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Endophytic Pestalotiopsis species associated with Rhododendron in Cangshan Mountain, Yunnan Province, China

Rhododendron is an essential ornamental plant that is abundant in Yunnan province. In Cangshan Mountain, Yunnan, China, 61 species of Rhododendron have been reported. Endophytic fungi are internal plant tissue inhabitants that do not harm the host. It has emerged as an exciting research topic as they have the potential to provide numerous secondary metabolites. This study is focused on taxonomic novelties and new host records of endophytic fungi associated with Rhododendron plants collected from Cangshan Mountain in Yunnan Province, China. Pestalotiopsis fungi are associated with a vast array of plant species worldwide. In this study, fresh leaves of Rhododendron cyanocarpum, Rhododendron decorum, and Rhododendron delavayi were collected from Cangshan Mountain, Yunnan Province, China. Endophytic Pestalotiopsis fungi associated with Rhododendron were characterized based on phylogenetic analyses of combined ITS, TEF1-α, and TUB genes along with morphological characteristics. Six new species (Pestalotiopsis appendiculata, Pestalotiopsis cangshanensis, Pestalotiopsis daliensis, Pestalotiopsis fusoidea, Pestalotiopsis rosarioides, and Pestalotiopsis suae) and a new host record (Pestalotiopsis trachicarpicola) are described. Detailed descriptions and color photo plates of these species are provided. It is the first time that the endophytic fungi of Rhododendron plants in Cangshan Mountain have been studied.


Introduction
Rhododendron is the largest genus of woody plants in the Northern Hemisphere and the largest genus in Ericaceae (Fang et al., 2005;Shrestha et al., 2018). Rhododendron is an important component of montane ecosystems, with more than 1,025 species and approximately 581 species in China (Cai et al., 2016;Ma et al., 2017;Cao et al., 2022). Moreover, Yunnan province in southwest China is a center of diversity for Rhododendron (Ma et al., 2021). Rhododendron is a popular landscape plant and a food source (Negi et al., 2013;Lin et al., 2021). Due to the economic significance of this plant, it is essential to evaluate the fungi associated with it (Chaiwan et al., 2022). Pathogenic fungi, mycorrhizal fungi, and endophytic fungi have been isolated from Rhododendron in previous research (Zhang et al., 2019); however, there is no record of endophytic fungi associated with Rhododendron in Cangshan Mountain, Yunnan, China.
As a part of the investigation on endophytic fungal diversity associated with Rhododendron plants in Cangshan Mountain, Yunnan province, China, we revealed seven Pestalotiopsis-like taxa from fresh leaves of Rhododendron. Their taxonomic positions were established based on morphological descriptions and multi-locus phylogenetic analyses. The endophytic fungal strain resources were stored for future study on their secondary metabolites.

Isolation and morphology
Fresh Rhododendron (Ericaceae) leaves were obtained from Cangshan Mountain, Yunnan Province, China. The gathered leaves were placed in a sterile polyethylene bag and stored at 4 • C. The symptomless leaves of each Rhododendron spp. were treated with gently running tap water to remove the surface debris. They were surface-sterilized by using 75% ethanol for 1 min, 0.1% HgCl 2 for 3 min, and washed five times using sterile distilled water, finally dried on sterile filter paper (Tao et al., 2013). The 5-mm diameter leaf discs treated as above were placed on potato dextrose agar (PDA) plates without antibiotics. The PDA plates were incubated in ambient light at 25 • C. When colonies appeared, they were transferred onto new PDA plates and further incubated in ambient light at 25 • C for morphological examination. Sporulation was induced on pine needle medium ("pine needle" and 1/10-strength PDA). Macromorphological characters of conidiomata on PDA were observed using an Optec SZ 760 compound stereomicroscope. Temporarily prepared microscope slides were placed under a Nikon ECLIPSE Ni-U compound stereomicroscope for observation and micro-morphological photography. Part of the pure culture that produced spores was removed and put into a water-agar medium (WA) with glycerol and air-dried at room temperature (De Silva et al., 2019). All endophytic isolates are stored at the Culture Collection of Kunming Institute of Botany, the Chinese Academy of Sciences (KUNCC), and the China General Microbiological Culture Collection Center (CGMCC). The Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (Herb. KUN-HKAS) housed the herbarium specimens. The MycoBank 1 number was registered (Crous et al., 2004).

DNA extraction, PCR amplification, and sequencing
Genomic DNA extraction was carried out from fresh mycelium growing on PDA at 25 • C using the Trelief TM Plant Genomic DNA Kit according to the manufacturer's instructions. The primer pairs ITS5/ITS4, EF1-728F/EF2, and Bt2a/Bt2b were used to amplify the ITS, TEF1-α, and TUB gene regions, respectively. The amplification was performed in a 25 µL reaction volume containing 12.5 µL of Master Mix (Tsingke Biotech, Yunnan, China), 1 µL of each primer (10 µm), 1 µL of template DNA, and 9.5 µL of deionized water. The PCR thermal cycles for three genes were performed under the following reaction conditions: an initial denaturing step for 94 • C for 3 min followed by 35 cycles of denaturation at 94 • C for 45 s, annealing at 55 • C for 45 s for ITS and TEF1-α, and 56 • C for 60 s for TUB, elongation at 72 • C for 1 min, and a final extension at 72 • C for 10 min. PCR products were verified on 1% agarose electrophoresis gels stained with ethidium bromide. Sequencing was carried out by Tsingke Biological Engineering Technology and Services Co., Ltd. (Yunnan, China).

Molecular phylogenetic analyses Sequence alignment
Sequences with high similarity indices were assembled in BioEdit, and those with low similarity indices were identified through a BLAST search for the closest matches with Pestalotiopsis taxa and from recently published data . All consensus and reference sequences were automatically aligned with MAFFT v.7 using the Auto strategy (Katoh and Standley, 2013). The aligned sequences from each gene region (ITS, TEF1α, and TUB) were combined and manually improved using Sequence Matrix (Hall, 1999). Uncertain regions were omitted from the alignment, and gaps were treated as data that was missing. Maximum likelihood (ML) and Bayesian inference were used to conduct phylogenetic analyses.

Phylogenetic analyses
Maximum likelihood analysis was performed at the CIPRES Science Gateway v.3.3 (Miller et al., 2010) using RAxML v.8.2.8 as part of the "RAxML-HPC2 on XSEDE" tool (Stamatakis, 2006;Stamatakis et al., 2008). The optimal ML tree search was conducted with 1,000 separate runs using the default algorithm of the programme from a random starting tree for each run. The final tree was selected amongst suboptimal trees from each run by comparing the likelihood scores using the GTR+GAMMA substitution model. Maximum likelihood bootstrap values equal to or greater than 60% were given as the first set of numbers above the nodes in the resulting ML tree.
Bayesian analysis was performed with MrBayes v.3.1.2 (Ronquist and Huelsenbeck, 2003) to evaluate posterior probabilities (Rannala and Yang, 1996) using Markov Chain Monte Carlo sampling (MCMC). The best-fit model of evolution was estimated using MrModeltest v.2.2 (Nylander, 2004). For Bayesian analysis, the best-fitting model of ITS, TEF1-α, and TUB was the GTR+I+G model. Posterior probabilities (PPs) (Rannala and Yang, 1996) were performed using Markov chain Monte Carlo sampling (BMCMC) in MrBayes v.3.1.2 (Liu et al., 2012). Six concurrent Markov chains were executed for 50 million generations, and samples of trees were taken every 5,000 generations (resulting in 10,000 trees). The initial 2,000 trees representing the burn-in phase of the analyses were discarded, while the remaining 8,000 trees were used to calculate PP in the majority rule consensus tree (Cai et al., 2006;Liu et al., 2012).

Phylogenetic analyses
The combined ITS, TEF1-α, and TUB sequence dataset included 154 ingroup taxa and two outgroup taxa (Neopestalotiopsis protearum and Pseudopestalotiopsis cocos) with 2,160 characters (ITS: 1-538 bp; TEF: 539-1477 bp; TUB: 1478-2160 bp) overall post-alignment, including the gaps. The RAxML and Bayesian analyses of the combined dataset resulted in phylogenetic reconstructions with largely identical topologies and a ML analysis with a final likelihood value of −17482.622268, as shown in Figure 1. The matrix exhibited 993 distinct alignment patterns, with 24.14% undetermined characters or gaps. The estimated base frequencies were as follows: A = 0.237874, C = 0.294954, G = 0.216783, T = 0.250389; substitution rates AC = 1.059763, AG = 3.258532, AT = 1.260093, CG = 0.980806, CT = 4.659318, GT = 1.000000; gamma distribution shape parameter α = 0.309168. The bootstrap support values for RAxML greater than 60% and the Bayesian posterior probabilities greater than 0.95 are given at each node (Figure 1).
In the phylogenetic analyses, all new strains were grouped with members of Pestalotiopsis. Pestalotiopsis rosarioides, Pestalotiopsis intermedia, and Pestalotiopsis linearis were grouped together; however, P. rosarioides has a separate branch with 84% ML and 0.96 BYPP support. Pestalotiopsis appendiculata established a distinct lineage with 98% ML and 0.99 BYPP bootstrap support. Pestalotiopsis suae was clustered as a sister taxon to Pestalotiopsis pinicola with a significant support (97% ML and 1 BYPP). Pestalotiopsis daliensis was MycoBank number: 845187, Figure 2.
Material Notes: P. appendiculata, P. intermedia, P. linearis, and P. rosarioides were grouped in the same clade in the phylogenetic analysis. Comparisons of ITS, TEF-1α, and TUB gene regions of P. appendiculata with P. linearis (2/538 in ITS, 12/398 in TEF-1α, and 23/450 in TUB), P. intermedia (3/537 in ITS, 17/398 in TEF-1α, and 13/479 in TUB), and P. rosarioides (3/553 in ITS, 22/553 in TEF-1α, and 22/458 in TUB) indicated significant differences. However, P. appendiculata can be distinguished from P. linearis and P. intermedia with its smaller conidia (Table 2). Moreover, the three-median cells of conidia in P. appendiculata are light gray; in contrast, they are brown in P. linearis, P. intermedia, and P. rosarioides. Based on the combined ITS, TEF-1α, and TUB sequence data, our phylogenetic analysis revealed that they are clearly distinct in the phylogram. P. appendiculata formed a separate branch with strong support values (98 ML/0.99 PP, Figure 1). Therefore, based on phylogenetic analysis and its morphological characteristics, P. appendiculata is introduced as a new species.

Discussion
Many fungal groups, such as Aspergillus, Ceratobasidium, Fusarium, Neocosmospora, Pestalotiopsis, Pyrenochaeta, Russula, Serendipita, Thanatephorus, and Trichoderma have been reported as endophytic fungi (Fu et al., 2022). As an ornamental plant, Rhododendron has achieved worldwide recognition (Cox and Cox, 1997). Recent research has isolated fungi from the leaf spots, mycorrhizae, and rhizosphere of Rhododendron (Medeiros et al., 2022). However, few studies have been conducted on the endophytic fungi of Rhododendron. Yunnan Province is one of the world's most significant distribution centers for Rhododendron (Tian et al., 2011). There are 61 species of Rhododendron in Cangshan Mountain, Yunnan Province, China (Zhang et al., 2021). Our investigation indicates high diversity of Pestalotiopsis species in Rhododendron. However, the current study collected the leaves of three Rhododendron species only. In future research, it is possible to expand the survey area and collect additional Rhododendron leaves to obtain more endophytic fungal resources.
Previous studies mentioned that the color intensities of the median conidial cell, differences in the size variation of conidia, and the presence or absence of basal appendages can be used as additional taxonomic characteristics for distinguishing Pestalotiopsis species (Jeewon et al., 2003;Liu et al., 2017).
Apical appendage characteristics, such as branching pattern, number, and attachment position, are also useful at the species level, but not at the generic level of a generic character (Crous et al., 2012). Therefore, it was proposed to implement additional morphological characteristics for species identification. ITS, TUB, and TEF1-α gene sequences can be combined to provide greater resolution for Pestalotiopsis . In our phylogenetic tree, branch lengths of Pestalotiopsis cangshanensis, P. fusoidea, and some other species in this genus were notably short and the support values were relatively low. Further studies of Pestalotiopsis are, therefore, required to reveal whether the less informative loci lead to the poorly resolved phylogram.

Funding
This work was mainly supported by the National Natural Science Foundation of China (Project ID: 32060005 and 31900020) and the Yunnan Fundamental Research Project (grant nos. 202101AU070137 and 202201AW070001).