**4. Materials and Methods**

### *4.1. Isolation of Endophytic Fungal Strains*

Endophytic fungi were isolated from leaves parts. Five healthy wild *D. nobile* plants were collected (from agricultural farm single location) from Jinshishi, Chishui, Guizhou, China and processed in the Bioresource Institute for Health Utilization, Zunyi Medical University, Zunyi, China, and then inoculated aseptically into PDA media. The samples were processed according to a method modified from Novotná et al. [8]. All the leaves segments of wild *D. nobile* were used for surface sterilization processing. All the leaves samples were washed with tap water to remove the dirt spots and connective tissue of stems. Thereafter, the leaves were washed with tap water to further clean the surface. After cleaning, all the plant samples were placed into laminar air flow and exposed to UV rays for 20 min. Under sterilized conditions in the laminar air flow, the samples (1.5 cm pieces) were inoculated onto PDA media plates. The sample plates were placed in a fungal incubator at 25 ◦C for 5 days. After 5 day of incubation, the fungal hyphal tips emerging from the samples were recovered and purified by sub-culturing from the grown hyphal tips.

#### *4.2. Morphological and Molecular Identification of Plant Endophytic Fungi*

The endophytic fungal genus was identified using lactophenol cotton blue staining according to the protocol followed by Barnett and Hunter [24] and Ellis [25]. The DNA was extracted from approximately 100 mg of fungal mycelia using the DNAsecure Plant Kit according to the manufacturer's instructions (Tianjin Biotech Co. Ltd., Beijing, China). PCR was performed using a standard protocol [26]. The reactions were prepared in 25 μL volumes constituting 2.0 μL primer, 12.5 μL 2× TsingKe (Blue) Master Mix (TSINGKE Biological Technology Co., Ltd, Beijing, China), 9.5 μL RNase-free distilled water, and template DNA (2 μL). A non-template tube was used as a negative control during PCR amplification to monitor purity. Amplifications were completed using a PCR thermal cycler equipment (Bio-Rad C1000 Thermal Cycler, Minnesota, USA). PCR cycling was performed using the conditions and primer sequences [27] presented in Table S2.

#### *4.3. Electrophoresis of PCR Products*

The PCR samples were analyzed using electrophoresis on a 1% (w/v) agarose gel (Agarose G-10, Gene Company Ltd., Hong Kong, China) [28]. A DNA marker (100 to 1000 bp molecular weights) (Beijing TransGen Biotech Co. Ltd., Beijing, China) was added to the first well of the gel. Electrophoresis was conducted in a horizontal electrophoresis system (Bio-Rad PowerPacTM Basic, Henderson, Singapore) for 30 min at 120 V and 250 mA using 1× TAE (Tris-acetate-EDTA) buffer. The gel was marked with ethidium bromide (0.1 μg/mL). A Molecular Imager Gel DocTM XR+ with Image Lab Software system (Bio-Rad Gel DocTM XR+ Imaging System, Hercules, CA, United States) was used to capture images using Image Lab software (version 4.1).
