*2.2. Evaluation of Protocorm and Seedling Pathogenicity*

The DME pathogenicity results with *D. nobile* and *D. o*ffi*cinale* protocorm revealed that *T. longibrachiatum* (ZF05) showed the least pathogenicity (11%) against the *D. nobile* protocorm after 7, 14, and 21 days, whereas it showed moderate pathogenic (33%) effects at 21 days of incubation against *D. o*ffi*cinale*. We found that most of the plant pathogens are latent pathogens or conditional pathogens, causing symptoms some weeks later when the plant defense mechanism has weakened. Many environmental factors function together to trigger the virulence factors or genes of the endophytic fungi. *T. longibrachiatum* (ZF05) started to change the color of the *D. o*ffi*cinale* protocorm from green to light yellowish at 14 days, which changed to brown to blackish at 21 days; whereas no color change symptoms were observed in the *D. nobile* protocorm. The second least pathogenic strain was *F. keratoplasticum* (ZF02), which was found to be non-pathogenic against *D. nobile* after 7 and 14 days of incubation, and least pathogenic (11%) against *D. o*ffi*cinale* after seven days of incubation, but showed moderate pathogenic effect after 21 days incubation on both *D. nobile* and *D. o*ffi*cinale*. The symptoms on the protocorms of *D. nobile* and *D. o*ffi*cinale* by *F. keratoplasticum* (ZF02) included the color of *D. o*ffi*cinale* protocorm starting to change from green to light yellowish at 14 days, then turning yellow at 21 days, whereas no color change symptoms were observed in the *D. nobile* protocorm due to *F. keratoplasticum* (ZF02). *F. oxysporum* (ZF03) and *F. solani* (ZF04) were moderate pathogens at seven days of incubation and strong pathogens (100%) at 21 days of incubation. *C. tropicicola* (ZF01) showed high pathogenicity

(77–100%) effects against both *D. nobile* and *D. o*ffi*cinale* seedlings. *F. oxysporum* (ZF03) produced no symptoms on the protocorms of *D. nobile* and *D. o*ffi*cinale* at 7 and 14 days, indicating these are latent pathogens because they demonstrated high pathogenicity at 21 days and their symptoms included color change of the green protocorm to brownish for both *D. nobile* and *D. o*ffi*cinale*. *F. solani* (ZF04) produced moderate symptoms on the protocorms of both *D. nobile* and *D. o*ffi*cinale* by changing their green color to light reddish to brown in the initial days and then turned dark reddish-brown at 21 days. *C. tropicicola* (ZF01) significantly affected the protocorms of *D. nobile* and *D. o*ffi*cinale* by changing their green color to dark brown at 7, 14, and 21 days (Figures 2 and 3).

**Figure 1.** Phylogenetic identification of the endophytic fungi based on ITS4 and ITS5 regions of DNA sequences. The evolutionary detachments were calculated using the Kimura two-parameter method.

**Figure 2.** Co-culturing pathogenic effects on protocorm of (**A**) *D. nobile* and (**B**) *D. o*ffi*cinale* with endophytic fungi. Means values are significantly different at *p* = 0.05 according to Duncan's multiple range test.

The DME pathogenicity results for *D. nobile* and *D. o*ffi*cinale* seedling revealed that *T. longibrachiatum* (ZF05) produced the least pathogenic effects on *D. o*ffi*cinale* protocorm after seven days and increased gradually with increasing incubation period (at 14 days; 33% disease index), which was stable at 21 days (33% disease index). For *D. nobile*, the fungus showed highly pathogenic (77% disease index) effects from the start to the end of the incubation period. The second least pathogenic strains were *F. keratoplasticum* (ZF02) and *F. solani* (ZF04), which showed least pathogenic effects against *D. o*ffi*cinale,* whereas *F. keratoplasticum* (ZF02) and *F. oxysporum* (ZF03) showed stable moderate pathogenic (33%) effects against *D. nobile* and *D. o*ffi*cinale*, respectively, throughout the incubation period. *C. tropicicola* (ZF01) showed high pathogenicity (77–100%) against both *D. nobile* and *D. o*ffi*cinale* seedlings. The seedlings symptoms included a color change of *D. nobile* seedlings from green to black spots and brownish to blackish spots on the *D. o*ffi*cinale* seedlings with *T. longibrachiatum* (ZF05) at 7, 14, and 21 days. *F. keratoplasticum* (ZF02) produced symptoms on the *D. nobile* and *D. o*ffi*cinale* seedlings including color change of the *D. o*ffi*cinale* seedlings from green to yellowish at 7 days, then to light brown at 14 days, then to black at 21 days; similar symptoms were observed in the *D. nobile* seedlings with *F. keratoplasticum* (ZF02). *F. oxysporum* (ZF03) produced light brown color symptoms on the seedlings of *D. nobile* and *D. o*ffi*cinale* at 7 and 14 days, which later changed to brownish to yellowish. *F. solani* (ZF04) produced moderate symptoms on the seedlings by changing its green color to light yellowish to brown at 7 days, changing to dark brown at 14 days, then to blackish brown at 21 days on the seedlings of *D. nobile* and *D. o*ffi*cinale*. *C. tropicicola* (ZF01) produced significant symptoms on the seedlings of *D. nobile* and *D. o*ffi*cinale* by changing the seedling color from green to brown at 7, 14, and 21 days (Figures 4 and 5).

**Figure 3.** Least pathogenicity by *Trichoderma longibrachiatum* (ZF05) against the *D. nobile* protocorm after 21 days. (**A**) Control (*D. nobile* protocorm with water agar media); (**B**) pure culture of *Trichoderma longibrachiatum* (ZF05); (**C**) *D. nobile* protocorm with *Trichoderma longibrachiatum* (ZF05) after 21 days; (**D**) Single *D. nobile* protocorm with *Trichoderma longibrachiatum* (ZF05) growth indicated by the arrow.

**Figure 4.** Co-culturing pathogenic effects of endophytic fungi on seedlings of *D. nobile* and *D. o*ffi*cinale*. Means values are significantly different at *p* = 0.05 according to Duncan's multiple range test.

**Figure 5.** Moderate pathogenicity by *Trichoderma longibrachiatum* (ZF05) against the *D. o*ffi*cinale* seedling after 21 days. (**A**) Control (*D. o*ffi*cinale* seedling with water agar media); (**B**) pure culture of *T. longibrachiatum* (ZF05); (**C**) *D. o*ffi*cinale* seedling with *T. longibrachiatum* (ZF05) after 21 days; (**D**) Single *D. o*ffi*cinale* seedling with *T. longibrachiatum* (ZF05) growth indicated by the arrow.
