Morphological and phylogenetic analyses reveal a new genus and two new species of Tubakiaceae from China

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

doi:10.3897/mycokeys.84.73940

Abstract

Species of Tubakiaceae have often been reported as plant pathogens or endophytes, 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/LSU/rpb2 and ITS/tef1/tub2, in conjunction with morphological characteristics. Five strains isolated from Lithocarpus fohaiensis and Quercus palustris in China represented a new genus of Tubakiaceae, Obovoideisporodochium and three species, viz. Obovoideisporodochium lithocarpi sp. nov., Tubakia lushanensis sp. nov. and T. dryinoides.

Keywords

multigene phylogeny, new genus, new species, taxonomy, Tubakia

Introduction

Diaporthales represents an important order in Sordariomycetes containing taxa that are mainly isolated as endophytes, saprobes or plant pathogens on various hosts (Fan et al. 2018). Tubakiaceae is a family in Diaporthales, which has been studied in recent years by Braun et al. (2018) by incorporating morphological and molecular data with appropriate genes to resolve species limitations in the family. Tubakiaceae currently comprises eight genera including Apiognomonioides U. Braun et al., Involutiscutellula U. Braun & C. Nakash., Oblongisporothyrium U. Braun & C. Nakash., Paratubakia U. Braun & C. Nakash., Racheliella Crous & U. Braun, Saprothyrium U. Braun et al., Sphaerosporithyrium U. Braun et al. and Tubakia B. Sutton (Braun et al. 2018).

Tubakia, the type genus of Tubakiaceae, was introduced by Sutton (1973). Species of Tubakia are endophytes in leaves and twigs of many tree species, but can also cause conspicuous leaf symptoms as plant pathogens (Harrington et al. 2012; Harrington & McNew 2016, 2018; Braun et al. 2018). The genus is characterised by unique pycnothyria, consisting of pigmented, radiating, seta-like cells (scutellum) on top of a columella, with small phialides on the underside of the scutellum producing ellipsoid, hyaline to brown conidia that are forced out from under the pycnothyrium for rain dispersal (Harrington & McNew 2018). Some species produce a second type of much smaller conidia (microconidia), either in “normal” pycnothyria or in separate, mostly smaller pycnothyria (Braun et al. 2018).

Saccardo (1913) introduced the genus Actinopelte for A. japonica, a scutellate fungus found in Japan on Castanea crenata (= C. pubinervis). Saccardo (1913) confused the large conidia of this species with asci, which was clarified and corrected by Theissen (1913) who provided a detailed discussion, description and illustration (Theissen 1913) of A. japonica. Von Höhnel (1925) revisited Actinopelte, added a new species, A. americana and introduced the new combination A. dryina, based on Leptothyrium dryinum. Yokoyama & Tubaki (1971) discussed the history of this genus in detail, published results of comprehensive examinations of Japanese collections in vivo and in vitro and described A. castanopsidis, A. rubra and A. subglobosa, based on Japanese collections. Since Saccardo’s Actinopelte turned out to be illegitimate (later homonym of Actinopelte Stitzenb. 1861), Sutton (1973) introduced the replacement name Tubakia and reallocated all species recognised and treated in Yokoyama & Tubaki (1971) to this genus. Twenty-one additional Tubakia species have subsequently been described including fifteen new Tubakia species and six combinations in Tubakia species (Yun & Rossman 2011; Harrington et al. 2012; Braun et al. 2014; Harrington & McNew 2018; Senanayake et al. 2017; Braun et al. 2018; Yun & Kim 2020).

During field trips to collect plant pathogens causing leaf spots symptoms in China, several specimens associated with typical diaporthalean symptoms were collected from various tree hosts, i.e. Betula dahurica (Betulaceae), Juglans regia (Juglandaceae), Prunus davidiana (Rosaceae), Lithocarpus fohaiensis, Quercus mongolica and Q. palustris (Fagaceae). Based on morphological analyses as well as phylogenetic data, this study presents a new genus of Tubakiaceae, Obovoideisporodochium and three species, viz. Obovoideisporodochium lithocarpi sp. nov., Tubakia lushanensis sp. nov. and T. dryinoides from diseased leaves of L. fohaiensis or Q. palustris.

Materials and methods

Isolation and morphological studies

The samples were collected from the Shandong and Yunnan Provinces, China. The strains were isolated from diseased leaves of Lithocarpus fohaiensis and Quercus palustris using tissue isolation methods. Tissue fragments (5 mm × 5 mm) were taken from the margin of leaf lesions and surface-sterilised by consecutively immersing in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s and then rinsing in sterile distilled water for 1 min. The pieces were dried with sterilised paper towels and placed on potato dextrose agar (PDA). All the PDA plates were incubated in a biochemical incubator at 25°C for 2–4 days. The colonies from the periphery were picked out and inoculated on to new PDA plates. Colony photos after 7 days and 15 days were taken by a digital camera (Canon Powershot G7X). 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. All fungal strains were stored in 10% sterilised glycerine at 4°C 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 and amplification

Genomic DNA was extracted from fungal mycelia grown 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), the partial large subunit (LSU) nrRNA gene, part of the beta-tubulin gene region (tub2), partial translation elongation factor 1-alpha (tef1) and partial RNA polymerase II second largest subunit (rpb2) genes were amplified and sequenced by using the primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Rehner & Samuels 1994; Vilgalys & Hester 1990), Bt2a/Bt2b (Glass & Donaldson 1995), EF1-728F/EF-2 (O’Donnell et al. 1998; Carbone & Kohn 1999) and frpb2-5F/frpb2-7cR (Liu et al. 1999; Sung et al. 2007).

The PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μl reaction volume, which contained 12.5 μl Green Taq Mix (Vazyme, Nanjing, China), 1 μl of each forward and reverse primer (10 μM stock) (Biosune, Shanghai, China) and 1 μl template genomic DNA in amplifier, adjusted with distilled deionised water to a total volume of 25 μl. The 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 50 s, extension at 72°C for 1 min and a final elongation step at 72°C for 10 min. The annealing temperatures for the genes were 55°C for ITS, 52°C for LSU, 53°C for tub2, 48°C for tef1 and 56°C for rpb2. The PCR products were separated with the 1% agarose gel, with added GelRed and UV light used to visualise the fragments. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA v. 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.

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

Species	Voucher¹	Host/Substrate	Country	GenBank accession number
Species	Voucher¹	Host/Substrate	Country	ITS	LSU	*tef1*	*tub2*	*rpb2*
Greeneria uvicola	FI12007	‒	Uruguay	HQ586009	GQ870619	‒	‒	‒
Involutiscutellula rubra	CBS 192.71*	Quercus phillyraeoides	Japan	MG591899	MG591993	MG592086	MG592180	MG976476
	MUCC 2303	Quercus phillyraeoides	Japan	MG591900	MG591994	MG592087	MG592181	MG976477
	ATCC 22473	Quercus phillyraeoides	Japan	MG591901	MG591995	MG592088	‒	MG976478
Oblongisporothyrium castanopsidis	CBS 124732	Castanopsis cuspidata	Japan	MG591849	MG591942	MG592037	MG592131	MG976453
Oblongisporothyrium castanopsidis	CBS 189.71*	Castanopsis cuspidata	Japan	MG591850	MG591943	MG592038	MG592132	MG976454
*Obovoideisporodochium lithocarpi*	SAUCC 0748*	*Lithocarpus fohaiensis*	China	MW820279	MW821346	MZ996876	MZ962157	MZ962155
*Obovoideisporodochium lithocarpi*	SAUCC 0745	*Lithocarpus fohaiensis*	China	MW820280	MW821347	MZ996877	MZ962158	MZ962156
Paratubakia subglobosa	CBS 124733	Quercus glauca	Japan	MG591913	MG592008	MG592102	MG592194	MG976489
Paratubakia subglobosa	CBS 193.71*	Quercus glauca	Japan	MG591914	MG592009	MG592103	MG592195	MG976490
Paratubakia subglobosoides	MUCC 2293*	Quercus glauca	Japan	MG591915	MG592010	MG592104	MG592196	MG976491
Racheliella wingfieldiana	CBS 143669*	Syzigium guineense	Africa	MG591911	MG592006	MG592100	MG592192	MG976487
Sphaerosporithyrium mexicanum	CPC 32258	Quercus eduardi	Mexico	MG591895	MG591989	MG592082	MG592176	‒
Sphaerosporithyrium mexicanum	CPC 33021*	Quercus eduardi	Mexico	MG591896	MG591990	MG592083	MG592177	MG976473
Tubakia americana	CBS 129014	Quercus macrocarpa	USA	MG591873	MG591966	MG592058	MG592152	MG976449
Tubakia californica	CPC 31505*	Quercus kelloggii	USA	MG591835	MG591928	MG592023	MG592117	MG976451
Tubakia dryina	CBS 112097*	Quercus robur	Italy	MG591851	MG591944	MG592039	MG592133	MG976455
Tubakia dryinoides	SAUCC 1924	*Quercus palustris*	China	MW784842	MW784852	MW842260	MW842263	MW842266
	CBS 329.75	Quercus sp.	France	MG591874	MG591967	MG592059	MG592153	MG976458
	MUCC2290	Castanea crenata	Japan	MG591876	MG591968	MG592061	MG592155	MG976459
	MUCC2291	Castanea crenata	Japan	MG591877	MG591969	MG592062	MG592156	MG976460
	MUCC2292*	Quercus phillyraeoides	Japan	MG591878	MG591970	MG592063	MG592157	MG976461
Tubakia hallii	CBS 129013	Quercus stellata	USA	MG591880	MG591972	MG592065	MG592159	MG976462
Tubakia iowensis	CBS 129012*	Quercus macrocarpa	USA	MG591879	MG591971	MG592064	MG592158	‒
Tubakia japonica	ATCC 22472*	Castanea crenata	Japan	MG591886	MG591978	MG592071	MG592165	MG976465
Tubakia koreana	KCTC46072	Quercus mongolica	South Korea	KP886837	‒	‒	‒	‒
Tubakia liquidambaris	CBS 139744	Liquidambar styraciflua	USA	MG605068	MG605077	MG603578	‒	‒
*Tubakia lushanensis*	SAUCC 1921	*Quercus palustris*	China	MW784677	MW784850	MW842262	MW842265	MW842268
*Tubakia lushanensis*	SAUCC 1923*	*Quercus palustris*	China	MW784678	MW784851	MW842261	MW842264	MW842267
Tubakia melnikiana	CPC 32255*	Quercus canbyi	Mexico	MG591893	MG591987	MG592080	MG592174	MG976472
Tubakia oblongispora	MUCC 2295*	Quercus serrata	Japan	MG591897	MG591991	MG592084	MG592178	MG976474
Tubakia paradryinoides	MUCC 2294*	Quercus acutissima	Japan	MG591898	MG591992	MG592085	MG592179	MG976475
Tubakia seoraksanensis	CBS 127490*	Quercus mongolica	South Korea	MG591907	KP260499	MG592094	MG592186	‒
Tubakia seoraksanensis	CBS 127491	Quercus mongolica	South Korea	HM991735	KP260500	MG592095	MG592187	MG976484
Tubakia sierrafriensis	CPC 33020	Quercus eduardi	Mexico	MG591910	MG592005	MG592099	MG592191	MG976486
Tubakia sp.	CBS 115011	Quercus robur	Netherlands	MG591912	MG592007	MG592101	MG592193	MG976488
Tubakia suttoniana	CBS 639.93	Quercus sp.	Italy	MG591921	MG592016	MG592110	MG592202	MG976493

Phylogeny

The generated consensus sequences for each gene were subjected to megablast searches to identify closely-related sequences in the NCBI’s GenBank nucleotide database (Zhang et al. 2000). For the ITS-LSU-rpb2 and ITS-tef1-tub2 analyses, subsets of sequences from the alignments of Braun et al. (2018) were used as backbones. 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 (ITS-LSU-rpb2 and ITS-tef1-tub2).

Phylogenetic analyses were based on Maximum Likelihood (ML) and Bayesian Inference (BI) for the multilocus 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 & Ronquist 2001; Ronquist & Huelsenbeck 2003; Ronquist et al. 2012), respectively. For the ML analyses, the default parameters were used and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included four parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 50 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. All resulting trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and the layout of the trees was done in Adobe Illustrator CC 2019.

Result

Phylogenetic analyses

ITS/LSU/rpb2 phylogeny

The alignment contained 37 isolates representing Tubakia and allied taxa and a strain of Greeneria uvicola (FI12007) was used as outgroup. The final alignment contained a total of 2459 characters used for the phylogenetic analyses, including alignment gaps, viz. ITS: 1–676, LSU: 677–1545, rpb2: 1546–2459. Of these characters, 1858 were constant, 115 were variable and parsimony-uninformative and 486 were parsimony-informative. MrModelTest recommended that the Bayesian analysis should use Dirichlet base frequencies for the ITS, LSU and rpb2. The GTR+I+G model was proposed for ITS, LSU and rpb2. The MCMC analysis of the three concatenated genes, run for 700,000 generations, resulted in 14,001 trees. The initial 3500 trees, representative of the analysis burn-in phase, were discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus trees (Fig. 1; first value: PP > 0.74 shown). The alignment contained a total of 744 unique site patterns (ITS: 266, LSU: 128, rpb2: 350). The topology of the ML tree confirmed the tree topology obtained from the Bayes analyses and, therefore, only the ML tree is presented (Fig. 1). Bayesian posterior probability (> 0.74) and ML bootstrap support values (> 74%) are shown as first and second position above nodes, respectively. The 37 strains were assigned to 25 species clades, based on the three-gene phylogeny (Fig. 1).

Figure 1.

Phylogram of Tubakiaceae, based on the concatenated ITS, LSU and rpb2 sequence alignment. The BI and ML bootstrap support values above 0.74 and 74% are shown at the first and second position, respectively. The tree is rooted to Greeneria uvicola (culture FI12007) and ex-type cultures are indicated in bold face. Strains from the current study are in red. Some branches were shortened for layout purposes – these are indicated by two diagonal lines with the number of times a branch was shortened indicated next to the lines.

ITS/tef1/tub2 phylogeny

The alignment contained 37 isolates representing Tubakia and allied taxa and a strain of Greeneria uvicola (FI12007) was used as outgroup. The final alignment contained a total of 1939 characters used for the phylogenetic analyses, including alignment gaps, viz. ITS: 1–676, tef1: 677–1358, tub2: 1359–1939. Of these characters, 1077 were constant, 136 were variable and parsimony-uninformative and 726 were parsimony-informative. MrModelTest recommended that the Bayesian analysis should use Dirichlet base frequencies for the ITS, tef1 and tub2 data partitions. The GTR+I+G model was proposed for ITS and HKY+I+G for tef1 and tub2. The MCMC analysis of the three concatenated genes, run for 170,000 generations resulted in 3401 trees. The initial 850 trees, representative of the analysis burn-in phase, were discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus trees (Fig. 2; first value: PP > 0.74 shown). The alignment contained a total of 997 unique site patterns (ITS: 266, tef1: 416, tub2: 315). The topology of the ML tree confirmed the tree topology obtained from the Bayes analyses and, therefore, only the ML tree is presented (Fig. 2). Bayesian posterior probability (> 0.74) and ML bootstrap support values (> 74%) are shown as first and second position above nodes, respectively. The 37 strains were assigned to 25 species clades, based on the three-gene phylogeny (Fig. 2).

Figure 2.

Phylogram of Tubakiaceae, based on the concatenated ITS, tef1 and tub2 sequence alignment. The BI and ML bootstrap support values above 0.74 and 74% are shown at the first and second position, respectively. The tree is rooted to Greeneria uvicola (culture FI12007) and ex-type cultures are indicated in bold face. Strains from the current study are in red. Some branches were shortened for layout purposes – these are indicated by two diagonal lines with the number of times a branch was shortened indicated next to the lines.

Based on phylogenetic data (Figs 1 and 2) and morphological analyses, the present study revealed a new genus of Tubakiaceae, Obovoideisporodochium and three species, viz. Obovoideisporodochium lithocarpi sp. nov., Tubakia lushanensis sp. nov. and T. dryinoides.

Taxonomy

Obovoideisporodochium Z. X. Zhang, J. W. Xia & X. G. Zhang, gen. nov.

MycoBank No: 841103

Type species

Obovoideisporodochium lithocarpi Z. X. Zhang, J. W. Xia & X. G. Zhang

Etymology

Composed of “obovoideisporo-” (obovoid spores) and “-dochium” (referring to the conidioma, i.e. sporodochium).

Description

Genus of Tubakiaceae. Living as endophyte in leaves and causing leaf spots. Asexual morph: mycelium consisting of septate, smooth and hyaline hyphae, thin-walled. Conidiomata sporodochial, appeared within 20 days or longer, formed on agar surface, slimy, pale bluish-green, semi-submerged. Sporodochial conidiophores densely and irregularly branched, bearing apical whorls of 2–3 phialides; sporodochial phialides monophialidic, subulate to subcylindrical, smooth, thin-walled, tapering towards apex, swelling at base. Conidia formed singly, obovoid to ellipsoid, smooth, thin walled, apex obtuse, base with inconspicuous to conspicuous hilum. Sexual morph: unknown.

Notes

In the two phylogenetic trees (Figs 1 and 2), Obovoideisporodochium is allied to Racheliella, Oblongisporothyrium and Paratubakia, but forms a separate lineage with full support (PP = 1, ML-BS = 100%), suggesting a genus of its own.

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

MycoBank No: 841104

Fig. 3

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Lithocarpus fohaiensis (Fagaceae), 11 Sep 2020, Z. X. Zhang, (holotype HSAUP0748, ex-type living culture SAUCC 0748).

Figure 3.

Obovoideisporodochium lithocarpi (SAUCC 0748). a infected leaf of Lithocarpus fohaiensis; b surface of colony after 15 days on MEA; c reverse of colony after 15 days on MEA; d conidiomata; e–g conidiophores, conidiogenous cells and conidia; h–i conidia. Scale bars: 10 μm (e–i).

Etymology

Name refers to the genus of the host plant Lithocarpus fohaiensis.

Description

Asexual morph: mycelium consisting of septate, smooth and hyaline hyphae, thin-walled, 1.0–2.0 μm. Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 5–6 mm/d and reaching 75–80 mm diam. in 14 d, formed some conspicuous concentric circles, aerial mycelium cottony, white initially, then becoming greyish-sepia. Conidiomata sporodochial, appeared within 20 days or longer, formed on agar surface, slimy, pale bluish-green, semi-submerged. Sporodochial conidiophores densely and irregularly branched, 12.0–26.5 × 1.5–3.0 μm, bearing apical whorls of 2–3 phialides; sporodochial phialides monophialidic, subulate to subcylindrical, 9.5–20.0 × 1.5–3.0 μm, smooth, thin-walled, tapering towards apex, swelling at base. Conidia formed singly, obovoid to ellipsoid, 5.5–8.0 × 2.5–4.0 μm, length/width ratio 1.7–3.1, hyaline, smooth, thin walled, apex obtuse, base with inconspicuous to conspicuous hilum, 0.4–0.9 μm diam. Sexual morph: unknown.

Culture characteristics

Cultures incubated on MEA at 25°C in darkness, attaining 52.0–58.0 mm diam. after 14 d (growth rate 3.5–4.0 mm diam./d), grey-white to creamy white with irregular margin, spread like petals from the inside and outside, reverse dark to light brown, distributed in an irregular circle. Conidial formation not observed.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Lithocarpus fohaiensis (Fagaceae), 11 Sep 2020, Z. X. Zhang, HSAUP0745; living culture SAUCC 0745.

Notes

In the two phylogenetic trees (Figs 1 and 2), Obovoideisporodochium lithocarpi is related to Racheliella wingfieldiana, Oblongisporothyrium castanopsidis, Paratubakia subglobosa and P. subglobosoides, but forms a separate single species lineage with full support (PP = 1, ML-BS = 100%). Furthermore, the conidia of O. lithocarpi (5.5–8.0 μm × 2.5–4.0 μm) are smaller than those of R. wingfieldiana (11.0–15.0 μm × 6.5–7.5 μm), Ob. castanopsidis (14.0–17.0 μm × 7.0–9.5 μm), P. subglobosa (10.0–13.0 μm × 8.0–11.0 μm) and P. subglobosoides (10.0–12.5 μm × 5.5–10.0 μm) and Racheliella, Oblongisporothyrium and Paratubakia spp. form crustose conidiomata and true pycnothyria.

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

MycoBank No: 841105

Fig. 4

Type

China, Shandong Province: Zibo Lushan National Forest Park, on diseased leaves of Quercus palustris Münchh (Fagaceae), 20 Sep 2020, Z. X. Zhang, (holotype HSAUP1923, ex-type living culture SAUCC 1923).

Figure 4.

Tubakia lushanensis (SAUCC 1923). a diseased leaf of Quercus palustris; b surface of colony after 15 days on MEA; c reverse of colony after 15 days on MEA; d conidiomata; e–i conidiogenous cells with conidia; j–k conidia. Scale bars: 10 μm (e–k).

Etymology

. Named after the type locality, Lushan National Forest Park.

Description

Asexual morph: Leaf spots irregular, occurring on leaf veins and at leaf edges. Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 5–7 mm/d and occupying an entire 90 mm Petri dish in 14 d, forming some conspicuous concentric circles, aerial mycelium cottony, white initially, then becoming greyish-sepia. Conidiomata pycnidial, usually globose or subglobose when viewed from above, formed on agar surface, black, semi-submerged, up to 200 μm diam. Pycnidial wall composed of an outer layer of yellow-brown, thick-walled textura angularis and an inner layer with hyaline, thin-walled cells. Conidiophores reduced to conidiogenous cells lining the inner cavity, ampulliform or flask-shaped, smooth, hyaline, 9.0–15.0 μm × 2.0–4.0 μm. Conidia solitary, globose to irregular globose, ellipsoid to broad ellipsoid, 10.0–18.0 μm × 7.5–16.0 μm, length/width ratio 1.0–1.7, slightly lighter and wall thin when immature, slightly darker and wall thickened when ripening, smooth, apex rounded, base with peg-like hila, 1.3–2.3 μm diam. Microconidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on MEA at 25°C in darkness, attaining 52.0–56.0 mm diam. after 14 d (growth rate 3.7–4.0 mm diam./d), creamy white to pale brown with regular margin, grey near the centre and hyphae clusters, reverse brown to dark brown rings, heterogeneous colour, with creamy-white edge. Conidial formation not observed.

Additional specimen examined

China, Shandong Province: Zibo Lushan National Forest Park, on diseased leaves of Quercus palustris Münchh. (Fagaceae), 20 Sep 2020, Z. X. Zhang, HSAUP1921; living culture SAUCC 1921.

Notes

The phylogenetic analysis of a combined three-gene alignment (ITS, tef1 and tub2) showed that T. lushanensis formed an independent clade and is phylogenetically distinct from its closest sister species T. seoraksanensis. This species can be distinguished from T. seoraksanensis by 65 different nucleotides in the concatenated alignment (21/628 in the ITS, 31/581 in the tef1 and 13/521 in the tub2). Morphologically, T. lushanensis differs from T. seoraksanensis in having smaller conidia (10.0–18.0 μm × 7.5–16.0 μm vs. 13.0–25.0 μm × 10.0–15.0 μm) (Yun & Rossman 2011). Furthermore, the MEA’s colony colour of T. lushanensis is different from T. seoraksanensis (surface: creamy white, pale brown to grey vs. whitish to pale yellow; reverse: creamy white, brown to dark brown vs. olive brown, light olive brown to yellow; Yun & Rossman 2011). Therefore, we describe this fungus as a novel species.

Tubakia dryinoides C. Nakash., Fungal Systematics and Evolution 1: 80 (2018)

Fig. 5

Description

Asexual morph: Living as endophyte in leaves, forming distinct leaf lesions, shape and size variable, subcircular to angular-irregular, pale brown to brown. Colonies on PDA incubated at 25°C in the dark with an average radial growth rate of 5–7 mm/d and occupying an entire 90 mm Petri dish in 14 d, forming some conspicuous concentric circles, aerial mycelium cottony, white initially, then becoming greyish-sepia. Conidiomata sporodochial, appeared within 14 days or longer, formed on agar surface, slimy, black, semi-submerged. Sporodochial conidiophores densely and irregularly branched, 11.0–24.0 μm × 1.5–5.0 μm, bearing apical whorls of 2–3 phialides; sporodochial phialides monophialidic, subulate to subcylindrical, 9.0–16.0 μm × 1.5–5.0 μm, smooth, thin-walled, apex obtuse to truncate, sometimes forming indistinct periclinal thickenings. Conidia solitary, ellipsoid to obovoid, 6.5–14.0 μm × 4.0–6.0 μm, wall thin, up to 1.0 μm, hyaline to subhyaline, smooth, apex and base broadly rounded, with inconspicuous to conspicuous basal hilum (frill), occasionally somewhat peg-like and truncate when conspicuous. Microconidia not observed. Sexual morph not observed.

Figure 5.

Tubakia dryinoides (SAUCC 1924). a diseased leaf of Quercus palustris; b surface of colony after 15 days on MEA; c reverse of colony after 15 days on MEA; d conidiomata; e–g conidiophores, conidiogenous cells with conidia; h–i conidia. Scale bars: 10 μm (e–i).

Culture characteristics

Cultures incubated on MEA at 25°C in darkness, attaining 38.0–42.0 mm diam. after 14 d (growth rate 2.7–3.0 mm diam./d), margin scalloped, at first creamy white, grey near the centre, reverse light brown to dark, with olivaceous edge. Conidial formation not observed.

Specimen examined

China, Shandong Province: Zibo Lushan National Forest Park, on diseased leaves of Quercus palustris (Fagaceae), 20 Sep 2020, Z. X. Zhang, HSAUP1924, living culture SAUCC 1924.

Notes

Braun et al. (2018) described Tubakia dryinoides, based on morphological and molecular data. The holotype of T. dryinoides (NBRC H-11618) was collected from Quercus phillyraeoides A. Gray (Braun et al. 2018). In our current research, isolate (SAUCC 1924) collected from diseased leaves of Quercus palustris clustered in the Tubakia dryinoides clade by strong support (Figs 1 and 2). We, therefore, consider the isolated strain (SAUCC 1924) as T. dryinoides. The conidiomata of T. dryinoides is only known from true pycnothyria and the sporodochial conidiomata of the isolated strain (SAUCC 1924) is new for T. dryinoides (Braun et al. 2018). Additionally, the conidia of our isolate (SAUCC 1924) is narrower than the original description of T. dryinoides (4.0–6.0 μm vs. 5.5–10.0 μm; Braun et al. 2018).

Discussion

In the study of the phylogenetic affinity and position of Tubakia in the Ascomycota hierarchical system, Senanayake et al. (2017) placed this genus in the newly-introduced family Melanconiellaceae. However, the recently-published phylogenetic analyses, including sequence data of the type species of Tubakia, confirmed that Tubakia warranted a family of its own, Tubakiaceae (Braun et al. 2018) and the description of eight genera including Apiognomonioides U. Braun et al., Involutiscutellula U. Braun & C. Nakash., Oblongisporothyrium U. Braun & C. Nakash., Paratubakia U. Braun & C. Nakash., Racheliella Crous & U. Braun, Saprothyrium U. Braun et al., Sphaerosporithyrium U. Braun et al. and Tubakia B. Sutton (Braun et al. 2018). The family comprises genera and species with sporodochia, crustose to pustulate pycnidioid stromatic conidiomata and superficial scutellate pycnothyria, monophialidic, colourless, conidiogenous cells, often with collarettes and conidia formed singly, mostly globose to broad ellipsoid-obovoid, aseptate, hyaline to pigmented, often with basal frill or truncate peg-like hilum.

The present study found two new species, one of which represents a novel genus in Tubakiaceae. In order to support the validity of the new species, we followed the guidelines of Braun et al. (2018). Based on ITS/LSU/rpb2 and ITS/tef1/tub2 molecular data, phylogenetic analyses revealed that two of the obtained isolates (SAUCC 0745 and SAUCC 0748) cluster in a separate lineage, fully supported at genus-level and related to the genera Racheliella, Oblongisporothyrium and Paratubakia. The new genus is named Obovoideisporodochium gen. nov. (type species: Obovoideisporodochium lithocarpi sp. nov.). The phylogenetic analyses also revealed that three isolates (SAUCC 1921, SAUCC 1923 and SAUCC 1924) pertain to the genus Tubakia. Owing to different nucleotides in the concatenated alignment and morphology, two isolates (SAUCC 1921 and SAUCC 1923) of Tubakia were identified as a new species, namely T. lushanensis sp. nov, whereas the third isolate (SAUCC 1924) was identified as T. dryinoides.

The centre of genetic diversity of Tubakia appears to be in East Asia, where Quercus and other genera of Fagaceae are the most common hosts (Harrington & McNew 2018). Our study supports this phenomenon well. Tubakia lushanensis (SAUCC 1921 and SAUCC 1923) and T. dryinoides (SAUCC 1924) were isolated from Quercus palustris (Fagaceae), thereby increasing the genetic diversity of Tubakia in East Asia.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 31900014, 31750001 and 31770016).

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