World Journal of Microbiology and Biotechnology (2019) 35:113 https://doi.org/10.1007/s11274-019-2686-x ORIGINAL PAPER Endophytic fungi from the branches of Camellia taliensis (W. W. Smith) Melchior, a widely distributed wild tea plant Xiaoxue Chen1 · Xulu Luo1 · Miaomiao Fan1 · Weilin Zeng1 · Chongren Yang2 · Jianrong Wu3 · Changlin Zhao3 · Yingjun Zhang2 · Ping Zhao1 Received: 28 July 2018 / Accepted: 29 June 2019 / Published online: 9 July 2019 © Springer Nature B.V. 2019 Abstract Camellia taliensis (W. W. Smith) Melchior is a wild tea plant endemic from the west and southwest of Yunnan province of China to the north of Myanmar and is used commonly to produce tea by the local people of its growing areas. Its chemical constituents are closely related to those of C. sinensis var. assamica, a widely cultivated tea plant. In this study, the α diver- sity and phylogeny of endophytic fungi in the branches of C. taliensis were explored for the frst time. A total of 160 fungal strains were obtained and grouped into 42 species from 29 genera, which were identifed based on rDNA internal transcribed spacer sequence analysis. Diversity analysis showed that the endophytic fungal community of the branches of C. taliensis had high species richness S (42), Margalef index D′ (8.0785), Shannon–Wiener index H′ (2.8494), Simpson diversity index DS (0.8891), PIE index (0.8947) and evenness Pielou index J (0.7623) but a low dominant index λ (0.1109). By contrast, that in the branches of C. taliensis had abundant species and high species evenness. Diaporthe tectonigena, Acrocalymma sp. and Colletotrichum magnisporum were the dominant endophytic fungi. The phylogenetic tree was established by maximum parsimony analysis, and the 11 orders observed for endophytic fungi belonging to Ascomycota and Basidiomycota were grouped into 4 classes. Graphic abstract Keywords Camellia taliensis · Wild tea plant · Endophytic fungi · Biodiversity · Phylogeny Extended author information available on the last page of the article Vol.:(0123456789)1 3 113 Page 2 of 15 World Journal of Microbiology and Biotechnology (2019) 35:113 Introduction Camellia taliensis (W. W. Smith) Melchior is a wild tea plant that belongs to genus Camellia sect. Thea, is endemic Endophytic fungi are microfungi growing in healthy plant from the western and southwestern areas of Yunnan prov- tissues for all or the most part of their life cycle with- ince, China to northern Myanmar and is used commonly to out causing disease symptoms in the host plants (Petrini produce tea beverage by the local people of China. One of 1991); these organisms play a key role in the terrestrial the most representative C. taliensis trees is located on the ecosystem and greatly afect the evolution, community Xiangzhuqing village, Fengqing county, Yunnan province, structure and ecological adaption of plants (Clay and China. This tree is known as the ancestor of tea tree called Holah 1999; Brundrett 2006). In 1930s, heavy losses in ‘Jin Xiu Cha Zu’ in Chinese by the local people because of animal husbandry lad by livestock poisoning were reported its hundreds of years of living cycle and its efcient growth to be caused by herbage infected with endophyte (Leucht- at 9.3 m in height and 5.8 m in girth. The long life cycle mann 1993). Taxomyces andreanae was frst discovered by of ‘Jin Xiu Cha Zu’ might be due to its adaptation to the Andrea Stierle as a microbial taxol producer from Pacifc environment for a long time, suggesting its strong resist- yew (Stierle et al. 1993). These fndings imply that as a ance to stress and adaptability to the habitat. The secondary potential source, endophytic fungi would be one of the metabolites of C. taliensis are closely related to those of most desirable components for novel medicinal compound the cultivated tea plant, C. sinensis var. assamica (Mast.) supply. Thus, studies on endophytes have been widely con- Kitamura (Gao et al. 2008; Zhu et al. 2012). However, its ducted. Endophytic fungi exist widely in major lineages endophytic microorganisms have never been explored. In of land plants, such as bryophytes, ferns, lichens, gym- this study, the α diversity and phylogeny of endophytic nosperms and angiosperms (Arnold et al. 2007; Joshee fungi associated with the branches of ‘Jin Xiu Cha Zu’ (C. et al. 2009) and are distinctly distributed throughout plant taliensis) were investigated for the frst time. This study will organs and tissues and associated with various plant struc- contribute to further evaluate the role of fungi in this treas- tures, such as leaves, branches, stems, roots and shoots ured tea resource and explore some new natural bioactive (Porras-Alfaro and Bayman 2011). Almost all the plants products from the isolated endophytic fungi. studied contain endophytic fungi (Aly et al. 2011), and more than 376 genera of endophytic fungi belonging to 83 families were isolated from the tissues of 212 species of Materials and methods medicinal plants, including Ascomycetes, Basidiomycetes and non-spore groups (Tan et al. 2015). Source of plant samples The studies on microorganism association with tea plants have recently increased. Endophytic fungi are richly A number of healthy and asymptomatic branches of ‘Jin diverse in tea plants and have formed unique communities Xiu Cha Zu’ (C. taliensis) were obtained in June 2017 at and produced various active substances during their long- Xiangzhuqing village (N24°35′51″, E100°04′53″, elevation term coevolution. Koide et al. (2005) reported the coloni- 2245 m), Xiaowan town, Fengqing county, Yunnan province, sation and lignin decomposition of Camellia japonica L. China. All tissues were collected, immediately brought to leaf litter by endophytic fungi. Su et al. (2010) observed the laboratory and stored at 4 °C in the refrigerator. All of that the mixed culture of endophytic fungi isolated from C. the samples were used to isolate endophytic fungi within sinensis (L.) O. Ktze can enhance the antagonistic efect 24 h after collection. on plant pathogenic fungi. Luo et al. (2012) isolated two new oxysporone derivatives, pestalrones A and B from the Isolation and preservation of endophytic fungi fermentation broth of the endophytic fungus Pestalotiopsis karstenii (Sacc. & Syd.) Stey. in the stems of C. sasan- The culture media used for the isolation of endophytic qua Thunb. Devi and Wahab (2012) evaluated the anti- fungi were Czapek dox agar (CDA) and potato dextrose microbial potential of endophytic fungi isolated from the agar (PDA). All the samples were washed with running tap leaves of C. sinensis plant. Zhou et al. (2013) researched water to remove dust or other attachments on the surface, the diversity and phylogenetic relationships of endophytic rinsed with distilled water and fnally air-dried. The plant fungi within the leaves of C. oleifera Abel. by using a samples were cut into sections of 5 cm, successively surface culture-independent method based on rDNA sequences. sterilised by soaking in 75% ethanol for 45 s and then 0.1% Other related reports (Kajula et al. 2010; Wang et al. 2015; mercuric chloride for 6 min and fnally washed three times Cai et al. 2016; Rabha et al. 2016; Yan et al. 2016; Gong with sterile water. Tissue blot was used to assess the efcacy et al. 2017) have also confrmed that tea endophytic fungi of surface sterilisation (Schulz et al. 1993; Stone et al. 2000). are a treasure trove of microbial resources to be developed. The blotted plates were observed after being incubated at 28 °C for 3–5 days. Each section was cut into small pieces 1 3 World Journal of Microbiology and Biotechnology (2019) 35:113 Page 3 of 15 113 of approximately 0.5 cm thick by using sterile clippers after same species (Jiang et al. 2016). The fungal sequences with sterilisation. These sample pieces were equally placed on the same BLAST results were divided into the same group 140 PDA plates and 140 CDA plates to avoid light and then in terms of species. One isolate in each group was selected kept at 28 °C in the incubator for 1–3 weeks until the out- as a representative, and its BLAST information was listed. growth of endophytic fungi was discerned. During incuba- The consensus sequence data of 42 endophytic fungal iso- tion, all the plant samples were observed every day. Hyphal lates were summarised and then submitted to NCBI. Their tips of the developing fungal colonies were transferred to a assigned GenBank accession numbers are shown in Table 1. fresh medium (Kumar and Kaushik 2013) and subcultured for 3–4 times until a purifed colony was obtained. The Clustering and phylogenetic analysis of endophytic resulting purifed fungal isolates were maintained on PDA fungi slants in cryovials at 4 °C and immersed in 30% glycerin in cryovials at − 80 °C. All operations were conducted under The taxonomic status of these endophytic fungi was classi- sterile conditions. fed according to phylum, class, order and family as shown in Table 2. One from each species of fungi was selected Identifcation of endophytic fungi to participate in the phylogenetic tree construction, aligned with reference sequences from GenBank using the Clustal All purifed isolates were successively obtained and frst X software and then trimmed by BioEdit Sequence Align- categorised based on their morphological characteristics. ment Editor 7.2.5.0. Clustering analysis was performed by The endophytic fungal isolates were identifed with the importing the sequences of each fungal species into the same morphotype if they possessed the same characteristics PAUP 4.0b10 (Swoford 2002). Maximum parsimony (MP) including colony colour, hyphal shape and structure, myce- analysis was performed with PAUP 4.0b10, and 1000 boot- lia, spore morphology and exudate colour (Li et al. 2016). strap replications were used to test the stability of clades in All the isolates were then subjected to molecular identifca- tree bisection–reconnection branch swapping.