*3.3. Screening for the Production of Extracellular Lytic Enzymes and Siderophore by the Antagonists*

All six actinobacterial isolates were able to produce at least 4 out of 5 hydrolytic enzymes to different degrees. All the isolates tested positive for amylase and cellulase.

The isolates AR10, AR26 and AL7 produced siderophore and AR1, AR10, AR26 and AL7 recorded chitinase activity. The isolates AL5 and AFE2 produced protease while all other isolates tested negative for protease activity. The results revealed that the isolates AR10, AR26 and AL7 were positive for siderophore, amylase, cellulase, and chitinase. The isolate AR26 was the most potent antagonist to produce prominently siderophore, cellulase and chitinase (Supplementary Figures S2–S7).

#### *3.4. Antifungal Activity of Volatile, Non-Volatile and Thermostable Compounds*

All of the four isolates AR10, AR26, AL5 and AL7 apparently produced volatile, non-volatile and thermostable compounds, and significant differences in the antifungal activity against the tested pathogens were observed among the isolates (Figure 6). The volatile compounds of isolates AR10, AR26, and AL5 were found to be more effective than non-volatile and thermostable compounds. The volatile organic compounds of isolate AR26 exhibited the maximum inhibitory effect against *C. scovillei* (77.04%), *C. truncatum* (72.63%) and *F. oxysporum* (69.53%). The thermostable compound of AR26 exhibited the strongest inhibitory action against *F. oxysporum,* followed by AR10.

**Figure 6.** Antifungal activity of volatile, non-volatile and thermostable compounds. Error bars represent the standard deviation of the data set.

#### *3.5. Assessment of In Vitro Antifungal Traits*

The results of the assessment for in vitro antifungal traits revealed that out of the six isolates screened, rhizospheric isolate AR26 showed the highest assessment value of 17 points followed by the isolate AR10 with 15 points. Hence, the actinobacterial isolate AR26 was selected as the most efficient antagonist for further studies (Table 1).

#### *3.6. Molecular Confirmation of Actinobacterial Isolates*

The results of the 16S rRNA sequence analysis of the actinobacterial isolates revealed that five isolates were closely affiliated to the genus *Streptomyces.* Isolates AR1, AR10, AL5, AR26 and AL7 exhibited the highest similarity with *Streptomyces rochei*, *Streptomyces deccanensis*, *Streptomyces azureus, Streptomyces tuirus*, and *S. geysiriensis*, respectively (Table 2). Phylogenetic analysis revealed that the isolates under current study formed five different clades (highlighted in red) and were supported with good bootstrap values (Figure 7). Isolate AR10 formed a distinct clade A with *S. deccanensis*, AL5 formed clade B with *S. azureus*, AR26 formed clade C with *S. tuirus,* AR1 formed clade D with *S. rochei,* and AL7 formed clade E with *S. geysiriensis,* with *Pseudomonas fluorescens* as the out group.


**Table 1.** Actinobacterial isolates for their ability to function as antagonist with various antifungal mechanisms.

Mycelial Growth inhibition percentage (1 = 30–54.9%; 2 = 55–74.9%; 3 = 75–95%); Lytic enzyme production was evaluated with 1 point and siderophore with 2 points each; *C.s*: *C. scoville*; *C.t*: *C. truncatum*; *F.o*: *F. oxysporum.*

**Table 2.** 16S ribosomal RNA partial sequence analysis of actinobacterial isolates and their closest BLASTN matches with NCBI database supplementary.

