Chiang Mai J. Sci. 2019; 46(4) : 626-638 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper Diversity of Culturable Endophytic Fungi Associated with Bryophytes, Pteridophytes and Spermatophytes from Dawei Mountain Nature Reserve, China Yong-Han Gao1 [a], Wei-Xiao Bai1 [a], Wei-Hong Sun1 [a], Wen-Na Zhou [a], Guang-Li Wu [a], Zheng-Qing Zhu [a], Jia-Yun Ma [b], Hai-Yan Li*[a] [a] Medical School, Kunming University of Science and Technology, Kunming 650500, Yunnan, China. [b] School of Statistics and Mathematics, Yunnan University of Finance and Economics, Kunming 650500, Yunnan, China. *Author for correspondence; e-mail: [email protected] 1, these authors contributed equally to this work Received: 26 October 2018 Revised: 7 February 2019 Accepted: 23 April 2019 ABSTRACT Bryophytes, pteridophytes and spermatophytes are each plants with different evolutionary degrees. In the present study, the diversity and community structure of culturable endophytic fungi (EF) associated with bryophytes, pteridophytes and spermatophytes collected from Dawei Mountain in China were investigated. A total of 2230 EF were isolated from 1440 segments of nine plant species. The colonization rate (CR) of bryophytes, pteridophytes and spermatophytes were 97.92%, 98.75% and 98.13%, respectively (P> 0.05, LSD test). Based on the morphological characteristics, 18S rDNA and internal transcribed spacer (ITS) analysis, the isolates were identified to 61 taxa, of which Colletotrichum and Xylaria were the most dominant genera, and their relative frequencies were 40.00% and 23.68%, respectively. Only 21 taxa were common between bryophytes, pteridophytes and spermatophytes, whereas, some endophytes showed host specificity or tissue preference, for example,Penicillium chrysogenum and P. daleae were found only in bryophytes. In addition to Colletotrichum and Xylaria, Trichoderma and Penicillium were the dominant genera in bryophytes. However, in pteridophytes and spermatophytes, Phomopsis was the dominant genus. The Shannon indices (H′) and the Sorenson’s coefficient similarity indices (CS) of EF from bryophytes, pteridophytes and spermatophytes ranged from 2.02 to 2.92 and 0.64 to 0.74, respectively. It was found that H′ of plants with different evolutional degree showed no significant difference P( > 0.05, LSD test). The CS of EF of two plant species with different evolutional degrees was not always lower than that of two plant species with the same evolutional degree. Key words: bryophyte, pteridophyte, spermatophyte, evolutionary degree, endophyte 1. INTRODUCTION The fungi causing asymptomatic infections fungi (EF) and they are a significant part of the in living plant tissues are known as endophytic plant microbiome [1]. They have been widely Chiang Mai J. Sci. 2019; 46(4) 627 studied in various geographic and climatic forest, moss evergreen broadleaved forest and zones and are ubiquitous within a wide range moss evergreen broad-leaved forest, there is of tissues of all examined plants with a rich a complete mountain forest ecosystem in the diversity of species [2-3]. Various studies have range of over 2000m above sea level. It was not demonstrated that endophytes can have profound affected by the “Quaternary Glacier” period effects on plant ecology, fitness and evolution, [11], thus many old plant species have been affecting the diversity and structure of the plant retained, and the diversity of plant species is community [4-6]. In plant fossils formed 400 high [12-13], especially bryophyte, pteridophytes million years ago, endophytes have been found and spermatophytes. To compare the diversity [7]. Thus, the interaction between host plants and community structure of EF associated with and endophytes, particularly the possible role bryophytes, pteridophytes and spermatophytes, of endophytes in the evolution of plants has nine plant species (three bryophytes, three gained considerable momentum. The study pteridophytes and three spermatophytes) were of diversity and community structure of EF collected from Dawei Mountain, and culturable associated with plants belonging to different endophytic fungi were studied in this work. evolutionary degrees is very important. Here, evolutionary degrees represent different 2. MATERIALS AND METHODS taxonomic levels of phylogenetic classification 2.1 Study Site and Sampling systems. In this system the evolutionary Three species of bryophytes (Marchantia relationships between the various organisms polymorpha., Marchantiaceae; Polytrichum form the basis of classification. Most botanists commune, Polytrichaceae; Hylocomium splendens, consider bryophytes and pteridophytes to be Hylocomiaceae), three species of pteridophytes the oldest living remnants of eukaryotic plants (Diacalpe aspidiodes, Peranemaceae; Coniogramme that colonized the land [8]. While bryophytes petelotii., Hemionitidaceae and Plagiogyria represent the basal clades, pteridophytes maxima., Plagiogyriaceae) and three species have been placed between bryophytes and of spermatophytes (Taiwania cryptomerioides., spermatophytes (gymnosperm and angiosperm) Cupressaceae; Embelia polypodioides., Mysinacease, in the sub divisions of the plant kingdom. and Rhododendron irroratum., Ericaceae) were Previous studies mainly focused on comparing collected from Dawei Mountain (22°28′-22°45′N, the diversity and community structure of EF 103°39′-103°51′E), Yunnan, southwest China of the same plant species growing in different in November 2012. For each plant species, 10 environments, or EF of different plant species healthy individuals at least 30m apart from growing in the same environment [9-10]. To our each other were chosen, and three healthy and knowledge, the differences of EF community separate branches were collected from each plant associated with different plant classes are still at random, and brought to the laboratory in unknown. sterile polythene bags and processed within 24 h. Dawei Mountain Nature Reserve, which is located in Yunnan Province, Southwest China, is 2.2 Fungal Isolation, Culture and Identification the most comprehensive region which has both For isolation of endophytic fungi, 20 evergreen and broad-leaved forests in China. And healthy leaves (or photosynthetic tissues) and the climate type is a humid subtropical climate, 20 healthy stems (or rhizoid) were selected from with annual precipitation exceeding 1500mm. each plant at random, washed under running From tropical humid rainforest to seasonal tap water and processed as follows: the samples rainforest, monsoonal evergreen broad-leaved were cut into segments (about 5 × 5 mm) and 628 Chiang Mai J. Sci. 2019; 46(4) surface-sterilized by sequentially dipping into isolates were sorted into different groups on 0.5% sodium hypochlorite for 2 min, followed the basis of colony surface texture, hyphal by 3 times washing with sterile distilled water, pigmentation, margin shapes and growth dipping into 70% ethanol for 2 min, rinsing 3 rates. All of the isolates were deposited in times with sterile distilled water, then drying the Medical School, Kunming University of on sterilized filter paper [14]. Then 80 leaf (or Science and Technology under the assigned photosynthetic tissue) segments and 80 stem numbers in this report. (or rhizoid) segments of each plant species were placed on a Petri dish containing potato 2.3 Statistical Analysis dextrose agar (PDA) medium amended with The colonization rate (CR) was calculated 0.5 g/l streptomycin sulfate. The plates were as the total number of segments colonized incubated at 25°C and checked every other day by endophytic fungi (EF) divided by the total for 45 d; the fungi growing out of the plant number of incubated segments [18]. The relative tissues were transferred to fresh PDA plates. frequency (RF) was calculated as the number The effectiveness of the surface sterilization was of isolates of one species divided by the total confirmed by making imprints of disinfected number of isolates [19]. plant fragments on PDA plates from which no The endophytic fungal diversity was fungal growth was observed [15]. evaluated using the Shannon index (H′), which Fungal identification was based on has two main components, evenness and the morphology, mechanism of spore production number of species. The Shannon index was and spore characteristics [16]. Some of the calculated according to the following formula: isolates, which posed difficulty in morphological k identification, were further examined based on H′ = −×∑ PPiiln , their ITS sequence analysis (The primers were i=1 ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and where k is the total species number of ITS4 (5’-TCCTCCGCTTATTGATATGC-3’)), one plot and Pi is the relative abundance of noting that sometimes matches in GenBank endophytic fungal species in one plot [20].To did not necessarily give correct names [17]. evaluate the degree of community similarity Sequences were submitted to GenBank and of the EF between two treatments, Sorenson’s assigned the accession numbers were from coefficient similarity index CS( ) was employed KM357284 to KM357350 (Table 1). Sterile and calculated according to the following formula: Table 1. The results of molecular identification. Isolate Accession no. of isolate Query Identity Most closely related species Numbers obtained in the obtained in the present Morphotype coverage (%) (accesssion no.) present study study (%) 1 1X-38 KM357284 Xylaria sp. 97 99 Xylaria sp. (JQ862679.1) 2 2X-97 KM357285 Colletotrichum gloeosporioides 97 100 Colletotrichum gloeosporioides (HQ845102.1) 3 9Y-69 KM357286 Colletotrichum