﻿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. 2017Senanayake et al. , 2018Fan 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, Tubakia californica 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(Braun et al. , 2018Zhu 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 Lithocarpus glaber 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).

Sample collection, fungal isolation and morphology
Diseased leaves of Lithocarpus glaber 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.

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 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.
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.  Notes. Phaeotubakia lithocarpicola is the sole species within the newly proposed genus, which is associated with leaf spot disease of Lithocarpus glaber. Two tubakiaceous species were reported from the host genus Lithocarpus before the present study, viz. Obovoideisporodochium lithocarpi from Lithocarpus fohaiensis in China and Tubakia californica from Lithocarpus densiflorus in the USA (Braun et al. 2018;Zhang et al. 2021). Phaeotubakia lithocarpicola 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(Senanayake et al. , 2018Fan 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 Phaeotubakia lithocarpicola 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(Voglmayr et al. , 2017Jiang 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). Obovoideisporodochium lithocarpi 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 Phaeotubakia lithocarpicola 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.