Morphological and Phylogenetic Analyses Reveal Two New Species of Sporocadaceae From Hainan, China

doi:10.21203/rs.3.rs-960719/v1

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Morphological and Phylogenetic Analyses Reveal Two New Species of Sporocadaceae From Hainan, China

https://doi.org/10.21203/rs.3.rs-960719/v1

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published 14 Apr, 2022

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Species of Sporocadaceae have often been reported as plant pathogens, endophytes or saprobic, commonly isolated from a wide range of plant hosts. The isolated fungi were studied through a complete examination based on multi-locus phylogeny of a combined dataset of ITS/TUB2/TEF1-α, in conjunction with morphological characteristics. Nine strains isolated from Schima superba, Ficus microcarpa and Ilex chinensis in Hainan Province, China, represented four species, viz, Monochaetia schimae sp. nov., Neopestalotiopsis haikouensis sp. nov., Neopestalotiopsis piceana and Pestalotiopsis licualacola.

Plant Physiology and Morphology

Sporocadaceae

Monochaetia

Neopestalotiopsis

Pestalotiopsis

multigene phylogeny

Xylariales, a group of fungi distributed worldwide, whose members show considerable variability in a number of characteristics, including position of ascomata, presence and type of sterile tissues, and habit (Barr 1990). The Sporocadaceae (type genus: Sporocadus Corda) is a well-defined family in the Xylariales (Liu et al. 2019). Based on phylogenetic analyses and morphological comparison, Sporocadaceae has delimited thirty genera including Monochaetia (Sacc.) Allesch., Neopestalotiopsis Maharachch. et al., Pestalotiopsis Steyaert, Pseudopestalotiopsis Maharachch. et al., etc., which is generally congruent with the classification system proposed by Nag Raj (1993) prior to the DNA phylogeny era. Species of Sporocadaceae are endophytic, plant pathogenic or saprobic, and associated with a wide range of host plants.

Initially, pestalotia-like asexual morphs were classified in Amphisphaeriaceae by Samuels et al. (1987), accommodating 36 genera (Hawksworth et al. 1995). Its ordinal level of classification, the Amphisphaeriales, was introduced by Eriksson & Hawksworth (1986), but treated as a synonym of Xylariales one year later by Eriksson & Hawksworth (1987). Hawksworth et al. (1995) followed and supported this classification by molecular data. The order was recently resurrected to include Amphisphaeriaceae, Clypeosphaeriaceae and another four novel families derived from Amphisphaeriaceae, however, the sequence dataset was largely incomplete and some of the introduced families were not well supported statistically (Senanayake et al. 2015). Subsequently, Bartaliniaceae, Discosiaceae, Pestalotiopsidaceae and Robillardaceae (Crous et al. 2015) were synonymized and revived the older family name Sporocadaceae to accommodate them, together with the Amphisphaeriaceae and Phlogicylindriaceae, Sporocadaceae was accommodated in the Xylariales, however, Amphisphaeriales was not accepted due to a lack of phylogenetic support in their analysis (Jaklitsch et al. 2016).

Presently, agreement on the classification and delimitation of the family itself seems to have been reached after intense debate. Fungi in the Sporocadaceae (e.g. Bartalinia, Pestalotia, Pestalotiopsis, Robillarda, Seimatosporium, Seiridium and Truncatella) possess common asexual morphological characters related to their acervular conidiomata, conidiogenesis and conidia (Liu et al. 2019). 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). Consequently, the taxonomic concept of, and generic delimitation within Sporocadaceae remain unclear.

Most of the genera of Sporocadaceae contain over-lapping morphological characters of conidia such as the number of median cells, colour of median cells, presence of apical and basal appendages (Jeewon et al. 2002). In this study, we made a collection of the established genera Pestalotiopsis, Neopestalotiopsis and Monochaetia species on leaves of Schima superba, Ficus microcarpa and Ilex chinensis in Hainan Province, China and the new species is established with descriptions, illustrations and molecular data based on ITS, TUB2 and TEF1-α loci.

Specimen collection and morphological descriptions

The samples were collected from Hainan Province, China. The strains were isolated from diseased leaves of Schima superba, Ficus microcarpa and Ilex chinensis using tissue isolation from surface sterilized leaf tissues were conducted following the protocol of Gao et al. (2014). Tissue fragments (0.5 × 0.5 cm) were taken from the margin of leaf lesions and surface-sterilized by consecutively immersing in 75% ethanol solution for 30 s, 5% sodium hypochlorite solution for 1 min, and then rinsing in sterile distilled water for 30 s. The pieces were dried with sterilized paper towels and placed on potato dextrose agar (PDA). All the plates were incubated at biochemical incubator 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 days and 15 days using a Powershot G7X mark Ⅱ digital camera. Micromorphological characters were observed using Olympus SZX10 stereomicroscope and Olympus BX53 microscope, all fitted with Olympus DP80 high definition colour digital cameras to photo-document fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 ℃ for further studies. The holotype specimens are deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Ex-type cultures are deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction, PCR amplification, and DNA sequencing

Genomic DNA was extracted from fungal mycelium on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). The internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS), part of the beta-tubulin gene region (TUB2), and partial translation elongation factor 1-alpha (TEF1-α) genes were amplified and sequenced by using primers pairs ITS4/ITS5 (White et al. 1990), T1/Bt2b (O’Donnell & Cigelnik 1997; Glass & Donaldson 1995), EF1-728F/EF2 (Carbone & Kohn 1999; O’Donnell et al. 1998), respectively.

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 in amplifier, and were adjusted with distilled deionized water to a total volume of 50 µL. 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 for each gene were 55°C for ITS, 54°C for TUB2, 52°C for TEF1-α. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Tsingke Biotechnology Company Limited (Shanghai, 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 in bold.
Species	Strain	Host/substrate	Country	GenBank accession number
Species	Strain	Host/substrate	Country	ITS	TUB2	TEF1
Bartalinia robillardoides	CBS 122705 T	Leptoglossus occidentalis	Italy	LT853104	LT853252	LT853202
Ciliochorella phanericola	MFLUCC 14-0984 T	Phanera purpurea	Thailand	KX789680	KX789682	–
Ciliochorella phanericola	MFLUCC 12-0310	Phanera purpurea	Thailand	KF827444	KF827478	KF827477
Monochaetia castaneae	CFCC 54354 = SM9-1 T	Castanea sp.	China	MW166222	MW218515	MW199741
Monochaetia castaneae	SM9-2	Castanea sp.	China	MW166223	MW218516	MW199742
M. dimorphospora	NBRC 9980	Castanea pubinervis	Japan	LC146750	–	–
M. ilexae	KUMCC 15-0520 T	Ilex sp.	China	KX984153	–	–
M. ilexae	CBS 101009	Air	Japan	MH553953	MH554612	MH554371
M. junipericola	CBS 143391 T	Juniperus communis, twig	Germany	MH107900	MH108045	MH108021
M. kansensis	PSHI2004Endo1030	Cyclobalaopsis myrsinaefolia	China	DQ534044	DQ534047	–
M. kansensis	PSHI2004Endo1031	Cyclobalaopsis myrsinaefolia	China	DQ534045	DQ534048	–
M. monochaeta	CBS 546.80	Culture contaminant	Netherlands	MH554056	MH554732	MH554491
	CBS 199.82 T	Quercus pubescens	Italy	MH554018	MH554694	–
	CBS 115004	Quercus robur	Netherlands	AY853243	MH554639	MH554398
M. quercus	CBS 144034 T	Quercus eduardi	Mexico	MH554171	MH554844	MH554606
M. schimae	SAUCC212201 T	Schima superba	China	MZ577565	OK104867	OK104874
	SAUCC212202	Schima superba	China	MZ577566	OK104868	OK104875
	SAUCC212203	Schima superba	China	MZ577567	OK104869	OK104876
M. sinensis	HKAS 10065 T	Quercus sp.	China	MH115995	MH115999	–
Neopestalotiopsis acrostichi	MFLUCC 17-1754 T	Acrostichum aureum	Thailand	MK764272	MK764338	MK764316
N. aotearoa	CBS 367.54 T	Canvas	New Zealand	KM199369	KM199454	KM199526
N. asiatica	CFCC 54339 = SM32	Castanea mollissima	China	MW166224	MW218517	MW199743
N. asiatica	SM7	Castanea mollissima	China	MW166225	MW218518	MW199744
N. brachiata	MFLUCC 17-1555 T	Rhizophora apiculata	Thailand	MK764274	MK764340	MK764318
N. brasiliensis	COAD 2166 T	Psidium guajava	Brazil	MG686469	MG692400	MG692402
	CFCC 54341 = ZY4	Castanea mollissima	China	MW166229	MW218522	MW199748
	ZY4-2D	Castanea mollissima	China	MW166230	MW218523	MW199749
N. cubana	CBS 600.96 T	leaf litter	Cuba	KM199347	KM199438	KM199521
N. egyptiaca	CBS 140162 T	Mangifera indica	Egypt	KP943747	KP943746	KP943748
N. eucalypticola	CBS 264.37 T	Eucalyptus globulus	–	KM199376	KM199431	KM199551
N. formicarum	CBS 362.72 T	dead ant	Ghana	KM199358	KM199455	KM199517
N. formicarum	CBS 115.83	Plant debris	Cuba	KM199344	KM199444	KM199519
N. haikouensis	SAUCC212271 T	Ilex chinensis	China	OK087294	OK104870	OK104877
N. haikouensis	SAUCC212272	Ilex chinensis	China	OK087295	OK104871	OK104878
N. honoluluana	CBS 114495 T	Telopea sp.	USA	KM199364	KM199457	KM199548
N. iraniensis	CBS 137768 T	Fragaria ananassa	Iran	KM074048	KM074057	KM074051
N. javaensis	CBS 257.31 T	Cocos nucifera	Indonesia	KM199357	KM199437	KM199543
N. mesopotamica	CBS 336.86 T	Pinus brutia	Iraq	KM199362	KM199441	KM199555
N. musae	MFLUCC 15-0776 T	Musa sp.	Thailand	KX789683	KX789686	KX789685
N. natalensis	CBS 138.41 T	Acacia mollissima	South Africa	KM199377	KM199466	KM199552
N. pandanicola	KUMCC 17-0175 T	Pandanaceae	China	–	MH412720	MH388389
N. petila	MFLUCC 17-1738 T	Rhizophora mucronata	Thailand	MK764276	MK764342	MK764320
N. phangngaensis	MFLUCC 18-0119 T	Pandanaceae	Thailand	MH388354	MH412721	MH388390
N. piceana	CBS 394.48 T	Picea sp.	UK	KM199368	KM199453	KM199527
	CBS 254.32	Cocos nucifera	Indonesia	KM199372	KM199452	KM199529
	SAUCC210112	Ficus microcarpa	China	OK149224	OK206434	OK206436
	SAUCC210113	Ficus microcarpa	China	OK149225	OK206435	OK206437
N. protearum	CBS 114178 T	Leucospermum cuneiforme cv. “Sunbird”	Zimbabwe	MK764278	MK764344	MK764322
N. rhizophorae	MFLUCC 17-1550 T	Rhizophora mucronata	Thailand	KM199360	KM199430	KM199524
N. rosae	CBS 124745	Paeonia suffruticosa	USA	KM199359	KM199429	KM199523
N. rosae	CBS 101057 T	Rosa sp.	New Zealand	KY885239	KY885245	KY885243
N. rosicola	CFCC 51992 T	Rosa chinensis	China	KY885239	KY885245	KY885243
	CFCC 51993	Rosa chinensis	China	KY885240	KY885246	KY885244
N. saprophytica	CBS 115452 T	Magnolia sp.	China	KM199345	KM199433	KM199538
N. sichuanensis	CFCC 54338 = SM15-1 T	Castanea mollissima	China	MW166231	MW218524	MW199750
N. sichuanensis	SM15-1C	Castanea mollissima	China	MW166232	MW218525	MW199751
N. sonneratae	MFLUCC 17-1745 T	Sonneronata alba	Thailand	MK764280	MK764346	MK764324
N. surinamensis	CBS 450.74 T	soil under Elaeis guineensis	Suriname	KM199351	KM199465	KM199518
N. thailandica	MFLUCC 17-1730 T	Rhizophora mucronata	Thailand	MK764281	MK764347	MK764325
N. vitis	MFLUCC 15-1265 T	Vitis vinifera cv. “Summer black"	China	KU140694	KU140685	KU140676
N. zimbabwana	CBS 111495 T	Leucospermum cunciforme cv. “Sunbird”	Zimbabwe	JX556231	KM199456	KM199545
Nonappendiculata quercina	CBS 116061 T	Quercus suber	Italy	MH553982	MH554641	MH554400
Nonappendiculata quercina	CBS 270.82	Quercus pubescens	Italy	MH554025	MH554701	MH554459
Pestalotiopsis australasiae	CBS 114126 T	Knightia sp.	New Zealand	KM199297	KM199409	KM199499
P. australis	CBS 114193 T	Grevillea sp.	Australia	KM199332	KM199383	KM199475
P. grevilleae	CBS 114127 T	Grevillea sp.	Australia	KM199300	KM199407	KM199504
P. hollandica	CBS 265.33 T	Sciadopitys verticillata	The Netherlands	KM199328	KM199388	KM199481
P. kenyana	CBS 442.67 T	Coffea sp.	Kenya	KM199302	KM199395	KM199502
P. knightiae	CBS 114138 T	Knightia sp.	New Zealand	KM199310	KM199408	KM199497
P. licualacola	HGUP4057 T	Licuala grandis	China	KC492509	KC481683	KC481684
	SAUCC210087	Ilex chinensis	China	OK087323	OK104872	OK104879
	SAUCC210088	Ilex chinensis	China	OK087324	OK104873	OK104880
P. oryzae	CBS 353.69 T	Oryza sativa	Denmark	KM199299	KM199398	KM199496
P. parva	CBS 278.35	Leucothoe fontanesiana	–	KM199313	KM199405	KM199509
P. portugalica	CBS 393.48 T	–	Portugal	KM199335	KM199422	KM199510
P. spathuliappendiculata	CBS 144035 T	Phoenix canariensis	Australia	MH554172	MH554845	MH554607
Pseudopestalotiopsis cocos	CBS 272.29 T	Cocos nucifera	Indonesia	KM199378	KM199467	KM199553
Pse. elaeidis	CBS 413.62 T	Elaeis guineensis	Nigeria	MH554044	MH554720	MH554479
Pse. indica	CBS 459.78 T	Rosa sinensis	India	KM199381	KM199470	KM199560
Seiridium papillatum	CBS 340.97 T	Eucalyptus delegatensis	Australia	LT853102	LT853250	MH554468
Seir. phylicae	CBS 133587 T	Phylica arborea	Tristan da Cunha	LT853091	LT853238	LT853188
Isolates marked with “T” are ex-type or ex-epitype strains.

Phylogenetic analyses

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 species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of three loci (ITS, TUB2 and TEF1-α). 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 (8.2.12) (Stamatakis 2014) and MrBayes on XSEDE (3.2.7a) (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003; Ronquist et al. 2012), respectively. Four Markov chains were run for two runs from random starting trees for 5 million generations (ITS + TUB2 + TEF1-α) 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).

Phylogeny

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-α has an aligned length of 2285 total characters (ITS: 1–616, TUB2: 617–1570, TEF1-α: 1571–2285) including gaps, of which 1119 characters are constant, 285 variable characters are parsimony-uninformative, and 881 are parsimony-informative. For the BI and ML analyses, the substitution model SYM+I+G for ITS, HKY+G for TUB2 and GTR+I+G for TEF1-α were selected and incorporated into the analyses. The MCMC analysis of the three concatenated genes run for 2,650,000 generations, resulting in 2651 trees. The initial 662 trees representative of the analysis burn-in phase was discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus trees (Fig. 1; first value: PP ≥ 0.50 shown). The alignment contained a total of 1453 unique site patterns (ITS: 269, TUB2: 670, TEF1-α: 514). The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig. 1).

ML bootstrap support values (≥ 50%) and Bayesian posterior probability (≥ 0.50) are shown as first and second position above nodes, respectively. The 81 strains were assigned to 59 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 Monochaetia schimae sp. nov., Neopestalotiopsis haikouensis sp. nov., Neopestalotiopsis piceana, Pestalotiopsis licualacola, respectively.

Taxonomy

Monochaetia schimae Z.X. Zhang, X.G. Zhang & J.W. Xia, sp. nov. Fig. 2.

MycoBank 841381

Etymology: Name refers to the genus of the host plant Schima superba.

Type: China, Hainan Province: East Harbour National Nature Reserve, on diseased leaves of Schima superba, 23 May 2021, Z.X. Zhang (holotype HSAUP212201; ex-type living culture SAUCC212201). GenBank deposition numbers of sequences derived from type: ITS, MZ577565; TUB2, OK104867; TEF1-α, OK104874.

Description: 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 diam/day, irregularly circular, raised, dense surface with lobate edge, zonate with different sector light brown at the margin, brown at the center; reverse brown at the margin, dark brown at the center. Sexual morph: Undetermined. Asexual morph: Leaf spots irregular, pale brown in center, brown to tan at margin. 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 18.0–24.0 × 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, conic, hyaline and smooth-walled; three median cells together 12.5–15.5 µm long, mean ± SD = 14.2 ± 0.7 µm, doliiform, brown, 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, central, tubular, filiform; basal appendage 2.5–5.0 µm long, single, tubular, filiform.

Additional specimen examined: China, Hainan Province: East Harbour National Nature Reserve, 23 May 2021, Z.X. Zhang. On diseased leaves of Schima superba (Theaceae), paratype HSAUP212202, ex-paratype culture SAUCC212202; on diseased leaves of Schima superba (Theaceae), paratype HSAUP212203, ex-paratype culture SAUCC212203.

Notes: Monochaetia schimae is introduced based on the multi-locus phylogenetic analysis, with three isolates clustering separately in a well-supported clade (ML/BI = 100/1). M. schimae is most closely related to M. ilexae (CBS 101009), but distinguished based on ITS, TUB2 and TEF1-α loci by 94 nucleotide differences in the concatenated alignment, in which 18/526 are distinct in the ITS region, 32/698 in the TUB2 region and 44/462 in the TEF1-α region. Morphologically, M. schimae differ from M. ilexae in its smaller conidia (18.0–24.0 × 4.5–6.0 vs. 20.0–27.0 × 3.0–5.0 µm). Therefore, we establish this fungus as a novel species.

Neopestalotiopsis haikouensis Z.X. Zhang, X.G. Zhang & J.W. Xia, sp. nov. Fig. 3.

MycoBank 841382

Etymology: Named after the host collection, Haikou City.

Type: China, Hainan Province, Haikou City: East Harbour National Nature Reserve, on diseased leaves of Ilex chinensis. 23 May 2021, Z.X. Zhang (holotype HSAUP212271; ex-type living culture SAUCC212271). GenBank deposition numbers of sequences derived from type: ITS, OK087294; TUB2, OK104870; TEF1-α, OK104877.

Description: Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 7.0–9.0 mm/day and occupying an entire 90 mm Petri dish in 7 d; edge undulate, white to grey white, with moderate aerial mycelium on the surface, with black, gregarious conidiomata; reverse similar in colour. Sexual morph: Undetermined. Asexual morph: Leaf spots irregular, grey white in center, brown to tan at margin. 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 conic with a truncate base, hyaline, rugose and thin-walled, 3.0–4.5µm long; three median cells doliiform, 11.5–15.0 µm long, mean ± SD = 13.2 ± 1.0 µm, wall rugose, versicoloured, 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.

Additional specimen examined: China, Hainan Province: East Harbour National Nature Reserve, 23 May 2021, Z.X. Zhang. On diseased leaves of Ilex chinensis, paratype HSAUP212272, ex-paratype culture SAUCC212272.

Notes: Phylogenetic analysis of a combined three gene showed that Neopestalotiopsis haikouensis formed an independent clade (Fig. 1) and is phylogenetically distinct from N. aotearoa (CBS 367.54), N. piceana (CBS 254.32), N. brachiate (MFLUCC 17-1555), N. phangngaensis (MFLUCC 18-0119), N. rhizophorae (MFLUCC 17-1550), N. petila (MFLUCC 17-1738) and N. rosicola (CFCC 51993). N. haikouensis can be distinguished from the phylogenetically most closely related species N. aotearoa by shorter and wider conidia (16.0–22.0 × 4.5–7.0 vs. 20.0–27.0 × 3.0–5.0 µm), and several loci (7/532 in the ITS region, 4/771 TUB2 and 6/487 TEF1-α). Therefore, we establish this fungus as a novel species.

Neopestalotiopsis piceana Maharachch., K.D. Hyde & Crous, Studies in Mycology 79:146. (2014) Fig. 4.

Description: Colonies on PDA reaching 70.0–80.0 mm diam after 7 d at 25°C, edge entire, whitish to pale honeycoloured, with sparse aerial mycelium on the surface, with black, gregarious conidiomata; reverse similar in colour. Sexual morph: Undetermined. Asexual morph: Leaf spots irregular, pale brown in center, brown to tan at margin. Conidiomata (on PDA) pycnidial, globose to clavate, solitary, 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 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.

Specimens examined: China, Hainan Province: Five Fingers Group Scenic Area, 20 May 2021, Z.X. Zhang. On diseased leaves of Ficus microcarpa, paratype HSAUP210112, ex-paratype culture SAUCC210112; on diseased leaves of Ficus microcarpa, paratype HSAUP210113, ex-paratype culture SAUCC210113.

Notes: In the present study, two strains (SAUCC210112 and SAUCC210113) from symptomatic leaves of Ficus microcarpa were similar to Neopestalotiopsis piceana (CBS 394.48) (Maharachch. et al. 2014) based on phylogeny (Fig. 1). Morphologically, our strains were similar to N. piceana, which was originally described with an asexual morph on wood of Picea sp., Cocos nucifera and fruit of Mangifera indica in China, but the sexual morph of N. piceana was undetermined. We therefore identify our strains as N. piceana.

Pestalotiopsis licualacola K. Geng, Y. Song, K.D. Hyde & Yong Wang bis, Phytotaxa 88 (3):51. (2013) Fig. 5.

Description: Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 7.0–9.0 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. Sexual morph: Undetermined. Asexual morph: Leaf spots irregular, pale brown in center, brown to tan at margin. Conidiomata (pycnothyria) solitary, scattered, black, raising above surface of culture medium, subglobose. Conidiophores cylindrical, hyaline, smooth-walled. Conidiophores most 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 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.

Specimens examined: China, Hainan Province: East Harbour National Nature Reserve, 23 May 2021, Z.X. Zhang. On diseased leaves of Ilex chinensis, paratype HSAUP210087, ex-paratype culture SAUCC210087; on diseased leaves of Ilex chinensis, paratype HSAUP210088, ex-paratype culture SAUCC210088.

Notes: In the present study, two strains (SAUCC210087 and SAUCC210088) from symptomatic leaves of Ilex chinensis were similar to Pestalotiopsis licualacola (HGUP4057) (Geng et al. 2013) based on phylogeny (Fig. 1). Morphologically, our strains were similar to P. licualacola, which was originally described with an asexual morph on leaves of Licuala grandis in China, but the sexual morph of P. licualacola was undetermined. We therefore identify our strains as P. licualacola.

In the study of its phylogenetic affinity and position in the Ascomycota hierarchical system, Liu et al. (2019) placed a series of coelomycetous fungi with appendage-bearing conidia in the newly introduced family Sporocadaceae (type genus: Sporocadus Corda). In most genera, conidiomata pycnidial, acervular or stromatic, semi-immersed or immersed, scattered, gregarious or confluent, glabrous; conidiophores branched or reduced to conidiogenous cells, mostly hyaline, smooth; conidiogenous cells ampulliform, lageniform, cylindrical or subcylindrical, hyaline, sometimes pale brown; conidia septate, smooth, undulate or verruculose, fusoid, subcylindrical or cylindrical, straight or curved; end cells mostly hyaline, or sometimes pale brown; median cells pale brown to dark brown, or sometime almost colourless; appendages on the end cells present, or absent in some genera, if present, tubular, filiform, straight or flexuous, attenuated or not, branched or unbranched.

In the previous study, Liu et al. (2019) revealed three major clades based on the ITS/LSU/RPB2 phylogeny, corresponding to the three previously proposed families “Bartaliniaceae, Discosiaceae and Pestalotiopsidaceae”. Because these family names were considered synonyms of Sporocadaceae by Jaklitsch et al. (2016), these groups are referred to as Clade 1 (Discosiaceae), Clade 2 (Pestalotiopsidaceae) and Clade 3 (Bartaliniaceae) for convenience. We picked Clade 2 of the family Sporocadaceae in this study, including Pestalotiopsis Steyaert, Neopestalotiopsis Maharachch. et al., Pseudopestalotiopsis Maharachch. et al., Monochaetia (Sacc.) Allesch., Ciliochorella Syd., Seiridium Nees and Nonappendiculata Liu et al. (Liu et al. 2019). Based on ITS/TUB2/TEF1-α molecular data, phylogenetic analyses revealed that the retrieved three isolates (SAUCC212201, SAUCC212202 and SAUCC212203) are include in Monochaetia, four isolates (SAUCC212271, SAUCC212272, SAUCC210112 and SAUCC210113) are include in Neopestalotiopsis and two isolates (SAUCC210087 and SAUCC210088) are include in Pestalotiopsis. Owing to different nucleotides in the concatenated alignment and morphology, three isolates (SAUCC212201, SAUCC212202 and SAUCC212203) of Monochaetia were delimited as a new species, namely M. schimae and two isolates (SAUCC212271 and SAUCC212272) of Neopestalotiopsis were delimited as a new species, namely N. haikouensis. Besides, two isolates (SAUCC210112 and SAUCC210113) of Neopestalotiopsis clustered in N. piceana and two isolates (SAUCC210087 and SAUCC210088) of Pestalotiopsis clustered in P. licualacola.

In addition, members of Sporocadaceae are of particular interest with regard to the production of secondary metabolites, e.g. Pestalotiopsis, Bartalinia and Morinia (Collado et al. 2006; Gangadevi & Muthumary 2008; Liu et al. 2009). Pestalotiopsis fici was shown to possess a very high number of gene clusters involved in bioactive compound synthesis (Wang et al. 2016). Because genera in this family of fungi share the same evolutionary history, it is important to found novel species and screen for novel metabolites in future studies.

Acknowledgments This work was supported by the National Natural Science Foundation of China (no. 31900014, U2002203).

Author contribution Conceived and designed the experiments: JW Xia. Performed the experiments: ZX Zhang. Analyzed the data: TC Mu, SB Liu and RY Liu. Wrote the paper: ZX Zhang, XG Zhang and JW Xia. All authors read and approved the final manuscript.

Data availability The data, including the sequences on GenBank and specimen data on MycoBank will be available to any researcher wishing to use them for non-commercial purposes, without breaching participant confidentiality.

Declarations Conflict of interest The authors declare no competing interests.

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Morphological and Phylogenetic Analyses Reveal Two New Species of Sporocadaceae From Hainan, China

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