Taxonomy and Phylogeny of Endophytic Fungi (Chaetomiaceae) Associated with Healthy Leaves of Mangifera indica in Yunnan, China

: Mangoes belong to Mangifera (Anacardiaceae), which contains 69 species, but only Mangifera indica is popularly cultivated and commercialized. Mango is one of the most important crops grown in China’s Yunnan Province and signiﬁcantly contributes to the economic security of these locals. Endophytic fungi have been recognized as beneﬁcial microbes that improve plant growth, productivity, and survivability under environmental stress. Nevertheless, many host plant-related endophytic fungi are yet to be identiﬁed, including the mango-related species. During this study, we recognized three different fungal species in the family Chaetomiaceae derived from healthy mango ( Mangifera indica ) leaves based on morphological examinations coupled with multi-gene phylogenetic analysis (ITS, LSU, rpb2 , and tub2 ). These species are Dichotomopilus funicola (KUNCC23-13347) and Humicola walleﬁi (KUNCC22-10759, 23-13348), derived from new hosts, and a new species of Arcopilus hongheensis (KUNCC22-10767, 23-13346).


Introduction
Mango is a deep-rooted, evergreen plant belonging to the genus Mangifera (Anacardiaceae), with 69 species and over 1500 verities [1,2].Nonetheless, among them, Mangifera indica is the most commonly cultivated species in worldwide farmlands and home yards [1,2].The cultivation of mango, particularly in China, has a history of over 1300 years, with more than 40 different varieties [3].China was the second largest mango producing country, where 3,483,500 metric tons of fruits were harvested from 359,600 hectares plantation in 2020 [3].Mango has been a significant source of income for rural communities in the main growing areas of Baoshan, Honghe, Huaping, Simao, and Yuanjiang in Yunnan [3,4].
Fungal endophytes are an intriguing group that resides within the intracellular and/or extracellular spaces of plant tissues without causing any adverse effects or visible disease symptoms among the hosts [5].Endophytic fungi offer a number of benefits to plants, such as protecting hosts from abiotic (e.g., drought, high temperature, and waterlogging) and biotic (e.g., pathogens and insect pests) stresses and promoting plant growth and germination [6][7][8][9].Moreover, owing to their numerous properties, such as anticancer, antidiabetic, biocontrol, immunosuppressive, and insecticidal potentials, endophytes are often used in agricultural, industrial, pharmaceutical, and medical sectors [10][11][12].Even though endophytes are among the most widely studied groups of fungi, studies of mangoassociated endophytes are comparatively limited.Nevertheless, according to the available Diversity 2023, 15, 1094 2 of 13 studies, a considerable number of endophytes associated with mango were recognized-for example, Vieira et al. [13] isolated 22 strains of endophytic Colletotrichum from leaves, stem fragments, and mature inflorescence images of sampled mango plants.In another study, Dashyal et al. [14] recognized 35 fungal endophytes only using the stems and leaves of mango.Similarly, Yang et al. [15] isolated 34 different fungal endophytic strains from mango leaves.Further, they found that some endophytes are capable of serving as bio-control agents.
Previous diversity studies have found that Chaetomiaceae could be the dominant group of endophytic fungi present in mango leaves [15].In this study, we isolated the Chaetomiaceae species from fresh and healthy leaves of mango.The new endophytic fungus viz.Arcopilus hongheensis, two new records of viz.Dichotomopilus funicola, and the known species viz.Humicola wallefii are described via detailed morphological comparisons and four-gene molecular phylogenetic analyses.

Samples Collection and Fungi Isolation
The fresh and healthy leaf samples were randomly picked from commercially cultured mango trees in Honghe Prefecture, Yunnan, China (102 • 50 11 E, 23 • 41 01 N); carefully arranged in separate polythene bags; and taken to the mycology laboratory for fungal endophyte isolation [16].Firstly, the samples were cleaned with tap water, and each leaf was cut into several small pieces (1 × 1 cm; middle parts of leaves).The leaf pieces were surface sterilized using 3% sodium hypochlorite (NaOCl) for 2 min, followed by three repeated washings with sterilized water.Further, surface sterilization was carried out via washing with 75% ethanol (C 2 H 6 O) for 2 min and three series of washing with sterile distilled water and allowed to dry under the laminar flow cabinet.Then, the edges of the leaf pieces were trimmed, transferred onto potato dextrose agar (PDA) plates, and incubated at 27 • C for 2-15 days [17,18].When individual hyphal tips grew out from the leaf blade, the mycelia were picked using sterilized needles, placed onto new PDA plates, and incubated at 27 • C for one month.Once endophytic fungi were sporulated in PDA, their dry cultures were deposited in the Kunming Institute of Botany, Academia Sinica (HKAS) herbarium, and the live cultures were deposited in the Kunming Institute of Botany Culture Collection (KUNCC).

Microscopic Examination
The morphology of Chaetomiaceae species was followed Wang et al. [19] and cultured on PDA media (1-3 months at 27 • C) to induce the development of ascomata.Mycelia masses, fruiting bodies, ascomatal hairs, ascomata, and ascospores were initially observed.The colony morphologies of Chaetomiaceae species were captured by measuring micromorphological characteristics in distilled water (some asci and ascospores were strained in Lugol's iodine solution), and slides were observed.The color codes in the manuscript followed colorhexa (http://www.colorhexa.com,accessed on 14 September 2023) [20].The fungi micro-structure sizes were measured using the Tarosoft (R) Image Frame Work program, and color photo-plates of the micro-structures were combined using Adobe Photoshop CS3 Extended v. 10.0 (Adobe ® , San Jose, CA, USA).

DNA Extraction, PCR Amplification, and Sequencing
Fungal mycelia were removed from the colonies on PDA using sterilized needles and transferred into 1.5 mL centrifugal tubes for DNA extraction.Genomic DNA extraction followed the instructions of the manufacturer of the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux ® , Beijing Bio Teke Corporation, Beijing, China).A part of the extracted DNA was used as a template to perform Polymerase Chain Reaction (PCR), while the remaining part of the DNA solution was stored at −20 • C for long-term preservation.The PCR reaction mixture was 25 µL in total, including 8.5 µL of double-distilled water (ddH 2 O), 12.5 µL of 2 × Power Taq PCR Master Mix (mixture of EasyTaqTM DNA Polymerase, dNTPs, and optimized buffer; Beijing Bio Teke Corporation-Bio Teke, Beijing, China), 2 µL of DNA template, and 2 µL of forward and reverse primers.The regions of internal transcribed spacer (ITS) and large subunit ribosomal RNA (LSU) were amplified using the primers ITS4/ITS5 and LR5/LROR [21,22].The PCR condition of ITS and LSU was created using an initial denaturation step of 3 min at 94 • C, followed by 35 cycles of 30 s at 94 • C, 50 s at 55 • C, and 1 min at 72 • C and a final denaturation step of 10 min at 72 • C [23].The partial RNA polymerase II subunit (rpb2) region was amplified using the primers fRPB2-5F and fRPB2-7cR [24].The PCR condition of rpb2 started with the initial denaturation at 95 • C for 3 min, followed by 35 cycles of denaturation at 95 • C for 45 s, annealing at 57 • C for 50 s, and extending 90 s at 72 • C, as well as an extension at 72 • C for 10 min [23].The beta-tubulin (tub2) was amplified using the primers T1 and T22 [25].The PCR condition of tub2 gene began with an initial denaturation step of 3 min at 94 • C, followed by 35 cycles of 30 s at 94 • C, 50 s at 52 • C, and 1 min at 72 • C and a final denaturation step of 10 min at 72 • C [26].The PCR products were sent to Beijing Bio Teke Corporation (Beijing, China) for purification and sequencing.

Phylogenetic Analyses
The forward and reverse sequences were combined in Geneious (Restricted) 9.1.2(https://www.geneious.com,accessed on 12 June 2023) and subjected to BLASTn searches in the GenBank (http://blast.ncbi.nlm.nih.gov/,accessed on 12 June 2023) [27] to select the species names, strains, and accession numbers of the most closely related genera/taxa by forming a spreadsheet of all involved species.Sequence alignments were carried out via the server version of MAFFT using default settings (http://mafft.cbrc.jp/alignment/server, accessed on 2 May 2023) [28], and we edited the beginning and end in BioEdit 7.2.3 [29].Ambiguous and uninformative regions were removed via trimAL v1.2 (http: //trimal.cgenomics.org,accessed on 12 June 2023), and sequences were manually combined using BioEdit.The alignment transformation environment (ALTER) online program was used to convert Fasta files to the PHYLIP (for ML) and NEXUS (for BI) formata [30].Maximum likelihood analysis (ML) was carried out by selecting RAxML-HPC2 via the XSEDE (8.2.12) [31] and GTRGAMMA substitution models at 1000 bootstrap iterations via the CIPRES Science Gateway platform v.3.3 (http://www.phylo.org/portal2,accessed on 12 June 2023, [32]).Bayesian analysis was conducted via MrBayes v. 3.1.2[31,33] in the CIPRES Science Gateway platform to evaluate posterior probabilities (PP) [34] using Markov Chain Monte Carlo sampling (MCMC).The GTR+I+G evolution model was also applied in the BI analyses.Bayesian analyses of six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled and printed to output at every 100th generation (resulting in 10,000 trees in total).Phylogenetic trees were visualized using FigTree v1.4.0 [35], and the final tree was formed via Microsoft PowerPoint 2006 version.
Known substratum: Isolated from soil [47]; isolated from fresh and healthy Mangifera indica leaves (This study).
Known substratum: Isolated from soil [47]; isolated from fresh and healthy Mangifera indica leaves (This study).
Our isolates were obtained in fresh and healthy mango leaves, and those species were probably voluntarily selected by mango hosts from soil or compost in plantations; however, this hypothesis needs to be proved via further experiments.Those isolated endophytic fungi could inoculate mango seedlings but require further testing.In addition, this study only isolated the endophytic fungi from leaf parts, as only leaves are available during the collecting time (December 2022), while endophytic fungi from roots, flowers, fruits, and stems need to be screened in future studies.We noted that the endophytic fungal diversity of plants can change with the seasons [63]; therefore, further systemic studies of endophytic fungi associated with different parts of mango and their diversity change in different seasons are necessary.
Most species of Arcopilus were previously recognized as Chaetomium until the genus Arcopilus was established by Wang et al. [36].Based on the phylogenetic analyses (Figure 1), 13 species were accepted in this genus, including four recently introduced species [19,37,41].Our strains (KUNCC22-10767 and KUMCC 23-13346) formed an independent group, separated from other species of Arcopilus; therefore, our isolates are considered a new species of Arcopilus.In addition, other strains, namely KUNCC23-13347 and KUNCC22-10759 (=KUNCC23-13348), were identified as Dichotomopilus funicola and Humicola wallefii, respectively, based on morphological comparisons and phylogenetic analyses.In addition, Wang et al. [19] suggested that oatmeal agar (OA) can be used as a standard medium to culture Chaetomiaceae fungi, which stimulates the production of sexual structures.Malt extract agar (MEA) and PDA are used for extrolite profiling but are not suitable for morphological studies as sexual morphs are generally poorly induced [19].In our study, Arcopilus hongheensis also formed very low numbers of ascomata in PDA, but Dwibedi et al. [64] reported asexual structures of Arcopilus aureus to be well formed in PDA.In addition, our Dichotomopilus funicola and Humicola wallefii isolates produced a relatively high number of ascomata in PDA.Chaetomium is a well-studied group in the family Chaetomiaceae, while other genera in Chaetomiaceae have been relatively poorly studied, especially in terms of their taxonomies, metabolites, and application prospects.Chaetomiaceae fungi were previously found to be a dominant group in mango leaves [15], but this is the first study to

Figure 1 .
Figure 1.The phylogram of partially related genera in Chaetomiaceae constructed based on combined LSU, ITS, rpb2, and tub2 sequences.The tree is rooted with Trichocladium tomentosum (CBS 144476) and T. uniseriatum (LC3756).The BI and ML bootstrap support values are equal to or above

Figure 1 .
Figure 1.The phylogram of partially related genera in Chaetomiaceae constructed based on combined LSU, ITS, rpb2, and tub2 sequences.The tree is rooted with Trichocladium tomentosum (CBS 144476) and T. uniseriatum (LC3756).The BI and ML bootstrap support values are equal to or above 0.95 BYPP, and 75% are shown above the nodes' first and second positions.Type strains are shown in bold, and newly generated strains are shown in red.