Re-Examination of Several Elsinoë Species Reported from Japan

Abstract

Elsinoë are plant pathogenic fungi that cause scabs, spotted anthracnose, and some morphological distortions on various plants, including woody plants, economically important crops, and ornamental plants. Taxonomical reexamination of Elsinoë species in Japan has not yet been conducted based on the modern species criteria. In this study, several Japanese isolates were reexamine based on the morphological and molecular-phylogenetic analysis of the internal transcribed spacer region (ITS), large subunit gene (LSU)m and protein-coding gene such as RNA polymerase II subunit (rpb2) and Translation elongation factor 1-alpha (tef). Japanese isolates were divided into four clades and three new species, Elsinoë hydrangeae, E. sumire, and E. tanashiensis were proposed. One species, Sphaceloma akebiae, was transferred to the genus Elsinoë.

1. Introduction

The genus Elsinoë (Myriangiales, Ascomycota) encompasses plant pathogenic fungi that cause disease on many plants, including crops, ornamental plants, and even woody plants. The symptoms of the disease caused by Elsinoë species can be seen by scabs that are often exhibited as raised, cork-like necrotic lesions on leaves and stems [1,2]. This genus was established by Raciborski [3] with the type species Elsinoë canavaliae Racib. Under the current code, the International Code of Nomenclature for Algae, Fungi, and Plants [4], the holomorphic name was decided to propose the generic name Elsinoë Racib. as a protection name over the generic name used for asexual morphs, Sphaceloma de Bary [5–8].

Scab is widely used as a disease name on leaves. Jenkins [9] recommended the alternative term “spot anthracnose” to refer to disease names caused by Elsinoë and Sphaceloma species instead of anthracnose, more broadly used by disease caused by Colletotrichum species [2]. However, the symptoms caused by Elsinoë species is not limited to only necrotic lesion on leaves and stem. In some hosts, it distorts infected organs, such as twisting of infected stems of Ipomea batatas (L.) Lam. [10] and Bidens spp. [11], and elongation of the stem in Manihot esculenta Crantz [12]. Although there was a lot of description of Elsinoë species causing diseases in crops, the impact of diseases caused by this fungus is more on the appearance of the harvested product rather than the crop productivity itself [13]. However, there are records of Elsinoë causing economically important diseases such as avocado scab by Elsinoë perseae (Jenkins) Rossman & W.C. Allen, citrus scab by Elsinoë fawcetti Bitanc. & Jenkins (Figure 1(a,b)) and Elsinoë australis Bitanc. & Jenkins, and grape spot anthracnose by Elsinoë ampelina (de Bary) Shear (Figure 1(c)).

Figure 1.

Elsinoë species causing citrus scab and anthracnose grape spot in Japan.

In the revision of Elsinoë taxonomy, a total of 79 species were accepted in the genus Elsinoë including new combinations transferred from the genus Sphaceloma [1,2,14]. In previous phylogenetic studies with multi genetic-loci, Elsinoë species appear to be host-specific fungus, as 77 out of 81 species are confined to only one host species or genus [2]. The identification of Elsinoë species is often difficult due to overlapping morphological characteristics, such as small conidia, similar conidiogenous cells, continuously expanding wide acervuli, and lacking fertile structure in nature [1]. Moreover, the establishment of pure culture was also challenging due to the slow growth of the isolates, and it is easily contaminated by other fungi [1]. On the other hand, scab symptom is considered a significant characteristic of Elsinoë infection. The isolates with similar cultural characteristics obtained from the typical symptoms can often be helpful for species identification [1].

In Japan, reports of Elsinoë and Sphaceloma infecting plants have been recorded infecting crops such as citrus, soybean, and grapevines. The Japanese isolates of Elsinoë species recorded are based on morphological characteristics and limited phylogenetic information obtained only from the Internal Transcriber Spacer (ITS) region of rDNA. As the advancement in molecular technique involves the usage of a polyphasic approach in species recognition by Consolidated Species Concept [15], a comprehensive study needs to be conducted to update the database of the Japanese isolates of Elsinoë. In this study, the isolates that were stored as Elsinoë and Sphaceloma in the culture collection of different research institutes in Japan were reexamined for their taxonomical position by using multi-locus phylogenetic analysis by using the regions, Internal Transcriber Spacer (ITS) and nuclear large subunit ribosomal (LSU) of the rDNA, RNA polymerase II subunit (rpb2) and Translation elongation factor 1-alpha (tef). Morphological characteristics were examined on host plants and media as well. This study aims to contribute to the phylogenetic backbone of Japanese isolates of Elsinoë species.

2. Materials and methods

2.1. Sample collection and morphological study

Specimens and culture were obtained from the National Agriculture and Food Research, Organization (MAFF, NARO), NITE Biological Resource Centre (NRBC), Herbarium of Forest Mycology and Pathology, Forestry and Forest Product Research Institute (TFM:FPH), Agriculture Promotion Office, Tokyo Metropolitan Government (Tachikawa, Tokyo, Japan), National Museum of Nature and Science (TNS), and Culture Collection of Laboratory of Phytopathology, Mie University (TSU) and examined in the framework of morphological and molecular-phylogeny approaches. The isolates were obtained from symptomatic plants of various hosts (Table 1). The isolates were cultured on malt agar (MA), oatmeal agar (OMA) (Difco, Becton Dickinson, Franklin, NJ), and potato dextrose agar (PDA) (Nissui Pharmaceutical Co., LTD., Tokyo, Japan) at 22 °C for 21 days. Colony colors were described after 3 weeks according to the color chart by Rayner [16] Observation of specimens’ collections (Table 2) were conducted by using the shear solution as a mounting medium.

Table 1.

List of Japanese Elsinoë isolates used in this study.

Fungal species	Isolate no.	Herbarium no.	Host family	Host species	Region	Previous identification
Elsinoë akebiae	MUCC 2982 (MAFF 243582)	TSU-MUMH 11977	Lardizabalaceae	Akebia trifoliata	Tokyo	Sphaceloma akebiae
Elsinoë bidentis	MUCC 2983 (MAFF 243588)		Asteraceae	Bidens pilosa	Gunma	Elsinoë bidentis
Elsinoë glycine	MUCC 2984 (MAFF 305214)		Fabaceae	Glycine max	Akita	Sphaceloma glycine
Elsinoë glycine	MUCC 2985 (MAFF 305611)		Fabaceae	Glycine max	Miyagi	Sphaceloma glycine
Elsinoë sp.	MUCC 2986 (MAFF 243587)		Fabaceae	Amphicarpaea edgeworthii	Gunma	Sphaceloma kurozawarnum
Elsinoë hydrangeae	MUCC 2988 (MAFF 243315)	TSU-MUMH 11976	Hydrangeaceae	Hyderangea serrata	Tokyo	Sphaceloma sp.
Elsinoë tilliae	MUCC 2989 (MAFF 243584)		Malvaceae	Tilia playtphyllos	Tokyo	Sphaceloma sp.
Elsinoë tilliae	MUCC 2990 (MAFF 243585)		Malvaceae	Tilia europaea	Tokyo	Sphaceloma sp.
Sphaceloma tsujii	MUCC 2991 (MAFF 410486)		Paulowniaceae	Paulownia tomentosa	Ibaraki	Sphaceloma tsujii
Elsinoë sumire	MUCC 2992 (MAFF 243579)	TSU-MUMH 11975	Violaceae	Viola sp.	Tokyo	Sphaceloma violae
Elsinoë fawcettii	MUCC 2993 (MAFF 675004)		Rutaceae	Citrus deliciosa (syn. Citrus unshiu)	Shizuoka	Elsinoë fawcettii
Elsinoë fawcettii	MUCC 2994 (MAFF 675008)		Rutaceae	Citrus medica (syn. Citrus hassaku)	Miyazaki	Elsinoë fawcettii
Elsinoë fawcettii	MUCC 2999 (MAFF 675005)		Rutaceae	Citrus deliciosa (syn. Citrus unshiu)	Wakayama	Elsinoë fawcettii
Elsinoë fawcettii	MUCC 3000 (MAFF 675006)		Rutaceae	Citrus deliciosa (syn. Citrus unshiu)	Kochi	Elsinoë fawcettii
Elsinoë ampelina	MUCC 2995 (MAFF 243580)		Vitaceae	Vitis sp.		Elsinoë ampelina
Elsinoë ampelina	MUCC 2996 (MAFF 244135)		Vitaceae	Vitis vinifera	Yamanashi	Elsinoë ampelina
Elsinoë ampelina	MUCC 2362		Vitaceae	Vitis vinifera		-
Elsinoë araliae	MUCC 2997 (MAFF 243589)		Araliaceae	Aralia elata	Gunma	Elsinoë araliae
Elsinoë araliae	MUCC 3490 (NBRC 6166)		Araliaceae	Aralia cordata	Ibaraki	Elsinoë araliae
Elsinoë corni	MUCC 2998 (MAFF 243590)		Cornaceae	Cornus florida	Tokyo	Elsinoë corni
Elsinoë tanashiemsis	MUCC3463 (MAFF 410340)		Juglandaceae	Juglans sp.		Sphaceloma sp.
Elsinoë tanashiensis	MUCC3466 (MAFF 410485)	TFM:FPH 01697	Salicaceae	Populus sp.	Tokyo	Sphaceloma populi

Table 2.

List of regions and primer set used in this study.

Region	Primer (forward)	Primer (reverse)	Annealing temperature
ITS	ITS5 (White et al. 1990)	ITS4 (White et al. 1990)	48 °C
LSU	LR0R (Rehner & Samuels, 1994)	LR5 (Vilgalys & Hester, 1990)	48 °C
RPB2	RPB2-5F (Sung et al. 2007)	fRPB2-7cR (Liu et al. 1999)	56 °C→ 53 °C→ 50 °C
TEF-1a	Elongation-1-F (Hyun et al. 2009)	Elongation-1-R (Hyun et al. 2009)	60 °C

2.2. DNA isolation, amplification, and sequencing

Genomic DNA was extracted from mycelia growing on MA by using DNeasy Ultra Clean Microbial Kit (Qaigen, Hilden, Germany) according to the manufacturer’s instructions. ITS and LSU region of the rDNA as well as TEF-1α (tef) and RPB2 (rpb2) were amplified via polymerase chain reaction using the T100 thermal cycler (Bio-Rad, Tokyo, Japan). PCR mixture of the final volume of 12.5 µL for all reaction was prepared as followed; 1–10 ng of genomic DNA, 0.1 uL of 0.25 U Taq DNA polymerase (Bioline, London, UK), 2.5 mM of MgCl₂, 1.25 µL of 10× NH₄ reaction buffer (Bioline), 40 µM dNTPs (Bioline), and 0.2 µM of each primer.

All PCR condition used in this study follows the previous study, where the PCR condition was as follows. For ITS and LSU [1]: Initial denaturation (95 °C, 2 min), 35 cycles of amplification (denaturation at 95 °C, 30 s; annealing at 48 °C, 1 min; extension at 72 °C, I minute), and final extension at 72 °C for 8 min; for tef [17]: Initial denaturation (95 °C, 2 min), 35 cycles of amplification (denaturation at 94 °C, 30 s; annealing at 60 °C, 1 min; extension at 72 °C, 2 min), and final extension at 72 °C for 10 min; for rpb2: Initial denaturation (95 °C, 5 min), touch-down amplification that consists of 5 cycles of 95 °C for 45 s, 56 °C for 45 s and 72 °C for 2 min, followed by 5 cycles of 95 °C for 45 s, 53 °C for 45 s and 72 °C for 2 min, and 30 cycles of 95 °C for 45 s, 50 °C for 45 s and 72 °C for 2 min, and final elongation of 72 °C for 8 min. Amplicons were sequenced in both directions by using BigDye Terminator version 3.1 cycle Sequencing Kit (Applied Biosystem, Foster City, CA) at Mie University Advance Science Research Promotion Center (Tsu, Mie, Japan). Primer sets used in this study are summarized in Table 2.

2.2.1. Phylogenetic analyses

The resulting sequences were assembled and aligned with 81 sequences of Elsinoë retrieved from the previous study by Marin-Felix et al. [2]and Fan et al. [1] on MEGA X software package [18] (Table 3). This matrix was aligned by using MAFFT online version [19] and edited manually using AliView [20]. Maximum-likelihood (ML) and Bayesian inference (BI) analyses were used in this study to estimate the phylogenetic relationship of the samples. ModelTest-NG [21] was used to estimate the best substitution model for each gene for ML and BI analysis and ML analysis was performed by using RAxML-NG [22]. Tree strength was tested by bootstrap analysis of 1000 replication [23]. BI analysis was performed by using MrBayes 3.2 [24] to estimate the posterior probability (PP) of the tree topologies based on the Metropolis-coupled Markov Chain Monte Carlo (MCMCMC) algorithm of four chains which run from a random tree for 150,000,000 generations with the evolutional model set as GTR model for ITS and LSU and HKY model for tef and rpb2. Trees were sampled and saved for each 1000 generation. First, 25% of the tree were discarded as a burn-in phase of analysis and the PP was determined by the remaining tree. Myriangium hispanicum (CBS 327.33) was selected as an outgroup in all analyses and trees were viewed by using FigTree v. 1.4.2 (Institute of Evolutionary Biology, University of Edinburgh, http://tree.bio.ed.ac.uk/software/figtree). The alignments and respective phylogenetic trees were deposited in TreeBASE, study number S30162.

Table 3.

Representative sequence used in this study.

Fungal species	Isolate number	Host	Country	Accession number
				ITS	LSU	RPB2	TEF-1a
Elsinoë abutilonis	CBS 510.40	Callianthe striata (syn. Abultilion striaum)	Brazil	KX887185	KX886949	KX887068	KX886831
Elsinoë akebiae	MUCC 2982	Akebia trifoliata	Japan	OQ504591	OQ504615	OQ472906	OQ472926
Elsinoë ampelina	CBS 208.25	Vitis vinifera	Brazil	KX887186	KX886950	KX887069	KX886832
Elsinoë ampelina	MUCC 2995	Vitis vinifera	Japan	OQ504587	OQ504611	OQ472902	OQ472922
Elsinoë ampelina	MUCC 2996	Vitis vinifera	Japan	OQ504588	OQ504612	OQ472903	OQ472923
Elsinoë ampelina	MUCC 2362	Vitis sp.	Japan	OQ504589	OQ504613	OQ472904	OQ472924
Elsinoë anacardia	CBS 470.62	Anacardium occidentale	India	KX887189	KX886953	KX887072	KX886835
Elsinoë annonae	CBS 228.64	Annona sp.	USA	KX887190	KX886954	KX887073	KX886836
Elsinoë arachidis	CBS 511.50	Arachis hypogaea	Brazil	KX887191	KX886955	KX887074	KX886837
Elsinoë araliae	MUCC 2997	Aralia elata	Japan	OQ504590	OQ504614	OQ472905	OQ472925
Elsinoë araliae	MUCC 3490	Aralia cordata	Japan	OQ504586	OQ504610	OQ472901	OQ472921
Elsinoë arrudai	CBS 220.50	Tournefortia breviflora	Brazil	KX887194	KX886958	KX887077	KX886840
Elsinoë asclepiadea	CPC 18544	Asclepias mellodora (syn. A. curassavica)	Brazil	KX887195	KX886959	KX887078	KX886841
Elsinoë australis	CBS 314.32	Citrus aurantium	Brazil	KX887198	KX886962	KX887081	KX886844
Elsinoë banksiicola	CBS 113734	Banksia prionote	Australia	KX887199	KX886963	KX887082	KX886845
Elsinoë barleriicola	CBS 471.62	Barleria gibsonii	India	KX887200	KX886964	KX887083	KX886846
Elsinoë bidentis	CBS 512.20	Bidens pilosa	Brazil	KX887201	KX886965	KX887084	KX886847
Elsinoë bidentis	MUCC 2983	Bidens pilosa	Japan	OQ504595	OQ504619	OQ472910	OQ472930
Elsinoë brasiliensis	CPC 18528	Chamaesyce hyssopifolia	Brazil	KX887204	N/A	KX887087	KX886850
Elsinoë caleae	CBS 221.50	Calea pinnatifida	Brazil	KX887205	KX886968	KX887088	KX886851
Elsinoë centrolobii	CBS 222.50	Centrolobium robustum	Brazil	KX887206	KX886969	KX887089	KX886852
Elsinoë citricola	CPC 18535 = RWB 1175	Citrus limonia	Brazil	KX887207	KX886970	KX887090	KX886853
Elsinoë corni	MUCC 2998	Cornus florida	Japan	OQ504596	OQ504620	OQ472911	OQ472931
Elsinoë coryli	CBS 275.76	Corylus avellana	France	KX887209	KX886972	KX887092	KX886855
Elsinoë diospyri	CBS 223.50	Diospyros kaki	Brazil	KX887210	KX886973	KX887093	KX886856
Elsinoë embeliae	CBS 472.62	Embelia ribes	India	KX887211	KX886974	N/A	KX886857
Elsinoë erythinae	CPC 18542	Erythrina sp.	Brazil	KX887214	KX886977	KX887096	KX886860
Elsinoë eucalypticola	CBS 124765	Eucalyptus sp.	Australia	KX887215	KX886978	KX887097	KX886861
Elsinoë eucalyptorum	CBS 120084	Eucalyptus propinqua	Australia	KX887216	KX886979	KX887098	KX886862
Elsinoë euphorbiae	CBS 401.63	Euphorbia pariflora (syn. Euphorbia pilulifera)	India	KX887217	KX886980	KX887099	KX886863
Elsinoë fagarae	CBS 514.50	Fagara riedelianum	Brazil	KX887218	KX886981	KX887100	KX886864
Elsinoë fawcettii	CBS 139.25	Citrus sp.	USA	KX887219	KX886982	KX887101	KX886885
Elsinoë fawcettii	MUCC 2993	Citrus deliciosa (syn. Citrus unshiu)	Japan	OQ504575	OQ504599	OQ472891	OQ472912
Elsinoë fawcettii	MUCC 2994	Citrus medica (syn. Citrus hassaku)	Japan	OQ504577	OQ504601	OQ472893	OQ472914
Elsinoë fawcettii	MUCC 2999	Citrus deliciosa (syn. Citrus unshiu)	Japan	OQ504578	OQ504602	OQ472894	OQ472915
Elsinoë fawcettii	MUCC 3000	Citrus deliciosa (syn. Citrus unshiu)	Japan	OQ504576	OQ504600	OQ472892	OQ472913
Elsinoë fici	CBS 515.50	Ficus luschnathiana	Brazil	KX887223	KX886986	KX887105	KX886869
Elsinoë fici-cariae	CBS 473.62	Ficus caria	India	KX887224	KX886987	KX887106	KX886870
Elsinoë flacourtiae	CBS 474.62	Flacourtia sepiaria	India	KX887225	KX886988	KX887107	KX886871
Elsinoë freyliniae	CBS 128204	Freylinia lanceolata	South Africa	KX887226	KX886989	KX887108	KX886872
Elsinoë genipae	CBS 342.39	Genipa americana	Brazil	KX887227	KX886990	KX887109	KX886873
Elsinoë genipae-americanae	CBS 516.50	Genipa americana	Brazil	KX887228	KX886991	KX887110	KX886874
Elsinoë glycines	CBS 389.64	Glycine soja	Japan	KX887229	KX886992	KX887111	KX886875
Elsinoë glycines	MUCC 2984	Glycine soja	Japan	OQ504593	OQ504617	OQ472908	OQ472928
Elsinoë glycines	MUCC 2985	Glycine soja	Japan	OQ504594	OQ504618	OQ472909	OQ472929
Elsinoë hederae	CBS 517.50	Herdera helix	Brazil	KX887231	KX886994	KX887113	KX886877
Elsinoë hydrangeae	MUCC 2988	Hydrangea serrata	Japan	OQ504583	OQ504607	OQ472898	N/A
Elsinoë ichnocarpi	CBS 475.62	Ichnocarpus frustescencs	India	KX887232	KX886995	KX887114	KX886878
Elsinoë jasminae	CBS 224.50	Jasminum sambac	Brazil	KX887233	KX886996	KX887115	KX886879
Elsinoë jasminicola	CBS 212.63	Jasminum malabaricum	India	KX887234	KX886997	N/A	KX886880
Elsinoë krugii	CPC 18531	Euphorbia heterophylla	Brazil	KX887235	KX886998	KX887116	KX886881
Elsinoë lagoa-santensis	CBS 518.50	Brysonima coccolobifolia	Brazil	KX887239	KX887002	KX887120	KX886885
Elsinoë ledi	CBS 167.33	Rhododendron neoglandulosum (syn. Ledum glandulosum)	USA	KX887240	KX887003	KX887121	KX886886
Elsinoë lepagei	CBS 225.50	Manikara zapota (syn. Achras sapota)	N/A	KX887241	KX887004	KX887122	N/A
Elsinoë leucospermi	CBS 111207	Leucospermum sp.	South Africa	KX887242	KX887005	KX887123	KX886887
Elsinoë lippiae	CBS 166.40	Phyla lancelolata (syn. Lippia lanceolate)	USA	KX887248	KX887011	KX887129	KX886893
Elsinoë mangiferae	CBS 226.50	Mangifera foetida (syn. M. indica)	Cuba	KX887249	KX887012	KX887130	KX886894
Elsinoë mattiroloanum	CBS 287.64	Arbutus unedo	Argentina	KX887250	KX887013	KX887131	KX886895
Elsinoë menthane	CBS 322.37	Mentha piperita	USA	KX887253	KX887016	KX887134	KX886898
Elsinoë mimosae	CPC 19478	Mimosa invisa	Brazil	KX887255	KX887018	KX887136	KX886900
Elsinoë oleae	CBS 227.59	Olea europaea	Italy	KX887256	KX887019	KX887137	KX886901
Elsinoë othonnae	CBS 139910	Othonna quinquedentate	South Africa	KR476726	N/A	MK540083	N/A
Elsinoë perseae	CBS 406.34	Persea americana	USA	KX887258	KX887021	KX887139	KX886903
Elsinoë phaseoli	CBS 165.31	Phaeseokus lunatus	Cuba	KX887263	KX887026	KX887144	KX886908
Elsinoë piri	CBS 163.29	Pyrus communis	N/A	KX887267	KX887030	KX887148	KX886912
Elsinoë pitangae	CBS 227.50	Eugenia pitanga	Brazil	KX887269	KX887032	KX887150	KX886914
Elsinoë poinsettiae	CBS 109333	Eugenia pulcherrima	Guatemala	KX887270	KX887033	KX887151	KX886915
Elsinoë pongamiae	CBS 402.63	Pongamia pinnata	India	KX887272	KX887035	KX887153	KX886917
Elsinoë populi	CBS 298.64	Populus deltoides subsp. deltoides (syn. P. serotine)	Argentina	KX887273	KX887036	KX887154	KX886918
Elsinoë protearum	CBS 113618	Protea sp.	Zimbabwe	KX887275	KX887038	KX887156	KX886920
Elsinoë punicae	CPC 19968	Punica granatum	South Africa	KX887276	KX887039	KX887157	KX886921
Elsinoë quercus-illcis	CBS 232.61	Quercus ilex	Italy	KX887277	KX887040	N/A	KX886922
Elsinoë randii	CBS 170.38	Carya sp.	Brazil	KX887278	KX887041	KX887158	KX886923
Elsinoë rhois	CBS 519.20	Toxicodendron vernix (syn. Rhus vernix)	Brazil	KX887280	KX887043	KX887160	KX886925
Elsinoë ricini	CBS 403.63 = ATCC15030	Ricinus communis	India	KX887281	KX887044	KX887161	KX886926
Elsinoë rosarum	CBS 212.33	Rosa sp.	USA	KX887283	KX887046	KX887163	KX886928
Elsinoë salicina	CPC 17824	Salix sp.	USA	KX887286	KX887049	KX887166	KX886931
Elsinoë samecarpi	CBS 477.62	Melanichyla caesia (syn. Semecarpus anacardium)	India	KX887287	KX887050	KX887167	KX886932
Elsinoë sesseae	CPC 18549	Ceatrum laevigatum?	Brazil	KX887288	KX887051	KX887168	KX886933
Elsinoë sicula	CBS 398.59	Prunus amygdalus	Italy	KX887289	KX887052	KX887169	KX886934
Elsinoë soludagnis	CBS 191.37	Solidago fistulosa	USA	KX887290	KX887053	KX887170	KX886935
Elsinoë sumire	MUCC 2992	Viola sp.	Japan	OQ504585	OQ504609	OQ472900	OQ472920
Elsinoë sp.	MUCC 2986	Amphicarpaea edgeworthii	Japan	OQ504592	OQ504616	OQ472907	OQ472927
Elsinoë tanashiensis	MUCC 3463	Juglans sp.	Japan	OQ504581	OQ504605	N/A	N/A
Elsinoë tanashiensis	MUCC 3466	Populus sp.	Japan	OQ504582	OQ504606	OQ472897	OQ472918
Elsinoë tectificae	CBS 124777 = CPC 14594		Australia	KX887292	KX887055	KX887172	KX886937
Elsinoë terminaliae	CBS 343.39	Terminalia catappa	Brazil	KX887293	KX887056	KX887173	N/A
Elsinoë theae	CBS 228.50	Cemellia sinensis (syn. Thea sinensis)	Brazil	KX887295	KX887058	KX887175	KX886939
Elsinoë tiliae	CBS 350.73 = ATCC 24510	Tilia cordata	New Zealand	KX887296	KX887059	KX887176	KX886940
Elsinoë tiliae	MUCC 2989	Tilia playtphyllos	Japan	OQ504579	OQ504603	OQ472895	OQ472916
Elsinoë tiliae	MUCC 2990	Tilia europaea	Japan	OQ504580	OQ504604	OQ472896	OQ472917
Elsinoë veneta	CBS 164.29 = ATCC 1833	Rubus sp.	N/A	KX887297	KX887060	KX887177	KX886941
Elsinoë verbenae	CPC 18561 = RWB 1232	Verbena bonariensis	Brazil	KX887298	KX887061	KX887178	KX886942
Elsinoë violae	CBS 336.35	Viola sp.	USA	KX887302	KX887065	KX887182	KX886946
Elsinoë zizyphi	CBS 378.62 = STCC 14656	Zizyphus rugisa	India	KX887303	KX887066	KX887183	KX886947
Sphaceloma tsujii	MUCC 2991	Paulownia tomentosa	Japan	OQ504584	OQ504608	OQ472899	OQ472919
Myriangium hispanicum	CBS 247.33	Acer monspessulanum	N/A	KX887304	KX887067	KX887184	KX886948

Isolates used in this study mark in boldface.

3. Results

3.1. Phylogeny

The sequencing results of all regions were combined and aligned in a data matrix of 104 OTU belonging to the genus Elsinoë. The final alignment contained a total of 2470 characters consisting of four regional sequences, ITS: 605 sites, LSU: 740 sites, rpb2: 747 sites, and tef: 371 sites, including alignment gaps. ML tree is shown in Figure 2, where the topologies of the generated tree from ML and BI analyses were congruent. Japanese isolates analyzed in this study formed four major clades with hitherto known or newly recognized species. These are clade 1 including Elsinoë bidentis (MUCC2983), clade 2 composed of most Japanese isolates including E. ampelina, clade 3 consisting Japanese isolates of E. glycine (MUCC2985, MUCC2984), E. akebiae (MUCC2982), Elsinoë sp. from Amphicarpeae edgeworthii (MUCC2986), and E. corni (MUCC2998), and clade 4 including cosmopolitan species such as E. fawcetti and E. tiliae.

Figure 2.

Phylogenetic tree of Elsinoë spp. constructed by ML using combined ITS, LSU, rpb2, and tef gene sequence datasets. ML bootstrap values and Bayesian PPs are given near branches (ML/PP).

For LSU, six Elsinoë species from different hosts isolated in Japan have identical sequences, this includes Elsinoë species isolated from Amphicarpha edgeworthii (MUCC2986), E. hydrangea (MUCC2988), E. tanashiensis (MUCC3466), E. sumirensis (MUCC2992), E. araliae (MUCC2997, MUCC3490), and Sphaceloma tsujii (MUCC2991).

In the same analysis of LSU gene, it was also found that E. asclepiadis (CPC 18544) from Brazil have identical sequences as E. akebiae (MUCC2982) and E. bidentis (MUCC2983) isolated from Japan. E. citricola (CPC 18535) from Brazil have identical sequences as E. ampelina of Japanese isolates (MUCC2362) and E. fawcetti of Japanese isolates (MUCC2993, MUCC2994, MUCC3000).

However, in our study, it shows that LSU genes were able to distinguish the USA isolates Elsinoë violae (CBS 336.35) from E. sumire (MUCC2992) as well as E. populi (CBS 298.64) from Argentina with E. tanashiensis (MUCC3463, MUCC3466) that share a same host plant of Viola sp. and Populus sp. Based on this analysis, it could be said that although most of the Japanese isolates have identical LSU genes, LSU can give good resolution based on geographical distribution.

3.2. Taxonomy

Elsinoë akebiae (Kuros. & Katsuki) A.H. Ujat & C. Nakash., comb. nov., Figure 3.

Figure 3.

Morphological feature of Elsinoë akebiae [A, D, F–H: TNS-F-99468; B–C, E: TSU-MUMH11977 (MUCC2982); I-L: TNS-F-99471]. (A) Specimen TNS-F-99468. (B–C) Isolate MUCC2982 on MA (B) and OMA (C). (D) Symptom of scab forming on the leaf of Akebia trifoliata. (E–G) Acervuli. (H) Conidia. (I) Symptom of scab forming on the leaf of Stauntonia hexaphylla. (J–K) Acervuli. (L) Conidia. Scale bars, 25 μm (F–H) and 50 μm (J–L).

MycoBank no: MB847771

Basionym: Sphaceloma akebiae Kuros. & Katsuki, Botanical Magazine Tokyo 70: 131, 1957.

Spots on the leaves scattered and aggregated near leaves veins, usually amphigenous, rounded or near-circular, 0.2–2 mm in diam., often coalescing and extending, flat or slightly depressed in the middle, first brown, then gray-white or grayish brown, with black-brown or purplish-brown margins. In stems, presented as more elongated, 3–5 mm on long side, depressed in the center.

Asexual morph: Acervuli dark brown of 2–3 mm, often coalescing. Conidiogenus cell subcuticular, subsequently erupting with piles of compact conidiophores 9–20 µm thick, hyaline, 5–7 × 2.6–4 µm, conidia hyaline, elliptical, 3.9–8 × 2–3.3 µm. [25].

Cultural characteristic: On MA: colony cerebriform, straw to yellow, folded toward the center of the colony; reverse, pale luteous with folding toward the center is observable. On PDA: surface; covered with white short dense mycelia, a pale luteous margin that turns salmon over time; Reverse, pale luteous that turns olivaceous buff over time. On OMA: surface; olivaceous with short aerial mycelia with folding toward the center, agar color around the colony changed to olivaceous buff; Reverse, rust around the center with citrine growth margin with folding toward the center.

Holotypus: on Akebia trifoliata (Thunb.) Koidz., Japan, Gunma, Tanigawa, 19 July 1936, by E. Kurosawa and S. Katsuki (TNS-F-99470 = SK 1498).

Herbarium specimens examined: on A. quinate (Thunb. ex Houtt.) Decne., Mt. Tsukuba, 15 August 1935, collected by E. Kurosawa (TNS-F-99468 = SK 1495); on A. trifoliata, see holotypus; Japan, Tokyo, Machida, July 2008, collected by T. Ono (epitype designated here, TSU-MUMH11977; ex-epitype culture MUCC2982); on Stauntonia hexaphylla (Thunb.) Decne., Japan, Kagoshima, Kagoshima University Botanical Garden, 26 October 1949, collected by S. Katsuki (TNS-F-99471 = SK1510, SK Herbarium collection housed in TNS).

Note: Present species was described by Kurosawa and Katsuki [25]. The morphological characteristics of holotype specimen were confirmed as the anamorphic state of the genus Elsinoë, the genus Sphaceloma. For the phylogenetic study, the epitype specimen was selected from the currently collected specimen. The nucleotide sequence of LSU gene is not enough to eliminate species of E. akebiae (MUCC2982), E. bidentis (MUCC2983), and E. asclepiadis (CPC 18544). A new combination E. akebiae is proposed in this study. It is inhabiting on Akebia sp. and Stauntonia hexophylla, which are of the family Lardizabalaceae whereby these plants are native to Far East Asia.

Elsinoë hydrangeae T. Ono, A. H. Ujat & C. Nakashima, sp. nov., Figure 4.

Figure 4.

Morphological feature of Elsinoë hydrangeae [A–G: TSU-MUMH11976 (MUCC2988)]. (A, D–E) Specimen TSU-MUMH11976. (B–C) Isolate MUCC2988 on MA (B) and OMA (C). (F) Conidia. (G) Acervuli. Scale bars, 50 μm.

MycoBank no: MB847772

Etymology: Named after the genus of the host plant (Hydrangea) from which the ex-type strain was obtained.

Leaf spot pale yellow to purple slate with brick margin, circular, 2 mm in diameter on the leaves, along the leaves vein, expanded toward adaxial surface and coalescent in a chain-like pattern, becoming grayish white in the middle, petiole and stems crinkled, often observed on newly developed leaves. Brown lesions with pale yellow scabs can be observed on the abaxial surface of the leaves when the spots coalescent. Mycelia dense on the irregular to globose acervuli.

Asexual morph: Acervuli dark brown, often coalescing, 7–27 µm in height, 1.4–6.8 mm in diam., subcuticular, subsequently erupting with compact conidiogenous cells. Conidiogenous cells hyaline, cylindrical monophialidic, sporulating enteroblastically, determinate, 15–27 × 3.2–4.5 µm. Conidia hyaline, elliptical to spindle-shaped, aseptate, 5–7.5 × 2–3.3 µm.

Cultural characteristic: On MA: surface raised, cerebriform, erumpent, salmon to sienna; reverse, red with white margin. On PDA: surface; erumpent and folded toward the center, yellow to salmon, sparse white aerial mycelium, reverse: dark red and folded in toward the center. On OMA: surface irregular, folded, yellow to red, dense short aerial mycelia, mucilaginous drops observable centrally. Reverse, flesh to rust with white margins and folded on the margins.

Holotypus: on Hydrangea serrata (Thunb.) Ser., Japan, Tokyo, Tachikawa, 25 August 2008, collected by T. Ono (TSU-MUMH11976, ex-type culture MUCC2988).

Host: Hydrangea serrata, H. serrata (Thunb.) Ser. var. yesoensis (Koidz.) H. Ohba, H. hirta (Thunb.) Siebold et Zucc., Herbarium specimen examined: see holotype.

Notes: This study also proposes Elsinoë hydrangeae as novel species of Elsinoë infecting Hydrangea species. Ono et al. [26] described this as Sphaceloma sp. on Hydrangea where it was first observed in Hydrangea serrata in the open field in Tachikawa and Hino in August 2008. The collected sample was brought back for pathogenicity test on other Hydrangea species to determine the host specificity. Based on the previous study, E. hydrangeae isolated from H. serrata shows a range of different pathogenicity on different Hydrangea species. While E. hydrangeae shows no pathogenicity on H. quercifolia W.Bartram, weak pathogenicity was observed on H. macrophylla (Thunb.) Ser. and H. arborescence L. Furthermore, it shows the same pathogenicity level as H. serrata and H. hirta (Thunb.) Siebold & Zucc. and H. serrata var. yesoensis (Koidz.) H.Ohba. This result shows that Elsinoë species is highly related to the specific host plant and its pathogenicity are differentiated within a host plant genus. Initial molecular analysis conducted by Ono et al. [26] by using rDNA ITS region shows that E. hydrangeae was closely related to E. araliae. As shown in Figure 1, although E. hydrangeae and E. araliae were placed in the same clade, the clade is composed of species on various host plants that are native to East Asia. Moreover, the morphology of isolates of E. araliae and E. hydrangeae grown on malt agar, oatmeal agar, and potato dextrose agar are very distinct from each other.

Elsinoë sumire T. Ono, A. H. Ujat & C. Nakashima, sp. nov., Figure 5.

Figure 5.

Morphological feature of Elsinoë sumire [A–I: TSU-MUMH11977 (MUCC2992)]. (A, D) Specimen TSU-MUMH11976. (B–C) Isolate MUCC2992 on MA (B) and OMA (C). (E–F) Conidiogenous cell. (G) Conidia. (H–I) Acervuli. Scale bars, 100 μm (E–F, H–I) and 10 μm (G).

MycoBank no: MB847783

Etymology: The name is derived from the generic name of Viola sp. in Japan, which means violet.

Lesions white to straw irregular, circular to angular, surrounded with citrine green margin, scabbed in numerous on adaxial and abaxial surface of the leaves, scattered along the vein, enlarged, overlapped, up to 3 mm eventually perforated.

Asexual morph: Acervuli pale brown, globose and oblate, enlarged and confluent eventually, solitary on the stems, up to 1 mm in width. Conidiogenous cells hyaline, cylindrical to ampulliform, monophialidic, sporulating enteroblastically, integrated, 0.1–0.2 × 1.0–1.4 µm. Conidia hyaline, aseptate, globular, ellipsoid to irregular, 0.1–0.15 × 0.1–1.0 µm.

Cultural characteristic: On MA: colony surface slightly raised, smooth, salmon with a white margin; aerial mycelia short and dense; reverse, red with a white margin, folded. On PDA: surface; folded and covered with white dense aerial mycelia sparse on the edges, salmon; Reverse, bay to umber with white margin and folded toward the center. On OMA: surface; smooth, bay to rust, and covered by white arial mycelia; reverse, red and folded with cracks forming toward the center.

Holotypus: Japan, Tokyo, Itabashi, on Viola sp., 11 June 2008, collected by T. Ono (TSU-MUMH11975; ex-type culture: MAFF 243579 = MUCC2992).

Host: Viola sp.

Herbarium specimens examined: see holotype; on Viola odorata L., Japan, Matsudo, Chiba, 3 October 1939, collected by E. Kurosawa (TNS-F-185408)

Notes: The type specimen had been identified as “Sphaceloma violae” and its isolate was deposited in the culture collection, at the Research Center of Genetic Resources, NARO, Tsukuba, Ibaraki, Japan (MAFF 243579 = MUCC2992). However, it is genetically and morphologically distinguishable from the ex-type strain of E. violae (CBS 336.35). The original description provided by Massey and Jenkins, [27] stated that the scab formed will enlarge into scabby, circular spot, often vinaceous buff although it may be ashen or white. While on E. sumire scab formed is epileptic and will overlap with each other when enlarged, often white to straw with citrine green margin.

Elsinoë sumire, which is the second Elsinoë species infecting Viola spp., was proposed as a new species in this study. Massey and Jenkins (1935) described another Elsinoë species, E. violae, from the scab disease of Viola spp. in the United States. According to the Fungal Databases, U.S. National Fungus Collections, ARS, USDA [28], the species habitats were mainly from North America and Europe, and on more than 15 Viola species. On the other hand, Kurosawa and Katsuki [25] reported the distribution of E. violae in Japan without the morphological descriptions of several voucher specimens. Two herbarium specimens of them were examined in this study. Those morphological characters and symptoms on the host plants were distinguishable, as mentioned above. In the phylogenetic tree (Figure 2), E. sumire and E. violae are placed in two different clades. While the host plant of E. violae and E. sumire are of the same genus, the symptoms on the host plant and the micromorphology varies greatly.

Elsinoë tanashiensis A.H. Ujat & C. Nakashima, sp. nov., Figure 6.

Figure 6.

Morphological feature of Elsinoë tanashiensis on Populus deltoides [A–F: TFM:FPH-01697 (MUCC3466)] and Juglans sp. [G, J–K: TFM:FPH-0330; H–I: TNS-F-185379]. (A) Specimen TFM:FPH-01697. (B–C) Isolate MUCC3466 on MA (B) and OMA (C). (D–E) Acervuli. (F) Conidiogenous cell. (G) Specimen TFM:FPH-0330. (H) Specimen TNS-F-185379 (I) conidiomata on Juglans sp. leaf (TNS-F-185379). (H) Conidia. (I) Acervuli. Scale bars, 100 μm (D) and 50 μm (E–F, J–K).

MycoBank no: MB847784

Etymology: The name reflects the locality of the holotype sample collected, Tanashi, Tokyo.

On the leaf's surface, lesions are observed as both black and white spots. Black spots, scattered, circular, depressed, and turned into grayish brown at the center, or developed as white spots and raised, scabbed in numerous on the adaxial and abaxial surface of the leaves, black spotted scab turned int light brown with no necrosis, on the abaxial surface, 1–2 mm in diameter. Light brown to yellow mucilaginous masses were observed on the lesion with grayish brown scab under humid conditions, and pale orange to brown mucilaginous masses observed on the white scab.

Asexual morph: Acervuli pale brown, globose and oblate, enlarged and confluent eventually, solitary on the stems, up to 1 mm in width. Conidiogenous cells hyaline, erumpent, cylindrical to ampulliform, monophialidic, sporulating enteroblastically, integrated, 70–200 mm. Conidia hyaline, aseptate, globular, ellipsoid to irregular, 4–5.5 × 1.5 µm.

Cultural characteristic: On MA: colony surface with short dense aerial mycelia, salmon, folded toward center of the colony; reverse, bay to flesh with rings. On PDA: surface; slightly raised and folded, covered with white aerial mycelia, saffron with pale luteous margin; reverse; pale luteous with rings. On OMA: surface; bay, covered by white arial mycelia with rings, agar around the colony changed to amber; Reverse, amber to umber with folding toward the center.

Holotypus: Japan, Tokyo, Tanashi, on Populus deltoides, October 1956 by O. Chiba and T. Kobayashi (TFM:FPH-01697, ex-type culture: MUCC3466 = MAFF 410485)

Host: Populus deltoides Marshall, P. nigra × P. trichocarpa, P. deltoides var. misouriensis, P. charkowiensis × P. candina, P euramericana I-154, P. euramericana × P. serotina, P. gelrica × P. robusta, P. “Leipzig”, P. eucalyptus [29]

Herbarium specimens examined: see holotype; on Juglans L., Japan, Akita, Funaoka, Kawabe, 26 June 1951, collected by K. Ito and O. Chiba, (TFM:FPH-0330); on Juglans regia L., Japan, Saitama, Hatogaya, 21 August 1938, collected by E. Kurosawa (TNS-F-185379).

Note: The isolate MCUU3466 was firstly described as Sphaceloma sp. on Populus [29] as the morphological description is different from E. populi described by Jenkins [30]. ITS region of MUCC3466 is identical with MUCC3463.

Elsinoë tanashiensis isolated from Populus deltoides from Japan is closely related to Elsinoë isolated from Juglans spp. Based on the phylogenetic analysis, ITS region is identical, while LSU gene are highly similar with 99% (1/1185) similarity. RPB2 and tef could not differentiate these two species as there are no sequences obtained from Elsinoë isolated from Juglans sp. As a pure culture of Elsinoë isolated from Juglans could not be established, morphological differences between isolates from Populus sp. and Juglans sp. could not be observed. However, the morphology of the conidiogenous cell of these two Elsinoë sp. on herbarium specimens are identical, whereby the size and shape of the conidiogenous cell is also same. Due to the similarity of the conidiogenous cell and conidia size, these two species could not be determined as different species although they infect different host plants.

In this study, novel species of Elsinoë tanashiensis is also proposed as the species infecting Populus sp. in Japan. Although there is already an established species of Elsinoë populi known to infect Populus species in South America [1] and Europe, [30], the first record of occurrence in Japan was only recorded as “Sphaceloma sp.” on Populus [29]. It was mentioned in the previous study by Chiba and Kobayashi [29] that this fungus could not be identified as Sphaceloma populi due to the difference in the morphological description with the protologue. Although Populus deltoides, the host plant of Elsinoë tanashiensis, is not native to Japan, it is widely planted in Japan.

Similarly, for Elsinoë sp. infecting the Juglans sp. in Japan, this study identifies the isolates as E. tanashiensis as the phylogenetic analysis and observation from specimens show high similarity with specimens from Populus sp. Although there are established species of Elsinoë randii known to infect Carya pecan in Brazil [31] and Juglans sp. in North and South America [32] and on Juglans regia L. and Juglans mandshurica Maxim. [33], this study shows that E. randii are placed in a different clade compared to E. tanashiensis.

Elsinoë sp., Figure 7.

Figure 7.

Morphological feature of Elsinoë sp. on Amphicarpaea edgeworthii [A–B: MUCC2986; C–F: TNS-F-185385] (A–B) Isolate MUCC2986 on MA (A) and PDA (B). (C) Specimen TNS-F-185385. (D) Conidiogenous cell. (E) Acervuli. (F) Conidia. (I) Acervuli. Scale bars, 50 μm (D–E) and 10 μm (F).

Cultural characteristic: On MA: colony surface cerebriform, ranging from blood color to bay to pale luteous. Older mycelia turn herbage green, folded toward the center of the colony; reverse, scarlet to pale luteous, folded toward the center, raised. On PDA: surface; folded toward the center, saffron to peach on the outer part, herbage green to yellowish green on the center.; reverse; raised, crack toward center, blood color to the bay. On OMA: surface; conidiomata olivaceous, mycelia grow into straw and turn citrine overtime; Reverse, buff and turn agar into translucent color overtime.

Asexual morph: Acervuli hyaline, coalescing, compact, up to 100 µm wide. Conidiogenous cells hyaline, erumpent, cylindrical to spindle-shape, monophialidic, sporulating enteroblastically, integrated, 15–20 mm. Conidia hyaline, aseptate, globular to ellipsoid, 1.0–2.3 × 1.0 µm.

Host: Amphicarpea edgeworthii Benth.

Herbarium specimen examined: on Amphicarpea edgeworthii (syn. Falcata japonica (Oliv.) Kom.), Japan, Tokyo, Saginomiya, 17 October 1937, collected by E. Kurosawa (TNS-F-185385).

Note: Sphaceloma kurozawarnum was first proposed as a new species infecting Amphicarpea edgeworthii (syn. Falcata japonica) by Kurata [34]. Although the host plant was of the same family Fabaceae as Glycine sp., the author noted that it was different from Elsinoë glycine. This species was treated as nomen nudem due to no description of morphological characteristics. In this study, the isolate used for phylogenetic analysis (MUCC2986) and the specimens observed (TNS-F-185385) are not linked. Both the bootstrap value of ML analysis and PP value of Bayesian analysis shows strong support of independent cladding, hence this isolate was treated as Elsinoë sp. on Amphicarpea edgeworthii.

Sphaceloma tsujii Hara, Figure 8.

Figure 8.

Morphological feature of Sphaceloma tsujii on Paulownia tomentosa [A–E: TFM:FPH-0448. (A) Specimen TFM:FPH-01697. (B) Conidiomata on leaf of Paulownia tomentosa. (D) Acervuli. (E) Conidia. (F) Isolate MUCC2991 on MA. Scale bars, 50 μm.

Paulownia leaves, buds, petioles, and stems are first observed with small, slightly amber spots on the leaf surface, which enlarged in round or conical shape, with dark brown margins of 0.8–1.2 mm in diameter, the center symptoms grayish red, slightly concave, eventually perforated. The abaxial symptoms were observed as reddish brown with light brown edges. On the petiole, veins, and shoots, the spots may be round or elliptical and reddish brown in color, but later turn grey and fall into a blotch [35].

Asexual morph: Mycelium branched with septate, colorless, 3–4 um width, acervuli scattered, subcuticular subsequently erumpent, 30–60 µm in diameter. Conidiogenous cell cylindrical, hyaline, monophialidic, 5–13.2 µm [35].

Cultural characteristic: On MA: colony surface cerebriform, salmon; reverse, bay to flesh. On PDA: surface; cerebriform, folded toward center, rust with buff margin; reverse; brick to cinnamon, folded toward center. On OMA: surface; amber, covered by white short dense arial mycelia folded toward center; Reverse, straw with folding toward the center.

Host: Paulownia tomentosa Steud.

Herbarium specimen examined: on Paulownia tomentosa Steud., Japan, Tokyo, Forest Experimental Station Meguro, 6 July 1959, collected by K. Ito, (TFM:FPH-0448).

Note: ITS region could not differentiate Elsinoë tsujii (MUCC2991) and Elsinoë rhois (CBS 519.50), rpb2 region could not differentiate Sphaceloma tsujii (MUCC2991) and Elsinoë tanashiensis (MUCC3466). Initially described as Gloeosporium sp. on Paulownia sp. [36], Sphaceloma tsujii was proposed as a new species by Hara [35] as a pathogen of Paulownia sp., however, in the protologue, there was no record of the specimens and isolates kept as holotypus. The isolate used in this study is not linked to any specimens available in records.

3.3. Taxonomical treatment based on the herbarium specimens

Elsinoë catalpae (Kuros. & Katsuki) A.H. Ujat & C. Nakash. comb. nov.

Basionym: Sphaceloma catalpae Kuros. & Katsuki, Botanical Magazine Tokyo 69: 316, 1956.

Holotype: Japan, Tokyo, Hinodai, on Catalpa speciosa E.Y. Teas, 2 September 1951, collected by E. Kurosawa (SK 1062).

Herbarium specimen examined: Japan, Chiba, Matsudo, on Catalpa speciosa E.Y. Teas, 12 September 1938, by E. Kurosawa (TNS-F-185400 = SK 1061).

Notes: The examined specimen was described in the protologue by Kurosawa and Katsuki [33].

Elsinoë japonicum (Kuros. & Katsuki) A.H. Ujat & C. Nakash. comb. nov.

Basionym: Sphaceloma japonicum Kuros. & Katsuki, Botanical Magazine Tokyo 70: 132, 1956.

Holotypus: Japan, Saitama, Hatogaya, on Ilex serrata var. sieboldii(Miq.) Rehder [= Ilex serrata Thunb.], 5 September 1938, by E. Kurosawa (SK 1484).

Herbarium specimen examined: Japan, Saitama, Hatogaya, on Ilex serrata var. sieboldii (Miq.) Rehder [= Ilex serrata Thunb.], 27 September 1938, collected by E. Kurosawa (TSN-F-185384 = SK 1486).

Notes: The examined specimen was described in the protologue by Kurosawa and Katsuki [25].

Elsinoë paederiae (Kuros. & Katsuki) A.H. Ujat & C. Nakash. comb. nov.

Basionym: Sphaceloma paederiae Kuros. & Katsuki, Annals of the Phytopathological Society of Japan 21: 15, 1956.

Holotype: Japan, Tokyo, Gotanda, on Paederia scandens (Lour.) Merr. [= P. foetida L.], 24 July 1938, collected by E. Kurosawa (TNS F-185381 = SK 1380).

Herbarium specimen examined: See holotype.

Notes: The examined specimen was described in the protologue by Kurosawa and Katsuki [37].

Elsinoë peucedani (Kuros. & Katsuki) A.H. Ujat & C. Nakash. comb. nov.

Basionym: Sphaceloma peucedani Kuros. & Katsuki, Botanical Magazine Tokyo 70: 134, 1956.

Holotype: Japan, Kanagawa, Mt. Ohkusu-yama, on Peucedanum decursivum (Miq.) Maxim. [= Angelica decursiva (Miq.) Franch. et Sav.], 15 September 1940, collected by E. Kurosawa (TNS F-185382 = SK 1512).

Herbarium specimen examined: Japan, Kanagawa, Mt. Ohkusu-yama, on Peucedanum decursivum (Miq.) Maxim. [= Angelica decursiva (Miq.) Franch. et Sav.], 15 September 1940, collected by E. Kurosawa (holotype TNS-F-185382 = SK 1512; TNS-F-185404).

Notes: The examined specimen was described in the protologue by Kurosawa and Katsuki [33].

Elsinoë zelkovae (Kuros. & Katsuki) A.H. Ujat & C. Nakash. comb. nov.

Basionym: Sphaceloma zelkovae Kuros. & Katsuki, Botanical Magazine Tokyo 69: 318, 1956.

Holotype: Japan, Saitama, Yorii, on Zelkova serrata (Thunb.) Makino, 30 July 1936, by E. Kurosawa (TNS-F-185380 = SK 1470).

Herbarium specimens examined: Japan, Saitama, Yorii, on Zelkova serrata (Thunb.) Makino, 30 July 1936, collected by E. Kurosawa (TNS-F-185380 = SK 1470); Mie, Tsu, on Z. serrata, 11 October 2022, collected by A.H. Ujat & C. Nakashima (TSU-MUMH11970).

Notes: The examined specimen was described in the protologue by Kurosawa and Katsuki [33].

4. Discussion

This study examined the taxonomical position of 22 fungal isolates of the genus Elsinoë from Japan based on their morphological and molecular phylogeny. Multi-locus phylogenetic analyses showed that isolates were divided into several different clades, and three isolates were recognized as new species. These are Elsinoë hydrangeae, E. tanashiensis, and E. sumire. In accordance with the “one fungus = one name” concept, the anamorphic genus Sphaceloma is being relocated under the teleomorphic genus Elsinoë [1,2]. In this study, one new combination of Japanese Sphaceloma species was proposed to transfer into Elsinoë based on the morphological characteristics of the type specimen and phylogenetic position of the ex-epitype isolate. Moreover, many Japanese isolates formed a clade supported by a high posterior probability value (BS/PP= 88/67) (Figure 2, Clade 2). The internal clade of clade 2 (Clade 2a; BS/PP = 89/97) is composed of several species on various host plants, including herbaceous and arboreal plants. On the other hand, it includes the ex-type strains of Elsinoë rhois (Bitanc. & Jenkins) X.L. Fan & Crous on Toxicodendron vernix from Brazil and sister to a cosmopolitan species Elsinoë ampelina (de Bary) Shear on Vitis vinifera from Brazil, forms another internal clade (clade 2b) with Japanese three isolates supported with a high BS/PP (99/100). In addition, another clade (Clade 2c; BS/PP = 90/100) was composed of E. ricini (Jenkins & C.C. Cheo) X.L. Fan & Crous from India, E. akebiae from Japan, Elsinoë sp. on A. edgeworthii from Japan, and E. glycines (Kurata & Kurib.) X.L. Fan & Crous from Japan. These results suggest that Elsinoë species acquire various host plants and speciated in Japan. it could be hypothesized that Japan might be one of the centers of the speciation and diversification of the genus Elsinoë.

Elsinoë populi (Sacc.) X.L. Fan & Crous from Argentina (Clade 4) and E. tanashiensis from Japan (Clade 2a) are placed in different subclade, even though they have a common host plant of genus Populus. Similar examples are observed on Elsinoë species on the plant genus Viola (E. violae (Massey & Jenkins) X.L. Fan & Crous (Clade 5) and E. sumire (Clade 2a)). It is suggested that these species obtained the host plant at different places independently. Elsinoë corni, isolated from Cornus florida in Japan are placed in Clade 3 as an independent species. In this study, morphological characteristic and the phylogenetic analysis comparison could not be conducted as there are no specimens linked to the isolate used in this study (MUCC2998) and no phylogenetic analysis were conducted on the holotypus material by Jenkins & Bitancourt [38].

Conversely, several species, widely distributed worldwide and having similar sequences on those generic loci regardless of the quite far geographical origin, were known in the previous study [1,2,17], and some are reconfirmed in this study, too. These are E. ampelina, E. bidentis (Bitanc. & Jenkins) Fan & Crous, E. fawcettii Bitanc. & Jenkins and E. tiliae Creelman. On Elsinoë species infecting Japanese citrus cultivar, only E. fawcetti was identified and not E. australis or E. citricola. Although E. australis is a phytopathogens of citrus, it remains restricted to Australia, Bolivia, Brazil, and Ethiopia [1]. E. citricola, on the other hand, was initially identified as Sphaceloma fawcettii, however it can be distinguished based on molecular data on rpb2 and tef region even though ITS and LSU region failed to distinguish between E. fawcetti and E. citricola [1]. This was confirmed in this study as well where the E. fawcetti isolated in Japan have similar ITS and LSU region with E. citricola but different rpb2 (63/745) and tef (2/370). The host plants of these species have been cropped or distributed around the world. From this situation, it is suggested that a specific strain of Elsinoë species has been spreading with the migration of the host plant.

Generally, each Elsinoë species has a narrow host range, which occurs on only one host species or genus [1,2]. In this study, despite having the same host plant, Viola sp., a new species of Elsinoë was proposed based on the morphological and phylogenetic analysis. Fan et al. [1] showed that E. violae has more than one host genus, Viola sp. and Symphocarpos sp. (Caprifoliaceae), from the phylogenetic analysis using a multi-locus combined matrix. On the other hand, our results suggested that E. sumire is a closely related species of E. rhois (Bitanc. & Jenkins) X.L. Fan & Crous on Rhus vernix L. (syn. Toxicodendron vernix Kuntze). According to Fan et al. [1], Elsinoë leucospermi, E. anacardia, E. violae and E. piri were found to occur on more than one host genus. Elsinoë piri (Woron.) Jenkins on different host genera, Pyrus (CBS 163.29) and Malus (Rosaceae) (CBS 179.82), has been recognized as one species of Elsinoë, having 6/370 cites changes of tef region sequence. In this study, the criteria of species delimitation of the genus Elsinoë were comprehensively judged based on the host plants, morphological characteristics, and phylogenetic relationship and did not synonymize the hitherto known species. Future discussion about Elsinoë species delimitation based on experimental host range or new barcode region sequence of this genus is needed.

In conclusion, the result of this study shows that many of the Japanese isolates are confined into one main clade which shows that Japanese isolates of Elsinoë could be endemic to East Asia. This study concurs with the previous study by Fan et al. [1] mentioning that Elsinoë species appear to be host specific. Although in our study there are lack of fresh specimens and isolates, type material and isolates designated from the previous study provide to be helpful in creating a workable taxonomy for Japanese isolates of Elsinoë.

Funding Statement

This study received financial support from JSPS KAKENHI [grant number 20K06146].

Disclosure statement

No potential conflict of interest was reported by the author(s).