**3. Results**

#### **3.1. Effect of imidacloprid on soil bacterial populations in laboratory studies**

The result of the laboratory study is given in Table 1. Application of imidacloprid at 125, 250, 500 and 1,000 ppm resulted in 9.80, 08.40, 6.73 and 5.60 × 10–6 colonies (*P* < 0.05) when compared to 11.05 × 10–6 in control plates (Graph 1).

#### **3.2. Effect of imidacloprid on soil bacterial populations in field studies**

In the field studies, imidacloprid was applied in both recommended and ×1.5 rates and the results are given in Graph 2. The results showed that imidacloprid-treated fields at recom‐ mended and ×1.5 rates showed significant (*P* < 0.05) decline in bacterial counts at different post-application intervals when compared to control, but the colony count increased with the time.

**Graph 1.** Effect of imidacloprid on bacterial populations in the soil under laboratory conditions

#### **3.3. Identification of soil isolate**

The bacterial strain SP-03 isolated from soil was a rod-shaped, Gram-positive bacterium, facultatively anaerobic, grows at 5–40°C, at pH 6–7; produce subterminal ellipsoidal endo‐ spores; white-colored colonies; positive for catalase activity, Voges-Proskauer, starch hydrol‐ ysis and oxidase; and negative for methyl red, gelatin liquefaction, production of indole and citrate. The 16S rDNA gene of SP-03 was isolated and sequenced. This 16S rDNA gene sequence was then compared with previously published 16S rDNA gene sequences and based on matches the strain was classified as a member of the genus Bacillus. The sequence of strain SP-03 displayed the highest identity (100%) with the 16S rDNA gene of Bacillus weihenste‐ phanensis KBAB4 (GenBank Accession Number: HG 486214.1) (Figure 1, 2). The Bacillus weihenstephanensis showed highest growth at 22°C and at pH of 7.0.

#### **3.4. Effect of imidacloprid on antioxidant enzymes in** *Bacillus weihenstephanensis*

On exposure of *Bacillus weihenstephanensis* to various molar concentrations (10–3 to 10–7) of imidacloprid for 24, 48, 72 and 96 h, there was a significant (*P* ≤ 0.05) increase in the activity of antioxidant enzymes studied. There was a significant increase (*P* ≤ 0.05) in the activity of superoxide dismutase (Graph 3), catalase (Graph 4) and peroxidase (Graph 5) in all the treated groups. The antioxidant enzyme activity increased with an increase in the concentration of imidacloprid.

#### **3.5. Gene isolation and sequencing of the stress enzymes of** *Bacillus weihenstephanensis* **on exposure to imidacloprid**

In the present gene sequencing study, different markers of 400, 600, 1,000 and 1,200 bp (Figure) 6) were run along with our test sample. The superoxide dismutase gene was corresponding to 624 bp. This gene was isolated, eluted and sent for sequencing. The sequence was received and this was subjected to NCBI BLAST which revealed that the nucleotide sequence was of 624 letters bearing accession number YP\_001648011.1 that showed 100% query coverage with *Bacillus weihenstephanensis* Fe/MnSOD (sod A) (Figure 3). The gene for catalase corresponded band at 800 bp. The isolated gene was eluted and sent for sequencing. The received sequence, subjected to NCBI BLAST revealed that the nucleotide sequence was of 800 letters bearing accession number YP\_001647388.1 and showed 100% query coverage with *Bacillus weihenste‐ phanensis* Hydroxyperoxidase HP(II) (*Kat E*) (Figure 4). The gene for peroxidase that corre‐ sponded to 480 bp was isolated, eluted and sent for sequence analysis (Table 2, 3). The sequence report was received, which was subjected to NCBI BLAST which revealed the nucleotide sequence of 480 letters bearing accession number YP\_001644822.1 and showed 100% query coverage with glutathione peroxidase of *Bacillus weihenstephanensis* (Figure 5)*.*
