*4.5. Collected Samples (Protocorm and Seedling) and Sample Processing*

Water agar (0.65% w/v) was used as media and relocated into the glass bottles for the coculturing experiments. No additional nutriments were added into the media; only twofold distilled water and agar were used. *D. nobile* and *D. o*ffi*cinale* (both protocorms aged 4 months) and seedlings (aged 8 months) were obtained from the Bioresource Institute for Healthy Utilization (BIHU), Zunyi Medical University (ZMU; Zunyi, Guizhou, China) for experiments and authenticated per previous work [29–32]. After the sterilization of the media bottles, the protocorms of the *D. nobile* and *D. o*ffi*cinale* were inoculated into separate bottles under aseptic conditions. For seedlings, we cut marks into the top leaf of the seedlings where it joined then stem using a sterilized scalpel blade over sterilized paper, as shown in Figure 8. Three seedlings were transferred into each bottle under aseptic conditions for pathogenicity and symptoms evaluation.

**Figure 8.** Different phases of coculturing experiment for pathogenicity evaluation. (**a**) Healthy seedlings; (**b**) cut marks at the top leaf and inoculation of mycelial plug of endophytic fungi; (**c**) symptoms developed for pathogenicity evaluation; and (**d**) histopathological examination of infected tissues.

### *4.6. In Vitro Inoculation for Seedling Pathogenicity Assay*

For pathogenicity experiments, the identified endophytic fungal mycelium was used for the pathogenicity evaluation. For protocorms, 0.5-cm fungal discs were transferred into the protocorm bottles of *D. nobile* and *D. o*ffi*cinale*. The control was set in triplicates under the same conditions without added any endophytic fungal cultures. After the inoculation, the test co-culturing bottles were transferred into the plant growth chamber instrument and maintained at 25 ◦C along with a 14/10 h light/dark cycle with 2000 lux light intensity. All the test bottles were examined in 7-day intervals up to 21 days. For *D. nobile* and *D. o*ffi*cinale* seedlings, the contents of endophytic fungal flask cultures were filtered to separate the mycelium suspension. The mycelium was concentrated and injected onto cut marks at terminal leaves at the link to the stem. The sterilized cotton plug served as a control for seedlings co-culturing pathogenicity and symptoms evaluation experiments. The disease index was measured using the disease scale and disease index formula. The scale of the disease index is described as follows: 0 indicates no disease (non-pathogens), 1 indicates small spots < 1% (pin brown spots: fewest pathogens), 3 indicates spots (1–11%: least pathogens), 5 indicates spots with 12–25% coverage (moderate pathogens), 7 indicates circular spots (26–55% coverage: moderate pathogens), and 9 indicates circular to irregular spots (>75% coverage: highly pathogenic) [33].

*Disease index percentage* <sup>=</sup> *Sum o f disease rating Total number o f rating* <sup>×</sup> *Maximum disease grade* <sup>×</sup> 100%
