# Genomic Insights into Two Endophytic Strains:
## 2 Materials and Methods
To quantify the siderophore produced by the endophytic bacteria isolates, endophytic culture filtrates that were previously grown in CAS medium were mixed in equal volume with freshly prepared CAS solution and incubated for 5 min before taking the spectrophotometric readings at 630 nm. Also, the assay protocol, as described by ([21], was used to assess the potentials of the endophytic bacterial isolates for promoting ACC deaminase activity. A freshly grown overnight endophytic culture was used as the inoculum by harvesting and centrifuging the broth culture to obtain a culture pellet. Thereafter, saline water that had been previously sterilized was used to wash the pellet thoroughly and was then resuspended in saline water before spot inoculation on three (3) mM ACC, incorporating minimal media Petri plates. A minimal media plate that had been incorporated with ammonium sulfate was used as the positive control, while the minimal media plates without any nitrogenous source served as the negative control. All plates were in triplicate and cultured for 72 h at 30 \({}^{\circ}\)C. The growth of the endophytic bacteria on the three (3) mM ACC, incorporating minimal media Petri plates, indicated the potential of the endophytic bacterial isolates to promote ACC deaminase activity. Furthermore, the endophytic isolates were assessed for their potency in producing ammonia [22]. This involved using a freshly grown overnight culture of endophytic bacteria isolates to inoculate 10 mL sterilized peptone water in test tubes and the incubation of this liquid medium on a rotary incubator for 48 h at 30 \({}^{\circ}\)C. The development of yellowish to brownish coloration after the addition of [PERSON]'s reagent (0.5 mL) was a positive indication of the potential of the isolates to produce ammonia. In addition, the ability of the endophytic isolates to produce exopolysaccharides was assessed using the methodology proposed by [23]. A 10% sucrose-supplemented Luria Bertani (LB) agar was prepared, and the pH of the medium was adjusted to 7 before sterilization. Also, freshly grown overnight cultures of the endophytic isolates were used to impregnate sterile filter paper and were placed carefully inside the LB medium plates before incubation for 48 h at 30 \({}^{\circ}\)C. The potential of the isolates to produce exopolysaccharides radiate was indicated by the formation of a mucoid colony on the filter paper. The ability of the isolated bacterial endophytes to produce hydrogen cyanide (HCN) was determined by the methods of [24]. The endophytic isolates were streaked on the LB agar medium that was incorporated with glycine (4.4 g/L). Thereafter, the sterilized filter paper was dipped into a solution of picric acid, then carefully placed on the lid of the petri-plates and sealed up with parafilm before incubation for 96 h at 30 \({}^{\circ}\)C. The isolates proved positive for hydrogen cyanide production when, after incubation, the filter paper changed color from yellowish to a reddish-brown color. For the molecular studies, a Quick-DNA Zymoclean bacterial and fungal miniprep extraction kit from (Zymo Research Corporation, Irvine, CA, USA) was used, and the directives from the manufacturer were meticulously followed. The extraction kit is efficient and simple for extracting DNA and contains the bashing beads, lysis tubes, buffer, the genomic lysis buffer, genomic DNA prewash, wash buffer, DNA elution buffer, zymo-spin filters, and collection columns. Prior to DNA extraction, the bacteria isolates were cultured in nutrient broth overnight. The ribosomal DNA (rDNA), containing the 16S rRNA gene for identifying the bacterial strains, as well as the selected key plant growth-promoting genes, was amplified by using both universal 16S primers 341 forward-AGAGTTTGATCCTGGCTCAG; 907-reverse--AAGGAGGTGATCCAGCCGA, while the specific primers for GCD were forward--GACCTGGGACATGGACGT; reverse-GTCCTTGCCGGTAGSTCATC.