Taxonomical re-examination of the genus Neofusicoccum in Japan

Abstract

Neofusicoccum is a genus of plant pathogenic fungi associated with various woody plants. Since Neofusicoccum has very similar morphological characteristics to the genus Botryospaheria, molecular phylogenetic analysis is essential to determine its taxonomic position. In Japan, a comprehensive taxonomic study of the genus Neofusicoccum has not been conducted. To elucidate the species diversity in Japan, we reexamined Japanese isolates of Neofusicoccum based on their morphology and molecular phylogenetic relationships, using the internal transcribed spacer (ITS) regions rpb2, tef1-α, and tub2. The Japanese isolates were divided into five clades recognized as the species. These species were N. parvum, other Neofusicoccum spp., and three new species proposed in this study, N. hyperici, N. miyakoense, and N. okinawaense. Furthermore, Physalospora laricina, which causes shoot blight of larch (Larix spp.), was transferred to the genus Neofusicoccum, and we propose its epitype and ex-epitype isolate.

1.Introduction

Neofusicoccum (Botryosphaeriaceae) is a genus of plant pathogenic fungi associated with various woody plants that causes shoot blight, canker, and dieback of fruit and forest trees (Phillips et al., 2008). The genus was established by Crous et al. (2006), based on the type species N. parvum (Pennycook & Samuels) Crous, Slippers & A. J. L. Phillips, and is closely related to the genus Fusicoccum. Neofusicoccum is characterized by fusiform to ellipsoid aseptate conidia and “dichomera-like” synanamorphs, which septate and turn brown with age, to form globose to pyriform conidia (Barber et al., 2005; Phillips, Rumbos, Alves, & Correia, 2005; Crous et al., 2006). The sexual morphs of Botryosphaeriaceae are similar in morphology, such that these structures are difficult to distinguish among the genera Botryosphaeria, Fusicoccum, and Neofusicoccum. These genera have been historically confused. The genus Fusicoccum, which had been regarded as an asexual morph of the genus Botryosphaeria, is known to be polyphyletic and synonymous with various other genera (Phillips, Fonseca, Povoa, Castilho, & Nolasco, 2002; Crous et al., 2006). Therefore, molecular phylogenetic analysis is essential to discriminate between Fusicoccum and other Fusicocum-like genera (Marin-Felix et al., 2017). Taxonomic studies of the genus Neofusicoccum and its related genera have been conducted based on worldwide multi-locus phylogenetic analyses (Dissanayake, Phillips, Hyde, & Li, 2016; Marin-Felix et al., 2017; Yang et al., 2017; Zhang, Lin, He, & Zhang, 2017). New generic criteria based on molecular phylogeny have resulted in an increased number of species, due to reclassification of species in the hitherto known Botryosphaeriaceae. According to the fungal names database Mycobank ( https://www.mycobank.org, accessed on 30 Sep 2020), forty-six species of Neofusicoccum have been described.

In Japan, several diseases caused by Neofusicoccum species have been reported: black leaf blight of Cymbidium spp. caused by N. parvum (Suzuki, Hattori, Motohashi, Natsuaki, & Okura, 2018); canker of Platanus × acerifolia caused by N. parvum (Motohashi et al., 2016); stem end rot of Mangifera indica by N. parvum (Takushi, Ajitomi, Arasaki, Ooshiro, & Sato, 2017) and Neofusicoccum sp. (Hara et al., 2016); fruit rot of chestnut caused by Neofusicoccum sp. (Sasaki, Nakamuyra, & Adachi, 2019). Etiological studies have been conducted in response to severe damage of important crops, including identifying the causal fungi; however, a comprehensive study on the diversity of Japanese Neofusicoccum species from various hosts has not yet been conducted. Moreover, our molecular phylogenetic analysis of Botryosphaeriaceae showed that many isolates previously identified as Botryosphaeria spp. belong to the Neofusicoccum clade (Hattori, Akiba, & Nakashima, 2019). In many Japanese isolates of the genus Neofusicoccum, the taxonomic positioning is unclear. The purpose of this study was to elucidate the species diversity of the genus Neofusicoccum in Japan. Therefore, we examined the morphological characteristics and molecular phylogenetic relationships of isolates preserved in culture collections as species of Botryosphaeria, Fusicoccum, and Guignardia, in addition to the isolates obtained during this study.

2. Materials and methods

2.1. Fungal isolation

Mango (M. indica) and Coffee (Coffea sp.) leaves or branches showing symptoms of leaf or shoot blight were collected from Miyako-jima Island, Okinawa, Japan. Isolates of Neofusicoccum spp. were obtained using the single conidia isolate method described by Nakashima et al. (2016). The isolates were cultivated on a potato dextrose agar (PDA) medium (Nissui Pharmaceutical Co., Ltd., Japan) and transferred to a pine needle agar (PNA) medium to observe conidiomata and conidia (Smith, Wingfield, Coutinho, & Crous, 1996). For accurate identification, the twelve isolates and two specimens that had been preserved as Botryosphaeria, Fusicoccum, or Guinardia species at the Laboratory of Forest Pathology, Forestry and Forest Products Research Institute (FFPRI, TFM: FPH) (Tsukuba, Ibaraki, Japan) and the culture collection of the Laboratory of Phytopathology, Mie University (TSU-MUCC) (Tsu, Mie, Japan) (Table 1) were revitalized. Herbarium specimens were borrowed from the Iwate University Museum (IUM) (Iwate, Japan) for the morphological studies. All isolates were cultured at room temperature (22 °C). All dried cultures and specimens for microscopic studies were kept in the mycological herbarium at Mie University (TSU-MUMH), and these established isolates have been maintained in the culture collection of TSU-MUMH and TSU-MUCC.

Table 1. List of JapaneseNeofusicoccum isolates and specimens used in this study.

Fungal species	Isolate No.	Original No.	Material No.	Host Family	Host species	Regions	Identified by previous study	Reference
N. hyperici	MUCC 241	–	MUMH 10423	Hypericaceae	Hypericum patulum	Aichi	Fusicoccum sp.	this study
N. hyperici	MUCC 2509	B1-2 (MAFF 410797)	–	Hypericaceae	Hypericum galioides	Tokyo	Botryosphaeria sp.	this study
N. hyperici	MUCC 650	–	MUMH 10606	‎Stachyuraceae	Stachyurus praecox	Aichi	Fusicoccum sp.	this study
N. laricinum	MUCC 2660	GC74 (MAFF 410183)	–	Pinaceae	Larix kaempferi	Hokkaido	Botryosphaeria laricina	Motohashi et al. (2009)
N. laricinum	MUCC 2662	GC59 (FFPRI 411215)	TFM: FPH-4038	Pinaceae	L. decidua	Ibaraki	Guignardia laricina	this study
N. laricinum	MUCC 2663	GC38 (FFPRI 411216)	–	Pinaceae	L. decidua	Iwate	G. laricina	this study
N. laricinum	MUCC 2666	GC14 (FFPRI 411217)	–	Pinaceae	L. kaempferi	Aomori	G. laricina	this study
N. laricinum	MUCC 2669	GC82 (FFPRI 411218)	–	Pinaceae	L. kaempferi	Iwate	G. laricina	this study
N. miyakoense	MUCC 2585	–	MUMH 11936	Coffeeae	Coffea sp.	Okinawa, Miyako-jima island	–	this study
N. miyakoense	MUCC 2586	–	MUMH 11937	Anacardiaceae	Mangifera indica	Okinawa, Miyako-jima island	–	this study
N. okinawaense	MUCC 789	MAFF 240624	MUMH 10839	Lythraceae	Lagerstroemia speciosa	Okinawa, Main island	Fusicoccum sp.	Nakashima (2008)
N. parvum	MUCC 2511	B1-10 (FFPRI 411214)	TFM: FPH-5589	Tamaricaceae	Tamarix tenuissima	Niigata	Botryosphaeria sp.	this study
N. parvum	MUCC 392	–	MUMH 10368	Ericaceae	Rhododendron hybrids	Aichi	Fusicoccum sp.	this study

2.2. Molecular and phylogenetic analysis

Genomic DNA was extracted from mycelial disks after culturing for seven days on PDA plates using a DNeasy UltraClean Microbial Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Targeted sequences of the rDNA ITS, rpb2, tef1-α, and tub2 gene-coding regions were amplified with a T100 Thermal Cycler (Bio-Rad, Tokyo, Japan) by polymerase chain reaction (PCR). The total volume of the PCR mixture was 12.5 μL. It consisted of 1 to 10 ng genomic DNA, 0.05 μL (ITS and tub2) or 0.1 μL (tef1-α and rpb2) of 0.25 unit Taq DNA polymerase (Bioline, London, UK), 1.25 μL of 10× NH₄ reaction buffer (Bioline), 1.9 mM (tub2) or 2.5 mM (ITS, rpb2, and tef1-α) MgCl₂ (Bioline), 2.5 mM (ITS) or 5.0 mM (tef1-α, tub2 and rpb2) each of deoxyribonucleotide triphosphate mixture (Bioline), 0.2 μM of each primer, 5.6% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA; added only for tef1-α amplification), and sterilized distilled water up to 12.5 μL. The rDNA ITS was amplified with the primers V9G ( (Bioline), 2.5 mM (ITS) or 5.0 mM (tef1-α, tub2 and rpb2) each of deoxyribonucleotide triphosphate mixture (Bioline), 0.2 μM of each primer, 5.6% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA; added only for tef1-α amplification), and sterilized distilled water up to 12.5 μL. The rDNA ITS was amplified with the primers V9G (de Hoog & Gerrits van den Ende, 1998) and ITS4 (White, Bruns, Lee, & Taylor, 1990); rpb2 with the primers RPB2-5f2 and fRPB2-7cR (Liu, Whenlen, & Benjamin, 1999); tef1-α with the primers EF1-728F, and EF1-986R (Carbone & Kohn, 1999); and tub2 with the primers BT2A and BT2B (Glass & Donaldson, 1995). The PCR conditions for ITS, tef1-α, and tub2 were: initial denaturation at 94 °C for 5 min; 40 cycles of amplification denaturation at 94 °C for 45 s; annealing at 48 °C (ITS), 52 °C (tef1-α) or 55 °C (tub2) for 30 s; and extension at 72 °C for 90 s; and final extension at 72 °C for 7 min. The PCR conditions for rpb2 were: initial denaturation at 95 °C for 5 min; touch-down amplification (5 cycles of 95 °C for 45 s, 60 °C for 45 s, and 72 °C for 120 s; 5 cycles of 95 °C for 45 s, 58 °C for 45 s, and 72 °C for 120 s; and 30 cycles of 95 °C for 45 s, 54 °C for 45 s, and 72 °C for 120 s), and final elongation at 72 °C for 8 min. The amplicon was sequenced in both directions using the respective PCR primers and the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an Applied Biosystems 3730xl DNA analyzer installed at the Mie University Advanced Science Research Promotion Center (Tsu, Mie, Japan). The sequences were assembled and aligned with 46 sequences of Neofusicoccum species recollected from GenBank, and aligned using MAFFT software version 7 (Katoh, Rozewicki, & Yamada, 2019). Maximum likelihood (ML) and Bayesian inference (BI) analyses were used to estimate phylogenetic relationships. ML analyses were performed using raxml HPC-PTHREADS software (Stamatakis, 2006). The strength of the internal branches from the resultant trees was tested by bootstrap (BS) analysis (Felsenstein, 1985) using 1,000 replications. BI analyses were performed using BEAST version 2.5.1 (Bouckaert et al., 2019) to estimate the posterior probabilities (PPs) of tree topologies based on metropolis-coupled Markov chain Monte Carlo (MCMC) searches, which used the MCMC algorithm of four chains in parallel from a random tree topology. The MCMC analysis lasted 10,000,000 generations. Trees were sampled and saved every 1,000 generations. The first 25% of saved trees were discarded as burn-in, and the PPs were determined from the remaining trees. Representative sequences for all taxa were uploaded to GenBank (Table 2). The sequence alignments prepared in this study were deposited in TreeBASE number S27074.

Table 2. List of Neofusicoccum species used for phylogenetic analysis.

Fungal species	Isolates No.	Host	Country	Accession numbers
				ITS	tef1	tub2	rpb2
Botryosphaeria dothidea	CBS 100564	Paeonia sp.	Netherlands	KX464085	KX464555	KX464781	KX463951
N. algeriense	CBS 137504	Vitis vinifera	Algeria	KJ657702	KJ657721	–	–
N. andinum	CBS 117453	Eucalyptus sp.	Venezuela	AY693976	AY693977	KX464923	KX464002
N. arbuti	CBS 116131	Arbutus menziesii	USA	AY819720	KF531792	KF531793	KX464003
N. australe	CBS 139662	Acacia sp.	Australia	AY339262	AY339270	AY339254	EU339573
N. batangarum	CBS 124924	Terminalia catappa	Cameroon	FJ900607	FJ900653	FJ900634	FJ900615
N. brasiliense	CMM 1285	Mangifera indica	Brazil	JX513628	JX513608	KC794030	–
N. buxi	CBS 116.75	Buxus sempervirens	France	KX464165	KX464678	–	KX464010
N. cordaticola	CBS 123634	Syzygium cordatum	South Africa	EU821898	EU821868	EU821838	EU821928
N. corticosae	CBS 120081	Eucalyptus corticosa	Australia	MN161920	KX464682	KX464958	KX464013
N. cryptoaustrale	CBS 122813	Eucalyptus sp.	South Africa	FJ752742	FJ752713	FJ752756	KX464014
N. eucalypticola	CBS 115679	–	–	AY615141	AY615133	AY615125	–
N. eucalyptorum	CBS 115791	E. grandis	South Africa	AF283686	AY236891	AY236920	–
N. grevilleae	CBS 129518	Grevillea aurea	Australia	JF951137	–	–	–
N. hellenicum	CERC 1947	Pistacia vera	Greece	KP217053	KP217061	KP217069	–
N. hongkongense	CERC 2973	Araucaria cunninghamii	China	KX278052	KX278157	KX278261	KX278283
N. hyperici	MUCC 241	Hypericum patulum	Japan	LC589125	LC589137	LC589147	LC589160
N. hyperici	MUCC 2509	H. galioides	Japan	LC589126	LC589138	LC589148	LC589161
N. hyperici	MUCC 650	Stachyurus praecox	Japan	LC589124	LC589136	–	LC589159
N. illicii	CGMCC 3.18310	Illicium verum	China	KY350149	–	KY350155	–
N. italicum	MFLUCC 15-0900	V. vinifera	Italy	KY856755	KY856754	–	–
N. kwambonambiense	CBS 123639	Syzygium cordatum	South Africa	EU821900	EU821870	EU821840	EU821930
N. laricinum	MUCC 2660	Larix kaempferi	Japan	LC589131	LC589142	LC589153	LC589166
N. laricinum	MUCC 2662	L. decidua	Japan	LC589129	LC589140	LC589151	LC589164
N. laricinum	MUCC 2663	L. decidua	Japan	LC589130	LC589141	LC589152	LC589165
N. laricinum	MUCC 2666	L. kaempferi	Japan	LC589132	LC589143	LC589154	LC589167
N. laricinum	MUCC 2669	L. kaempferi	Japan	LC589128	LC589139	LC589150	LC589163
N. lumnitzerae	CMW 41469	Lumnitzera racemosa	South Africa	KP860881	KP860724	KP860801	KU587925
N. luteum	CBS 562.92	Actinidia deliciosa	New Zealand	KX464170	KX464690	KX464968	KX464020
N. macroclavatum	CBS 118223	E. globulus	Australia	DQ093196	DQ093217	DQ093206	KX464022
N. mangiferae	CBS 118531	M. indica	Australia	AY615185	DQ093221	AY615172	–
N. mangroviorum	CMW 41365	Avicennia marina	South Africa	KP860859	KP860702	KP860779	KU587905
N. mediterraneum	CBS 121718	Eucalyptus sp.	Greece	MH863145	–	–	KX464024
N. microconidium	CERC 3497	E. urophylla × E. grandis	China	KX278053	KX278158	KX278262	MF410203
N. miyakoense	MUCC 2585	Coffea sp.	Japan	–	LC589146	LC589157	LC589170
N. miyakoense	MUCC 2586	Mangifera indica	Japan	LC589133	LC589144	LC589155	LC589168
N. nonquaestitum	CBS 126655	Umbellularia californica	USA	GU251163	GU251295	GU251823	KX464025
N. occulatum	CBS 128008	E. grandis hybrid	Australia	EU301030	EU339509	EU339472	EU339558
N. okinawaense	MUCC 789	Lagerstroemia speciosa	Japan	LC589134	LC589145	LC589156	LC589169
N. pandanicola	MFLUCC 17-2270	Pandanus sp.	China	MH275072	MH412778	–	–
N. parvum	CBS 138823	Populus nigra	New Zealand	AY236943	AY236888	AY236917	EU821963
N. parvum	MUCC 2511	Tamarix tenuissima	Japan	LC589127	–	LC589149	LC589162
N. parvum	MUCC 392	Rhododendron hybrids	Japan	LC589123	LC589135	–	LC589158
N. pennatisporum	MUCC 510	Allocasuarina fraseriana	Australia	EF591925	EF591976	EF591959	–
N. pistaciae	CBS 595.76	Pistacia vera	Greece	KX464163	KX464676	KX464953	KX464008
N. pistaciarum	CBS 113083	P. vera	USA	KX464186	KX464712	KX464998	KX464027
N. pistaciicola	CBS 113089	P. vera	USA	KX464199	KX464727	KX465014	KX464033
N. protearum	CBS 114176	Leucadendron salignum x Leucadendron laureolum cv. Silvan Red	South Africa	AF452539	KX464720	KX465006	KX464029
N. pruni	CBS 121112	Prunus salicina	South Africa	EF445349	EF445391	KX465016	KX464034
N. ribis	CBS 115475	Ribes sp.	USA	AY236935	AY236877	AY236906	EU339554
N. sinense	CGMCC 3.18315	Unknown woody plant	China	KY350148	KY817755	KY350154	–
N. sinoeucalypti	CGMCC 3.18752	E. urophylla × E. grandis	China	KX278061	KX278166	KX278270	KX278290
N. stellenboschiana	CBS 110864	V. vinifera	South Africa	AY343407	AY343348	KX465047	KX464042
N. terminaliae	CBS 125263	Terminalia sericea	South Africa	GQ471802	GQ471780	KX465052	KX464045
N. umdonicola	CBS 123645	Syzygium cordatum	South Africa	EU821904	EU821874	EU821844	EU821934
N. ursorum	CBS 122811	Eucalyptus sp.	South Africa	FJ752746	FJ752709	KX465056	KX464047
N. variabile	CBS 143480	Mimusops caffra	South Africa	MH558608	–	MH569153	–
N. versiforme	CBS 118101	E. camaldulensis	Australia	KF766154	KX464757	KF766128	KX464041
N. viticlavatum	CBS 112878	V. vinifera	South Africa	AY343381	AY343342	KX465058	KX464048
N. vitifusiforme	CBS 110887	V. vinifera	South Africa	AY343383	AY343343	KX465061	KX464049

Ex-type strains are in bold.

3. Results

3.1. Phylogeny

A total of 13 isolates were obtained from the samples in this study (Table 1). The ITS + rpb2 + tef1-α + tub2 combined dataset consisted of 60 sequences with a total of 1,829 characters (ITS: 551, rpb2: 593, tef1-α: 301, tub2: 384), including alignment gaps. The out-group taxon was B. dothidea (CBS 100564). The two trees estimated using ML and BI had the same species grouping. As a result of the phylogenetic analysis, the 13 Japanese Neofusicoccum isolates in this study were divided into five clades. The first clade was composed of isolates from Hypericum patulum (MUCC 241), H. galioides (MUCC 2509), and Stachyurus praecox (MUCC 650). The second clade was composed of an ex-type isolate of N. parvum (CBS 138823), isolates from Rhododendron hybrids (MUCC 392), and Tamarix tenuissima (MUCC 2511). The third clade was composed of isolates from Larix decidua (MUCC 2662; MUCC 2663) and Larix kaempferi (MUCC 2660; MUCC 2666; MUCC 2669). The fourth clade was from Coffea sp. (MUCC 2585) and M. indica (MUCC 2586). The fifth clade was from Lagerstroemia speciosa (MUCC 789) (Fig. 1).

Fig. 1 – Phylogenetic tree of Neofusicoccum spp. The tree was constructed by maximum likelihood (ML) analysisusing combined ITS, rpb2, tef1-α, and tub2 gene region datasets. ML bootstrap values (ML-BS) and posterior probability in Bayesian inference analyses (BI-PP) are given near the branches (ML-BS/BI-PP). Ex-type strains are in bold. Species identified in this study are shown in red.

3.2. Taxonomy

Neofusicoccum hyperici Y. Hattori & C. Nakash., sp. nov. Fig. 2.

MycoBank no.: MB 837717.

Fig. 2 – Morphological features of Neofusicoccum hyperici (MUMH 10423 and MUCC 241). A: Specimen MUMH 10423. B: Symptoms with pycnidia forming on Hypericum patulum leaf. C: Colony on PDA (MUCC 241). D: Vertical section of pycnidium in leaf tissue. E: Conidia and conidiophores. F–G: Conidia. H: Pycnidia forming on PNA. Bars: D 50 μm; E 20 μm; F, G 10 μm.

Etymology: Named after the genus name of the host plant genus (H. patulum) from which the type strain was obtained.

Sexual morph: Unknown.

Asexual morph formed on the host and PNA: Leaf spots yellowish-brown to brown, small at the edge, later enlarged and coalescent, expanded toward the whole of a leaf. Conidiomata pycnidial, epidermal, merged, superficial on PNA, solitary, globose, dark brown to black, ellipsoid, unilocular, with a central ostiole, 63–110 × 46–89 µm; pycnidial wall composed of depressed or irregular cells in three to four layers, dark brown to black. Conidiophores reduced to conidiogenous cells; conidiogenous cells discrete, hyaline, cylindrical to ampulliform, determinate, with periclinal thickening, rarely proliferating percurrently, 4.4–7 × 1.6–2.3 μm. Paraphyses not seen. Conidia holoblastic sporulation for first conidia, phialidic sporulation for following conidia, hyaline, smooth, aseptate, various in shape, cylindrical to fusiform, reniform, straight or slightly curved, granulate, subtruncate to bluntly rounded at the base, acute to rounded at the apex, 15–18 × 5.3–6.4 μm, 15.96 × 6.00 μm on average, L/W = 2.66 (n = 50).

Cultural characteristics on PDA: Colony is white to dark gray, with floccose and dense aerial mycelia, and reaching 90 mm diam at 14 d after inoculation at room temperature (22 °C).

Holotypus: JAPAN, Aichi, Nagoya, on H. patulum, 18 Jul 2006, collected by I. Araki (MUMH 10423, ex-type culture MUCC 241).

Host: Hypericum galioides, H. patulum, and S. praecox.

Other materials examined: JAPAN, Tokyo, Hachioji, on H. galioides, 4 Jul 1963, collected by T. Kobayashi (culture MUCC 2509 = MAFF 410797); JAPAN, Aichi, Nagoya, on S. praecox, 17 Jul 2007, collected by I. Araki (MUMH 10606, culture MUCC 650).

Note: In molecular phylogenetic analysis, two strains isolated from the genus Hypericum (MUCC 241; MUCC 2509) and one strain from the genus Stachyrus (MUCC 650) formed a clade strongly supported by a 93% ML-BS value. In addition, MUCC 241 and MUCC 2509 formed an internal clade within the clade, but no difference in morphological characteristics was found among the three isolates. Neofusicoccum hyperici is phylogenetically closely related to N. parvum (CBS 138823) and N. pandanicola Tibpromma & K.D. Hyde (MFLUCC 17-2270). However, the conidia size of N. pandanicola (15–26 × 8–12 µm) (Tibpromma et al., 2018) and N. parvum (11–)14–18(–23) × (7–)8–10(–11) µm (Pennycook & Samuels, 1985) were larger than N. hyperici (15–18 × 5.3–6.4 μm). Neofusicoccum hyperici showed almost no difference in morphological characteristics between the host plants and the PNA.

Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash., comb. nov. Fig. 3.

MycoBank no.: MB 837720.

≡ Physalospora laricina Sawada Bull. Gov. For. Exp. Stn. Tokyo 46: 126, 1950.

≡ Guignaridia laricina (Sawada) W. Yamam. & Kaz. Itô, Science Reports of the Hyogo University of Agriculture 5: 9, 1961.

≡ Botryosphaeria laricina (Sawada) Shang, Acta Mycologia Sinica 6: 249, 1987.

Fig. 3 – Morphological features of Neofusicoccum laricinum (FPH-4038 and MUCC 2662). A: Specimen FPH-4038. Symptoms with pycnidia forming on Larix decidua leaf. B: Colony on PDA (MUCC 2662). C: Vertical section of pycnidium in leaf tissue. D: Conidia and conidiophores. E, F: Conidia. Bars: C 100 μm; D–F 10 μm.

Sexual morph: Physalospora laricina (fide Sawada, 1950): Caulicolous. Diseased twigs defoliated from the middle to the tip, with exudate resin. Fruit bodies lined, erumpent. Ascomata epidermal, blackish, globose, 368 µm diam; ostiole erumpent, 60 µm diam; paraphyses developed, intricate, 3 µm. Ascus clavate, rounded at the apex, stipitate at the base, hyaline, 114–135 × 22–26 µm. Ascospores ellipsoid, smooth, hyaline, 24–27 × 13 µm.

Asexual morph formed on the host: Conidiomata pycnidial, epidermal, merged, solitary, globose, dark brown, subglobose, unilocular, with a central ostiole, 204–246 × 207–212 µm; pycnidial wall composed of depressed or irregular cells in three to four layers, brown to dark brown. Conidiophores reduced to conidiogenous cells; conidiogenous cells discrete, hyaline, cylindrical to ampulliform, determinate, with periclinal thickening, or proliferating percurrently, 9–23 × 2.4–5 μm. Paraphyses not seen. Conidia holoblastic sporulation for first conidia, phialidic sporulation for following conidia, hyaline, smooth, aseptate, slightly colored and septate with age, ellipsoid to fusiform, granulate, subtruncate to bluntly rounded at the base, rounded to subacute at the apex, with a short frill at the both ends, 23–38 × 7–12 μm, 29.85 × 8.50 μm on average, L/W = 3.57 (n = 33)..

Cultural characteristics on PDA: Colony is white to gray, with dense aerial mycelia, reaching 90 mm diam at 14 d after inoculation at room temperature.

Syntypus: JAPAN, Aomori, Sanbongi, on L. kaempferi, 27 Sep 1949, collected by K. Sawada (IUM-FS515), ibid, on L. kaempferi, 27 Sep 1949, collected by K. Sawada (IUM-FS516); ibid, on L. kaempferi, 27 Sep 1949, collected by K. Sawada (IUM-FS517); JAPAN, Aomori, Yokohama, on L. kaempferi, 3 Oct 1949, collected by K. Sawada & S. Murai (IUM-FS518); ibid, on L. kaempferi, 3 Oct 1949, collected by K. Sawada & S. Murai (IUM-FS519); ibid, on L. kaempferi, 3 Oct 1949, collected by K. Sawada & S. Murai (IUM-FS520).

Epitype for anamorphic state (designated here): JAPAN, Ibaraki, Mito, on L. decidua, 14 Jun 1973, collected by H. Kondo (TFM: FPH-4038, ex-epitype culture FFPRI 411215 = MUCC 2662).

Host: L. kaempferi (Sawada, 1950) and L. decidua.

Other materials examined: JAPAN, Hokkaido, Tomakomai, on L. kaempferi, 27 Jul 1971, collected by S. Yokota (culture MUCC 2660 = MAFF 410183); JAPAN, Ibaraki, Mito, on L. decidua, 14 Jun 1973; JAPAN, Iwate, Morioka, on L. decidua, 18 Aug 1965, collected by Y. Yokosawa (culture MUCC 2663 = FFPRI 411216); JAPAN, Aomori, Kamikita, on L. kaempferi, 9 Jun 1961, collected by T. Uozumi (culture MUCC 2666 = FFPRI 411217); JAPAN, Iwate, Iwate, on L. kaempferi, 3 Jul 1986, collected by T. Shoji (culture MUCC 2669 = FFPRI 411218).

Note: Physalospora laricina has been recognized as an important pathogenic fungus causing larch shoot blight disease in Japan and has been extensively studied for its ecology and pathogenicity (Uozumi, 1961; Sato, Yokozawa, & Shoji, 1963), but it was identified only on the basis of its morphological characteristics. In the observation of syntype specimens of P. laricina (IUM-FS515, IUM-FS516, IUM-FS517, IUM-FS518, IUM-FS519, and IUM-FS520), only the sexual morph was observed; there is no mention of the asexual morph in the description by Sawada (1950)However, many studies of this disease and its causal fungus have been conducted in Japan because Larix species are very important for silviculture in northern Japan; from these studies, the asexual morphs of P. laricina and Macrophoma sp. have been reported. Inoculation tests on the sexual and asexual morphs have confirmed isogenicity among the isolates of both states (Uozumi, 1961; Sato et al., 1963). As mentioned above, we examined several isolates of present species. However, we did not observe ascomata or conidiomata on artificial media, although Uozumi (1961) reported their formation on such media. The morphological characteristics of the asexual morph of herbarium specimen TFM: FPH-4038 (origin of isolate MUCC 2662) are identical to Macrophoma sp. recorded by Uozumi (1961), and under the current criteria belong to the genus Neofusicoccum. Moreover, the examined isolates formed a single clade in the phylogenetic tree; therefore, we suggest that these isolates should be treated as an independent species. This species was transferred to the genus Guignardia by Yamamoto (1961) and later transferred to Botryosphaeria by Shang (1987). Based on the results of molecular analysis in this study, we propose to transfer the genus to Neofusicoccum and designate the epitype FPH-4038 (ex-epitype culture MUCC 2662).

Neofusicoccum miyakoense Y. Hattori & C. Nakash., sp. nov. Fig. 4.

MycoBank no.: MB 837718.

Fig. 4 – Morphological features of Neofusicoccum miyakoense (MUMH 11936 and MUCC 2585). A: Specimen MUMH 11936. Symptoms with pycnidia forming on Coffea sp. branch. B: Colony on PDA (MUCC 2585). C: Vertical section of pycnidium in the leaf tissue. D: Conidia and conidiophores. E: Conidia. Bars: C 50 μm; D­–E 10 μm.

Etymology: Name refers to Miyako-jima, the island in Japan where this fungus was collected.

Sexual morph: Unknown.

Asexual morph formed on the host: Caulicolous or foliicolous. Lesions turned to pale brown to brown. Conidiomata pycnidial, scattered, visible as small dots on the lesion, epidermal, immersed, solitary, globose to subglobose, suppressed, dark brown to black, ellipsoid, unilocular, with a central ostiole, 118–185 × 102–132 µm; pycnidial wall composed of depressed or irregular cells in three to four layers, dark brown to black. Conidiophores reduced to conidiogenous cells; conidiogenous cells discrete, hyaline, cylindrical to ampulliform, determinate, with periclinal thickening, 4.2–9.3 × 1.5–2.8 μm. Paraphyses not seen. Conidia holoblastic sporulation for first conidia, phialidic sporulation for following conidia, hyaline to slightly colored, smooth, with granular contents, aseptate, ellipsoid, rounded at both ends, or truncated at the base, rounded at the apex, 10–14.6 × 5–7.5 μm, 12.11 × 6.18 μm on average, L/W = 1.96 (n = 100).

Cultural characteristics on PDA: Colony is dark gray to olivaceous gray, with floccose aerial mycelia, dense at the center, reaching 90 mm diam at 14 d after inoculation at room temperature.

Holotypus: JAPAN, Okinawa, Miyako-jima, on Coffea sp., 28 Jul 2018, collected by Y. Hattori & C. Nakashima (MUMH 11936, ex-type culture MUCC 2585).

Host: Coffea sp. and M. indica

Other material examined: JAPAN, Okinawa, Miyako-jima, on M. indica, 28 Jul 2018, collected by Y. Hattori & C. Nakashima (MUMH 11937, culture MUCC 2586).

Note: As a result of phylogenetic analysis, an independent clade was formed with strongly supported statistical values (ML-BS: 92%, and BI-PP: 1). Although this species is phylogenetically closely related to N. mangiferae (Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips (CBS118531) and N. microconidium G.Q. Li & S.F. Chen (CERC 3497), the conidia of N. mangiferae (13.6 × 5.4 μm) (Phillips et al., 2013) and N. microconidium (12.3 × 5 μm) (Li, Liu, Li, Liu, & Chen, 2018) are on average longer and narrower than those of N. miyakoense (12.11 × 6.18 μm). Furthermore, the conidia of N. miyakoense are ellipsoidal in shape, whereas those of N. microconidium are narrower and fusiform, and those of N. mangiferae are ellipsoid to nearly fusiform. See also N. okinawaense. We did not observe ascomata or conidiomata on the PNA.

Neofusicoccum okinawaenseY. Hattori & C. Nakash., sp. nov. Fig. 5.

MycoBank no.: MB 837719.

Fig. 5 – Morphological features of Neofusicoccum okinawaense (MUMH 10839 and MUCC 789). A: Specimen MUMH 10839. B: Symptoms with pycnidia forming on Lagerstroemia speciosa. C: Colony on PDA (MUCC 789). D: Vertical section of pycnidium in the leaf tissue. E: Conidia and conidiophores. F: Conidia. Bars: D 50 μm; E, F 10 μm.

Etymology: Name refers to Okinawa, the island in Japan where this fungus was collected.

Sexual morph: Unknown.

Asexual morph formed on the host: Leaf spots grayish-white to brown, small at the edge, later enlarged and coalescent, expanded toward the whole of a leaf. Conidiomata pycnidial, epidermal, submerged, solitary, globose to subglobose, dark brown to black, ellipsoid, unilocular, with a central ostiole, 149–164 × 120–149 µm; pycnidial wall composed of depressed or irregular cells in three to four layers, dark brown to black. Conidiophores reduced to conidiogenous cells; conidiogenous cells discrete, hyaline, cylindrical, with periclinal thickening, 4.7–8.7 × 2.7–3.8 μm. Paraphyses not seen. Conidia holoblastic sporulation for first conidia, phialidic sporulation for following conidia, hyaline to pale yellowish-brown, smooth with granular contents, unicellular, aseptate, ellipsoid to wider fusiform, rounded at both ends, or subtruncate at the base, 11.8–15 × 5.2–6.7 μm, 13.60 × 5.99 μm on average, L/W = 2.28 (n = 18).

Cultural characteristics on PDA: Colony is white to gray, covered with floccose aerial mycelia, reaching 90 mm diam at 14 d after inoculation at room temperature.

Holotypus: JAPAN, Okinawa, Nakagami, on Lagerstroemia speciosa, 19 Nov 2007, collected by C. Nakashima (MUMH 10839, ex-type culture MUCC 789 = MAFF 240624).

Host: Lagerstroemia speciosa.

Note: As a result of phylogenetic analysis, MUCC 789 formed a clade with N. miyakoense (MUCC 2585 and MUCC 2586). The clade was strongly supported by ML-BS (99%) and BI-PP (1). Although this species is phylogenetically closely related to N. miyakoense, later species formed an independent clade composed of two isolates obtained from different host plants. Furthermore, these two species were included within a basal clade of the genus Neofusicoccum, with N. mangiferae in the genus Mangifera from Australia and N. microconidium in Eucalyptus from China. The host plants for Japanese Neofusicoccum species were three foreign species introduced for cultivation in recent years, Coffea sp. and M. indica for N. miyakoense, and Lagerstroemia speciosa for N. okinawaense. Based on the genetic distance among the species, they might be strongly related to the common host plant genera Mangifera and Eucalyptus for speciation and host jumping. The average conidial size and L/W ratio of N. okinawaense (13.60 × 5.99 μm, L/W = 2.28; n = 7) is larger than N. miyakoense (12.11 × 6.18 μm, L/W = 1.96; n = 100). We did not observe ascomata or conidiomata on the PNA.

Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Studies in Mycology 55: 248.

Description and illustration: See Pennycook and Samuels (1985).

Host in Japan: Cymbidium spp. (Suzuki et al., 2018), M. indica (Takushi et al., 2017), Platanus × acerifolia (Motohashi et al., 2016), Rhododendron hybrids, and T. tenuissima (this study).

Materials examined: JAPAN, Aichi, Nagoya, on Rhododendron hybrids, 9 May 2006, collected by Unknown collector (MUMH 10368, culture MUCC 392); JAPAN, Niigata, Iwafune, on T. tenuissima, 22 Jul 1983, collected by T. Kobayashi (TFM: FPH-5589, culture MUCC 2511 = FFPRI 411214).

Note: According to the USDA fungal host database (https://nt.ars-grin. gov/fungaldatabases/fungushost/FungusHost.cfm, accessed on 6 Oct 2020), there are no reports of N. parvum on the genus Tamarix, suggesting that T. tenuissima is a new host.

Discussion

In this study, the taxonomical positions of 13 Japanese isolates of the genus Neofusicoccum were examined based on their morphology and molecular phylogeny. As a result, these isolates were divided into five clades. Based on their phylogenetic position and morphological characteristics, four of them were described as new species: N. hyperici, N. laricinum, N. miyakoense, and N. okinawaense. Neofusicoccum hyperici was closely related to N. parvum and N. pandanicola, however it was morphologically distinct. This species is characterized by small conidia and an independent phylogenetic position. The size and L/W ratio of conidia are useful characteristics for differentiation and delimitation among Neofusicoccum species (Crous et al., 2006; Marin-Felix et al., 2017; Yang et al., 2017; Li et al., 2018). These criteria were supported in the present study. However, species limitations by other phenotypic characteristics, such as the host range and geological distribution, remain unclear.

Neofusicoccum parvum is distributed worldwide and is known to have an extremely broad host range as a pathogen or endophyte (Slippers & Wingfield, 2007). For example, this species has been reported in the following plants: grapevine (Úrbez-Torres & Gubler, 2009; Chacón, Gramaje, Izquierdo, Martínez, & Mena, 2020), walnut (Yu, Tang, Peng, Chen, & Zhai, 2015), and mango (Slippers et al., 2005; Takushi et al., 2017). Additionally, plural species or strains can be found on the same leaf (Slippers & Wingfield., 2007). These studies suggest that extensive, global sampling will be necessary to fully understand the host associations and distribution of Botryosphaeriaceae, including the genus Neofusicoccum (Jami, Slippers, Wingfield, & Gryzenhout, 2014). In this study, two isolates of N. parvum were identified from two plants, including a newly recorded host, T. tenuissima. The expansion of the known host range following expanded sampling appears to be a common pattern of recent studies on the Botryosphaeriaceae, and is drastically changing the perceptions of host association (Jami et al., 2014). Future efforts are still needed to collect and isolate the genus Neofusicoccum from various host plants in Japan and around the world.

The current species delimitation by morphology and phylogenetic relationships has resulted in an explosive increase in the number of species of Neofusicoccum. In this study, we examined isolates of N. laricinum originating from both sexual and asexual morphs, and collected from various places and dates in Japan. Sequence analyses showed that only a few nucleotides differed among isolates on the same host genus (2/551 nucleotides in ITS). Furthermore, all regions other than ITS were the same. Conversely, sequence analyses of N. parvum showed 15 nucleotide differences in the ITS region among the isolates in different genera. Furthermore, the isolates of N. hyperici showed two nucleotide differences in the tef1-a region among the different host genera. These results support the previous studies. The homology of the sequences used in this study was not suitable for identifying the species level. The phylogenetic analyses based on a combination of the four regions (ITS, tef1-a, tub2 and rpb2) indicated that the species form independent phylogenetic clades supported by high BS values (Li et al., 2018). The multiple species concepts that comprise morphological features, host range, and geographical distribution, as well as phylogenetic relationships, are considered important to species recognition.

In this study, the new combination N. laricinum was transferred from the genus Physalospora to the genus Neofusicoccum, due to its morphological characteristics and molecular phylogenetic position. This study reveals, for the first time, the molecular phylogenetic position of N. laricinum. Similar taxonomic problems exist among many Japanese tree diseases and their pathogens, such as blight disease of coniferous trees (Kobayashi, 1957, 1962) and dieback in Paulownia (Itô & Kobayashi, 1951).

Neofusicoccum miyakoense collected from Miyako-jima Island, and N. okinawaense collected from the main island of Okinawa, are proposed as new species in this study. Although the two species have similar morphological characteristics, such as ellipsoidal conidia, they have been recognized as separate clades in the molecular phylogenetic tree. Both Miyako-jima Island and the main island of Okinawa belong to the Ryukyu Islands in the Okinawa Prefecture, Japan, but they have different geographical backgrounds. While the Ryukyu Islands are oceanic Islands formed by volcanic activity, Miyako-jima Island is located about 300 km from the main island of Okinawa and is known to have a unique ecosystem based on its geological history of having been once submerged. Several new species of tree pathogens have been reported on Miyako-jima Island in previous studies (Kobayashi & Kawabe, 1992, Nonaka, Omura, Masuma, & Kaifuchi, 2013), and these geographic isolations and differences might be involved in the unique evolution of the species.

In conclusion, the results of re-examination of the genus Neofusicoccum based on morphological characteristics and phylogenetic analysis revealed a high degree of diversity in this genus in Japan. However, the strains reexamined in this study are part of the genus Neofusicoccum collected in Japan, and many isolates may still be treated as other genera. Therefore, further study is required to understand the full diversity of the genus Neofusicoccum in Japan.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Acknowledgments

We thank Prof. Keiichi Motohashi, Faculty of International Agricultural Development, Tokyo University of Agriculture (TUA) and Prof. Hidehiko Kikuno, Miyako Sub-tropical Farm, TUA, for their guidance in collecting specimens on Miyako-jima Island. This study received financial support from a JSPS KAKENHI grant (no. 20J12025 to YH, and no. 20K06146 to CN).

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