Using Culture-Dependent and Molecular Techniques to Identify Endophytic Fungi Associated with Tea Leaves ( Camellia spp.) in Yunnan Province, China

: The association of endophytic fungi with the host plant is called a symbiotic relationship. Studies of the endophytic fungi from tea have been reported in numerous documents, but researchers still largely focus on tea endophytic fungi as they have ability to produce bioactive compounds which have numerous applications. The present work characterizes the fungal endophytic communities associated with healthy tea leaves in Yunnan Province, China. A total of 287 fungal strains were isolated from healthy leaf tissues of tea plants using a culture-dependent approach. Based on nuclear ribosomal DNA internal transcribed spacer (ITS) sequence analyses taken from the fungal cultures, strains were classiﬁed into 28 fungal genera with high similarity matches to known sequences in GenBank. The majority of genera (98.25%) belong to the phylum Ascomycota and most of the dominating fungal endophytes are from the genera Colletotrichum and Clonostachys


Introduction
Tea surged in global popularity in the 21st century, and nowadays nearly half of the world's population drinks tea [1]. Currently, tea is cultivated in over 52 countries, with China and India being the two largest producers [2][3][4]. In southwestern China, Yunnan Province is one of China's most important tea-growing regions, and several species of Camellia are used for tea production in this region [5]. Moreover, Yunnan Province has long been assumed to be one of the original tea domestication centers of the world [6,7].
Endophytic fungi, also known as mycosymbionts, live asymptomatically within various tissues of host plants [8,9]. Endophytic fungi are a diverse polyphyletic group of microorganisms that can thrive in various plant tissues above and/or underground, including stems, leaves, and/or roots [10]. In general, leaves feature a more diverse fungal endophytic community compared to other parts of the plant [11,12]. According to Faeth and Fagan [13], it is estimated that there are more than one million species of endophytic fungi. These endophytic organisms confer a number of benefits to the host plant, such as improving resilience to abiotic stress, tolerance to fungal pathogens, and growth performance of the host plant [14][15][16][17][18][19][20][21][22].
Studying endophytes is largely based on molecular techniques. However, the reliability of molecular findings can be verified using Bill's concept, which is a culture-dependent approach [23,24]. Guo et al. [25,26] mentioned that the use of morphological characteristics alone is not sufficient to identify fungal endophytes, especially when they do not sporulate, and thus, DNA data are needed for identification. The internal transcribed spacer (ITS) region is the most widely sequenced genetic marker for fungi and has also been formally proposed as the standard fungal barcode marker [27,28]. Thus, depending on the objectives of the endophytic fungal study, some authors only use the ITS region to identify endophytic fungi [29,30]. However, multi-gene analyses are more accurate for identifying endophytic fungal species [31,32].
Tea plants are rich in endophytic fungi. The systematic study of tea endophytes began in the early 21st century [1,33]. Endophytic fungi reported from tea have been isolated from several parts of the plant, viz. flowers, leaves, roots, shoots, and stems [34]. Past studies relating to tea endophytes have focused mainly on the composition, diversity, and distribution of endophytes in tea plants, as well as studies relating to the impact of climate and the age of tea plants on the make-up of tea endophyte communities [33,[35][36][37][38]. Despite an increase in attention, tea endophytic fungal studies remain limited in scope compared to other economically important crops [1]. One reason for this could be since the number and types of endophytic fungi existing in tea plants vary with time as well as across different altitudinal locations, tissue types, and cultivars [38,39]. Furthermore, different types of tea plant tissues have their own dominant microflora [35]. In China, endophytic fungal studies in tea have been carried out in several areas and most of the endophytic fungi were isolated from leaves and branches of tea plants [35,38,40,41].
The current study was designed to determine the endophytic fungal communities in tea leaf (Camellia spp.) tissues collected in Yunnan Province, China. Using culture and DNA-barcode (ITS) methods, we isolated and identified endophytic fungal communities from 11 tea plantations in different areas of Yunnan Province.

Study Site and Sampling
The study was conducted in Baoshan, Honghe, Lijiang, and Xishuangbanna of Yunnan Province, China, from 2019 to 2020 ( Figure 1 and Table 1). Healthy tea leaves were randomly collected from different kind of tea plantations, viz. shade tea, wild tea, terraced tea, and mix planting, and immediately placed in plastic bags, ice boxes, labeled and subjected to fungal isolation within 48 h.

Isolation and Identification of Endophytic Fungi
At each site, 20 leaves randomly selected from healthy tea plants were first washed in running tap water to remove soil and dust. Tibpromma et al. [32] was followed for the surface sterilization procedures. Leaf pieces (0.5 cm size pieces) were placed on potato dextrose agar (PDA) dishes with amoxicillin added to prevent bacterial growth (50 mg of amoxicillin per 1 L of PDA). All dishes were incubated at room temperature (20-25 • C) for five days and periodically checked. Mycelia emerging from the leaf bits were aseptically transferred to new PDA dishes and incubated at 28 • C. A total of 287 strains were isolated from the Yunnan tea leaf samples.

DNA Extraction, PCR Amplification and DNA Sequencing
The pure mycelia of endophytic fungal cultures grown on PDA at room temperature for four weeks were used for DNA extraction. The pure fungal mycelia were scraped off with a sterile scalpel and transferred to 1.5 mL micro-centrifuge tubes under aseptic conditions. The Biospin Fungal Genomic DNA Extraction Kit (BioFlux, China) was used to perform DNA extraction on the fungal cultures following the manufacturer's protocols. Polymerase chain reaction (PCR) was used to amplify partial gene regions of Internal Transcribed Spacers (ITS) using ITS5 and ITS4 primer [49]. The total volume of PCR mixtures for amplifications was set as described in Tibpromma et al. [50]. Purification and sequencing of PCR products were carried out by Sangon Biotech Co., Shanghai, China. ITS sequence data produced in this study were checked for the quality of chromatograms, and raw forward and reverse sequences were assembled using Geneious Pro.v4.8.5. Assembled sequences were trimmed out from the LSU and SSU sequence regions with an online program (https://plutof.ut.ee, accessed 6 January 2022), leaving only the ITS1-5.8S-ITS2 sequence region, and the size of the ITS gene was approximately 400-500 bp. ITS sequences were used in a BLAST search of the GenBank (http://blast.ncbi.nlm.nih.gov, accessed 15 January 2022) database to determine their most-probable closely related genus. Identifications of ITS sequences were made using the highest hit score of listed species (Supplementary  Table S1). By this method, each isolate was identified and assigned to a specific genus.
The dominant fungal genera derived from each collecting site and among all isolates, belonged to the phylum Ascomycota (98.25%), 0.70 belonged to the phylum Basidiomycota, and 1.05% could not be identified and were thus marked as unknown fungal endophytes ( Figure 2). In order level, Glomerellales was reported as the predominant order, while Xylariales and Hypocreales were the most diverse orders with other isolates from the orders Agaricales, Amphisphaeriales, Botryosphaeriales, Capnodiales, Coniochaetales, Diaporthales, Eurotiales, Mycosphaerellales, Phomatosporales, Pleosporales, and Polyporales. At the genus level, Colletotrichum and Clonostachys were the dominant genera (42.16 and 21.25%, respectively) and other isolates within 1-10% included Diaporthe, Fusarium, Nemania, Nigrospora, Pestalotia, Phomatospora, Phyllosticta and unidentified fungal endophytes (Figure 3). Nineteen endophytic genera with <1% were regarded as rare genera, viz. Alternaria, Annulohypoxylon, Aquapteridospora, Cercospora, Cladosporium, Coniochaeta, Daldinia, Epicoccum, Fomitopsis, Gliomastix, Kretzschmaria, Melanconiella, Neosetophoma, Penicillium, Pestalotiopsis, Psathyrella, Pseudopestalotiopsis, Trichoderma, and Xylaria (Figure 3). Different fungal groups among tea plantation types are shown as: (1) Clonostachys was the most abundant genus in terraced tea plantations; (2) in the three shade tea sites (AMB, AMC, ML), the most abundant genus was found to be Colletotrichum and the other shade tea site (HT), it is Clonostachys; (3) in the two wild tea sites, the dominant fungal groups were Colletotrichum and Nigrospora; (4) and the most abundant genus in the mixed planting site was Colletotrichum (Figure 2). When assessing the diversity of species according location, in Baoshan, Clonostachys was most abundant; in Honghe, Colletotrichum was most abundant; whereas in Lijiang, abundance was split according to the two sites, Colletotrichum was most abundant in one site (LJ) and Nigrospora is the other site (SG); and finally, in Xishuangbanna, the most abundant genus was also found to be Colletotrichum. Furthermore, members of Colletotrichum were found in all types of tea plantations) (Figure 2).
Colletotrichum comprises fungi that are classed as endophytes, saprobes, entomopathogens, as well as many species of phytopathogens [51][52][53][54]. Several Colletotrichum species have been reported as endophytes in living plant tissues (with the majority from C. boninense, gloeosporioides, and graminicola species complexes) [51,52,[54][55][56][57]. Further complicating the issue regarding Colletotrichum (species in the C. gloeosporioides species complex) is that these species are able to switch their lifestyle from endophytic to pathogenic modes [58,59]. According to Tibpromma et al. [60], Colletotrichum acutatum and Colletotrichum camelliaem are candidate pathogens of emergent diseases on the tea plant, with the added potential of shifting to novel areas or hosts under future climate change scenarios. Members of the genus Clonostachys, which has a global distribution, are known as mycoparasites, lichenicolous fungi, endophytes, and saprobes [61,62]. Many species of Clonostachys have been studied for their secondary metabolites [61]. For example, Clonostachys rosea is an excellent biocontrol agent that can control a wide range of plant pathogens [63]. Clonostachys rosea is also commonly found as an endophyte of healthy palm trees and is a good candidate for further study as a potential biological control agent of date palms diseases [64].

Discussion
The 287 fungal endophytes isolated from 11 tea plantations in Yunnan Province, China, were successfully identified at the genus level. The results indicate a high diversity of endophytic fungi with consisting of 28 genera, the majority belonging to Sordariomycetes in the phylum Ascomycota with Colletotrichum (related to C. gloeosporioides species complex) and Clonostachys (related to Clonostachys rosea) being the most dominant ( Figure 3).
Our results confirm the work of Lu and Wu [40] and Wu et al. [41] who reported a high diversity of fungal endophytes in tea plants from China. These authors noted that tea leaves maintained a high level of endophytic diversity, and no endophytes were found in tea seeds. Similar to our findings, Rodriguez et al. [15] analyzed endophytic fungi from woody plants and reported a high diversity of non-clavicipitaceous endophytes, most of which belonged to the phylum Ascomycota, while only a few were Basidiomycota. Xie et al. [1] categorized endophytic fungi previously reported from tea into three phyla, five classes, 14 orders, 24 families, and 34 genera. Members of Pleosporales (Dothideomycetes), Diaporthales, Glomerellales, Hypocreales, and Xylariales (Sordariomycetes) were reported as the dominant strains. Our results also showed similar fungal groups in Ascomycota to the results of Xie et al. [1] but different groups in Basidiomycota. Lu and Wu [40] isolated endophytic fungi from tea trees in southern Henan Province and found that Colletotrichum sp., Pestalotiopsis sp., Phomopsis sp., and Macrophoma sp. were the predominant fungi which is consistent with our results as Colletotrichum sp. is the dominant fungal group, however we found a strong presence of Clonostachys in our study, which was not reported by Lu and Wu [40].
In this study, we cultured endophytic fungi, and the results will facilitate further research into valuable bioactive compounds and the biocontrol potential of tea fungal endophytes for sustainable agricultural development. However, our work lacked enough rigorous sampling between sites to allow for in depth statistical analyses on the distribution of fungal endophytes and how this may be influenced by environmental factors. Future work should make added efforts to include such data.