Research Article |
Corresponding author: Ning Xie ( shainin@msn.cn ) Academic editor: Marc Stadler
© 2018 Indunil C. Senanayake, Rajesh Jeewon, Erio Camporesi, Kevin D. Hyde, Yu-Jia Zeng, Sheng-Li Tian, Ning Xie.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Senanayake IC, Jeewon R, Camporesi E, Hyde KD, Zeng Y-J, Tian S-L, Xie N (2018) Sulcispora supratumida sp. nov. (Phaeosphaeriaceae, Pleosporales) on Anthoxanthum odoratum from Italy. MycoKeys 38: 35-46. https://doi.org/10.3897/mycokeys.38.27729
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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.
Combined gene analysis, Dothideomycetes , graminicolous fungi, new species, spore septation
Phaeosphaeriaceae is a highly diverse and large family in the order Pleosporales (
Sulcispora was proposed by
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.
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
Fresh fungal mycelium grown on MEA for four weeks at 20°C was used for DNA extraction (
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 |
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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 |
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
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 (
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
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;
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.
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.
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.
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).
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.
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).
Species name | Herbarium type data | Host | No of septa | Swollen cell | Reference |
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Sulcispora pleurospora | FH 196419 (isotype) | Deschampsia cespitosa (Poaceae) | 5–6 | 3rd |
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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 |
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Sulcispora supratumida | ZT (6 collections) | Seleria caerulea (Poaceae)Carex firma (Cyperaceae) | 6 | 2nd |
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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.
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 |
Indunil C. Senanayake thanks to Shaun Pennycook (Landcare Research, Christchurch, New Zealand) for helping to correct this manuscript.