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BtA and BtK strains were isolated from commercially available Bt-based biopesticides, Zentari WG (NongHyup Chemical, Seongnam, Korea) and Nabangmyul WP (Daeyu, Seoul, Korea), respectively. A 0.5 g of each biopesticide formulation was transferred into a 1.5 ml E-tube (Eppendorf, Seoul, Korea) containing 1 ml of sterile distilled water and the mixture was vortexed to homogenize. The E-tubes containing the suspensions were heat-treated at 60°C for 1 h using a heating block (Allsheng, Hangzhou, China). Following heat treatment, 100 µl of each suspension was spread onto Tryptic Soy Agar (TSA) (BD, Franklin, USA). The plates were incubated at 28°C for 72 h to isolate single colonies of BtA and BtK, which were subsequently selected, streaked using a loop and stored at -80°C in 10% glycerol stocks for accurate identification.

For molecular identification of the isolated strains, each strain was cultured on TSA medium for 48 h, followed by 16S rRNA gene analysis. Genomic DNA was extracted using a 5% Chelex solution (BIO-RAD, Seoul, Korea). PCR amplification was performed using the CFX Duet Real-Time PCR System (BIO-RAD, Seoul, Korea). The PCR reaction mixture consisted of AccuPower® HotStart PCR PreMix (Bioneer, Daejeon, Korea), 1 µl each of the 27F and 1492R primers (Bioneer, Daejeon, Korea), and template DNA, adjusted to a final volume of 20 µl with sterile distilled water. The PCR was performed under the followng conditions: An initial denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. A final extension was performed at 72°C for 5 min. The amplified PCR products were sequenced by a commercial service provider (Macrogen, Seoul, Korea), and sequence homologies were analyzed using the NCBI BLAST tool (https://blast.ncbi.nlm.nih.gov). Using the MEGA 11.0 program (www.megasoftware.net), the phylogenetic trees were built using the Neighbor-joining approach and the Poisson correction model (1,500 bootstrap repetitions to support branching clusters). In addition, biochemical characteristics based on carbon source utilization of each isolated strain were analyzed using GEN III MICROPLATE (Biolog, Hayward, CA, USA).

Each strain previously cultured on TSA plates was inoculated into 50 ml of Tryptic Soy Broth (TSB) (BD, Franklin, USA) from frozen glycerol stocks and incubated at 28°C with shaking at 120 rpm for 48 h to measure the growth rates of BtA and BtK strains. Subsequently, 10 µl of each Bt culture (1 × 10⁵ colony forming units (CFU)/μL) was transferred into 100 ml of fresh TSB and subcultured under the same incubation conditions. To generate growth curves, the optical density (OD) at 600 nm was measured at 4 h intervals using a spectrophotometer (Ubi-600 UV/Vis, MicroDigital, Seongnam, Korea), and bacterial cell counts were determined using a hemocytometer (Paul Marienfeld, Lauda-Königshofen, Germany). The experiment was conducted using three independent TSB cultures for each strain.

Each Bt strain was inoculated into 100 ml of TSB and incubated at pre-culture condition (28°C for 48 h) under the same conditions used in the growth curve experiment based on previous study (Seo and Kim, 2011; Eom et al., 2014). Cultures were incubated for 96 h at various temperatures (0, 5, 10, 20, 28, 40, 50, 60, and 70°C). Time-course experiments also were conducted at 28°C for various time points (0, 6, 12, 24, 48, 72, and 96 h). Effects of temperature stress on sporulation of BtA and BtK were measured by count the numbers of spores.

Based on the previous experiment, 2 temperature conditions (0°C and 70°C), which showed the highest levels of spore formation, were selected for further time-course analysis. Spore and crystal formations were examined at various time points (0, 6, 12, 24, 48, 72, and 96 h) under these 2 temperatures. The numbers of spores and crystals were determined using a hemocytometer. The experiments were conducted using three independent TSB cultures per strain, and the measurements of spores and crystals were performed in triplicate for each culture.

Plutella xylostella, which had been reared for more than 10 generations under laboratory conditions, were used for bioassay. The insect was obtained from the Insect Molecular Physiology Laboratory at Gyeongkuk National University. The P. xylostella population was maintained at 26 ± 2°C, with a photoperiod of 16:8 h (L:D) and 60% of RH. Sterilized cabbage leaves were provided as the larval diet after washing with tap water. BtA and BtK cultures were first incubated under pre-culture condition in TSB and then subjected to temperature treatment at 0°C and 70°C for 96 h. Cabbage leaves were cut into 4 cm diameter leaf disks and immersed in the treated culture broth for 20 min. Subsequently disks were air-dried for 10 min in 9 cm of Petri dishes lined with filter paper. Each dried leaf disk was then exposed to 2^nd instar larvae after 6 h starvation. Larval mortality rate was recorded every 24 h for 96 h. The control group was treated under the same conditions using cabbage leaves immersed in sterile TSB without bacterial inoculation. Each experiment was conducted with 10 larvae per replicate, and three replicates were performed.

All experimental results were analyzed by ANOVA and treatment means were compared using PROC GLM in SAS (SAS Institute Inc., 1989).

The amplified PCR products obtained from the DNA of the isolated strains yielded sequences of 1,404 bp for BtA and 1,360 bp for BtK. Sequence analysis using NCBI BLAST revealed high genetic similarity of isolated bacteria compared with B. thuringiensis (Fig. 1). Two isolates showing 99.86% identity with B. thuringiensis subsp. aizawai (Accession No. KY819034.1) for BtA (Fig. 1A) and 100% identity with B. thuringiensis subsp. kurstaki (Accession No. EF117854.1) for BtK (Fig. 1B). Additionally, carbon utilization profiling using Biolog GEN III plates showed that both strains metabolized key carbon sources such as D-serine, guanidine HCl, and aztreonam, demonstrating identical patterns to B. thuringiensis as standard in the Biolog Station database. Therefore, both isolates were finally identified as BtA and BtK (Table 1).

Fig. 1 
Phylogenetic analysis of 16S rRNA of the isolated Bacillus thuringiensis with other Bacillus genus. (A); Isolate from commercial Bt pesticide (Xentari) is clustered by B. thringiensis subsp. aizawai (BtA). (B); Isolate from commercial Bt pesticide (Nabangmyeol) is clustered by B. thringiensis subsp. kurstaki (BtK). The phylogenetic tree was generated by Neighbor-joining method using the software package MEGA11.0. Each node contains bootstrap value after 1,500 replications to support branching and clustering. Accession numbers follow the scientific names.

Both BtA and BtK strains followed the typical bacterial growth curve model (Fig. 2). In bacterial cell count measurements using a hemocytometer, BtA entered the exponential phase from 2 to 16 h after the lag phase, reaching a peak of 10.6 × 10⁷ cells, followed by a decline in cell number. BtK showed exponential growth from 4 to 24 h. Both strains then transitioned into the stationary phase, during which the rates of cell proliferation and death were balanced, and subsequently entered the death phase after 24 h of incubation, showing a decrease in viable cell numbers (Fig. 2A). Statistically significant differences in bacterial counts over incubation time were observed for both BtA (F =24.23; df: 7,16; P < 0.0001) and BtK (F =219.58; df: 7,16; P < 0.0001). In optical density analysis using a spectrophotometer, BtA showed a continuous increase in absorbance, reaching 1.576 at 16 h post-inoculation, after which it plateaued during the stationary phase. BtK showed a rapid increase in absorbance up to 8 h, with no further significant change after 16 h (Fig. 2B). There are significant differences in absorbance on incubation time for both BtA (F =45.52; df: 7,16; P < 0.0001) and BtK (F =116.22; df: 7,16; P < 0.0001), indicating that the increase in optical density corresponded to an increase in bacterial cell numbers.

Fig. 2 
Cell growth of Bacillus thuringiensis subsp. aizawai (BtA) and B. thringiensis subsp. kurstaki (BtK). Bacteria were inoculated in TSB medium at 28℃ in shaking incubator (120 rpm). (A); Number of live cells of BtA and BtK. (B); Absorbance of BtA and BtK and optical density (OD) were measured at 600 nm. Each treatment was replicated three times per replication. Different letters indicate significant differences among means at (Type I error = 0.05, LSD test). Uppercase and lowercase letters indicate statistical analysis results for BtA and BtK, respectively.

The isolated BtA and BtK strains showed a positive correlation between treatment duration and sporulation under both low and high-temperature conditions following constant temperature incubation (Fig. 3). In the experiment conducted to determine the optimal temperature for spore formation after 72 h, BtA produced 4.5 × 10⁶ spores at 28°C, whereas it produced 1.4× 10⁷ and 1.5 × 10⁷ spores at 0°C and 70°C, respectively, indicating the highest spore counts at the extreme temperatures. The number of spores formed over time increased until 48 h and plateaued thereafter (Fig. 3A and 3B). For BtA, temperature (F =174.38; df: 8,18; P < 0.0001) and incubation time (F =191.88; df: 6,14; P < 0.0001) had statistically significant effects on spore formation. BtK produced the highest number of spores as 1.5 × 10⁷ each at both 0°C and 70°C, and spore numbers increased with longer incubation times. Statistically significant differences were also found for BtK based on temperature (F =131.28; df: 8,18; P < 0.0001) and time (F =12.06; df: 6,14; P < 0.0001). Spores formed by both BtA and BtK strains were observed within the bacterial cell wall under microscope (Fig. 3C).

Fig. 3 
Sporulation of Bacillus thuringiensis subsp. aizawai (BtA) and B. thringiensis subsp. kurstaki (BtK) by incubation temperatures and times. (A); The numbers of spores of BtA and BtK after subsequent incubation at different temperatures for 96 h following pre-culture (28℃, 48 h). (B); The numbers of spores of BtA and BtK after subsequent incubation at 28℃ for 96 h following pre-culture (28℃, 48 h). (C); Spore and crystal in BtA and BtK strains after subsequent incubation at 28℃ for 96 h following pre-culture. S and C mean spore and crystal, respectively. Each treatment was replicated three times per replication. Different letters indicate significant differences among means at (Type I error = 0.05, LSD test). Uppercase and lowercase letters indicate statistical analysis results for BtA and BtK, respectively.

After initial pre-culture, subsequent incubation at 0°C and 70°C showed that BtA produced over 3 times, and BtK over 4 times more spores compared to subsequent incubation at 28°C (Fig. 4). For BtA, spore numbers at 0°C and 70°C reached 5.5 × 10⁶ and 4.3 × 10⁶, respectively, within the first 6 h of subsequent incubation (F =29.40; df: 2,6; P < 0.001), and peaked at 1.4× 10⁷ and 1.3×10⁷ at 48 h (F =111.00; df: 2,6; P < 0.0001). However, no further increase in spore numbers was observed after 48 h (Fig. 4A). BtK formed 5.3 × 10⁶ and 4.5 × 10⁶ spores at 0°C and 70°C, respectively, at 6 h (F =44.33; df: 2,6; P < 0.001), with a maximum of 1.5×10⁷ spores at 48 h under both conditions (F =52.55; df: 2,6; P < 0.001). No significant increase in spore numbers was observed similar to BtA (Fig. 4B).

Fig. 4. 
Sporulation of Bacillus thuringiensis subsp. aizawai (BtA) and B. thringiensis subsp. kurstaki (BtK) by high and low temperatures. (A); The numbers of spores of BtA after subsequent incubation at 0℃ and 70℃ for 96 h following pre-culture (28℃, 48 h). (B); The numbers of spores of BtK after subsequent incubation at 70℃ for 96 h following pre-culture (28℃, 48 h). Each treatment was replicated three times per replication. Two asterisks above the bars indicate statistically significant differences between temperatures at each incubation time (P < 0.0001).

The production of crystals in BtA and BtK under various temperatures showed a similar trend to sporulation, with higher levels observed as the incubation time increased under both low and high temperatures (Fig. 5). After pre-culture, BtA showed 6 times and 4 times higher crystal production at 0°C compared to 28°C and 70°C, respectively, during 12 h of subsequent culture. At 48 h, BtA produced 1.4× 10⁷ crystals at 0°C, which was higher than the 4.8 × 10⁶ at 28°C and 5.8 × 10⁶ at 70°C (F =490.74; df = 2,6; P < 0.0001)(Fig. 5A). BtK produced 5-fold and 2-fold higher levels of Cry proteins at 0°C compared to 28°C and 70°C, respectively, during 12 h of subsequent incubation. After 48 h at 0°C, BtK produced 1.3 × 10⁷ crystal, which was higher than 6.8×10⁶ at 70°C and 6.2 × 10⁶ at 28°C (F =46.87; df = 2,6; P < 0.001)(Fig. 5B). Additional incubation beyond 48 h did not further increase crystal production in either strain.

Fig. 5 
Crystallization of Bacillus thuringiensis subsp. aizawai (BtA) and B. thringiensis subsp. kurstaki (BtK) by high and low temperatures. (A); The numbers of crystals of BtA after subsequent incubation at 0℃ and 70℃ for 96 h following pre-culture (28℃, 48 h). (B); The numbers of crystals of BtK after subsequent incubation at 70℃ for 96 h following pre-culture (28℃, 48 h). Each treatment was replicated three times per replication. Asterisk above the bars indicate statistically significant differences between temperatures at each incubation time (P < 0.001).

When BtA and BtK strains were pre-culture at 28°C for 48 h and then subjected to subsequent culture at 0°C, both strains showed rapid and high insecticidal activity to 2^nd instar larvae of P. xylostella (Fig. 6). After subsequent culture at 0°C, BtA showed over 60% higher mortality rate at 24 h compared to those incubated at 28°C and 70°C (F =84.69; df = 2,6; P < 0.0001), and reached to 100% mortality rate by 48 h. However, BtA under subsequent culture at 28°C reached 100% mortality rate at 96 h, while subsequent culture at 70°C showed 80% mortality rate (Fig. 6A). For BtK, subsequent culture at 0°C showed about 80% and 40% higher mortality rate at 24 h compared to subsequent culture at 28°C and 70°C, respectively (F =198.02; df = 2,6; P < 0.0001). BtK required to reach 100% mortality rate was 24 h faster than that of the incubated at 28°C (Fig. 6B). Notably, BtK showed approximately 13% higher mortality rate than BtA at 24 h after treatment, and differences in insecticidal activity between the two strains at 96 h.

Fig. 6. 
Insecticidal effect of Bacillus thuringiensis subsp. aizawai (BtA) and B. thringiensis subsp. kurstaki (BtK) to Plutella xylostella 2^nd instar larvae. (A); mortality rate of BtA after subsequent incubation at 0℃ and 70℃ for 96 h following pre-culture (28℃, 48 h). (B); mortality rate of BtK after subsequent incubation at 70℃ for 96 h following pre-culture (28℃, 48 h). Each treatment was replicated three times with 10 larvae per replication. Two asterisks above the bars indicate statistically significant differences between temperatures at each incubation time (P < 0.0001).