*3.2. Secondary Screening for the Antifungal Activity of Actinobacterial Isolates and Scanning Electron Microscopic Assay*

The six isolates (Figure 4) which showed the strongest antagonism against the three pathogens were further subjected to secondary screening by paired culture antibiosis to further confirm their antagonistic ability against *C. scovillei*, *C. truncatum* and *F. oxysporum*. The results of this assay indicated that all six isolates were capable of inhibiting the growth of *C. scovillei*, *C. truncatum* and *F. oxysporum*. The isolate AR26 was found to be significantly superior to other isolates in inhibiting mycelial growth of *C. scovillei* (59.63%), *C. truncatum* (61.18%) and *F. oxysporum* (63.58%), respectively followed by the isolate AR10 (Supplementary Figure S1). The isolate AFE2 recorded the lowest percentage of mycelial growth inhibition of the tested pathogens, relative to the other five isolates. Scanning Electron Microscopy (SEM) observations indicated a clear evidence for antifungal activity of isolate AR26 against *C. scovillei*, *C. truncatum* and *F. oxysporum*. The antifungal activity was observed as distinct morphological deformities in pathogen hyphae in the presence of antagonist and hyphae were found to be twisted and shrunk in *C. scovillei*, disintegrated in *C. truncatum*, aggregated into clusters in *F. oxysporum* with reduction in mycelial mat (Figure 5). In contrast, the hyphae in the control plate were dense, intact with regular structure.


**Figure 1.** Heat map showing the in vitro antagonism of actinobacterial isolates against chilli fruit rot pathogens as represented by zone of inhibition.

**Figure 2.** Venn diagram representing the actinobacterial isolates exhibiting strong antagonism against fruit rot pathogens.

**Figure 3.** Antifungal activity of actinobacterial isolates against chilli fruit rot pathogens. (**A**) Control *C. scovillei* (**D**) Control *C. truncatum* (**G**) Control *F. oxysporum.* Antifungal activity of actinobacterial isolate AR26 against (**B**) *C. scovillei* (**E**) *C. truncatum* (**H**) *F. oxysporum*. Antifungal activity of actinobacterial isolate AR10 against (**C**) *C. scovillei* (**F**) *C. truncatum* (**I**) *F. oxysporum*.

**Figure 4.** Actinobacterial isolates with potent antagonistic activity against fruit rot pathogens.

**Figure 5.** Scanning Electron Micrographs showing the interaction of antagonist. *S. tuirus* AR26 with *C. scovillei*, *C. truncatum* and *F. oxysporum*; *(***A**) intact mycelium of *C. scovillei* in the absence of antagonist; (**B**) twisted and shrunken hyphae of *C. scovillei* in the presence of antagonist; (**C**) mycelium of *C. truncatum* in absence of antagonist; (**D**) distorted hyphae of *C. truncatum* in the presence of antagonist; (**E**) dense and intact mycelium of *F. oxysporum* in absence of antagonist; (**F**) aggregated hyphae of *F. oxysporum* in presence of antagonist.
