Although D. chinense is a waterlogging-resistant medicinal plant, its endophytic community is rarely known. Considering the new roles of endophytic fungi in plant development, growth, adaptability and diversity, we need to fill this gap in order to exploit of endophytes for a better understanding of D. chinense plant and its important metabolites found in the TGR. Therefore, one of the purposes of this study was to examine the community composition of fungal endophytes from TGR. To our knowledge, our study is the first report on isolation and identification of fungal endophytes. Here, we took a culture-dependent approach, since our final goal is to build a working collection of fungal endophytes that can be explored for their potentially beneficial properties in D. chinense plant. In this work, a total of 154 endophytic fungi were isolated from D. chinense in the TGR and classified into 27 different taxa according to their morphological characteristics and unique phenotypic characters. Among them, the fungi that belong to Phomopsis, Diaporthe, Fusarium and Irpex, have been reported as the main endophytes of wetland shrub Myricaria laxiflora in the TGR  and riparian plant species . This is also in accordance with the report that Fusarium, Phomopsis and Irpex are not sensitive to ﬂooding stress . Previously, it has been reported that the assembly of land plant endophytic fungi is composed of representatives Sordariomycetes, Dothideomycetes and Pezizomycetes [67, 68], while plants from water or moist environments are more often parasitized by Eurotiomycetes . In the current study, Sordariomycetes was the most prevalent class with relative frequency of 50%, while Dothideomycetes and Eurotiomycetes had a relative frequency of 33.8% and 1.3%, respectively. Thus, our data indicated that both terrestrial and aquatic fungi are present in the D. chinense plant. Our results showed similarity to those of Kandalepas et al.,, who discovered high numbers of Sordariomycetes and low numbers of Dothideomycetes and Eurotiomycetes in wetland plants from Louisiana . Additionally, out of 27 taxa detected, 7 taxa were darkly pigmented with thickly-walled septate hyphae that belongs to Diaporthales, Phomopsis sp., Lasiodiplodia theobromae, Neofusicoccum parvum,Irpex lacteus, Periconia sp., Botryosphaeria dothidea, which were referred to as dark septate fungi (DSE) . Among the 154 isolates, 20 (13.0%) belongs to this group. The result showed that D. chinense were colonized by abundant DSEs, as some other researchers reported their occurrence in wetland plant species [71, 72]. Jumpponen and Trappe suggested that DSE frequently play unique roles in terrestrial ecosystems . Therefore, these special endophytic fungal communities not only revealed the apparent environmental specificity of the TGR area, but also helped to understand the special ecological functions shown by these fungal group.
Interestingly, many isolates from the genera Phomopsis, Fusarium, Diaporthe, Neofusicoccum parvum, Xylaria venosula, Lasiodiplodia theobromae and Botryosphaeria dothidea were common and well-known pathogens but also common endophytes existing asymptomatically [74, 75]. Among them, Diaporthe and Phomopsis complex are the causes of seed decay and cause soybean blight and canker diseases . Neofusicoccum parvum was reported as one of the most aggressive causal agents of the trunk disease Botryosphaeria dieback . Botryosphaeria and its anamorph complex are particularly important for symptoms such as fruit rot, shoot blight, dieback and canker of numerous woody hosts . However, it is incredible that symptoms of the disease did not appear in plants collected by D. chinense. This phenomenon suggests that fungal species may represent an evolutionary transition, or simply fungi have achieved remarkable ecological plasticity, thus ensuring the optimal growth and reproduction of various hosts, which ultimately leads to the expansion of their bio-geographic distribution . On the other hand, most of the transitions from a mutualistic to a parasitic interaction are characterized by imbalance in nutrient exchange  or by environmental variations [81, 82]. Furthermore, if plants are under physiological stress, the type of interaction between endophytes and host plants are also regulated . In this regard, the mutualistic interactions between fungal invaders and host plants are deciphered as a balance, which is considered as a combination of environmental and physiological effects that benefit both sides . The adaptive benefits of mutualistic fungi promote or are responsible for plant adaptation to biotic and abiotic stress by increasing resistance to drought and water stress [84, 85]. In addition, several other genera were also isolated from D. chienense, including Penicillium ochrochloron, Mycorrhizal basidiomycete, Ceriporia lacerta, Diaporthe longicolla, Diaporthe eres, Flavodon flavus, Irpex sp., Parphoma sp. and Phoma medicaginis. Although they were obtained with low relative abundant, those minor genera may play an important ecological role in their host plants, or may be able to synthesize bioactive compounds . Therefore, fungal isolates reported here may have a positive impact on the health of D. chinense by maintaining a balance in the composition of the associated microbiome, serving as a defense or helping to deal with water stress.
Another objective of this study was to assess the potentially beneficial properties of endophytic fungi to humans. All the endophytes extracts were screened for antioxidant, antimicrobial and anticancer activities and they showed at least one biological activity. Among the screened isolates, 99 (64.3%) isolates exhibited remarkable antioxidant activity, of which 18 (11.7%) had very notable activity with IC50 value of ≤ 3 μg/mL, suggesting that it may protect D. chinense from oxidative stress in the flooding environment as suggested by Zeng et al . Because of the protective effect of antioxidants, they are essential for plant survival and fitness and presumably selection have leaded to both redundant and highly specific pathways that address ROS production and stress mediation . For example, Mirzahosseini et al. have reported that endophytic fungi can alleviate the oxidative damage produced by ROS accumulation in plant cells such as F. arundinacea [89, 90]. Regarding antimicrobial activity, 31.2%, 11.7%, 19.5%, 69.5% and 29.9% extracts of endophytes showed activity against Penicillium, Candida albicans, Aspergillus niger, Staphylococcus aureus and Escherichia coli respectively, which was comparable and even exceeded some results reported by other authors in similar studies [91, 92]. For example, from the 39 endophytic fungal extracts of Viguiera arenaria and Tithonia Diversifolia plants, Guimaraes et al. found only 5.1% and 25.6% extracts to be active against Staphylococcus aureus and Escherichia coli respectively . Unexpectedly, Pseudomonas aeruginosa was most sensitive to the fungal extracts among the tested bacterial though it was reported to be drug resistant towards many antibiotics . Usually, the fungal extracts also showed higher activity against the Gram-negative than the Gram-positive ones. This different sensitivity has been suggested to be attributed to the high level of lipopolysaccharides that are contained in the Gram-positive bacteria membrane, which could make the cell wall impermeable to bioactive compounds . As for anticancer activity, 27 out of 154 fungal exacts (17.5%) showed activity against IHH4/CFPAC–1 cell line, in which 11 fungal extracts were active against both tested celllines. Statistically, 18 out of 27 anticancer isolates were exclusively isolated from the roots, 9 were only recovered from stems. Generally, for the same fungal species e.g. Neofusicoccum parvum, the isolates from roots showed stronger bioactivity compared to those from the stems regardless of antimicrobial, antioxidant or anticancer bioactivities. Such data well supported the traditional practice of native people who often used the extracts from roots to relieve analgesic, antirheumatic and diuretic .
Of these isolates screened, a high proportion of bioactivities were mostly detected from the fungal extracts belonging to Phomopsis sp. (24.7%), Neofusicoccum parvum (23.4%) and Xylaria venosula (9.1%), which was attributed to their high separation rate. As did here, Phomopsis sp. have been reported as dominant member of the endophytic community . Phomopsis is a dominant member of the endophytic community because it grows rapidly, thus inhibiting slow growing endophytes, which might be one of the reasons for the low number of species detected in this study . Additionally, Phomopsis and related taxa contain important endophytic and are known to produce a series of bioactive secondary metabolites in vitro with a variety of different chemical structures . However, few studies conducted on the active metabolites of Neofusicoccum parvum, and its antioxidant activity accounted for the highest proportion in the current study, which has never been reported in previous studies [99, 100]. Besides, Xylaria species are widely distributed on the temperate to the tropical zones in the terrestrial globe, and fungi of this genus have been proved to be potential sources of novel secondary metabolites, and many of them have biological activities related to drug discovery, including cytotoxic, antimalarial, and antimicrobial activities . In terms of bioactivity, active extracts of DS16–1 (Phomopsis sp.), DR28–1 (Phomopsis sp.), DS35–1 (Ceriporia lacerata),, DR41–2 (Ceriporia lacerata) and R46–1 (Phomopsis sp.) were found promising. In particular, the strain DR10–1(Irpex lacteus) showed wide spectrum bioactivities, suggesting that possible use of one endophyte could be a valuablecandidate as new antioxidant, antimicrobial and anticancer agents.
Finally, we isolated two known compounds including indole–3-carboxylic acid and indole–3-carboxylic acid derivatives from the wide spectrum bioactive strain Irpex lacteus DR10–1. As far as we know, this is the first time that indole–3-carboxylic acid (1) and indole–3-carboxaldehyde (2) have been isolated from endophytic fungus Irpex lacteus. It was previously demonstrated that indole–3-carboxylic acid isolated from endophytic fungal strain of Epicoccum nigrum associated with Entada abyssinica had remarkable activity against Gram-negative strains (Staphylococcus aureus) with MIC values of 6.25 μg/mL . This finding is consistent with literature report on the antibacterial activity of indole–3-carboxylic acid, from which a novel series of indole–3-carboxylic acid derivatives were previously reported to possess potent antibacterial activity against Enterococcus faecalis . In addition, it has been reported that indole–3-carboxylic acid had weak cytotoxic effects on both normal and tumor cells, and its antioxidant activity is weak . Recently, the indole–3-carboxylic acid (IAA) and other auxins have been shown to stimulate cell elongation, resulting in root growth initiation or an enhancement of nutritional elements absorption by the hosts [104, 105]. Besides, IAA was supposed to improve the adaptability of plant microbe interaction .