Sample collection
The P. fullo larvae to be used in the study were collected from vineyards, gardens and fields in some cities in the Turkey where they are seen densely. The larvae collected from the field were kept in plastic containers with some soil on them. The larvae, which were kept in 11 L plastic containers as 6 pieces, are fed regularly with fruits. Collected larvae samples were obtained in September 2023.
Isolation of Bacillus zhangzhouensis OBB from P. fullo
P. fullo larvae brought to the laboratory and surface sterilization was performed with 70% ethyl alcohol in sterile petri dishes. Then, they were placed in petri dishes containing sterile pure water, washed 2–3 times, and the alcohol was removed, and the insects' intestines were used in isolation. The intestines were crushed with 10 ml of phosphate buffer (PB, 50 mM, pH 7.4). 1 liter of MYPGP (10 g Mueller Hinton, 10 g Yeast extract, 3 g K2HP04, 2 g Glucose, 0.5 g trehalose) medium was prepared (Sharpe et al., 1970). Then, 500 µl of the crushed intestine sample was taken and inoculated into 30 ml of MYPGP medium. After overnight incubation (30oC, 180 rpm), the bacterial culture was incubated in a water bath at 70oC for 15 minutes. 100 µl of bacterial culture was inoculated into 30 ml of fresh MYPGP medium containing vancomycin (150 ug/ml). After 48 hours of incubation at 30oC, the bacterial culture was diluted to 10− 9 and then inoculated into 50 µl of MYPGP agar medium with appropriate diluents. After overnight incubation (30oC), the colony formed was collected and inoculated onto fresh MYPGPV agar medium (Kang et al., 2012). The purified colony was identified using morphological, biochemical and molecular methods (16S rRNA).
Gram Staining, Spore Staining and Crystal Protein Structure Staining of Bacillus zhangzhouensis OBB isolates
For Gram staining, bacterial samples were spread on a clean slide with pure water and detected in a burner flame. First, they were treated with crystal violet and washed with pure water. Then, they were treated with Lugol and washed again with pure water. They were washed again with alcohol and stained with safranin. Finally, they were washed with pure water, dried and examined under a light microscope. Bacteria with purple colored gram-positive bacillus morphology were observed (Claus, 1992).
For endospore staining, a small drop of water was left in the middle of a clean slide and a small amount of bacterial culture was spread with the help of a loop. The slide was detected by passing it through a flame. The preparation placed on a boiling water bath apparatus was treated with malachite green for 5 minutes. Then, after being washed with pure water, it was treated with safranin for 30 seconds (Temiz, 2000; Uçan and Erganiş, 2005). The presence of green stained spores was observed (Reynolds et al., 2009).
In order to determine whether the isolates contained parasporal crystal proteins, Coomassie Brilliant Blue solution, which is used for protein staining, was used in the identification of the isolates. After each isolate was incubated in petri dishes for a period of 5–10 days sufficient for the production of endospore and toxin protein, it was spread on the slide by placing 1 drop of water and dried as a thin film, then Coomassie Brilliant Blue solution was dropped on the slide, waited for 1 minute, washed with distilled water and dried, and then examined (Rampersad et al., 2002). Microscopically, the presence of endospore and endotoxin was examined with both light microscope (OLYMPUS-CX31) and Phase Contrast Microscope (OLYMPUS-CKX41). In addition, images were taken by conducting scanning electron microscope (SEM) examinations.
Molecular identification of bacterium
Genomic DNA isolation was performed using a DNA purification kit (Genomic DNA Miniprep Kit, BioBasic). 16S rRNA sequences of bacterial isolates whose genomic DNAs were isolated were amplified by PCR method using 5' ATGGTACCGTGTGTGACGGGCGGTGTGA-3' (forward) and 5'-ATTCTAGAGTTTGATCATGGCTCA 3' (reverse) primers (William et al., 1991). Taq 2X PCR master mix was used (Applied Biological Materials-G888). PCR, 25 µl Taq 2X PCR master mix, 1 µl 10 µM forward primer, 1 µl 10 µM reverse primer, 1 µl (5 ng/µl) DNA, 23 µl pure water were used to set up a total of 25 µl reaction. Samples were left to react in a thermocycler (Sensoquest). The reaction steps are given in Table 1. PCR products were visualized on a 1.2% agarose gel. The visible band on the gel was sent to MACROGEN (The Netherlands) for sequencing. 16S rRNA sequencing was performed using universal primers. Sequences were verified with BLAST in the NCBI GenBank database.
Table 1
PCR conditions used for amplification of the16S rRNA gene region
PCR Cycle | Temperature | Time | Number of cycles |
Initial denaturation | 95°C | 30 s. | 1 |
Denaturasyon | 95°C | 30 s. | |
Annealing | 55°C | 30 s. | 35 |
Extension | 72°C | 1 dk. | |
Final extension | 72°C | 5 dk. | 1 |
Bacillus zhangzhouensis OBB phylogeny
Sequence assembly was performed using Bioedit software (Hall et al., 2011 ). The sequence of the isolate was uploaded to the GenBank database. Sequence was deposited in GenBank under accession number PQ100677. The sequences were compared with the RefSeq database using the nBLAST search tool from NCBI GenBank. The sequence of the isolate and its closely related species were used for phylogenetic analysis. Phylogeny tree analyses were performed online with BLAST.
Determination of cry genes of Bacillus zhangzhouensis OBB isolates by PCR
The presence of cry genes was determined by PCR using the primers specified in Table 2. It was performed with the PCR conditions specified in Table 1.
Table 2
Primers used to screen for the presence of cry genes
Gene | Primer sequences (5' -> 3') | Tm (°C) |
cry1 (277 bp) | CATGATTCATGCGGCAGATAAAC (f) TTGTGACACTTCTGCTTCCCATT (r) | 59 |
cry2 (701 bp) | GTTATTCTTAATGCAGATGAATGGG (f) CGGATAAAATAATCTGGGAAATAGT (r) | 55 |
cry3 (604bp) | CGTTATCGCAGAGAGATGACATTAAC (f) CATCTGTTGTTTCTGGAGGCAAT (r) | 59 |
cry4 (439 bp) | GCATATGATGTAGCGAAACAAGCC (f) GCGTGACATACCCATTTCCAGGTCC (r) | 59 |
SDS Polyacrylamide Gel Electrophoresis
The bacterial isolate is grown in TSB (Tryptic Soy Broth) for 10 days. When the sporulation is more than 90%, it is precipitated by centrifugation at 14000 rpm at + 4 oC. Since the proteins (cry) are large, they remain in the pellet after centrifugation. The pellet is washed several times with pure water and used directly for sds. Protein concentrations of the spore-crystal mixture were obtained according to the procedures outlined by Bradford (1976). Total proteins were screened by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (10% PAGE) (Mehtap, 2022). The determined amounts of proteins were taken at equal concentrations from the samples and the samples were kept in boiling water for 10 minutes after the treatment buffer (60 mM Tris-HCl (pH 6.8), 25% Glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.1% bromophenol blue) was added. Then, it was loaded onto 10% SDS-PAGE as described by Laemmli (1970). The separation process was carried out by applying 130 V current to the gel. After the running process was completed, the gel was stained with Coomassie Brilliant Blue dye for 1 night and then washed with pure water. The gel image was transferred to the computer with a scanner.