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 taliensis (W. W. Smith) Melchior, a widely distributed wild

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 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 was established by maximum parsimony analysis, and the 11 orders observed for endophytic fungi belonging to and Basidiomycota were grouped into 4 classes. Graphic abstract

Keywords Camellia taliensis · Wild tea plant · Endophytic fungi · Biodiversity · Phylogeny

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Introduction Camellia taliensis (W. W. Smith) Melchior is a wild tea plant that belongs to 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 (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 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 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. Neighbour- tion by analysing the internal transcribed spacer (ITS) region joining (NJ) trees (Fig. 1) were built using a Kimura two- of the nuclear ribosomal DNA. parameter (K2P) model in PAUP 4.0b10. The robustness of The endophytic fungi were cultured until the colonies the internal branches was also assessed with 1000 bootstrap reached sufcient mass for DNA extraction. Mycelia were replications (Yuan et al. 2010). scraped from the surface of medium and ground into powder in liquid nitrogen by using a tissue grinder. Then, 500 mg of Diversity analyses of endophytic fungi mycelia powder was subjected to genomic DNA extraction using E.Z.N.A.™ High Performance (HP) Fungal DNA Kit Counting the number of endophytic fungal isolates (N) and (Omega Bio-Tek, US) according to CTAB-based extraction using species as the statistical unit are necessary to evaluate method (O’Donnell et al. 1997). the endophytic composition, dominant species and abun- Fungal DNA was analysed by sequencing the ITS region dance of endophytic fungi in the tissue masses of branches of the rDNA, which utilised the universal primers ITS1 (5′- of C. taliensis. The isolation frequency (IF), which refers to TCC​GTA​GGT​GAA​CCT​GCG​G-3′) and ITS4 (5′-TCC​TCC​ the frequency of occurrence of certain endophytic fungi in GCT​TAT​TGA​TAT​GC-3′) (White et al. 1990). The frag- total isolates (Sun et al. 2014), of endophytic fungi can be ments corresponding to the ITS-1, 5.8S and ITS-2 regions calculated based on the number of isolates (N). The IF of were amplifed by polymerase chain reaction (PCR). The each endophytic fungal species from CDA and PDA media PCR mixture containing 12.5 μL 2 × Taq PCR mater Mix, and total IF are summarised in Table 3. The relative abun- 4.0 μL of template DNA, 1.0 μL of ITS1 primer, 1.0 μL of dance (RA) (Liu et al. 2013) of endophytic fungal at the ITS4 primer and 6.5 μL of distilled water. Amplifcations class, order and family levels is shown in Fig. 2, and the were performed an initial denaturation at 94 °C for 3 min, IF at the family level is shown in Fig. 3. Diversity indices, followed by 35 cycles at 94 °C for 30 s, 51.5 °C for 30 s and including the species richness index (S), Margalef index 70 °C for 1 min with a fnal extension at 70 °C for 10 min. (D′), Shannon–Wiener index (H′), Simpson’s diversity index All the PCR products of endophytic fungi were sent to (DS), Simpson’s dominant index (λ), the probability of inter- Tsingke Biological Technology Co, Ltd. (Kunming, China) specifc encounter (PIE) index and Pielou’s evenness index for sequencing. Basic local alignment search tool (BLAST) (J), were used to refect the diversity, richness and evenness was used to search for the best match in the National Center of species in the community (Schoch et al. 2006; Santos for Biotechnology Information (NCBI) database (https​:// et al. 2010; Hu et al. 2013; Walther et al. 2013; Zhang et al. www.ncbi.nlm.nih.gov/) to identify the endophytic fungi. 2013, 2014; Bråthen et al. 2015; Fernandes et al. 2015). The Sequences with similarity over 94% belonged to the same calculation method and results of diversity index are shown genus, and those with similarity over 97% belonged to the in Table 4.

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Table 1 The identifcation of endophytic fungi from C. taliensis by BLAST in the GenBank Isolate Fungal code GenBank BLAST match results number access (N) number Max score Query Identity (%) E value Closest species GenBank References coverage access (%) number

30 Ct-BC20 MH305493 721 98 95 0.0 Acrocalymma fci NR137953 Trakunyingcharoen et al. (2014) 4 Ct-BC78 MH305494 917 100 100 0.0 Alternaria alter- MF462315 – nata 1 Ct-BC64 MH305495 1037 99 99 0.0 Bjerkandera adusta KY703409 – 1 Ct-BC54 MH305496 887 100 100 0.0 Cladosporium KY400091 – cladosporioides 1 Ct-BC65 MH305497 952 100 100 0.0 Colletotrichum KX243296 – acutatum 18 Ct-BP17 MH305530 907 98 99 0.0 Colletotrichum NR132056 Liu et al. (2014) magnisporum 1 Ct-BC91 MH305498 761 99 93 0.0 Colletotrichum JQ247631 Peng et al. (2013) simmondsii 3 Ct-BC83 MH305499 920 99 99 0.0 Coniochaeta sp. KF367558 Oliveira et al. (2013) 2 Ct-BP67 MH305531 833 99 98 0.0 Cryptosporiopsis HQ157902 Kernaghan and ericae Patriquin (2011) 1 Ct-BP61 MH305523 941 99 99 0.0 Daldinia childiae KU683757 U’Ren et al. (2016) 4 Ct-BC57 MH305500 881 95 99 0.0 Daldinia sp. JX658450 Stadler et al. (2014) 1 Ct-BP78 MH305528 931 100 99 0.0 Diaporthe sp. KC357558 Huang et al. (2013) 1 Ct-BP41 MH305525 830 99 97 0.0 Diaporthe eucalyp- NR120157 Crous et al. (2012) torum 1 Ct-BP29 MH305527 723 99 93 0.0 sp. DQ780461 Promputtha et al. (2007) 37 Ct-BP79 MH305526 854 99 97 0.0 Diaporthe tectoni- KX986782 Gao et al. (2017) gena 1 Ct-BP77 MH305524 730 100 92 0.0 Phomopsis sp. JQ954648 Huang et al. (2013) 3 Ct-BC92 MH305501 942 100 100 0.0 Glomerella cin- KJ934362 – gulata 1 Ct-BC80 MH305502 761 99 95 0.0 Lecythophora sp. KU702600 Hesse et al. (2016) 5 Ct-BC63 MH305503 752 99 95 0.0 Leptosphaeria sp. KX100373 Blanchette et al. (2016) 2 Ct-BC55 MH305504 948 100 99 0.0 Microdiplodia JN198395 Wu et al. (2013) hawaiiensis 1 Ct-BC70 MH305505 963 78 99 0.0 Nemania bipapil- GU292818 Hsieh et al. (2010) lata 1 Ct-BP71 MH305521 734 97 94 0.0 Neohendersonia KX820257 Giraldo et al. (2017) kickxii 1 Ct-BC50 MH305506 883 98 98 0.0 Neosetophoma KJ812297 Overy et al. (2014) samarorum 1 Ct-BC76 MH305507 841 99 99 0.0 Nigrograna sp. MF399065 – 3 Ct-BC38 MH305508 928 100 100 0.0 Paraconiothyrium EU295637 Damm et al. (2008) brasiliense 9 Ct-BC37 MH305509 909 99 99 0.0 Paraconiothyrium JX496020 Verkley et al. (2014) fungicola 1 Ct-BC85 MH305510 942 98 99 0.0 Paraphaeosphaeria JX496038 Verkley et al. (2014) neglecta 1 Ct-BC61 MH305511 989 100 99 0.0 Pestalotiopsis KJ934363 – guepinii 2 Ct-BC52 MH305512 885 99 100 0.0 Peyronellaea KM030324 – pinodella

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Table 1 (continued) Isolate Fungal code GenBank BLAST match results number access (N) number Max score Query Identity (%) E value Closest species GenBank References coverage access (%) number

2 Ct-BC59 MH305513 867 99 99 0.0 Phaeosphaeria NR137933 – podocarpi 3 Ct-BC44 MH305514 839 100 98 0.0 EU035984 Sutton et al. (2008) curvatum 2 Ct-BC29 MH305515 915 100 99 0.0 Phomopsis amyg- HQ632014 Dai et al. (2012) dali 1 Ct-BP28 MH305529 795 100 97 0.0 Phomopsis KC153100 Gao et al. (2014) lithocarpus 1 Ct-BP40 MH305522 896 100 99 0.0 Phomopsis sp. FJ042518 Yuan et al. (2009) 1 Ct-BC87 MH305516 1048 100 100 0.0 Schizophyllum MF061788 – commune 1 Ct-BC77 MH305517 894 100 99 0.0 Trichocladium sp. KU556534 Novinscak et al. (2016) 1 Ct-BC72 MH305518 693 100 100 0.0 Trichoderma cit- KJ174203 Hsieh et al. (2010) rinoviride 2 Ct-BC46 MH305519 961 100 98 0.0 Xylaria curta GU322444 Sandoval-Denis et al. (2014) 1 Ct-BC60 MH305520 909 99 99 0.0 Xylaria sp. EU678663 Hsieh et al. (2010) 1 Ct-BP42 MH305492 545 99 86 4E−151 Diaporthe pseu- KM100721 – domangiferae 1 Ct-BP48 MH305491 468 100 85 7E−128 Phomopsis capsici KY488351 – 5 Ct-BP81 MH305490 664 88 87 0.0 Magnicama- NR153445 Tanaka et al. (2015) rosporium iriomotense Total: 160

Results the query coverage and identity of seven isolates were both under 90%. The identities of Ct-BP42 and Ct-BP48 were Identifcation of endophytic fungi 86% and 85%, respectively, and they were close to Diaporthe sp. and Phomopsis sp., respectively. The identities of fve A total of 160 endophytic fungal isolates were obtained isolates represented by Ct-BP81 were also less than 90%. from the branches of C. taliensis. Specifcally, 90 isolates originated from the CDA plates, and 70 were from the PDA Clustering and phylogenetic analyses of endophytic plates. According to their morphological characteristics, fungi the isolates from the CDA and PDA plates were prelimi- narily categorised according to morphotypes and recorded As presented in Table 2, 153 out of 160 isolates had been as Ct-BC and Ct-BP. The code of same morphotypes was classifed. The status of the other seven isolates with iden- adjacent. All isolates were molecularly identifed by ana- tities under 90% had not been specifed. Thus, they were lysing their ITS rDNA regions ranging from 500 to 600 bp treated as unknown species but were still included in the using the BLAST in the NCBI GenBank. On the basis of calculation of RA. Among the 153 explicit isolates, 151 morphology observation and molecular identifcation, 42 isolates of endophytic fungi were included in Ascomycota isolates represented 42 results and were summarised in a list and within three classes: Leotiomycetes, of identifcation results for endophytic fungi isolates, includ- and . Only two isolates, namely, Polypo- ing their GenBank accession number in NCBI, closest spe- rales (one species, one isolate) and Agaricales (one spe- cies, query coverage and genetic similarity to the sequences cies, one isolate) were clustered in Basidiomycota within deposited (Table 1). The genetic identities of 113 isolates Agaricomycetes (two species, two isolates). The dominant were above 97%, those of 37 isolates were 94% to 97% and group of endophytic fungi was Sordariomycetes (23 species, those of 10 isolates were under 94%. Among these isolates, 87 isolates), which contains Glomerellales (4 species, 23

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Table 2 Taxon of all endophytic fungi isolates from C. taliensis Phylum Class Order Family Species Isolate number(N)

Basidiomycota Agaricomycetes Polyporales Meruliaceae Bjerkandera adusta 1 Agaricales Schizophyllaceae Schizophyllum commune 1 Ascomycota Leotiomycetes Helotiales Dermateaceae Cryptosporiopsis ericae 2 Dothideomycetes Capnodiales Cladosporiaceae Cladosporium cladosporioides 1 Phaeosphaeriaceae Neosetophoma samarorum 1 Phaeosphaeria podocarpi 2 Pleosporales incertae sedis­ a Nigrograna sp. 1 Pleosporales incertae sedis­ a Neohendersonia kickxii 1 Didymosphaeriaceae Microdiplodia hawaiiensis 2 Paraconiothyrium brasiliense 3 Paraconiothyrium fungicola 9 Paraphaeosphaeria neglecta 1 Acrocalymmaceae Acrocalymma sp. 30 Didymellaceae Peyronellaea pinodella 2 Leptosphaeriaceae Leptosphaeria sp. 5 Pleosporaceae Alternaria alternata 4 Sordariomycetes Glomerellales Glomerellaceae Colletotrichum acutatum 1 Colletotrichum magnisporum 18 Colletotrichum simmondsii 1 Glomerella cingulata 3 Hypocreales Hypocreaceae Trichoderma citrinoviride 1 Coniochaetales Coniochaetaceae Coniochaeta sp. 3 Lecythophora sp. 1 Diaporthaceae Diaporthe sp. 1 Diaporthe eucalyptorum 1 Phomopsis sp. 1 Diaporthe tectonigena 37 Phomopsis sp. 1 Phomopsis amygdali 2 Phomopsis lithocarpus 1 Phomopsis sp. 1 Chaetomiaceae Trichocladium sp. 1 Xylariales Hypoxylaceae Daldinia childiae 1 Daldinia sp. 4 Sporocadaceae Pestalotiopsis guepinii 1 Xylariaceae Nemania bipapillata 1 Xylaria curta 2 Xylaria sp. 1 3 a Units with no clear classifcation status isolates), Hypocreales (1 species, 1 isolate), Coniochaetales isolates) belonged to Leotiomycetes, Helotiales. In total, 37 (2 species, 3 isolates), Diaporthales (6 species, 43 isolates), species of endophytic fungi distributed through 29 genera, Sordariales (1 species, 1 isolate) and Xylariales (7 species, 21 families, 11 orders and 4 classes were obtained from this 13 isolates); followed by Dothideomycetes (13 species, 62 count. isolates), which includes Capnodiales (1 species, 1 isolate) As shown in Fig. 1, the endophytic fungal phylogenetic and Pleosporales (12 species, 61 isolates). Only one species clustering was consistent with their identifcation at the spe- of endophytic fungi Cryptosporiopsis ericae Sigler (two cies level. The orders of the endophytic fungi were signed

1 3 World Journal of Microbiology and Biotechnology (2019) 35:113 Page 7 of 15 113 on the right side of the phylogenetic tree. The morpholo- Keissler, Trichoderma citrinoviride Bisset, Trichocladium gies of Ct-BC20 and Ct-BP40 were completely diferent, but sp., Pestalotiopsis guepinii (Desm) Stey and Phialemonium they grouped together in the phylogenetic tree because they curvatum, were found. were genetically close to each other. Ct-BC20 belonged to Acrocalymmaceae, which was introduced as a novel family to accommodate the members of this genus in Dothideo- Diversity analysis of endophytic fungi mycetes (Trakunyingcharoen et al. 2014). Acrocalymma Alcorn & J.A.G. Irwin was phylogenetically closely to Table 3 summarises the species of endophytic fungi, N and phoma-like taxa. Furthermore, Diaporthe Nitschke was the IF for each species from two kinds of media and IF. Fig- teleomorph of Phomopsis. The same fungus has diferent ure 3 shows the diference of IF among each family from generic names for anamorph and teleomorph according to a CDA and PDA media. Those seven isolates that could not genetic standpoint. Clustering analysis was performed using be classifed were also listed. In Table 3, Diaporthe tec- two fungi, Syncephalis depressa (GenBank Access Num- tonigena (Schw.) Miura had the highest IF (23.13%), fol- ber: KY001683, Benny et al. 2016) and Rhizomucor pusil- lowed by Acrocalymma sp. (18.75%) and Colletotrichum lus (Lindt) Schipper (GenBank Access Number: JN206312, magnisporum (11.25%). Acrocalymma sp. had the highest Walther et al. 2013) as out-groups. These species belonged IF (25.56%) on CDA plates, whereas D. tectonigena had the to Zygomycota because most endophytic fungi (98.75%) highest (38.57%) on PDA plates. belonged to Ascomycota. Consistent with the results for RA analyses, Dia- porthaceae (60.48%), Acrocalymmaceae (35.56%) and Relative abundance analyses of endophytic fungi Glomerellaceae (30.95%) had the highest IF (Fig. 3). Thir- teen families of endophytic fungi were observed from the Numerous culturable endophytic fungal isolates were CDA plates, and two families were found from the PDA obtained from the branches of C. taliensis tissues and were plates. The isolate number of mutual families was still dis- classifed in 2 phyla, 4 classes, 11 orders, 21 families, 29 tinct for the two types of media, indicating that endophytic genera, 39 species and 3 unknown species (7 isolates). Asco- fungi species show evident richness and culture selectivity. mycota was the main phylum for the isolates and in Basidi- Furthermore, the dominant population in the branches of C. omycota endophytic fungi with a low proportion. The RA taliensis included Diaporthe tectonigena, Acrocalymma sp. (%) of endophytic fungi at the class, order and family levels and Colletotrichum magnisporum. is presented in Fig. 2. The diversity indices and the calculation method of each As shown in Fig. 2, 95.62% of the total isolates were result of endophytic fungi species associated with C. talien- identifed. At the class level (Fig. 2a), Sordariomycetes sis are summarised in Table 4. The species richness (S) and accounted for 54.38% with the highest abundance, followed Margalef index (D′) are two important parameters for α by Dothideomycetes (38.75%). Agaricomycetes and Leotio- diversity analysis. Species richness index (S) was obtained mycetes were the smallest and accounted for 1.25%. Eleven by counting the number of endophytic fungal species in the orders of endophytic fungi were classifed without consider- tissue. These two indices can refect the richness of endo- ing the taxon-undetermined isolates (4.38%). Pleosporales phytic fungi species. Large values of S and D′ indicate the (38.13%) and Diaporthales (28.13%) were the main com- richness of the endophytic fungi (Jiang et al. 2016). Spe- munity members (Fig. 2b). Pleosporales had eight families, cies diversity can be evaluated by the Shannon–Wiener (H′), and Acrocalymma sp. was only one genus of Acrocalym- Simpson diversity (DS) and inter specifc encounter (PIE) maceae (18.75%), which was the largest family in Pleospo- indices, which consider the heterogeneity and homogene- rales. Diaporthaceae was the only family from Diaporthales ity of the species frequencies. Generally, Shannon’s diver- and consisted of six species, of which the most abundant is sity index commonly ranges from 1.5 to 4.5. The higher the Diaporthe sp. At the order level, Glomerellales (14.38%) Shannon’s diversity index is and the closer the Simpson’s was the third largest. Polyporales, Agaricales, Capnodi- diversity index is to 1, the more intensifed the heritable ales, Hypocreales and Sordariales had the least abundance variation and the stronger the adaptive capacity for micro- at 0.63%. The RA of endophytic fungi at the family level environmental change of the communities are because they is shown in Fig. 2c. Fourteen families each with only one tend to expand the distribution range and enter new envi- species of endophytic fungi, such as Bjerkandera adusta ronments. The probability of inter specifc encounter (PIE) (Willd.) P. Karst., Schizophyllum commune Fr., Crypto- index was used to evaluate the encountering probability of sporiopsis ericae, Cladosporium cladosporioides (Fries) the individuals belonging to diferent species. As shown in de Vries, Nigrograna sp., Neohendersoniakickxii (Wes- Table 4, the endophytic fungi colonising in the branches of tend.) B. Sutton & Pollack, Acrocalymma sp., Peyronel- C. taliensis showed the highest species richness and diver- laeapinodella, Leptosphaeria sp., Alternaria alternate (Fr.) sity of S (42), D′ (8.0785), H′ (2.8494), DS (0.8891) and

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◂Fig. 1 Cluster analysis of endophytic fungi associated with the and its hemi-parasitic plant, and the RA of Pleosporales was branches of C. taliensis. An individual of each taxon isolated was 2.5%. However, the value was 38.18% in the present study. used in the construction of the clustering together with a reference C. sequence retrieved from GenBank. The bootstrap numbers shown The diversity of endophytic fungi in the branches of on the left were obtained using 1000 replicates. Fungi Syncephalis taliensis is relatively higher than in other tea trees. Chen depressa and Rhizomucor pusillus were used as outgroup for the con- et al. (2006) reported that Colletotrichum gloeosporioides struction of the tree. Filled triangle the similarity is less than 90%, and Guignardia mangiferae are the dominant population of flled rectangle the similarity is between 90 and 94%, flled circle the C. sinensis Dia- similarity is between 94 and 97% the widely cultivated tea tree . By contrast, porthe tectonigena, Acrocalymma sp. and Colletotrichum magnisporum were found to be the dominant endophytic PIE (0.8947). The varied trends of H′, DS and PIE should fungal isolates in the titled plant. Among these, Glomer- be kept consistent. ellaceae, Diaporthaceae and Acrocalymmaceae were the The Pielou index (J) can refect the evenness of species most frequent species with high RAs of 14.38%, 28.13% and can be evaluated on the basis of the Shannon–Wiener and 18.75%, respectively. The endophytic fungi of tea plants index (H′) and the size of samples with a value of 0.7623. were reported to be mainly Colletotrichum, Pestalotiopsis, The dominant index (λ) was used to evaluate the ecological Guignardia, Phomopsis, Macropho, Aspergillus, Tricho- dominance of a community and was inversely proportional derma, Penicillium, Alternaria and Fusarium (Gong et al. to Simpson’s diversity index (DS). A high λ indicates that the 2017). For example, Zhang et al. (2013) isolated 625 strains community might have low species diversity and evenness. of endophytic fungi from C. oleifera and identifed 59 taxa The endophytic fungal community showed a low degree of as 31 Deuteromycota, 19 Ascomycota and 9 Basidiomy- ecological dominance with a λ value of 0.1109. Overall, the cota. Rabha et al. (2016) isolated 30 endophytic fungi from endophytic fungal communities from the branches of C. healthy asymptomatic leaves of C. sinensis and identifed taliensis showed high species richness and diversity but a all as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. low degree of ecological dominance. Colletotrichum magnisporum being the dominant endo- phytic fungi in the branches of C. taliensis was consistent with previous reports. In some cases, the abundance distribution of endophytic Discussion fungal species was skewed with many frequent species and several incidental species; this fnding might be related to the Plant endophytic fungi are important for the conservation sampling size and method (Photita et al. 2001). Wang et al. and sustainable utilisation of rare and endangered plant (2016) investigated the endophytic fungi in the branches resources due to their interaction with host plants and their of three varieties of C. sinensis cultivars, from which 29 coevolution (Guo 2018). Wild tea plants are the most valu- species from Fuding Dabaicha with H′ 1.07 and D′ 0.41, able primary germplasm for tea preservation and research. 34 species from E-cha No.1 with H′ 2.14 and D′ 2.56 and Considering the archaic specifcity of ‘Jin Xiu Cha Zu’ (C. 31 species from E-cha No.5 with H′ 1.66 and D′ 1.41 were taliensis) in Xiangzhuqing village, this study explored for obtained. A study on C. oleifera collected from fve diferent the frst time the α diversity and phylogeny of endophytic areas of Hubei province, China (Zhang et al. 2017) indi- fungi in this tree by using a culture-dependent method. cated that the endophytic fungi from Huanggang showed A total of 160 endophytic fungal isolates, mostly belong- the highest diversity with H′ 2.44, and the highest indices ing to Ascomycota and within in the classes of Sordari- were in the stem (2.52) and autumn (2.58), respectively. In omycetes and Dothideomycetes, were obtained from the the present study, the high H′ 2.8494 and D′ 8.0785 revealed branches of C. taliensis. The results are consistent with the that C. taliensis hosted rich and diverse endophytic fungi. reference that Sordariomycetes and Dothideomycetes are the Although several genera such as Bjerkandera, Schizophyllum main groups of endophytic fungi from other plants (Zhang and Nigrograna were isolated with low relative abundance, et al. 2014). Additionally, two fungi species belonging to these minor genera may have an important ecological role Basidiomycota were obtained. The isolated endophytic fungi for their host plants or could be capable of synthesising bio- could be categorised into 39 species, 29 genera, 21 families, active compounds. 11 orders, 4 classes and 2 phyla. Although the anamorph Research on endophytic fungi has been widely promoted and teleomorph of several fungi had diferent taxonomic for the past 30 years, but some disputes still exist (Chen status, they were genetically identical and thus classifed at and Sun 2015; Fernandes et al. 2015; Huang et al. 2017). A the same level in the phylogenetic tree. All the isolated endo- large number of endophytic fungi could not be cultured on phytic fungi belonging to Pleosporales were clustered as an artifcial synthetic medium. Even the use of various media independent branch and separated from other orders. Zhou cannot assure that all endophytic fungi are isolated. Many et al. (2014) isolated the endophytic fungi of C. oleifera factors, such as the sample number, plant age and water

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Table 3 Isolation frequency Endophytic fungal species CDA PDA Total (IF) of each endophytic fungal species from CDA and PDA N IF N IF N IF medium Bjerkandera adusta 1 1.11 – – 1 0.63 Schizophyllum commune 1 1.11 – – 1 0.63 Cryptosporiopsis ericae – – 2 2.86 2 1.25 Cladosporium cladosporioides 1 1.11 – – 1 0.63 Neosetophoma samarorum 1 1.11 – – 1 0.63 Phaeosphaeria podocarpi 2 2.22 – – 2 1.25 Nigrograna sp. 1 1.11 – – 1 0.63 Neohendersonia kickxii – – 1 1.43 1 0.63 Microdiplodia hawaiiensis 2 2.22 – – 2 1.25 Paraconiothyrium brasiliense 2 2.22 1 1.43 3 1.88 Paraconiothyrium fungicola 7 7.78 2 2.86 9 5.63 Paraphaeosphaeria neglecta 1 1.11 – – 1 0.63 Acrocalymma sp. 23 25.56 7 10.00 30 18.75 Peyronellaea pinodella 1 1.11 1 1.43 2 1.25 Leptosphaeria sp. 5 5.56 – – 5 3.13 Alternaria alternata 4 4.44 – – 4 2.50 Colletotrichum acutatum 1 1.11 – – 1 0.63 Colletotrichum magnisporum 1 1.11 17 24.29 18 11.25 Colletotrichum simmondsii 1 1.11 – – 1 0.63 Glomerella cingulata 3 3.33 – – 3 1.88 Trichoderma citrinoviride 1 1.11 – – 1 0.63 Coniochaeta sp. 3 3.33 – – 3 1.88 Lecythophora sp. 1 1.11 – – 1 0.63 Diaporthe sp. – – 1 1.43 1 0.63 Diaporthe eucalyptorum – – 1 1.43 1 0.63 Phomopsis sp. – – 1 1.43 1 0.63 Diaporthe tectonigena 10 11.11 27 38.57 37 23.13 Phomopsis sp. – – 1 1.43 1 0.63 Phomopsis amygdali 2 2.22 – – 2 1.25 Phomopsis lithocarpus – – 1 1.43 1 0.63 Phomopsis sp. – – 1 1.43 1 0.63 Trichocladium sp. 1 1.11 – – 1 0.63 Daldinia childiae – – 1 1.43 1 0.63 Daldinia sp. 4 4.44 – – 4 2.50 Pestalotiopsis guepinii 1 1.11 – – 1 0.63 Nemania bipapillata 1 1.11 – – 1 0.63 Xylaria curta 2 2.22 – – 2 1.25 Xylaria sp. 1 1.11 – – 1 0.63 Phialemonium curvatum 3 3.33 – – 3 1.88 Others 2 2.22 5 7.14 7 4.38 Total 90 100.00 70 100.00 160 100.00

“–” means the corresponding endophytic fungal species was not observed N number of isolates status and nutrition level, can infuence isolation. Whether isolated endophytic fungi do not sporulate in culture and are or not classical morphological classifcation criteria can designated as mycelia sterilia (Peng et al. 2013). The rDNA accurately refect the taxonomic status of fungi is worth ITS region is designated as a fungal barcode and regarded as exploring. Identifcation using morphological is the best region for identifcation at the fungal species level hampered only by the diversity of endophytic fungi. Many (Schoch et al. 2012).

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Fig. 2 Relative abundance (RA, %) of endophytic fungi at the level of class (a), order (b) and family (c)

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Fig. 3 Isolation frequency (IF) of endophytic fungi from CDA and PDA medium on the family level

Table 4 Diversity analyses of Diversity index Equation Result endophytic fungi from branch of C. taliensis Species richness (S) S = number of endophytic fungal species 42 D� S Margalef index (D′) = ( − 1)∕ ln Nt 8.0785 H′ S Shannon–Wiener index ( ) H� 2.8494 =− Pi ln Pi,Pi = Ni∕Nt i∑=1 D S Simpson’s diversity index ( S) D 2 0.8891 S = 1 − Pi i∑=1 λ S Simpson’s dominant index ( ) 2 0.1109 = Pi i∑=1 S PIE index (PIE) N 0.8947 PIE = i (N − N )∕ N − 1 � N � t i t i∑=1 t � � J H� H H Pielou index (J) = ∕ max, max = ln S 0.7632

Yunnan, China is the original place of tea trees and is strong resistance of older and wild tea trees may be related where most of the wild tea plants are distributed together to the endogenous fora. Further studies are necessary to with many cultivars. Some wild tea trees contained rich understand the relationship between the wild tea plant and tea polyphenols (up to 40%), whilst others had high amino their endophytic fungi. acids ( ~ 6.5%) (Wang and Yu 2002) which is diferent from surrounding tea varieties. C. taliensis contains rich and Acknowledgements This work was supported by the National Natural Science Foundation of China [Grant Number 31470429], the Fund specifc tea polyphenols (Gao et al. 2008; Zhu et al. 2012). of State Key Laboratory of Phytochemistry and Plant Resources in Endophytes greatly afect plant nutrition, development and West China [Grant Number P2017-KF10], and the Yunnan Agricultural stress resistance. The specifc secondary metabolites and Foundation Projects [Grant Number 2017FG001-016].

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Afliations

Xiaoxue Chen1 · Xulu Luo1 · Miaomiao Fan1 · Weilin Zeng1 · Chongren Yang2 · Jianrong Wu3 · Changlin Zhao3 · Yingjun Zhang2 · Ping Zhao1

* Yingjun Zhang 2 State Key Laboratory of Phytochemistry and Plant [email protected] Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, * Ping Zhao People’s Republic of China [email protected] 3 Key Laboratory of Forest Disaster Warning and Control 1 Key Laboratory for Forest Resources Conservation of Yunnan Province, Southwest Forestry University, and Utilization in the Southwest Mountains of China, Kunming 650224, People’s Republic of China Ministry of Education, Southwest Forestry University, Kunming 650224, People’s Republic of China

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