Phaeotubakialithocarpicola gen. et sp. nov. (Tubakiaceae, Diaporthales) from leaf spots in China

Abstract

Tubakiaceae represents a distinct lineage of Diaporthales, including its type genus Tubakia and nine additional known genera. Tubakiaceous species are commonly known as endophytes in leaves and twigs of many tree species, but can also be plant pathogens causing conspicuous leaf symptoms. In the present study, isolates were obtained from diseased leaves of Lithocarpusglaber collected in Guangdong Province, China. The identification was conducted based on morphology and phylogeny of combined loci of 28S nrRNA gene (LSU), internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) of the nrDNA operon, translation elongation factor 1-alpha (tef1) and beta tubulin (tub2). As a result, a distinct clade in Tubakiaceae was revealed named Phaeotubakialithocarpicolagen. et sp. nov., which was distinguished from the other tubakiaceous taxa by its dark brown conidiogenous cells and conidia.

Introduction

The fungal order Diaporthales contains members usually inhabiting plant tissues as pathogens, endophytes and saprophytes (Rossman et al. 2007; Senanayake et al. 2017, 2018; Fan et al. 2018; Jiang et al. 2021a; Udayanga et al. 2021). Tubakiaceae was proposed as a diaporthalean family based on its type genus Tubakia, and the other seven genera, namely Apiognomonioides, Involutiscutellula, Oblongisporothyrium, Paratubakia, Racheliella, Saprothyrium and Sphaerosporithyrium (Braun et al. 2018). Subsequently, Ellipsoidisporodochium and Obovoideisporodochium were added to this family based on morphological and phylogenetical evidence (Zhang et al. 2021; Liu et al. 2022). Hence, ten genera have been accepted in Tubakiaceae before the present study.

Species of Tubakiaceae are usually characterized by forming pycnothyria composed of convex scutella with radiating threads of cells fixed to the substratum by a central columella, mostly surrounded by a sheath of small fertile cells that give rise to one-celled, phialidic conidiogenous cells (Harrington et al. 2012; Braun et al. 2018). However, some species also form crustose or pustulate pycnidioid conidiomata, for example, Tubakiacalifornica is known to only have crustose pycnidioid conidiomata during its lifecycle (Braun et al. 2018). Moreover, conidia of tubakiaceous species are globose, subglobose, ellipsoid, broad ellipsoid-obovoid to subcylindrical or somewhat irregular in shape, aseptate, hyaline, subhyaline to pigmented (Braun et al. 2018; Zhang et al. 2021). Conidia of Apiognomonioides, Ellipsoidisporodochium, Oblongisporothyrium, Obovoideisporodochium and Saprothyrium species are known to be hyaline (Braun et al. 2018; Zhang et al. 2021; Liu et al. 2022). Conidia of Involutiscutellula, Paratubakia and Sphaerosporithyrium species are hyaline to slightly pigmented (Braun et al. 2018), while conidia of Racheliella and Tubakia species are hyaline to pigmented (Braun et al. 2014, 2018; Zhu et al. 2022).

Tubakiaceae species are known to be endophytes in leaves and twigs of many tree species, but can also cause conspicuous symptoms on host leaves as plant pathogens (Harrington et al. 2012; Braun et al. 2018; Zhu et al. 2022). Nearly all tubakiaceous species are reported from Fagaceae, such as species of Castanea, Castanopsis, Fagus, Lithocarpus and Quercus (Braun et al. 2018; Morales-Rodríguez et al. 2021). In addition, these fungi are also discovered from the other plant families, i.e., Altingiaceae, Anacardiaceae, Nyssaceae, Oleaceae, Rosaceae, Sapindaceae and Ulmaceae (Braun et al. 2018; Liu et al. 2022).

The aim of the present study is to identify two isolates obtained from diseased leaves of Lithocarpusglaber from Guangdong Province by morphological characters and phylogeny based on combined loci of 28S nrRNA gene (LSU), internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) of the nrDNA operon, translation elongation factor 1-alpha (tef1) and beta tubulin (tub2).

Materials and methods

Sample collection, fungal isolation and morphology

Diseased leaves of Lithocarpusglaber were collected from Guangdong Province, China. The leaf samples were packed in paper bags and transferred to the laboratory for isolation. The leaves were firstly surface-sterilized for 2 min in 75% ethanol, 4 min in 1.25% sodium hypochlorite, and 1 min in 75% ethanol, then rinsed for 2 min in distilled water and blotted on dry sterile filter paper. Then diseased tissues were cut into 0.5 cm × 0.5 cm pieces using a double-edge blade, and transferred onto the surface of potato dextrose agar (PDA, 200 g potatoes, 20 g dextrose, 20 g agar per L), and incubated at 25 °C to obtain cultures. The hyphal tips were then transferred to clean plates of PDA, malt extract agar (MEA, 30 g malt extract, 5 g mycological peptone, 15 g agar per L) and synthetic low nutrient agar (SNA, 1 g KN2PO4, 1 g KNO3, 0.5 g MgSO4-7H2O, 0.5 g KCl, 0.2 g glucose, 0.5 g gucrose per L) under a dissecting stereomicroscope with sterile needles. The cultures were deposited in China Forestry Culture Collection Center (CFCC, http://cfcc.caf.ac.cn/; accessed on 6 December 2022), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF, http://museum.caf.ac.cn/; accessed on 6 December 2022).

Morphology of the new taxa was studied based on conidiomata formed on PDA plates under a dissecting microscope (M205 C, Leica, Wetzlar, Germany). The conidiogenous cells and conidia were immersed in tap water, then the microscopic photographs were captured with an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany) equipped with an Axiocam 506 color camera, using differential interference contrast (DIC) illumination. More than 50 conidia were randomly selected for measurement. Culture characters were recorded from PDA, MEA and SNA after 10 days at 25 °C in the dark.

DNA extraction, PCR amplification and phylogenetic analyses

The fungal genomic DNA was extracted from mycelia grown on PDA palates after 10 days following the method in Doyle and Doyle (1990). Four partial loci, ITS and LSU regions, tef1 and tub2 genes were amplified by the following primer pairs: ITS1 and ITS4 for ITS (White et al. 1990), LR0R and LR5 for LSU (Vilgalys and Hester 1990), EF1-688F and EF2 for tef1 (Carbone and Kohn 1999), and Bt2a and Bt2b for tub2 (Glass and Donaldson 1995).

The polymerase chain reaction (PCR) conditions were set as follows: an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 50 s at 48 °C (ITS and LSU) or 54 °C (tef1 and tub2), and 1 min at 72 °C, and a final elongation step of 10 min at 72 °C. PCR products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminator Kit v.3.1 (Invitrogen, Waltham, MA, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

The sequences obtained in the current study were assembled using SeqMan v. 7.1.0, and reference sequences were retrieved from the website of the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov; accessed on 15 October 2022), based on sequences from Braun et al. (2018) and Zhang et al. (2021). The sequences were aligned using MAFFT v. 7 and corrected manually using MEGA v. 7.0.21 (Katoh et al. 2019).

The phylogenetic analyses of combined matrixes of ITS-LSU-tef1-rpb2 were performed using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) methods. MP analysis was run using a heuristic search option of 1000 search replicates with random-additions of sequences with a tree bisection and reconnection (TBR) algorithm in PAUP v. 4.0b10 (Swofford 2003). Maxtrees were set to 5 000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). ML was implemented on the CIPRES Science Gateway portal (https://www.phylo.org) using RAxML-HPC BlackBox 8.2.10 (Miller et al. 2010; Stamatakis 2014), employing a GTR-GAMMA substitution model with 1000 bootstrap replicates. Bayesian inference was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist and Huelsenbeck 2003). Two MCMC chains, starting from random trees for 1000000 generations and trees, were sampled every 100^th generation, resulting in a total of 10000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP > 0.9) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v. 1.4.2 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank, and the GenBank accession numbers of all accessions included in the phylogenetic analyses are listed in Table 1.

Table 1.

Isolates and GenBank accession numbers used in the phylogenetic analyses.

Species	Isolatea	Host	Location	GenBank accession number
				ITS	LSU	tef1	tub2
Apiognomonioidessupraseptata	CBS 632.92*	Quercusglauca	Japan	MG976447	MG976448	NA	NA
Ellipsoidisporodochiumphotiniae	SAUCC 210421*	Photiniaserratifolia	China	OK175559	OK189532	OK206440	OK206442
Ellipsoidisporodochiumphotiniae	SAUCC 210423	Photiniaserratifolia	China	OK175560	OK189533	OK206441	OK206443
Involutiscutellularubra	CBS 192.71*	Quercusphillyraeoides	Japan	MG591899	MG591993	MG592086	MG592180
Involutiscutellularubra	MUCC2303	Quercusphillyraeoides	Japan	MG591900	MG591994	MG592087	MG592181
Involutiscutellularubra	MUCC2305	Quercusphillyraeoides	Japan	MG591902	MG591996	MG592089	MG592182
Melanconisgroenlandica	CBS 116540*	Betulanana	Greenland	KU878552	KU878553	KU878554	KU878555
Oblongisporothyriumcastanopsidis	CBS 124732	Castanopsiscuspidata	Japan	MG591849	MG591942	MG592037	MG592131
Oblongisporothyriumcastanopsidis	CBS 189.71*	Castanopsiscuspidata	Japan	MG591850	MG591943	MG592038	MG592132
Obovoideisporodochiumlithocarpi	SAUCC 0748*	Lithocarpusfohaiensis	China	MW820279	MW821346	MZ996876	MZ962157
Paratubakiasubglobosa	CBS 124733	Quercusglauca	Japan	MG591913	MG592008	MG592102	MG592194
Paratubakiasubglobosa	CBS 193.71*	Quercusglauca	Japan	MG591914	MG592009	MG592103	MG592195
Paratubakiasubglobosoides	MUCC2293*	Quercusglauca	Japan	MG591915	MG592010	MG592104	MG592196
Phaeotubakialithocarpicola	CFCC 54422*	Lithocarpusglaber	China	OP951017	OP951015	OQ127584	OQ127586
Phaeotubakialithocarpicola	RK7CX	Lithocarpusglaber	China	OP951018	OP951016	OQ127585	OQ127587
Racheliellawingfieldiana	CBS 143669*	Syzigiumguineense	South Africa	MG591911	MG592006	MG592100	MG592192
Saprothyriumthailandense	MFLUCC 12-0303*	Decaying leaf	Thailand	MF190163	MF190110	NA	NA
Sphaerosporithyriummexicanum	CPC 31361	Quercuseduardi	Mexico	MG591894	MG591988	MG592081	MG592175
Sphaerosporithyriummexicanum	CPC 32258	Quercuseduardi	Mexico	MG591895	MG591989	MG592082	MG592176
Sphaerosporithyriummexicanum	CPC 33021*	Quercuseduardi	Mexico	MG591896	MG591990	MG592083	MG592177
Tubakiaamericana	CBS 129014	Quercusmacrocarpa	USA	MG591873	MG591966	MG592058	MG592152
Tubakiacalifornica	CPC 31496	Quercusagrifolia	USA	MG591829	MG591922	MG592017	MG592111
Tubakiacalifornica	CPC 31499	Quercuswislizeni	USA	MG591832	MG591925	MG592020	MG592114
Tubakiadryina	CBS 112097*	Quercusrobur	Italy	MG591851	MG591944	MG592039	MG592133
Tubakiadryina	CBS 114912	Quercus sp.	Netherlands	MG591853	MG591946	MG592041	MG592135
Tubakiadryina	CBS 129016	Quercusalba	USA	MG591870	MG591963	MG592056	MG592150
Tubakiadryinoides	CBS 329.75	Quercus sp.	France	MG591874	MG591967	MG592059	MG592153
Tubakiadryinoides	CBS 190.71	Castaneacrenata	Japan	MG591876	MG591968	MG592061	MG592155
Tubakiahallii	CBS 129013*	Quercusstellata	USA	MG591880	MG591972	MG592065	MG592159
Tubakiahallii	CBS 129015	Quercusstellata	USA	MG591881	MG591973	MG592066	MG592160
Tubakiajaponica	CBS 191.71	Castaneacrenata	Japan	MG591885	MG591977	MG592070	MG592164
Tubakialiquidambaris	CBS 139744	Liquidambarstyraciflua	USA	MG605068	MG605077	MG603578	NA
Tubakiamelnikiana	CPC 32249	Quercuscanbyi	Mexico	MG591889	MG591983	MG592076	MG592170
Tubakiaoblongispora	MUCC2295*	Quercusserrata	Japan	MG591897	MG591991	MG592084	MG592178
Tubakiaparadryinoides	MUCC2294*	Quercusacutissima	Japan	MG591898	MG591992	MG592085	MG592179

Note: NA, not applicable. Ex-type strains are marked with *, and strains from the present study are in black bold. ^a CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CFCC: China Forestry Culture Collection Center, Beijing, China; CPC: Culture collection of P. W. Crous, housed at CBS; MFLUCC: Mae Fah Luang University Culture Collection, Thailand; MUCC: Lab. of Plant Pathology, Mie University, Japan; SAUCC: Shandong Agricultural University Culture Collection, China.

Results

Phylogenetic analyses

The alignment based on the sequence dataset (ITS, LSU, tef1 and tub2) included 35 ingroup taxa, comprising 2736 characters in the aligned matrix. Of these, 1721 characters were constant, 206 variable characters were parsimony-uninformative and 809 characters were parsimony informative. The MP analysis resulted in two equally most parsimonious trees (TL = 2708, CI = 0.615, RI = 0.804, RC = 0.385) and the first tree is shown in Fig. 1. The topologies resulting from MP, ML and BI analyses of the concatenated dataset were congruent. Isolates from the present study formed an individual clade in Tubakiaceae representing a new genus and species named Phaeotubakialithocarpicola.

Figure 1.

Phylogram of Tubakiaceae based on combined ITS, LSU, tef1 and tub2 loci. Numbers above the branches indicate maximum parsimony bootstrap (MP BP ≥ 50%), ML bootstrap values (ML-BS ≥ 50%) and Bayesian Posterior Probabilities (BPP ≥ 0.9). The tree is rooted with Melanconisgroenlandica (CBS 116540). Ex-type strains are marked with *, and strains from the present study are marked in bold blue.

Taxonomy

. Phaeotubakia

Ning Jiang gen. nov.

24795432-F4C2-5879-85F3-C7B99BE789D1

MB846813

Etymology.

Named derived from phaeo (= pigmented) and its morphological similarity to Tubakia.

Type species.

Phaeotubakialithocarpicola Y.Q. Zhu & Ning Jiang.

Description.

Sexual morph: Unknown. Asexual morph in vitro: Conidiomata sporodochial, slimy, black, semi-submerged. Conidiophores reduced to conidiogenous cells. Conidiogenous cells brown, smooth, guttulate, cylindrical to ampulliform, attenuate towards apex, phialidic. Conidia blastic, subglobose, broad ellipsoid to ellipsoid, seldom irregular, brown to dark brown, walls smooth, becoming thicker with age, base rounded or with truncate basal hilum.

Notes.

Phaeotubakia is proposed as the eleventh genus of Tubakiaceae based on morphological features and phylogeny of combined ITS, LSU, tef1 and tub2 loci (Fig. 1). Phaeotubakia is distinguished from Apiognomonioides, Ellipsoidisporodochium, Involutiscutellula, Oblongisporothyrium, Obovoideisporodochium, Paratubakia, Racheliella, Saprothyrium and Sphaerosporithyrium by having brown to dark brown conidia (Braun et al. 2018; Zhang et al. 2021). Several species of Tubakia are known to have brown conidia, which is similar to Phaeotubakialithocarpicola (Braun et al. 2018; Zhu et al. 2022). However, they are phylogenetically distinct (Fig. 1).

. Phaeotubakia lithocarpicola

Y.Q. Zhu & Ning Jiang sp. nov.

3033F3C5-37E1-5D83-910E-65A6A9188207

MB846814

Fig. 2

Figure 2.

Morphology of Phaeotubakialithocarpicola (CFCC 54452) A colonies on PDA, MEA and SNA after 10 days at 25 °C B conidiomata formed on PDAC conidiogenous cells giving rise to conidia D–G conidia. Scale bars: 200 μm (B); 10 μm (C–G).

Etymology.

Named after the host genus, Lithocarpus.

Description.

From leaf spots, circular to subcircular, margin distinct, brown to fuscous. Sexual morph: Unknown. Asexual morph in vitro: Conidiomata sporodochial, appeared after 10 days on PDA surface, slimy, black, semi-submerged, 50–350 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells brown, smooth, guttulate, cylindrical to ampulliform, attenuate towards apex, phialidic, 6–15.5 × 3.5–5 μm. Conidia blastic, subglobose, broad ellipsoid to ellipsoid, seldom irregular, brown to dark brown, walls smooth, becoming thicker with age, base rounded or with truncate basal hilum, (13.5–)14–16.5(–18) × (5.5–)7–8.5(–9) μm (n = 50), L/W = 1.7–3.2.

Culture characters.

Colonies on PDA flat, spreading, with flocculent aerial mycelium, white to pale luteous, with age forming concentric zones, reaching a 90 mm diameter and forming abundant black conidiomata after 10 days at 25 °C; on MEA flat, spreading, with flocculent aerial mycelium and crenate edge, pale luteous to pale grey, reaching a 45 mm diameter after 10 days at 25 °C; on SNA flat, spreading, with flocculent aerial mycelium forming concentric rings and entire edge, pale luteous, reaching a 60 mm diameter after 10 days at 25 °C.

Specimens examined.

China, Guangdong Province, Qingyuan City, Yangshan County, Guangdong Nanling Nature Reserve, on diseased leaves of Lithocarpusglaber, 4 December 2019, Yong Li (holotype CAF 800071; ex-holotype culture CFCC 54422). Guangdong Province, Qingyuan City, Yangshan County, Guangdong Nanling Nature Reserve, on diseased leaves of Lithocarpusglaber, 3 December 2019, Dan-ran Bian (culture RK7CX).

Notes.

Phaeotubakialithocarpicola is the sole species within the newly proposed genus, which is associated with leaf spot disease of Lithocarpusglaber. Two tubakiaceous species were reported from the host genus Lithocarpus before the present study, viz. Obovoideisporodochiumlithocarpi from Lithocarpusfohaiensis in China and Tubakiacalifornica from Lithocarpusdensiflorus in the USA (Braun et al. 2018; Zhang et al. 2021). Phaeotubakialithocarpicola represents the third tubakiaceous species discovered from the host genus Lithocarpus. However, P.lithocarpicola differs from O.lithocarpi and T.californica by brown conidiogenous cells and brown to dark brown conidia (Braun et al. 2018; Zhang et al. 2021).

Discussion

Diaporthales is a well-resolved fungal order based on evidence of both morphology and phylogeny (Senanayake et al. 2017, 2018; Fan et al. 2018; Jiang et al. 2020). Tubakia was placed in Melanconiellaceae of Diaporthales (Senanayake et al. 2017), and subsequently transferred to the newly established family of its own Tubakiaceae (Braun et al. 2018). Meanwhile, some species were removed from Tubakia, and seven new genera were proposed based on these species (Braun et al. 2018). Soon after, Ellipsoidisporodochium and Obovoideisporodochium were added to Tubakiaceae (Zhang et al. 2021; Liu et al. 2022). In the present study, the eleventh genus Phaeotubakia is proposed to be included in this family.

Members of Tubakiaceae are quite similar in morphology, but phylogenetically distinct (Braun et al. 2018; Senanayake et al. 2018; Zhang et al. 2021). The sexual morph of Tubakiaceae is not prominent, hence genera and species are distinguished mainly based on their asexual morphology and molecular data.

The newly proposed genus and species Phaeotubakialithocarpicola in the present study produce brown to dark brown conidia on the PDA plates, which is morphologically different from the other tubakiaceous taxa, but similar to Melanconis-like taxa of Diaporthales (Voglmayr et al. 2012, 2017; Jiang et al. 2021b). Four families of Diaporthales are known to contain Melanconis-like genera and species, namely Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae (Jiang et al. 2018; Fan et al. 2018; Senanayake et al. 2018). Hence, traditional morphological identification of diaporthalean fungi is insufficient.

The center of genetic diversity of Tubakia appears to be in East Asia, e.g. China and Japan, where Fagaceae hosts are the most common hosts (Harrington and McNew 2018). Obovoideisporodochiumlithocarpi and several new Tubakia species (T.cyclobalanopsidis and T.quercicola) recently discovered from trees of Fagaceae (Zhang et al. 2021; Zhu et al. 2022), and Phaeotubakialithocarpicola proposed in the present study support this phenomenon well. More taxa of Tubakiaceae may be revealed by more investigations of fungal diversity on Fagaceae in the future.

Citation

Jiang N, Zhu Y-Q, Xue H, Piao C-G, Li Y (2023) Phaeotubakia lithocarpicola gen. et sp. nov. (Tubakiaceae, Diaporthales) from leaf spots in China. MycoKeys 95: 15–25. https://doi.org/10.3897/mycokeys.95.98384

Funding Statement

This research was funded by the National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (NMRC-2021-7).