*3.1. Identification and Characterization of the Isolate ADR1*

Amplification of the 16S rRNA gene from the genome of ADR1 produced a sequence of 1452 nucleotides. Blast analysis revealed 99.17% sequence identity of the ADR1 sequence with *S. californicus* strains with a query coverage of 99%. The phylogenetic relationship of the strain ADR1 can be seen in Figure 1, where it showed closest relationship with *S. californicus* strains. The 16S rRNA gene sequence obtained in this study was submitted as '*Streptomyces californicus* strain ADR1' to NCBI GenBank with accession no. KU299789.1.

A few strains of *S. californicus* had been reported earlier from the soil [41–44]. However, there are no reports of any endophytic strain of *S. californicus* till date to the best of our knowledge, making the present isolate as a new endophytic actinobacterial strain, designated as *S. californicus* strain ADR1.

The strain ADR1 was characterized for its cultural attributes on ISP media 1 to 7. The results (Table 1) suggested that the extent of growth of the culture varied from scanty to abundant on different ISP media. Further, differences with respect to the colour of substrate and aerial mycelia, and production of diffusible pigments were also noted as described in the Table 1. When compared with the cultural characteristics of non-endophytic *S. californicus* strains JCM 6910, MNM-1400 and G16, it was observed that ADR1 shared a few similarities, for example, colour of aerial mycelium on ISP 2, 3 and diffusible pigments on 4, with *S. californicus* strain JCM 6910, a soil isolate from Japan [41,44,45]. However, the growth of ADR1 was abundant on ISP3, while that of JCM 6910 was poor. No diffusible pigment was produced by ADR1 in ISP5, while violet pigment was produced by JCM 6910. Other soil isolates of *S. californicus,* strain MNM-1400 and strain G16, were morphologically very different from the strain ADR1 [44,45] Thus, the endophytic *S. californicus* strain ADR1 was evidently distinct from the soil isolates.

**Figure 1.** Phylogenetic analysis of isolate ADR1. Neighbour-joining phylogenetic tree showed maximum likelihood model showing the phylogenetic relationship of selected isolate (highlighted in blue) based on 16S rRNA gene sequence alignments. The numbers at the branching points are the percentages of occurrence in 500 bootstrapped trees. Bar indicated 0.0008 substitutions per nucleotide position.


**Table 1.** Cultural characteristics of the isolate ADR1 on international *Streptomyces* project (ISP) media.

A detailed view of the morphology was obtained through the study of single colonies (Figure 2; Panel A and B). Growth on different ISP media produced differences in colour and appearance of the colonies, which appeared as dense, depressed and rocky on ISP1, while on ISP2, 3 and 5 the aerial mycelia appeared to be fluffy and dusty. Clear exudates can be seen over the colony on ISP6. The colonies on ISP7 appeared scantly grown lacking distinct structures that were observed on other media. Prominent differences in the extent of growth, structure and pigmentation of the colonies on different ISP media were consistent with the earlier reports [46]. Such media-dependent phenotypic variations suggested that the primary and secondary metabolism of the culture varied significantly with changes in composition of the medium, which is in agreement with the current understanding of the physiology of the genus *Streptomyces* [47,48]. The microscopic observations showed highly branched flexuous mycelium and the arrangement of spores in a chain inside mycelium as shown in (Figure 2, panel C and D). These are some explicit features of *Streptomyces* species [32].

**Figure 2.** Morphological characterization of *S. californicus* ADR1. Colony observations were made on different ISP media. Panel (**A**) represented the magnified view showing exterior structures of the single colony while panel (**B**) showed interior view of the colony as observed under a Nikon Stereo Zoom Microscope SMZ1270; the mycelial network of branched hyphae could be observed in panel (**C**) and arrangement of spores in chains were viewed in panel (**D**).
