Morphological and Phylogenetic Analyses Reveal Three New Species of Pestalotiopsis (Sporocadaceae, Amphisphaeriales) from Hainan, China

Species of Pestalotiopsis were mainly introduced as endophytes, plant pathogens or saprobes from various hosts. In this study, ten strains were isolated from Ficus macrocarpa, Phoebe zhennan and Spatholobus suberectus in China. Based on multilocus phylogenies from the internal transcribed spacer (ITS), the partial translation elongation factor 1-alpha gene (tef1α) and the partial beta-tubulin gene (tub2), in conjunction with morphological characteristics, we describe three new species, viz., Pestalotiopsis ficicola sp. nov., P. phoebes sp. nov. and P. spatholobi sp. nov.


Introduction
Pestalotiopsis Steyaert, belonging to Sporocadaceae (Amphisphaeriales, Ascomycota), was introduced by Steyaert in 1949 (type species: Pestalotiopsis guepinii De Not.) [1]. Species of Pestalotiopsis are endophytic, plant pathogenic or saprobic and are associated with a wide range of host plants [2][3][4][5][6]. Currently, a total of 402 names are documented for Pestalotiopsis in the Index Fungorum (http://www.indexfungorum.org/, accessed on 16 May 2023). Initially, Pestalotiopsis resembling those taxa having affinities with Pestalotia were also known as pestalotioid fungi. Pestalotioid fungi are easily characterized by multiseptate and more or less fusiform conidia with appendages at one or both ends, frequently with some melanized cells.
Pestalotia, isolated from Vitis sp., was introduced by De Not. in 1841 [7]. Subsequently, Steyaert split Pestalotia into three genera, viz., Pestalotia (with six-celled conidia), Pestalotiopsis (with five-celled conidia) and Truncatella (with four-celled conidia), based on the number of cells [1]. Although Guba [8,9] initiated some controversy, Steyaert [10][11][12] provided further evidence in support of splitting Pestalotia. Sutton [13] accepted most of the genera discussed here (Pestalotia, Pestalotiopsis and Truncatella) which fit into fairly welldefined groups and cited the electron microscope investigation of Griffiths and Swart [14] to support Steyaert's division of Pestalotiopsis. Molecular phylogenetic analysis largely promoted the development of taxonomy. Jeewon et al. [15] established a phylogenetic tree by analyzing some ITS sequences of Pestalotiopsis species and found that Pestalotiopsis species could be divided into three large branches, with the color type of colored cells and the morphology of the end of the apical accessory filaments as the main distinguishing characteristics. Hu et al. analyzed the ITS and tubulin gene of Pestalotiopsis, combined with morphological characteristics and molecular data, and analyzed the relationship between species of Pestalotiopsis [16]. Maharachch. et al. split Pestalotiopsis sensu lato into three genera-Pestalotiopsis sensu stricto, Neopestalotiopsis and Pseudopestalotiopsis-based on phylogeny of multiple genes and conidial morphology; Pseudopestalotiopsis and Pestalotiopsis Microorganisms 2023, 11, 1627 2 of 12 can be easily distinguished from Neopestalotiopsis by its versicolorous median cells [6]. Liu et al. included Pestalotiopsis into Sporocadaceae using morphological characteristics and on the basis of a multilocus phylogenetic analysis [5]. Some genera of the Sporocadaceae can be divided into three categories based on the number of appendages, viz., genera with a single apical and basal appendage (Monochaetia, Seiridium), other genera which do not form appendages (Nonappendiculata) or genera which have 2-4 appendages (Pestalotiopsis, Ciliochorella, Neopestalotiopsis and Pseudopestalotiopsis) [17]. Recently, Jiang et al. obtained 43 isolates of Pestalotiopsis from diseased leaf tissues of Fagaceae based on combined morphology and phylogeny between 2016 and 2021 [18].
In this study, we produced a collection of the genera Pestalotiopsis species from leaves of Ficus macrocarpa, Phoebe zhennan and Spatholobus suberectus in East Harbour National Nature Reserve, Hainan Province, China. Three new species were described based on unique morphological characters and distinct phylogenetic placement, viz., Pestalotiopsis ficicola sp. nov., P. phoebes sp. nov. and P. spatholobi sp. nov.  [19]. Fragments (5 × 5 mm) were taken from the edges of the leaf lesions, surface sterilized for 30 s in 75% ethanol, rinsed in sterile deionized water for 30 s, rinsed in 5% sodium hypochlorite solution for 1 min and then rinsed four times in sterile deionized water for 30 s [17]. The pieces were blotted on sterile filter paper to dry, transferred onto PDA flats (PDA medium: potato 200 g, agar 15-20 g, dextrose 15-20 g, deionized water 1 L, pH~7.0, available after sterilization) and incubated at 25 • C for 2-3 days. The edges of hyphal growth were then transferred to new PDA flats to obtain pure cultures; simultaneously, they were inoculated on PDA and incubated at 23 • C under continuous near-ultraviolet light to promote sporulation [20].

Samples of
All Pestalotiopsis plates were incubated at 25 • C for 14 days and morphological characters including graphs of the colonies were documented on the 14th day using a digital camera (Canon G7X, Canon, Tokyo, Japan). Morphological characters of conidiomata were studied using a stereomicroscope (Olympus SZX10, Olympus Corporation, Tokyo, Japan) while the micromorphological structures were observed using a microscope (Olympus BX53, Olympus Corporation, Tokyo, Japan). All cultures were deposited in 10% sterilized glycerin and sterile water at 4 • C for future studies. Micromorphological structural measurements were taken using the Digimizer software v. 5.6.0 (https://www.digimizer.com/, accessed on 16 May 2023), with 20 measurements taken for each structure [17]. Voucher specimens were deposited in the Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS) and the Herbarium of the Department of Plant Pathology, Shandong Agricultural University, Taian, China (HSAUP). Ex-holotype living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org, accessed on 16 May 2023).

DNA Extraction and Amplification
Genomic DNA was extracted from the colonies grown on PDA using a kit method (OGPLF-400, GeneOnBio Corporation, Changchun, China) [21]. Gene sequences were obtained from five loci including the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the partial translation elongation factor 1-alpha gene (tef1α) and the partial beta-tubulin gene (tub2). These were amplified by the primer pairs and polymerase chain reaction (PCR) programs listed in Table 1. Amplification reactions were performed in a 25 µL reaction volume which contained 10 µL 2 × Hieff Canace ® Plus PCR Master Mix (With Dye) (Yeasen Biotechnology, Cat No. 10154ES03, Shanghai, China), 0.5 µL of each forward and reverse primer (10 µM) (TsingKe, Qingdao, China) and 1 µL template genomic DNA, adjusted with distilled deionized water to a total volume of 25 µL. PCR amplification products were visualized on 2% agarose electrophoresis gel. DNA sequencing was performed using an Eppendorf Master Thermocycler (Hamburg, Germany) at the TsingKe Company Limited (Qingdao, China) bidirectionally. Consensus sequences were obtained using MEGA 7.0 [22]. All sequences generated in this study were deposited in GenBank (Table S1: See Supplementary File S1).

DNA Extraction and Amplification
Novel sequences obtained in this study and related sets of sequences from Jiang et al. [18] were aligned with MAFFT v. 7 and corrected manually using MEGA 7 [27]. Multilocus phylogenetic analyses were based on the algorithms maximum likelihood (ML) and Bayesian inference (BI) methods. The ML was run on the CIPRES Science Gateway portal (https://www.phylo.org, accessed on 16 May 2023) [28] using RAxML-HPC2 on XSEDE v. 8.2.12 [29] and employed a GTRGAMMA substitution model with 1000 bootstrap replicates. Other parameters were default. For Bayesian inference analyses, the best model of evolution for each partition was determined using ModelTest v. 2.3 [30] and included the analyses. The BI was performed using MrBayes on XSEDE v. 3.2.7a [31][32][33] and two Markov chain Monte Carlo (MCMC) chains were run, starting from random trees, for 2,000,000 generations (average standard deviation of split frequencies < 0.01). Additionally, a sampling frequency of 100 generations was used. The first 25% of trees were discarded as burn-in and BI posterior probabilities (PPs) were conducted from the remaining trees. The consensus trees were optimized using

Phylogenetic Analyses
The alignment contained 182 strains representing Pestalotiopsis and the strain MFLUCC 12-0652 of Neopestalotiopsis magna was used as outgroup [6]. The dataset had an aligned length of 2011 characters including gaps, viz., ITS: 1-565, tef1α: 566-1149 and tub2: 1150-2011 (Supplementary File S1). Of these, 1172 were constant, 281 were parsimony uninformative and 558 were parsimony informative. The ModelTest suggested that the BI used the Dirichlet base frequencies, the GTR + I + G evolutionary mode for ITS and tub2, and HKY + I + G for tef1α.

Discussion
In the present study, ten strains from three host genera (Ficus macrocarpa, Phoebe zhennan and Spatholobus suberectus) were split into three new species (Pestalotiopsis ficicola sp. nov., P. phoebes sp. nov. and P. spatholobi sp. nov.) based on phylogeny and morphology. The Global Biodiversity Information Facility (https://www.gbif.org/, accessed on 16 May 2023) contains 4174 georeferenced records of Pestalotiopsis species reported around the world. Most species are distributed in countries such as the United States of American, Europe, Australia and China where suitable climates and the environment favors growth of unusual microbial species [38].
Historically, traditional identification of Pestalotiopsis species has long been a complicated endeavor [8][9][10][11][12]. Fortunately, with the development of molecular technology and the efforts of many taxonomists, the taxonomic status of Pestalotiopsis (Sporocadaceae, Amphisphaeriales) has become increasingly apparent [5,6,13,18,19]. Recent research showed that the same Pestalotiopsis species can be found on hosts belonging to multiple plant families, for example, P. chamaeropis was isolated from Chamaerops humilis (Arecaceae), Quercus sp., Castanopsis sp. (Fagaceae) and Camellia sp. (Theaceae) [6,18,39]. On the other hand, a variety of Pestalotiopsis species were isolated from the same plant host, for example, Pestalotiopsis chamaeropis, P. kenyana, P. nanjingensis and P. rhodomyrtus were isolated from the Quercus aliena (Fagaceae) [18]. These findings indicate that Pestalotiopsis species have the ability to infect a wide range of host plants rather than showing restricted host preferences.
In the pestalotioid species, apical or basal appendages differ in number, origin, position, number of branches and branching pattern, and these characters can be divided into some genera of the Sporocadaceae [5,17]. Monochaetia and Seiridium possess single apical and basal appendages, Nonappendiculata do not possess any appendages, and Pestalotiopsis, Ciliochorella, Neopestalotiopsis, Pseudopestalotiopsis possess 2-4 appendages. In previous studies, some members of Sporocadaceae were shown to contain secondary metabolites with significant biological activity, e.g., the sterol constituents of the marine fungus Pestalotiopsis sp. XWS03F09 exhibit selective inhibitory activities against antitumor cells [40][41][42][43]. Therefore, it is necessary to carry out genome sequencing of Pestalotiopsis species and fully explore, through structural and functional annotation, the diversity of secondary metabolites which might be useful for various biotechnological applications. This shows the importance of taxonomy and deposition of specimens.