytretic, proliferating sympodially, 7–46 × 3–7.5 μm, with pores up to 1 μm wide, surrounded by scars 3–4.5 μm wide. Conidia mostly clavate, straight to curved, pale olivaceous brown, often paler towards both ends, smooth to asperulate, 3(–5)-distoseptate, often constricted at the septa, 30–57 × 11– 19 μm, with a protruding hilum, 1.5–3 μm wide.

Notes — This fungus was originally described as an atypical species of Exserohilum (Sivanesan 1987), and examination of the holotype revealed that its conidia produce hila which are delimited from the basal cell by a septum, different from other Exserohilum species. This kind of hilum is produced by many Curvularia species (Alcorn 1991, Zhang et al. 2004, Madrid et al. 2014), and therefore this is the correct genus for E. sorghicola. Morphologically, this fungus resembles members of the trifolii-clade of Curvularia in producing predominantly 4-celled conidia with a strongly protruding hilum (Madrid et al. 2014). However, there is no culture available for DNA sequence analyses, and its exact phylogenetic placement in Curvularia still has to be assessed.

Sporidesmiella novae-zelandiae (S. Hughes) Madrid, Hern.-Restr. & Crous, comb. nov. — MycoBank MB822998; Fig. 27

Fig. 27.

Sporidesmiella novae-zelandiae (DAOM 159962 isotype). a–h. Conidiophores and conidia; i–l. conidia. — Scale bars: a = 20 μm; b–h = 10 μm.

Basionym. Sporidesmium hyalospermum var. novae-zelandiae S. Hughes, New Zealand J. Bot 16: 349. 1978.

Synonym. Sporidesmiella hyalosperma var. novae-zelandiae (S. Hughes) P.M. Kirk, Trans. Brit. Mycol. Soc. 79: 479. 1982.

Exserohilum novae-zelandiae (S. Hughes) H.P. Upadhyay & Mankau, Mycologia 83: 373. 1991.

Type material. New Zealand, Canterbury Province, Okuti Valley, near Little River, on rotten wood, 17 May 1963, S.J. Hughes (PDD 304 20 holotype (of Sporidesmium hyalospermum var. novae-zealandiae), not seen; DAOM 159962 isotype).

Vegetative hyphae septate, branched, subhyaline to pale brown, smooth and thin-walled, 1–2.5 μm wide. Conidiophores macronematous, mononematous, septate, unbranched, straight or flexuous, showing either percurrent or sympodial proliferations and sometimes both, dark brown, becoming paler towards the apex, smooth to asperulate, thick-walled, length undetermined, 4.5–9 μm wide, often with a bulbous base up to 13 μm wide. Conidiogenous cells integrated, terminal, mostly subcylindrical, often flexuous, holoblastic, mono- and polyblastic, 7.5–38 μm long, with flat or slightly convex, non-darkened scars 3.5–5 μm wide. Conidia solitary, mostly clavate with an obconically truncate base, pale olivaceous to pale golden brown, smooth, 3–4-distoseptate, 20.5–28 × 11.5–15 μm, 3–5.5 μm wide at the base, basal cell cut-off by a dark-brown septum.

Additional material examined. Mexico, Laguna de Zempoala, Morelos, plant debris (of grass), 22 Feb. 1989, R. Mankau (CBS 135842).

Notes — This species was originally described as Sporidesmium hyalospermum var. novae-zelandiae by Hughes ( 1978). This and the type variety were reallocated to Sporidesmiella by Kirk ( 1982) and var. novae-zelandiae was later transferred to Exserohilum by Upadhyay & Mankau ( 1991) as E. novae-zelandiae. The combination proposed by the latter authors is not appropriate since the conidiogenous cells in the holotype lack the darkened scars and conspicuous pores typical of Exserohilum spp. and produce conidia with an obconically truncate base, not with a truly protruding hilum. Although DNA sequence data are lacking for the type specimens of the varieties of S. hyalosperma, morphological differences indicate that S. hyalosperma var. novae-zelandiae and S. hyalosperma var. hyalosperma are different fungi. The latter differs from the variety novae-zelandiae in having conidiophores that proliferate almost always percurrently, with numerous conspicuous annellations and narrower (9–12 μm wide) conidia (Morgan-Jones & Cole 1964, Kirk 1982, Wu & Zhuang 2005). Furthermore, the isolate CBS 135842, identified as Exserohilum novae-zelandiae by Upadhyay & Mankau ( 1991), proved to be related to Annulatascaceae, Sordariomycetes (unpubl. data), revealing that the fungus is phylogenetically distant from Exserohilum (Pleosporaceae, Dothideomycetes). Based on morphological and molecular evidence we consider that ‘Exserohilum’ novae-zelandiae should be, at least provisionally, retained in Sporidesmiella. Currently, Sporidesmiella comprises 24 species (Seifert et al. 2011). However, Sporidesmiella fusiformis is the only species with molecular data available in this genus, and it belongs to Didymosphaeriaceae (Dothideomycetes) (Shenoy et al. 2006) rather than Annulatascaceae, like S. novae-zelandiae. Further studies are needed in order to establish generic boundaries in Sporidesmiella.

DISCUSSION

In this study, we have reviewed the taxonomic circumscription of Exserohilum (= Setosphaeria) using molecular and morphological data. This is the first study that presents a robust phylogeny using a broad distribution of Exserohilum isolates from different hosts and geographic origins. Furthermore, we also studied the holotype material of several species and protologues of species listed in the genus to provide a statement of their placement in the genus. The phylogenetic analysis based on four genes gapdh, rpb2, LSU and ITS (Fig. 1), shows that the morphologically atypical species E. paspali is conspecific with B. micropus, as previously noticed by Alcorn ( 1991), but belongs in Curvularia rather than in Exserohilum or Bipolaris. This phylogenetic analysis also confirms that C. crassiseptum and C. heteropogonicola, previously known as Exserohilum species, are correctly placed in Curvularia (Alcorn 1991, Zhang et al. 2004). Furthermore, excluding those species or others such as E. novae-zelandiae, Exserohilum is highly supported as a monophyletic clade, which is clearly different from other helminthosporoid genera such as Bipolaris, Curvularia, Johnalcornia, Porocercospora and Pyrenophora.

Exserohilum species are defined as pathogenic fungi to humans and plants, mainly grasses, as well as, saprobic, endophytic and soil-borne fungi. Species of this genus are frequently found as asexual morphs in nature, although the sexual morph was often obtained by combining compatible strains, except for E. minor and E. khartoumensis (Drechsler 1923, Luttrell 1963a, Nelson 1965, Alcorn 1978, 1986, El Shafie & Webster 1981). The setosphaeria-like sexual morph of different Exserohilum species can be morphologically very similar and therefore morphology of the asexual morph is considered more reliable for identification purposes (Leonard 1976, Alcorn 1986, Sivanesan 1987). The traditional morphological attributes of conidia that have been used as taxonomical criteria at the generic rank for Bipolaris, Curvularia, Exserohilum and Pyrenophora are mainly the germination patterns of the conidia, septum ontogeny and hilum morphology (Leonard & Suggs 1974, Alcorn 1983, 1990, 1991, Sivanesan 1987). Among those features, the hilum morphology is the most valuable criteria to delineate Exserohilum species. The hilum is characterised by an enveloping structure around the hilar protrusion which is often thickened basally or laterally (Alcorn 1983). However, in some Curvularia species, a structure resembling that of the hilum in Exserohilum can be present, but in Curvularia, the hilum is separated from the conidial body by a septum.

By combining morphological data with multi-locus analysis from ITS, act, gapdh, his, rpb2, tef1 and tub2 sequences, we were able to delimit 11 species in Exserohilum, one of which is formally proposed as a new species, E. corniculatum, in addition to other previously described species, i.e., E. holmii, E. khartoumensis, E. minor, E. monoceras, E. neoregeliae, E. oryzicola, E. pedicellatum, E. protrudens, E. rostratum and E. turcicum. The phylogenetic position of E. monoceras is still unresolved, since molecular data from type material was not available. The phylogenetic tree shows a clade of E. turcicum, closely related to strains identified as E. monoceras; however, more study is needed on members of these clades in order to resolve their relationship (Fig. 2). This study demonstrated that some species can be morphologically highly variable, but molecularly they are very closely related and they are treated here as synonyms, E. curvatum with E. holmii and E. fusiforme with E. oryzicola. Exserohilum rostratum was an exceptional case, where this species is shown as conspecific to E. antillanum, E. gedarefense, E. longirostratum, E. macginnisii, E. prolatum and H. leptochloae. Morphological variability in conidial shape, size and pigmentation of E. rostratum has been already noticed in natural substrate (Drechsler 1923), as well as in culture, which is influenced by external factors like carbon source, glucose concentration, type of culture media, light exposure and pH, among others (Mitra 1931, Kafi & Tarr 1966, Tarr & Kafi 1968, Leonard 1976, Anahosur & Sivanesan 1978, Honda & Aragaki 1978). Previously, Leonard & Suggs ( 1974) observed morphological similarities among E. rostratum, H. leptochloae and E. longirostratum. More recently, Da Cunha et al. ( 2012) demonstrated that E. rostratum, E. longirostratum and E. macginnisii are very closely related based on multigene sequence analysis of clinical isolates, but did not propose any synonymy. Here we propose them as synonyms of E. rostratum.

For an accurate species identification of Exserohilum species, a molecular analysis is required. Among the nine loci used in this study, ITS, act and rpb2 were able to resolve 11, 13 and 12 OTUs, respectively, with varying statistical support. Although each of these loci proved to be suitable barcoding markers for species identification, a combined analysis is highly recommended. The gapdh gene is recommended for species resolution in Bipolaris and Curvularia (Da Cunha et al. 2012, Manamgoda et al. 2012,) but in Exserohilum this region showed E. rostratum (including E. corniculatum) as a polyphyletic group. However, the other nine species were well-supported.

Besides the 11 phylogenetic species recognised here, the taxonomic placement at species and generic level of other 15 species listed as ‘doubtful’ (i.e., E. curvisporum, E. echinochloae, E. elongatum, E. frumentacei, E. glycinea, E. heteromorphum, E. israeli, E. lagenarioides, E. longisporum, E. oryzae, E. oryzinum, E. parlierense, E. phragmitis, E. psidii and E. sodomii) have to be confirmed as members of Exserohilum by molecular data.

The present investigation significantly extends the knowledge of the taxonomy of Exserohilum. Nevertheless, extensive sampling of uncommon species and inclusion of additional data like chemical compounds (i.e., secondary metabolites), ecology and host range for species are needed in order to resolve some taxonomic and phylogenetic aspects.