# Genomic Insights into Two Endophytic Strains:
## 2 Materials and Methods  
* The isolation, morphogenomic authentication, and plant growth-stimulating screening of cowpea endophytic bacterial isolates. Standardized microbiological protocols were followed in the course of isolating, characterizing, and assessing growth improvement under in vitro conditions. In brief, the two endophytic bacterial strains were isolated from cowpea root tissue; the isolates were then subjected to morphological, biochemical, and molecular, as well as plant growth-improving, protocols. * Isolation protocol. Several surface sterilization protocols were deployed in preparing the cowpea root tissues for the isolation of the endophytic bacterial strains. Firstly, the root tissues were rinsed under running tap water to remove any attached debris. Further surface sterilization was carried out by washing the cowpea plant samples with 70% ethanol and then (3%) sodium hypochlorite for three minutes. This was followed again by sterilizing the samples with 70% ethanol and then washing it thoroughly three times with sterilized water to remove epiphytic micro-organisms from it. The efficacy of the surface sterilization was assessed by plating the final rinse water on growth media. The sterilized tissues were crushed with a mortar and pestle and diluted using a phosphate buffer saline; then, it was subsequently plated on varied microbiological growth media. The Petri plates were replicated and incubated [17]. Further purification of the isolated endophytes was achieved through several subculturing processes, with the pure isolates finally being stored for further analysis on storage media incorporating 20% glycerol. * In vitro PGP screening assay. The potency of the bacterial endophytic isolates to solubilize phosphate was assessed using [18] protocol. Freshly grown 18 h culture of endophytic bacterial strains in Luria Bertani (LB) broth was used as an aliquot to inoculate the National Botanical Research Institute's Phosphate (NBRIP) solubilization agar plate containing the insoluble calcium triphosphate at 0.5 g/L. The plates were replicated thrice and incubated for 7 days at 30 \({}^{\circ}\)C. The appearance of a transparent halo zone around the culture colony confirmed that the isolates did indeed have the potential to solubilize phosphate. Likewise, the quantitative phosphate solubilizing potency using the (NBRIP) solubilizing liquid medium was assessed by the phosphomolybate blue method at an optical density of 820 nm. For assessing the indole acetic acid (IAA)-producing abilities of the two endophytic isolates, the [19] protocol was applied. An overnight culture of the endophytic bacterial isolates was used to inoculate peptone water that contained five (5) mM L-tryptophan, and the liquid media was incubated on a rotary shaker at 150 rpm for 48 h and at 30 \({}^{\circ}\)C. Thereafter, the cultures were processed and centrifuged to obtain a supernatant; Salkowski reagent was then added to the supernatant in a ratio of 2:1, and the resultant solution was incubated in the dark for 30 min before the absorbance was measured with the aid of a spectrophotometer at 530 nm. A standard curve of IAA was also plotted using various concentrations of indole acetic acid in order to quantify the amount of IAA produced by the endophytic isolates. The ability of the endophytic bacterial isolates to produce siderophore was assessed qualitatively on Chrome Azural S (CAS) agar plates [20]. The endophytic isolates were freshly grown in the Luria Bertani (LB) medium to obtain 0.5 concentration at OD600. A diffusible disc was then placed on a freshly prepared CAS-blue agar plate. Thereafter, 10 \(\upmu\)L aliquots of each endophytic culture was inoculated onto the plate and incubated for 72 h. The appearance of a yellowish-orange halo around the culture colony indicated a positive potency to produce siderophore.