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. 2022 Apr 14;88:171–192. doi: 10.3897/mycokeys.88.82229

Morphological and phylogenetic analyses reveal two new species of Sporocadaceae from Hainan, China

Zhaoxue Zhang 1, Rongyu Liu 1, Shubin Liu 1, Taichang Mu 1, Xiuguo Zhang 1, Jiwen Xia 1,✉
PMCID: PMC9023435  PMID: 35585932

Abstract

Species of Sporocadaceae have often been reported as plant pathogens, endophytes or saprophytes and are commonly isolated from a wide range of plant hosts. The isolated fungi were studied through a complete examination, based on multilocus phylogenies from combined datasets of ITS/tub2/tef1, in conjunction with morphological characteristics. Nine strains were isolated from Ficusmicrocarpa, Ilexchinensis and Schimasuperba in China which represented four species, viz., Monochaetiaschimaesp. nov., Neopestalotiopsishaikouensissp. nov., Neopestalotiopsispiceana and Pestalotiopsislicualicola. Neopestalotiopsispiceana was a new country record for China and first host record from Ficusmacrocarpa. Pestalotiopsislicualicola was first report from Ilexchinensis in China.

Keywords: Monochaetia , multigene phylogeny, Neopestalotiopsis , Pestalotiopsis

Introduction

The family Sporocadaceae was established by Corda in 1842 (type genus: Sporocadus). Species of Sporocadaceae are endophytic, plant pathogenic or saprobic, and associated with a wide range of host plants (Maharachch. et al. 2013; Jayawardena et al. 2015; Liu et al. 2019). Currently, the family comprises 35 genera including Monochaetia (Sacc.) Allesch., Neopestalotiopsis Maharachch. et al., Pestalotiopsis Steyaert, Pseudopestalotiopsis Maharachch.et al., and etc. Most genera have multi-septate and more or less fusiform conidia with appendages at one or both ends, frequently with some melanised cells. Also known as pestalotioid fungi, resembling those taxa having affinities with Pestalotia (Liu et al. 2019).

Steyaert (1949) segregated two novel genera from Pestalotia, namely Pestalotiopsis (with 5-celled conidia) and Truncatella (with 4-celled conidia) based on the conidial forms. This resulted in apparent controversy from Guba (1956, 1961). He emphasised that there was no point in assembling species with similar numbers of conidial septa into distinct genera. Subsequently, Steyaert (1953, 1961, 1963) provided further evidence in support of splitting Pestalotia. Sutton (1980) accepted most of the genera discussed here (Pestalotia, Pestalotiopsis, Truncatella) which fitted into fairly well-defined groups and cited the electron microscope investigation of Griffiths and Swart (1974), which examined the conidial wall of Pestalotiapezizoides and two species of Pestalotiopsis (P.funerea and P.triseta) to support Steyaert’s division of Pestalotiopsis. Maharachch. et al. 2014 segregated two novel genera from Pestalotiopsis, namely Neopestalotiopsis and Pseudopestalotiopsis, based on conidia pigment colour, conidiophores and molecular phylogeny. Neopestalotiopsis can be easily distinguished from Pseudopestalotiopsis and Pestalotiopsis by its versicolourous median cells (Maharachch. et al. 2014). Saccardo (1884) introduced Monochaetia as a subgenus of Pestalotia (as Pestolozzia). The genus Monochaetia was introduced by Allescher (1902), which included 23 species. Allescher (1902) designated the type Monochaetiamonochaeta which has a single apical appendage (Guba 1961; Maharachch. et al. 2014; Senanayake et al. 2015). Steyaert (1949) transferred numerous Monochaetia species to Pestalotiopsis or Truncatella. More than 40 species of Monochaetia were recognised by the monograph of Guba (1961). There are 127 Monochaetia epithets in the Index Fungorum (accession date: 31 March 2022) and most have been transferred to other genera such as Sarcostroma, Seimatosporium and Seiridium (Nag Raj 1993; Maharachch. et al. 2011, 2014, 2016). Seridium and Monochaetia have obvious morphological differences and show separate clades (de Silva et al. 2017).

To date, most phylogenetic studies addressing genera of Sporocadaceae have been based solely on ITS and LSU sequences (Barber et al. 2011; Tanaka et al. 2011; Jaklitsch et al. 2016), or on concatenated datasets of more genes but with incomplete datasets (Senanayake et al. 2015; Wijayawardene et al. 2016). In this study, we made a collection of the established genera Monochaetia, Neopestalotiopsis and Pestalotiopsis species from leaves of Ficusmicrocarpa, Ilexchinensis and Schimasuperba in Hainan Province, China. The inventories allowed establishing two new species that are described here.

Materials and methods

Isolation and morphological studies

The samples were collected from Hainan Province, China. The strains were isolated from diseased leaves of Ficusmicrocarpa, Ilexchinensis and Schimasuperba using surface disinfected tissue fragments (0.5 × 0.5 cm) taken from the margin of leaf lesions (Gao et al. 2014; Jiang et al. 2021a). Surface disinfection consisted of steps including immersion in 75% ethanol for 30 s, 5% sodium hypochlorite (Aladdin, Shanghai, China) for 1 min, and sterile distilled water for 30 s. The pieces were dried with sterilized paper towels and placed on potato dextrose agar (PDA). All plates were incubated at 25 °C for 3–4 days. Then, hyphae were picked out of the periphery of the colonies and inoculated onto new PDA plates. Photographs of the colonies were taken at 7 and 15 days using a Powershot G7X mark II digital camera. Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, all fitted with Olympus DP80 high definition colour digital cameras to photo-document fungal structures. The size of conidia was measured by software Digimizer (https://www.digimizer.com/), and thirty individual measurements were obtained for each character. All fungal strains were stored in 10% sterilised glycerin at 4 °C for further studies. The holotype specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Ex-type cultures were deposited in theShandong Agricultural University Culture Collection (SAUCC). Taxonomic information on the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification

Genomic DNA was extracted from fungal mycelium grown on PDA using cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). The internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS) and partial beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced by using primers pairs ITS5/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997), and EF1-728F/EF-2 (O’Donnell et al. 1998; Carbone and Kohn 1999).

PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 50 μL reaction volume, which contained 25 μL Green Taq Mix (Vazyme, Nanjing, China), 2 μL of each forward and reverse primer (10 μM) (Tsingke, Beijing, China), and 2 μL template genomic DNA, to which distilled deionized water was added. PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 7 min. Annealing temperature was 55 °C for ITS, 54 °C for tub2, 52 °C for tef1. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Tsingke Biotechnology Company Limited (Qingdao, China). Consensus sequences were obtained using MEGA 7.0 or MEGA-X (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.

Species and GenBank accession numbers of DNA sequences used in this study. New sequences are in bold.

Species Strain Host/substrate Country GenBank accession number Reference
ITS tef1 tub2
Bartaliniarobillardoides CBS 122705 T Leptoglossusoccidentalis Italy LT853104 LT853202 LT853252 Bonthond et al. 2018
Ciliochorellaphanericola MFLUCC 14-0984 T Phanerapurpurea Thailand KX789680 – KX789682 Jiang et al. 2021b
MFLUCC 12-0310 Phanerapurpurea Thailand KF827444 KF827477 KF827478 Jiang et al. 2021b
Monochaetiacastaneae CFCC 54354 = SM9-1 T Castaneamollissima China MW166222 MW199741 MW218515 Jiang et al. 2021b
SM9-2 Castaneamollissima China MW166223 MW199742 MW218516 Jiang et al. 2021b
M.dimorphospora NBRC 9980 Castaneapubinervis Japan LC146750 – – Liu et al. 2019
M.ilicis KUMCC 15-0520 T Ilex sp. China KX984153 – – de Silva et al. 2017
CBS 101009 Air Japan MH553953 MH554371 MH554612 Liu et al. 2019
M.junipericola CBS 143391 T Juniperuscommunis Germany MH107900 MH108021 MH108045 Crous et al. 2018
M.kansensis PSHI2004Endo1030 Cyclobalaopsismyrsinaefolia China DQ534044 – DQ534047 Liu et al. 2006
PSHI2004Endo1031 Cyclobalaopsismyrsinaefolia China DQ534045 – DQ534048 Liu et al. 2006
M.monochaeta CBS 546.80 Culture contaminant Netherlands MH554056 MH554491 MH554732 Liu et al. 2019
CBS 199.82 T Quercuspubescens Italy MH554018 – MH554694 Liu et al. 2019
CBS 115004 Quercusrobur Netherlands AY853243 MH554398 MH554639 Liu et al. 2019
M.quercus CBS 144034 T Quercuseduardi Mexico MH554171 MH554606 MH554844 Liu et al. 2019
M.schimae SAUCC212201 T Schimasuperba China MZ577565 OK104874 OK104867 This study
SAUCC212202 Schimasuperba China MZ577566 OK104875 OK104868 This study
SAUCC212203 Schimasuperba China MZ577567 OK104876 OK104869 This study
M.sinensis HKAS 10065 T Quercus sp. China MH115995 – MH115999 de Silva et al. 2018
Neopestalotiopsisacrostichi MFLUCC 17-1754 T Acrostichumaureum Thailand MK764272 MK764316 MK764338 Norphanphoun et al. 2019
N.alpapicalis MFLUCC 17-2544 T Rhizophoramucronata Thailand MK357772 MK463547 MK463545 Kumar et al. 2019
N.aotearoa CBS 367.54 T Canvas New Zealand KM199369 KM199526 KM199454 Maharachch. et al. 2014
N.asiatica MFLUCC 12-0286 T Unidentified tree China JX398983 JX399049 JX399018 Maharachch. et al. 2012
CFCC 54339 = SM32 Castaneamollissima China MW166224 MW199743 MW218517 Jiang et al. 2021b
N.brachiata MFLUCC 17-1555 T Rhizophoraapiculata Thailand MK764274 MK764318 MK764340 Norphanphoun et al. 2019
N.brasiliensis COAD 2166 T Psidiumguajava Brazil MG686469 MG692402 MG692400 Bezerra et al. 2018
CFCC 54341 = ZY4 Castaneamollissima China MW166229 MW199748 MW218522 Jiang et al. 2021b
ZY4-2D Castaneamollissima China MW166230 MW199749 MW218523 Jiang et al. 2021b
N.chiangmaiensis MFLUCC 18-0113 T Dead leaves Thailand – MH388404 MH412725 Tibpromma et al. 2018
N.chrysea MFLUCC 12-0261 T Pandanus sp. China JX398985 JX399051 JX399020 Maharachch. et al. 2012
N.clavispora MFLUCC 12-0281 T Magnolia sp. China JX398979 JX399045 JX399014 Maharachch. et al. 2012
N.cocoes MFLUCC 15-0152 T Cocosnucifera Thailand KX789687 KX789689 – Norphanphoun et al. 2019
N.coffea-arabica HGUP 4019 T Coffeaarabica China KF412649 KF412646 KF412643 Song et al. 2013
N.cubana CBS 600.96 T Leaf litter Cuba KM199347 KM199521 KM199438 Maharachch. et al. 2014
N.dendrobii MFLUCC 14-0106 T Dendrobiumcariniferum Chiang Rai, Thailand MK993571 MK975829 MK975835 Ma et al. 2019
N.egyptiaca CBS 140162 T Mangiferaindica Egypt KP943747 KP943748 KP943746 Crous et al. 2015
N.ellipsospora MFLUCC 12-0283 T Dead plant materials China JX398980 JX399047 JX399016 Maharachch. et al. 2012
N.eucalypticola CBS 264.37 T Eucalyptusglobulus – KM199376 KM199551 KM199431 Maharachch. et al. 2014
N.foedans CGMCC 3.9123 T Mangrove plant China JX398987 JX399053 JX399022 Maharachch. et al. 2012
N.formicidarum CBS 362.72 T Dead ant Ghana KM199358 KM199517 KM199455 Maharachch. et al. 2014
CBS 115.83 Plant debris Cuba KM199344 KM199519 KM199444 Maharachch. et al. 2014
N.hadrolaeliae COAD 2637 T Hadrolaeliajongheana Minas Gerais, Brazil MK454709 MK465122 MK465120 Freitas et al. 2019
N.haikouensis SAUCC212271 T Ilexchinensis China OK087294 OK104877 OK104870 This study
SAUCC212272 Ilexchinensis China OK087295 OK104878 OK104871 This study
N.honoluluana CBS 114495 T Telopea sp. USA KM199364 KM199548 KM199457 Maharachch. et al. 2014
N.iraniensis CBS 137768 T Fragariaananassa Iran KM074048 KM074051 KM074057 Ayoubi et al. 2016
N.javaensis CBS 257.31 T Cocosnucifera Indonesia KM199357 KM199543 KM199437 Maharachch. et al. 2014
N.macadamiae BRIP 63737c T Macadamiaintegrifolia Australia KX186604 KX186627 KX186654 Akinsanmi et al. 2017
N.magna MFLUCC 12-0652 T Pteridium sp. France KF582795 KF582791 KF582793 Maharachch. et al. 2012
N.mesopotamica CBS 336.86 T Pinusbrutia Iraq KM199362 KM199555 KM199441 Maharachch. et al. 2014
N.musae MFLUCC 15-0776 T Musa sp. Thailand KX789683 KX789685 KX789686 Norphanphoun et al. 2019
N.natalensis CBS 138.41 T Acaciamollissima South Africa KM199377 KM199552 KM199466 Maharachch. et al. 2014
N.pandanicola KUMCC 17-0175 T Pandanaceae China – MH388389 MH412720 Tibpromma et al. 2018
N.pernambucana URM 7148-01 T Vismiaguianensis Brazil KJ792466 KU306739 – Silvério et al. 2016
N.petila MFLUCC 17-1738 T Rhizophoramucronata Thailand MK764276 MK764320 MK764342 Norphanphoun et al. 2019
N.phangngaensis MFLUCC 18-0119 T Pandanaceae Thailand MH388354 MH388390 MH412721 Tibpromma et al. 2018
N.piceana CBS 394.48 T Picea sp. UK KM199368 KM199527 KM199453 Maharachch. et al. 2014
CBS 254.32 Cocosnucifera Indonesia KM199372 KM199529 KM199452 Maharachch. et al. 2014
SAUCC210112 Ficusmicrocarpa China OK149224 OK206436 OK206434 This study
SAUCC210113 Ficusmicrocarpa China OK149225 OK206437 OK206435 This study
N.protearum CBS 114178 T Leucospermumcuneiforme cv. “Sunbird” Zimbabwe JN712498 KM199542 KM199463 Maharachch. et al. 2014
N.rhizophorae MFLUCC 17-1550 T Rhizophoramucronata Thailand MK764278 MK764322 MK764344 Norphanphoun et al. 2019
N.rosae CBS 124745 Paeoniasuffruticosa USA KM199360 KM199524 KM199430 Maharachch. et al. 2014
CBS 101057 T Rosa sp. New Zealand KM199359 KM199523 KM199429 Maharachch. et al. 2014
N.rosicola CFCC 51992 T Rosachinensis China KY885239 KY885243 KY885245 Norphanphoun et al. 2019
CFCC 51993 Rosachinensis China KY885240 KY885244 KY885246 NNorphanphoun et al. 2019
N.samarangensis MFLUCC 12-0233 T Syzygiumsamarangense Thailand JQ968609 JQ968611 JQ968610 Maharachch. et al. 2012
N.saprophytica MFLUCC 12-0282 T Magnolia sp. China KM199345 KM199538 KM199433 Maharachch. et al. 2014
N.sichuanensis CFCC 54338 = SM15-1 T Castaneamollissima China MW166231 MW199750 MW218524 Jiang et al. 2021b
N.sonneratae MFLUCC 17-1745 T Sonneronataalba Thailand MK764280 MK764324 MK764346 Norphanphoun et al. 2019
N.steyaertii IMI 192475 T Eucalytpusviminalis Australia KF582796 KF582792 KF582794 Maharachch. et al. 2012
N.surinamensis CBS 450.74 T soil under Elaeisguineensis Suriname KM199351 KM199518 KM199465 Maharachch. et al. 2014
N.thailandica MFLUCC 17-1730 T Rhizophoramucronata Thailand MK764281 MK764325 MK764347 Norphanphoun et al. 2019
N.umbrinospora MFLUCC 12-0285 T unidentified plant China JX398984 JX399050 JX399019 Maharachch. et al. 2012
N.vitis MFLUCC 15-1265 T Vitisvinifera cv. “Summer black” China KU140694 KU140676 KU140685 Jayawardena et al. 2016
N.zimbabwana CBS 111495 T Leucospermumcunciforme cv. “Sunbird” Zimbabwe JX556231 KM199545 KM199456 Maharachch. et al. 2014
Nonappendiculataquercina CBS 116061 T Quercussuber Italy MH553982 MH554400 MH554641 Liu et al. 2019
CBS 270.82 Quercuspubescens Italy MH554025 MH554459 MH554701 Liu et al. 2019
Pestalotiopsisaustralasiae CBS 114126 T Knightia sp. New Zealand KM199297 KM199499 KM199409 Maharachch. et al. 2014
P.australis CBS 114193 T Grevillea sp. Australia KM199332 KM199475 KM199383 Maharachch. et al. 2014
P.grevilleae CBS 114127 T Grevillea sp. Australia KM199300 KM199504 KM199407 Maharachch. et al. 2014
P.hollandica CBS 265.33 T Sciadopitysverticillata The Netherlands KM199328 KM199481 KM199388 Maharachch. et al. 2014
P.kenyana CBS 442.67 T Coffea sp. Kenya KM199302 KM199502 KM199395 Maharachch. et al. 2014
P.knightiae CBS 114138 T Knightia sp. New Zealand KM199310 KM199497 KM199408 Maharachch. et al. 2014
P.licualicola HGUP4057 T Licualagrandis China KC492509 KC481684 KC481683 Geng et al. 2013
SAUCC210087 Ilexchinensis China OK087323 OK104879 OK104872 This study
SAUCC210088 Ilexchinensis China OK087324 OK104880 OK104873 This study
P.oryzae CBS 353.69 T Oryzasativa Denmark KM199299 KM199496 KM199398 Maharachch. et al. 2014
P.parva CBS 278.35 Leucothoefontanesiana – KM199313 KM199509 KM199405 Maharachch. et al. 2014
P.portugalica CBS 393.48 T – Portugal KM199335 KM199510 KM199422 Maharachch. et al. 2014
P.spathuliappendiculata CBS 144035 T Phoenixcanariensis Australia MH554172 MH554607 MH554845 Liu et al. 2019
Pseudopestalotiopsiscocos CBS 272.29 T Cocosnucifera Indonesia KM199378 KM199553 KM199467 Maharachch. et al. 2014
Pse.elaeidis CBS 413.62 T Elaeisguineensis Nigeria MH554044 MH554479 MH554720 Liu et al. 2019
Pse.indica CBS 459.78 T Rosasinensis India KM199381 KM199560 KM199470 Maharachch. et al. 2014
Seiridiumpapillatum CBS 340.97 T Eucalyptusdelegatensis Australia LT853102 MH554468 LT853250 Bonthond et al. 2018
Seir.phylicae CBS 133587 T Phylicaarborea Tristan da Cunha LT853091 LT853188 LT853238 Bonthond et al. 2018
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Isolates marked with “T” are ex-type or ex-epitype strains.

Phylogeny

Newly generated sequences in this study were aligned with additional related sequences downloaded from GenBank (Table 1) using MAFFT 7 online service with the Auto strategy (Katoh et al. 2019, http://mafft.cbrc.jp/alignment/server/). To establish the identity of the isolates at the species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of three loci (ITS, tub2 and tef1). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) (Miller et al. 2012) using RaxML-HPC2 on XSEDE v. 8.2.12 (Stamatakis 2014) and MrBayes on XSEDE v. 3.2.7a (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2012), respectively. Four Markov chains were run for two runs from random starting trees for 10,000,000 generations (ITS + tub2 + tef1) until the split deviation frequency value < 0.01, and trees were sampled every 1000 generation. The first quarter generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. The resulting trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CC 2019. New sequences generated in this study were deposited at GenBank (https://www.ncbi.nlm.nih.gov; Table 1). The final concatenated sequence alignments were deposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S29480).

Result

Phylogenetic analyses

Nine strains of Sporocadaceae isolated from plant hosts from Hainan, China, were grown in culture and used for analyses of molecular sequence data. The combined dataset of ITS-tub2-tef1 has an aligned length of 2285 total characters (ITS: 1–638, tub2: 639–1558, tef1: 1559–2285) including gaps, of which 869 characters are constant, 292 variable and parsimony-uninformative, and 1124 parsimony-informative. For the BI and ML analyses, the substitution model GTR+G for ITS, HKY+I+G for tub2 and GTR+I+G for tef1 were selected and incorporated into the analyses. The MCMC analysis of the three concatenated genes run for 7,795,000 generations, resulting in 7796 trees. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig. 1).

Figure 1.

Figure 1.

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Phylogram of Sporocadaceae based on combined ITS, tub2 and tef1 sequences. The BI and ML bootstrap support values above 0.90 and 70% are shown at the first and second position, respectively. The tree is rooted to Bartaliniarobillardoides (CBS 122705), ex-type or ex-epitype cultures are indicated in bold face. Strains from the current study are in red. Some branches were shortened according to the indicated mulipliers.

Bayesian posterior probability (≥ 0.90) and ML bootstrap support values (≥ 70%) are shown as first and second position above nodes. The 96 strains were assigned to 75 species clades based on the three gene loci phylogeny (Fig. 1). Based on the multi-locus phylogeny and morphology, nine isolates were assigned to four species, including Monochaetiaschimae sp. nov., Neopestalotiopsishaikouensis sp. nov., Neopestalotiopsispiceana and Pestalotiopsislicualicola.

Taxonomy

. Monochaetia schimae

Z. X. Zhang, J. W. Xia & X. G. Zhang sp. nov.

5ACD3395-C104-58D5-8EC2-53136E31A7FE

MycoBank No: 841381

Fig. 2

Figure 2.

Figure 2.

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Monochaetiaschimae (SAUCC212201, ex-type) a diseased leaf of Schimasuperbab surface of colony after 15 days on PDAc reverse of colony after 15 days on PDAd conidiomata e, f conidiogenous cells with conidia g–j conidia. Scale bars: 10 μm (e–j).

Type.

China, Hainan Province: East Harbour National Nature Reserve, on diseased leaves of Schimasuperba, 23 May 2021, Z.X. Zhang (holotype HSAUP212201; ex-type living culture SAUCC212201).

Etymology.

Name refers to the genus of the host plant Schimasuperba.

Description.

Leaf spots irregular, pale brown in centre, brown to tan at margin. Sexual morph not observed. Asexual morph on PDA: Conidiomata solitary, scattered, black, raising above surface of culture medium, subglobose, exuding black conidial droplets from central ostioles after 10 days in light at 25 °C. Conidiophores cylindrical, hyaline, smooth-walled. Conidiogenous cells 9.0–16.5 × 1.2–2.2 μm, phialidic, ampulliform, discrete, hyaline, smooth, thin-walled. Conidia 18–24 × 4.5–6.0 μm, mean ± SD = 20.5 ± 1.1 × 5.5 ± 0.4 μm, fusiform, tapering at both ends, 4-septate; apical cell 2.0–4.0 μm long, conical, hyaline and smooth-walled; three median cells doliiform, 12.5–15.5 μm long, mean ± SD = 14.2 ± 0.7 μm, olivaceous, rough-walled, upper second cell 3.8–5.3 μm long, upper third cell 3.4–5.0 μm long, upper fourth cell 4.4–5.4 μm long; basal cell 2.2–4.5 μm long, conical, hyaline and smooth-walled; apical appendage 7.0–12.5 μm long (mean = 9.2 μm), single, unbranched, central, tubular, filiform; basal appendage 2.5–5.0 μm long, single, unbranched tubular, filiform.

Culture characteristics.

Colonies on PDA 39.0–45.0 mm in diameter after 15 days at 25 °C in darkness, growth rate 2.5–3.0 mm/day, irregularly circular, raised, dense surface with lobate edge, zonate in different sectors, light brown at the margin, brown at the centre; reverse brown at the margin, dark brown at the centre.

Additional specimen examined.

China, Hainan Province: East Harbour National Nature Reserve, 23 May 2021, Z.X. Zhang. On diseased leaves of Schimasuperba, paratype HSAUP212202, living culture SAUCC212202; on diseased leaves of Schimasuperba, paratype HSAUP212203, living culture SAUCC212203.

Notes.

Monochaetiaschimae is introduced based on the multi-locus phylogenetic analysis, with three isolates clustering separately in a well-supported clade (BI/ML = 0.99/96). Monochaetiaschimae is phylogenetically close to M.castaneae from leaves of Castaneamollissima, M.ilicis from leaves of Ilex sp., and M.junipericola from twigs of Juniperuscommunis. However, Monochaetiaschimae differs from M.castaneae by 148 nucleotides (11/463 in ITS, 89/743 in tub2 and 48/403 in tef1), from M.ilicis by 94 nucleotides (18/526 in ITS, 32/698 in tub2 and 44/456 in tef1), and from M.junipericola by 91 nucleotides (10/524 in ITS, 40/411 in tub2 and 41/304 in tef1). Furthermore, they are distinguished by hosts and conidial sizes (18.0–24.0 × 4.5–6.0 μm in M.schimae vs. 18.8–27.3 × 4.7–6.6 μm in M.castaneae vs. 20.0–27.0 × 5.0–8.0 μm in M.ilicis vs. 22.0–28.0 × 5.0–7.0 μm in M.junipericola). In morphology, Monochaetiacastaneae differs from M.schimae by the colour of colonies (cinnamon vs. brown), Monochaetiailicis differs from M.schimae by the colour of median cells (brown vs. olivaceous), and M.junipericola differs from M.schimae by longer conidiogenous cells (10.0–30.0 μm vs. 9.0–16.5 μm) (de Silva et al. 2017; Crous et al. 2018; Jiang et al. 2021b).

. Neopestalotiopsis haikouensis

Z. X. Zhang, J. W. Xia & X. G. Zhang sp. nov.

258515C1-6141-59F9-8114-3C1274776E97

MycoBank No: 841382

Fig. 3

Figure 3.

Figure 3.

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Neopestalotiopsishaikouensis (SAUCC212271, ex-type) a diseased leaf of Ilexchinensisb surface of colony after 7 days on PDAc reverse of colony after 7 days on PDAd conidiomata e–g conidiogenous cells with conidia h–j conidia. Scale bars: 10 μm (e–j).

Type.

China, Hainan Province, Haikou City: East Harbour National Nature Reserve, on diseased leaves of Ilexchinensis. 23 May 2021, Z.X. Zhang (holotype HSAUP212271; ex-type living culture SAUCC212271).

Etymology.

Named after the host location, Haikou City.

Description.

Leaf spots irregular, grey white in centre, brown to tan at margin. Sexual morph not observed. Asexual morph on PDA: Conidiomata globose to clavate, solitary or confluent, embedded or semi-immersed to erumpent, dark brown, exuding globose, dark brown to black conidial masses. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells discrete, subcylindrical to ampulliform, hyaline, 5.0–10.0 × 2.0–6.0 μm, apex 1.0–2.0 μm diam. Conidia fusoid, ellipsoid, straight to slightly curved, 4-septate, 16.0–22.0 × 4.5–7.0 μm, mean ± SD = 20.0 ± 1.8 × 5.5 ± 0.4 μm; basal cell conical with a truncate base, hyaline, rugose and thin-walled, 3.0–4.5 μm long; three median cells doliiform, 11.5–15.0 μm long, mean ± SD = 13.2 ± 1.0 μm, wall rugose, septa darker than the rest of the cell, second cell from the base pale brown, 3.5–5.5 μm long; third cell honey-brown, 4.0–6.0 μm long; fourth cell brown, 3.8–5.7 μm long; apical cell 2.5–5.5 μm long, hyaline, cylindrical to subcylindrical, thin- and smooth-walled; with 2–3 tubular apical appendages (mostly 3), arising from the apical crest, unbranched, filiform, 13.5–24.0 μm long, mean ± SD = 19.1 ± 3.5 μm; basal appendage 2.0–7.0 μm long, single, tubular, unbranched, centric.

Culture characteristics.

Colonies on PDA occupying an entire 90 mm petri dish in 7 days at 25 °C in darkness, growth rate of 7.0–14.0 mm/day, edge undulate, white to grey white, with moderate aerial mycelium on the surface, with black, gregarious conidiomata; reverse similar in colour.

Additional specimen examined.

China, Hainan Province: East Harbour National Nature Reserve, 23 May 2021, Z.X. Zhang. On diseased leaves of Ilexchinensis, paratype HSAUP212272, living culture SAUCC212272.

Notes.

Phylogenetic analysis of a combined three-gene ITS-tub2-tef1 showed that Neopestalotiopsishaikouensis formed an independent clade with full-supported (BI/ML = 1/100, Fig. 1) and is phylogenetically distinct from N.cocoes (MFLUCC 15-0152), N.formicidarum (CBS 362.72) and N.sichuanensis (CFCC 54338). Neopestalotiopsishaikouensis can be distinguished from the phylogenetically most closely related species N.cocoes by narrower conidia (4.5–7.0 vs. 7.5–9.5 μm), N.formicidarum by smaller conidia (16.0–22.0 × 4.5–7.0 vs. 20.0–29.0 × 7.5–9.5 μm), and N.sichuanensis by shorter conidia (16.0–22.0 vs. 23.2–32.8 μm). Furthermore, some species were reported from the same host genus Ilex, including Pestalotianeglecta, Pestalotiopsisannulata, P.humicola and P.ilicis. After comparison, P.humicola was closest to N.haikouensis in morphology, but with 78/588 differences in the ITS region (Maharachch. et al. 2014; Liu et al. 2019; Jiang et al. 2021b).

. Neopestalotiopsis piceana

S.S.N. Maharachch., K.D. Hyde & P.W. Crous, Studies in Mycology 79:146. (2014)

6B8AB555-F733-5E02-8311-780850EB95F6

Fig. 4

Figure 4.

Figure 4.

Open in a new tab

Neopestalotiopsispiceana (SAUCC210112) a diseased leaf of Ficusmicrocarpab surface of colony after 7 days on PDAc reverse of colony after 7 days on PDAd conidiomata e–g conidiogenous cells with conidia h–j conidia. Scale bars: 10 μm (e–j).

Description.

Leaf spots irregular, pale brown in centre, brown to tan at margin. Asexual morph on PDA: Conidiomata solitary, globose to clavate, semi-immersed, brown to black; exuding globose, dark brown to black conidial masses. Conidiophores reduced to conidiogenous cells. Conidiogenous cells discrete, ampulliform to lageniform, hyaline, smooth and thin walled, simple, 4.0–12.0 × 2.0–10.0 μm, apex 2.0–5.0 μm diam. Conidia ellipsoid to clavate, straight to slightly curved, 4-septate, 19.5–26.5 × 5.5–7.0 μm, mean ± SD = 22.7 ± 0.8 × 6.1 ± 0.4 μm; somewhat constricted at septa; basal cell obconic with truncate base, rugose and thin-walled, 2.7–5.0 μm long; three median cells 12.0–16.0 μm long, mean ± SD = 14.7 ± 0.9 μm, doliiform, verruculose, versicoloured, septa darker than the rest of the cell, second cell from base pale brown, 4.0–5.7 μm long; third cell dark brown, 3.5–5.2 μm long; fourth cell brown, 3.8–5.8 μm long; apical cell obconic, hyaline, thin and smooth-walled, 2.5–5.2 μm long; with 1–3 tubular apical appendages, arising from the apical crest, flexuous, unbranched, 21.0–32.0 μm long, mean ± SD = 24.8 ± 3.5 μm; basal appendage single, tubular, unbranched, centric, 2.7–6.5 μm long.

Culture characteristics.

Colonies on PDA incubated at 25 °C in the dark with an average radial growth rate of 9.0–14.0 mm/day and occupying an entire 90 mm petri dish in 7 d, with edge undulate, whitish, aerial mycelium on surface, fruiting bodies black, concentric; reverse of culture yellow to pale brown.

Specimen examined.

China, Hainan Province: Five Fingers Group Scenic Area, 20 May 2021, Z.X. Zhang. On diseased leaves of Ficusmicrocarpa, HSAUP210112, living culture SAUCC210112; on diseased leaves of Ficusmicrocarpa, HSAUP210113, living culture SAUCC210113.

Notes.

In the present study, two strains (SAUCC210112 and SAUCC210113) from symptomatic leaves of Ficusmicrocarpa were clustered with Neopestalotiopsispiceana clade (Maharachch. et al. 2014) based on phylogeny (Fig. 1). Morphologically, our strains were the same as N.piceana, which was originally described with an asexual morph on wood of Picea sp., Cocosnucifera and fruit of Mangiferaindica. The sexual morph of N.piceana was undetermined yet. Neopestalotiopsispiceana was a new record for China and first reported from Ficusmacrocarpa (Moraceae).

. Pestalotiopsis licualicola

K. Geng, Y. Song, K.D. Hyde & Yong Wang bis, Phytotaxa 88 (3):51. (2013)

CE5D32D9-599D-5EF1-BF8F-16BB35C4CE4F

Fig. 5

Figure 5.

Figure 5.

Open in a new tab

Pestalotiopsislicualicola (SAUCC210087) a diseased leaf of Ilexchinensisb surface of colony after 7 days on PDAc reverse of colony after 7 days on PDAd conidiomata f, g, j, k conidiogenous cells with conidia e, h, i, l, m conidia. Scale bars: 10 μm (e–m).

Description.

Leaf spots irregular, pale brown in centre, brown to tan at margin. Asexual morph on PDA: Conidiomata solitary, scattered, black, raising above surface of culture medium, subglobose. Conidiophores cylindrical, hyaline, smooth-walled. Conidiophores often indistinct. Conidiogenous cells discrete, hyaline, simple, filiform, 5.5–10.0 μm long. Conidia 18.0–24.5 × 4.0–5.5 μm, mean ± SD = 20.5 ± 1.9 × 5.3 ± 0.3 μm, fusiform, straight to slightly curved, 4-septate, smooth, greyish brown; basal cell conical, hyaline, thin-walled, 2.8–6.0 μm long; with three median cells, dark brown, concolorous, septa and periclinal walls darker than the rest of the cell, together 11.5–16.0 μm long, mean ± SD = 13.2 ± 1.2 μm; second cell from base 3.4–5.5 μm; third cell 3.3–4.7 μm; fourth cell 3.5–5.1 μm; apical cell hyaline, conic to subcylindrical, 3.1–5.3 μm; with 1–3 tubular apical appendages (mostly 1) without knobs, arising from the apex of the apical cell, 10.0–20.5 μm long, mean ± SD = 16.0 ± 4.0 μm; basal appendage filiform, short.

Culture characteristics.

Colonies on PDA reaching 70.0–80.0 mm diam after 7 d at 25 °C, growth rate 9.0–12.0 mm/day, edge entire, whitish to pale honey coloured, with sparse aerial mycelium on the surface, with black, gregarious conidiomata; reverse similar in colour.

Specimen examined.

China, Hainan Province: East Harbour National Nature Reserve, 23 May 2021, Z.X. Zhang. On diseased leaves of Ilexchinensis, HSAUP210087, living culture SAUCC210087; on diseased leaves of Ilexchinensis, HSAUP210088, living culture SAUCC210088.

Notes.

In the present study, two strains (SAUCC210087 and SAUCC210088) from symptomatic leaves of Ilexchinensis were clustered to Pestalotiopsislicualicola clade (Geng et al. 2013) based on phylogeny (Fig. 1). Morphologically, our strains were the same as P.licualicola, which was originally described with an asexual morph on leaves of Licualagrandis in China. The sexual morph of P.licualicola was undetermined yet. This is the first time this species has been reported in Ilexchinensis (Aquifoliaceae) in China.

Discussion

Based on phylogeny and morphology, nine strains from three host species (Ficusmicrocarpa, Ilexchinensis and Schimasuperba) were described as well as two new species (Monochaetiaschimae sp. nov. and Neopestalotiopsishaikouensis sp. nov.) and two known species (Neopestalotiopsispiceana and Pestalotiopsislicualicola). In the genus Monochaetia, most species were found on Fagaceae hosts, including Castaneapubinervis (Monochaetiadimorphospora), Castaneamollissima (Monochaetiacastaneae), Quercuspubescens (Monochaetiamonochaeta) and etc. In our study, the species of Monochaetia (M.schimae) was first reported from Schimasuperba (Theaceae). Ilex was widely grown as an evergreen tree all over the world and isolated many pathogens, endophytes or saprophytes (Alfieri et al. 1984; Maharachch. et al. 2014; de Silva et al. 2017; Solarte et al. 2018). More than 100 strains (Xylariales) have been isolated from the genus Ilex. Among these, there was 13 pestalotia-like fungi, and we compare morphology with my new collection. In morphology, the conidia size of Pestalotiopsishumicola is similar to Neopestalotiopsishaikouensis. Phylogenetic analyses of Maharachch. et al. (2014) and the current study show Neopestalotiopsis and Pestalotiopsis are different genus. The known species Neopestalotiopsispiceana was described from Picea sp. (Pinaceae) in United Kingdom (Maharachch. et al. 2014) and Pestalotiopsislicualicola was described from Licualagrandis (Palmaceae) in China (Geng et al. 2013). In this study, Neopestalotiopsispiceana was a new record for China and first reported from Ficusmacrocarpa (Moraceae), Pestalotiopsislicualicola was first reported from Ilexchinensis (Aquifoliaceae) in China, so we described and illustrated N.piceana and P.licualicola again. Species in genera have multi-septate and more or less fusiform conidia with a single apical and basal appendage (Monochaetia, Seiridium); other genera do not form appendages (Nonappendiculata) or have 2–4 appendages (Pestalotiopsis, Ciliochorella, Neopestalotiopsis, Pseudopestalotiopsis) (Maharachch. et al. 2014; Bonthond et al. 2018; Liu et al. 2019). Our study supported this phenomenon.

As many pestalotioid species have overlapping morphological traits, sequence data is essential to resolve these three genera and introduce new species (Jeewon et al. 2002; de Silva et al. 2017; Norphanphoun et al. 2019). Combined gene sequences of ITS, tub2 and tef1 can provide a better resolution for Monochaetia. However, more genes are needed to provide better resolution and support in Neopestalotiopsis. In the previous studies, members of Sporocadaceae are of particular interest with regard to the production of secondary metabolites, e.g. Bartalinia, Morinia and Pestalotiopsis (Collado et al. 2006; Gangadevi and Muthumary 2008; Liu et al. 2009). Pestalotiopsisfici was shown to possess a very high number of gene clusters involved in bioactive compound synthesis (Wang et al. 2016). Owing to Pestalotiopsis and other genus in this family sharing the same evolutionary history, it is important to report novel species and screen for novel metabolites in future studies.

Supplementary Material

XML Treatment for Monochaetia schimae
XML Treatment for Neopestalotiopsis haikouensis
XML Treatment for Neopestalotiopsis piceana
XML Treatment for Pestalotiopsis licualicola

Acknowledgements

This work was jointly supported by the National Natural Science Foundation of China (nos. 31900014, U2002203, 31750001) and National Science and Technology Fundamental Resources Investigation Program of China (2019FY100704).

Citation

Zhang Z, Liu R, Liu S, Mu T, Zhang X, Xia J (2022) Morphological and phylogenetic analyses reveal two new species of Sporocadaceae from Hainan, China. MycoKeys 88: 171–192. https://doi.org/10.3897/mycokeys.88.82229

Funding Statement

This work was supported by the National Natural Science Foundation of China (nos. 31900014, U2002203, 31750001).

Supplementary materials

Supplementary material 1

The combined ITS, tub2 and tef1 sequences

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Zhaoxue Zhang, Rongyu Liu, Shubin Liu, Taichang Mu, Xiuguo Zhang, Jiwen Xia

Data type

phylogenetic

Explanation note

The combined ITS, tub2 and tef1 sequences.

mycokeys-88-171-s001.fas (218.3KB, fas)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Monochaetia schimae
mycokeys.88.82229-treatment1.xml (19KB, xml)
XML Treatment for Neopestalotiopsis haikouensis
mycokeys.88.82229-treatment2.xml (17.3KB, xml)
XML Treatment for Neopestalotiopsis piceana
mycokeys.88.82229-treatment3.xml (12.2KB, xml)
XML Treatment for Pestalotiopsis licualicola
mycokeys.88.82229-treatment4.xml (11.7KB, xml)
Supplementary material 1

The combined ITS, tub2 and tef1 sequences

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Zhaoxue Zhang, Rongyu Liu, Shubin Liu, Taichang Mu, Xiuguo Zhang, Jiwen Xia

Data type

phylogenetic

Explanation note

The combined ITS, tub2 and tef1 sequences.

mycokeys-88-171-s001.fas (218.3KB, fas)

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