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Research Article
Sulcispora supratumida sp. nov. (Phaeosphaeriaceae, Pleosporales) on Anthoxanthum odoratum from Italy
expand article infoIndunil C. Senanayake§|, Rajesh Jeewon, Erio Camporesi#¤«, Kevin D. Hyde»|, Yu-Jia Zeng§, Sheng-Li Tian§, Ning Xie§
‡ Kunming Institute of Botany, Chinese Academy of Science, Kunming, China
§ Shenzhen University, Shenzhen, China
| Mae Fah Luang University, Chiang Rai, Thailand
¶ University of Mauritius, Reduit, Mauritius
# A.M.B. Gruppo Micologico Forlivese “Antonio Cicognani”, Forlì, Italy
¤ A.M.B. Circolo Micologico “Giovanni Carini”, Brescia, Italy
« Società per gli Studi Naturalisticidella Romagna, Bagnacavallo, Italy
» Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan, Thailand
Open Access

Abstract

Sulcispora is typified by S. pleurospora. We collected a sulcispora-like taxon on leaves of Anthoxanthum odoratum L. in Italy and obtained single ascospore isolates. Combined ITS, LSU, SSU and tef1 sequence analyses suggested that Sulcispora is placed in the family Phaeosphaeriaceae and a newly collected Sulcispora species is introduced here as S. supratumida sp. nov. Detailed descriptions and illustrations are provided for Sulcispora supratumida and it is compared with the type species, S. pleurospora.

Keywords

Combined gene analysis, Dothideomycetes , graminicolous fungi, new species, spore septation

Introduction

Phaeosphaeriaceae is a highly diverse and large family in the order Pleosporales (Hyde et al. 2013) with more than 42 accepted genera (Hyde et al. 2017; Karunarathna et al. 2017; Wanasinghe et al. 2018). Members of Phaeosphaeriaceae are pathogens or hyper-parasites on living plants and humans and saprobes of decaying plant matter (Tennakoon et al. 2016; Ahmed et al. 2017).

Sulcispora was proposed by Shoemaker and Babcock (1989) as a monotypic genus to accommodate Sulcispora pleurospora (≡ Phaeosphaeria pleurospora Niessl). Some morphological characters of Phaeosphaeria pleurospora did not fit within species concepts of Phaeosphaeria and Shoemaker and Babcock (1989), therefore, introduced the genus Sulcispora. The genus name refers to the numerous furrows on the ascospore wall (Shoemaker and Babcock 1989). Sulcispora pleurospora has been reported on monocotyledonous hosts in genera such as Anthoxanthum, Carex, Deschampsia, Sesleria and Tofieldia (Leuchtmann 1984; Shoemaker and Babcock 1989).

In this study, we collected sulcispora-like species associated with leaf spots of Anthoxanthum odoratum in Italy. We compared the morphological characters of our collection with the isotype of Sulcispora pleurospora. Morphologically, our collection differs from the type species of Sulcispora, S. pleurospora. Therefore, we introduce our collection as a new species. Combined ITS, LSU, SSU and tef1 sequence analysis including taxa in Phaeosphaeriaceae indicates that the here-studied fungus grouped with “Phaeosphaeria pleurospora” (CBS 460.84) with high support value.

Methods

Sample collection, specimen examination and single spore isolation

Specimens were collected from Anthoxanthum odoratum L. from Italy in 2013. They were examined and photographed using a Carl Zeiss Discovery V8 stereo-microscope fitted with Axiocam. Sections of ascomata were taken by hand under a stereo-microscope. Sections and other micro-morphological characters were photographed using a Nikon Eclipse 80i compound microscope fitted with a Canon 450D digital camera. All microscopic measurements were made with Tarosoft image framework (v. 0.9.0.7). Colony characteristics were recorded from cultures grown on Malt Extract Agar (MEA).

Single spore isolation was carried out following the method described by Chomnunti et al. (2014). Germinated ascospores were aseptically transferred into fresh MEA plates and incubated at 20 °C to obtain pure cultures and later transferred to MEA slants and stored at 4 °C for further study. The holotype and paratype specimens were deposited at the Mae Fah Luang University (MFLU) fungaria and the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), respectively. Living cultures were deposited at the Mae Fah Luang Culture Collection (MFLUCC). MycoBank (http://www.mycobank.org/) and Facesoffungi (Jayasiri et al. 2015) numbers were obtained for the new strain. The new species was established based on recommendations outlined by Jeewon and Hyde (2016).

DNA extraction, PCR amplification and DNA sequencing

Fresh fungal mycelium grown on MEA for four weeks at 20°C was used for DNA extraction (Jeewon et al. 2002). Genomic DNA extraction and PCR reactions were carried out using ITS4/ITS5 for internal transcribed spacer nrDNA (ITS), LR5/LROR for large subunit nrDNA (LSU), NS1/NS4 for large subunit nrDNA (SSU) and 983F/2218R for translation elongation factor 1 (tef1) genes according to the same protocol of Maharachchikumbura et al. (2012). The PCR products were observed on 1% agarose electrophoresis gel stained with ethidium bromide. Purification and sequencing of PCR products were carried out at the Kunming Institute of Botany, Chinese Academy of Science, Kunming, China. Sequence quality was checked and sequences were condensed with DNASTAR Lasergene v.7.1. Sequences derived in this study were deposited in GenBank (Table 1).

Table 1.

Isolates used in this study and their GenBank and culture accession numbers. The strain of Sulcispora supratumida sp. nov. is set in bold font and all ex-type strains are annotated with “T”.

Taxon Culture accession no ITS LSU SSU tef-1
Allophaeosphaeria muriformia MFLUCC 13-0349T KP765680 KP765681 KP765682
A. subcylindrospora MFLUCC 13-0380T KT314184 KT314183 KT314185
Amarenographium ammophilae MFLUCC 16-0296T KU848196 KU848197 KU848198 MG520894
Ampelomyces quisqualis CBS 129.79T HQ108038 JX681064 EU754029
Bhatiellae rosae MFLUCC 17-0664T MG828873 MG828989 MG829101
Chaetosphaeronema hispidulum CBS 216.75 KF251148 KF251652 EU754045
Dactylidina dactylidis MFLUCC 14-0963T MG828887 MG829003 MG829114 MG829199
D. shoemakeri MFLUCC 14-0966T MG828886 MG829002 MG829113 MG829200
Dematiopleospora mariae MFLUCC 13-0612T KJ749653 KJ749652 KJ749655
Didymella exigua CBS 183.55T GU237794 EU754155 EU754056
Didymocyrtis caloplacae CBS 129338 JQ238641 JQ238643
D. ficuzzae CBS 128019 KP170647 JQ238616
D. cladoniicola CBS 128026 JQ238626
Embarria clematidis MFLUCC 14-0976T MG828871 MG828987 MG829099 MG829194
Entodesmium rude CBS 650.86 GU301812 GU349012
Equiseticola fusispora MFLUCC 14-0522T KU987668 KU987669 KU987670 MG520895
Galliicola pseudophaeosphaeria MFLUCC 14-0527T KT326692 KT326693 MG829203
Hawksworthiana clematidicola MFLUCC 14-0910T MG828901 MG829011 MG829120 MG829202
H. lonicerae MFLUCC 14-0955T MG828902 MG829012 MG829121 MG829203
Italica achilleae MFLUCC 14-0959T MG828903 MG829013 MG829122 MG829204
Juncaceicola alpine CBS 456.84 KF251181 KF251684
J. luzulae MFLUCC 16-0780 KX449529 KX449530 KX449531 MG520898
Leptospora rubella CPC 11006 DQ195780 DQ195792 DQ195803
Loratospora aestuarii JK 5535B GU301838 GU296168
L. luzulae MFLUCC 14-0826 KT328497 KT328495 KT328496
Melnikia anthoxanthii MFLUCC 14-1010T KU848205 KU848204
Muriphaeosphaeria galatellae MFLUCC 14-0614T KT438333 KT438329 KT438331 MG520900
Neosetophoma italica MFLUCC14-0826T KP711356 KP711361 KP711366
N. samarorum CBS 138.96T FJ427061 KF251664 GQ387517
Neostagonospora caricis CBS 135092/S616T KF251163 KF251667
N. eligiae CBS 135101T KF251164 KF251668
Nodulosphaeria hirta MFLUCC 13-0867 KU708849 KU708845 KU708841 KU708853
N. senecionis MFLUCC 15-1297 KT290257 KT290258 KT290259
Ophiobolus cirsii MFLUCC 13-0218T KM014664 KM014662 KM014663
O. disseminans AS2L14-6 KP117305
Ophiosphaerella agrostidis MFLUCC 11-0152T KM434271 KM434281 KM434290 KM434299
Paraleptosphaeria dryadis CBS 643.86 J F740213 GU301828 KC584632 GU349009
Paraphoma chrysanthemicola CBS 522.66 FJ426985 KF251670 GQ387521
P. radicina CBS 111.79T KF251172 KF251676 EU754092
Parastagonospora nodorum CBS 110109T KF251177 KF251681 EU754076
P. poagena CBS 136776T KJ869116 KJ869174
Phaeosphaeria chiangraina MFLUCC 13-0231T KM434270 KM434280 KM434289 KM434298
P. oryzae CBS 110110T KF251186 KF251689 GQ387530
P. papayae S528 KF251187 KF251690
Phaeosphaeria pleurospora CBS 460.84 AF439498
Phaeosphaeriopsis glaucopunnctata MFLUCC 13-0265T KJ522473 KJ522477 KJ522481 MG520918
P. triseptata MFLUCC 13-0271T KJ522475 KJ522479 KJ522484 MG520919
Poaceicola arundinis MFLUCC 15-0702T KU058716 KU058726 MG520921
P. italica MFLUCC 13-0267T KX926421 KX910094 KX950409 MG520924
Populocrescntia forlicesesensis MFLU 15-0651T KT306948 KT306952 KT306955 MG520925
Premilcurensis senecionis MFLUCC 13-0575T KT728365 KT728366
Sclerostagonospora sp. CBS 123538 FJ372393 FJ372410
Scolicosporium minkeviciusii MFLUCC 12-0089T KF366382 KF366383
Septoriella leuchtmannii CBS 459.84T KF251188 KF251691
Setomelanomma holmii CBS 110217 GU301871 GQ387572 GU349028
Setophoma sacchari CBS 333.39T KF251245 KF251748 GQ387525
S. terrestris CBS 335.29T KF251246 KF251749 GQ387526
Sulcispora supratumida MFLUCC 14-0995 KP271443 KP271444 KP271445 MH665366
Tintelnotia destructans CBS 127737T NR_147684 NG_058274 KY090698
T. destructans CBS 137534 KY090663 KY090697
Vagicola chlamydospora MFLUCC 15-0177T KU163658 KU163654
V. vagans CBS 604.86 KF251193 KF251696
Vrystaatia aloeicola CBS 135107 KF251278 KF251781
Wojnowicia dactylidis MFLUCC 13-0735T KP744470 KP684149 KP684150
W. lonicerae MFLUCC 13-0737T KP744471 KP684151 KP684152
Wojnowiciella eucalypti CPC 25024T KR476741 KR476774 LT990617
Xenoseptoria neosaccardoi CBS 128665T KF251281 KF251784
X. neosaccardoi CBS 120.43 KF251280 KF251783
Yunnanensis phragmitis MFLUCC 17-0315T MF684862 MF684863 MF684867 MF683624
Y. phragmitis MFLUCC 17-1365T MF684869 MF684865 MF684864 MF683625

Sequence alignment and phylogenetic analysis

BLASTn searches were made using the newly generated sequences to assist in taxon sampling for phylogenetic analyses. In addition, representatives of the Phaeosphaeriaceae were selected following Tennakoon et al. (2016) and Wanasinghe et al. (2018) (Table 1). Combined multi-locus sequence data of ITS, LSU, SSU and tef1 regions were aligned using default settings of MAFFT v.7 (Katoh et al. 2017) and manually adjusted using BioEdit 7.1.3 (Hall 1999) to allow maximum alignment and minimum gaps. Maximum likelihood analysis was performed by RAxML (Stamatakis and Alachiotis 2010) implemented in raxmlGUIv.1.3 (Silvestro and Michalak 2012). The search strategy was set to rapid bootstrapping and the analysis carried out using the GTRGAMMAI model of nucleotide substitution with 1000 replicates. The model of evolution was estimated by using MrModeltest 2.2 (Nylander 2004).

For the Bayesian inference (BI) analyses of the individual loci and concatenated ITS, LSU, SSU and tef-1 alignment, the above mentioned model test was used to determine the best fitting nucleotide substitution model settings for MrBayes v. 3.0b4. A dirichlet state frequency was predicted for all three data partitions and GTR+I+G as the best model for all single gene and combined datasets. The heating parameter was set to 0.2 and trees were saved every 1000 generations (Ronquist and Huelsenbeck 2003). The Markov Chain Monte Carlo (MCMC) analysis of four chains started in parallel from a random tree topology. The Bayesian analysis lasted 10,000,000 generations (average standard deviation of split frequencies value = 0.0098) and the consensus trees and posterior probabilities were calculated from the 9,998,000 trees sampled after discarding the first 20% of generations as burn-in. Trees obtained in this study were deposited in TreeBASE under accession number S22938. The phylogram was visualised in FigTree v. 1.2.2 (Rambaut and Drummond 2008).

Results

Phylogenetic inferences

The combined ITS, LSU, SSU and tef-1 sequence data set comprised 69 strains of Phaeosphaeriaceae with Didymella exigua as the outgroup taxon. All individual trees generated under different criteria and from single gene datasets were essentially similar in topology and not significantly different from the tree generated from the concatenated dataset. Maximum likelihood analysis with 1000 bootstrap replicates yielded a tree with the likelihood value of ln: -13019.593920 and the following model parameters: alpha: 0.144187; Π(A): 0.245356, Π(C): 0.229408, Π(G): 0.267562 and Π(T): 0.257674. The best scoring RAxML tree is shown in Figure 1. Maximum likelihood bootstrap values ≥50% and Bayesian inference (BI) ≥0.9 are given at each node.

Figure 1. 

Maximum likelihood majority rule consensus tree based on a combined dataset of ITS, LSU, SSU and tef-1 sequences. Bootstrap support values ≥50% and Bayesian inference (BI) ≥0.9 are given at the nodes. The tree is rooted to Didymella exigua (CBS 183.55). The culture accession numbers are given after the species names. All ex-type strains are in bold. The newly introduced species from this study is in bold red.

The phylogenetic trees obtained from maximum likelihood were topologically congruent to previous studies on Phaeosphaeriaceae (Phookamsak et al. 2014; Thambugala et al. 2014; Tennakoon et al. 2016; Karunarathna et al. 2017; Wanasinghe et al. 2018). This phylogenetic analysis showed the placement of 45 genera within Phaeosphaeriaceae. The here-studied strain clustered with CBS 460.84 (one of Leuchtmann’s Swiss strains of S. pleurospora from Carex firma) with 100% bootstrap support value. The ITS sequence of the CBS 460.84 is almost identical to our strain (MFLUCC 14–0995). However no LSU, SSU and tef-1 sequences were obtained from CBS 460.84 in GenBank. The herbarium specimen of CBS 460.84 is in Westerdijk Fungal Biodiversity Institute (CBS) under accession number CBS H-15991 (SWITZERLAND, Kt. Graubünden, Zügenschlucht near Davos, Carex firma, A. Leuchtmann). However, CBS has presently stopped sending specimens on loan, hence we could not compare morphological characters of the here studied strain with CBS 460.84. Additionally Sulcispora sisterly clustered with the type species of Loratospora, L. aestuarii with low support and the second species of Loratospora, L. luzulae. was distantly clustered.

Taxonomy

Sulcispora supratumida Senan., Camporesi & K.D. Hyde, sp. nov.

MycoBank No: 826887
Facesoffungi No: FoF 04782
Figure 2

Etymology

The species epithet is based on the two Latin words “supra” meaning upper and “tumidus” meaning swollen, referring to the position of swollen cells of ascospores.

Type

ITALY. Province of Forli-Cesena, Premilcuore, Passodella Valbura, on dead leaves of Anthoxanthum odoratum L. (Poaceae), 25 May 2013, Erio Camporesi, IT 1306 (MFLU 15–0038, holotype; HKAS 83865, paratype): living cultures, MFLUCC 14–0995.

Description

Saprobic on leaves of Anthoxanthum odoratum L., visible as black spots, occurring on the upper surface of entire leaf. Sexual morph. Ascomata 110–150 × 90–140 µm (x– = 140–125 µm, n = 10), scattered, solitary, immersed, uniloculate, globose, black. Ostiole 35–40 µm (x– = 39 µm, n = 10) wide, papillate, central, periphysate. Periphyses 15–20 µm long, hyaline. Peridium comprising 2–4 layers of brown to dark brown, thick-walled, cells of textura angularis to textura globularis. Hamathecium comprising 2–4 µm wide, cellular, hyaline, branched, septate, pseudoparaphyses, constricted at the septa, anastomosing mostly above the asci and embedded in a mucilaginous matrix. Asci 85–125 × 20–35 µm (x– = 100 × 30 µm, n = 20), 8-spored, few, bitunicate, fissitunicate, subglobose to clavate, short pedicellate, apically rounded, with an ocular chamber, arising from the base of the ascoma and attached to parenchymatous cell matrix at base. Ascospores 30–35 × 6–9 µm (x– = 35 × 7 µm, n = 25), bi-seriate to tri-seriate, narrowly fusiform, narrowing towards the end cells, reddish to dark brown, 6-septate, second septum supra-median, slightly constricted, not constricted at other septa, second segment swollen, straight, with 12–16 longitudinal furrows on surface, lacking a mucilaginous sheath. Asexual morph. Undetermined.

Figure 2. 

Sulcispora supratumida (MFLU 15–0038). a Leaves of Anthoxanthum odoratum b Appearance of ascomata on host surface c Cross section of ascoma d Peridium e Pseudoparaphyses f–i Asci j–nAscosporeso Upper surface of the culture p Lower surface of the culture. Scale bars: 200 µm (b), 50 µm (c), 20 µm (d–i), 10 µm (j–n).

Culture characteristics

2 cm diameter after 4 weeks incubated in dark at 25 °C on MEA, pinkish-white, circular, slightly woolly, margin lobate, effuse, lacking aerial mycelium, tightly attached to the media.

Discussion

Shoemaker and Babcock (1989) observed type specimens of Phaeosphaeria pleurospora and found that the ascospores of P. pleurospora with striated ornamented walls are different to those of other genera in Phaeosphaeriaceae. Hence, they introduced the genus Sulcispora to accommodate P. pleurospora and placed it in Phaeosphaeriaceae. Sulcispora pleurospora has some similarities with Phaeosphaeria exarata Shoemaker & C.E. Babc., in having very large cells in the peridium, ascospores with a continuous sheath and ornamented wall of ascospores with coarse, longitudinal ridges (Shoemaker and Babcock 1989).

In this study, a combined gene sequence analysis of taxa amongst the Phaeosphaeriaceae provides substantial evidence to support Sulcispora as a distinct genus in Phaeosphaeriaceae. Sulcispora differs from other genera in having immersed ascomata with a relatively thin wall, cellular pseudoparaphyses, short pedicellate asci and brown ascospores (Phookamsak et al. 2014).

Leuchtmann (1984) reported variation of ascospore septation amongst several collections of Phaeosphaeria pleurospora from different host plants. Phaeosphaeria pleurospora, collected from Sesleria caerulea (L.) Ard. and Carex firma Mygind ex Host, usually formed 6-septate ascospores and the second segment was swollen. Our collection is morphologically identical to Leuchtmann’s collection. However, the isotype and some of Leuchtmann’s collections from other host plants had 5–8-septate ascospores and the third or fourth segment was swollen (Table 2). Therefore Leuchtmann (1984) characterised Phaeosphaeria pleurospora as a species with 5–8 septate ascospores. However, Leuchtmann’s collection of Sulcispora pleurospora is likely to comprise more than a single species and possibly constitutes a species complex.

Table 2.

Ascospore morphology comparison of Sulcispora species

Species name Herbarium type data Host No of septa Swollen cell Reference
Sulcispora pleurospora FH 196419 (isotype) Deschampsia cespitosa (Poaceae) 5–6 3rd Shoemaker and Babcock 1989
F6952, F6949, F6951 (isotype) Deschampsia cespitosa (Poaceae) 6 3rd In this study
M (1 collection), ZT (8 collections) 6 monocotyledonous hosts,1 dicotyledonous host 6–8 3rd or 4th Leuchtmann 1984
Sulcispora supratumida ZT (6 collections) Seleria caerulea (Poaceae)Carex firma (Cyperaceae) 6 2nd Leuchtmann 1984
MFLU 15-0038 (holotype) Anthoxanthum odoratum (Poaceae) 6 2nd In this study

Based on the morphology, we identified our collection as different from the isotype of Sulcispora pleurospora. Hence, we introduced a new species as Sulcispora supratumida sp. nov. However, the ITS sequence of our strain clustered with that of CBS 460.84 (one of Leuchtmann’s Swiss strain of S. pleurospora from Carex firma) with 100% bootstrap support value. There are only two base pair differences between the ITS regions of both strains. Since there are no sequence data of other DNA regions of Sulcispora pleurospora deposited in GenBank, we could not confirm whether or not CBS 460.84 is Sulcispora supratumida. However, it would eventually be practical to obtain the living strain of CBS 460.84 and generate further sequence data.

Keys for species in Sulcispora

1 Ascomata erumpent, long papillate, 5–8-septated, ascospores with 3rd swollen cell S. pleurospora
Ascomata immersed, short papillate, 6-septated, ascospores with 2nd swollen cell S. supratumida

Acknowledgements

Indunil C. Senanayake thanks to Shaun Pennycook (Landcare Research, Christchurch, New Zealand) for helping to correct this manuscript.

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