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• cry1Aa/Ad F 246 52 5’-TTATACTTGGTTTCAGGCCC-3’ Ceron et al. (1994) R 53 5’-TTGGAGCTCTCAAGGTGTAA-3’ cry1Ab/Ac F 216 47 5’-AACAACTATCTGTTCTTGAC-3’ Ceron et al. (1994) R 42 5’-CTCTTATTATACTTACACTAC-3’ cry1Ac F 180 42 5’-GTTAGATTAAATAGTAGTGG-3’ Ceron et al. (1994) R 48 5’-TGTAGCTGGTACTGTATTG-3’ cry1Ad F 171 48 5’-CAGCCGATTTACCTTCTA-3’ Ceron et al. (1994) R 53 5’-TTGGAGCTCTCAAGGTGTAA-3’ cry1B F 367 49 5’-CTTCATCACGATGGAGTAA-3’ Ceron et al. (1994) R 48 5’-CATAATTTGGTCGTTCTGTT-3’ cry1C F 130 49 5’-AAAGATCTGGAACACCTTT-3’ Ceron et al. (1994) R 46 5’-CAAACTCTAAATCCTTTCAC-3’ cry2 F 1556 50 5’-TAAAGAAAGTGGGAGTCTT-3’ Masson et al. (1998) R 47 5’-AACTCCATCGTTATTTGTAG-3’ cry5 F 322 55 5’-TAAGCAAAGCGCGTAACCTC-3’ Poojitkanont et al. (2008) R 55 5’-GCTCCCCTCGATGTCAATG-3’ spe-cry9A F 571 55 5’-GTTGATACCCGAGGCACA-3’ Bravo et al. (1998) R 51 5’-CCGCTTCCAATAACATCTTTT-3’ spe-cry9C F 306 50 5’-CTGGTCCGTTCAATCC-3’ Bravo et al. (1998) R 51 5’-CCGCTTCCAATAACATCTTTT-3’
• F: forward; R: reverse; Tm: melting temperature; bp: base pair. PCR amplification was performed under the following conditions: initial denaturation at 95 °C for 2.5 min followed by 34 cycles at 94 °C for 1 min, 48 °C for 1 min, and 72 °C for 1 min, and a final extension step at 72 °C for 5 min (Bravo et al. 1998). Melting temperatures for each primer pair are given in Table 1. Following amplification, the PCR products (15 µL) were electrophoresed (at 80 V for 2 h) on 1X Tris-acetate-EDTA (TAE with ethidium bromide) buffer in 1% agarose gel. The specific PCR products were excised from the gel and purified for further analysis using a Fermentas DNA extraction kit (K0513) according to the manufacturer’s instructions.
• Obtaining Bt spore–crystal mixture Bt isolates were grown in 150 mL T3 medium (3 g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.005 g MnCl 2 , 6 g NaH 2 PO 4 , 1 g Na 2 HPO 4 ) and incubated for 7 days at 30 °C (Travers et al. 1987). Later, the cell suspensions were centrifuged at 15,000 × g for 10 min at 4 °C. Pellets were washed twice in 20 mL sterile dH 2 O and centrifuged for 10 min at 15,000 × g.
• Freeze-drying and scanning electron microscope view Bt spore–crystal mixtures were freeze-dried using a Labconco-Welch freeze-dryer according to the manufacturer’s instructions and were stored at 4 °C until further use. Spore–crystal samples were spread on a microscope slide and fixed after air-drying at room temperature. Later, they were sputter-coated with 10 nm Au/Pd using a SC7620 mini-sputter coater (Quorum
• Technologies) and viewed using a LEO 440 scanning electron microscope at 20 kV beam current. Protein electrophoresis Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was conducted as described by Valicente et al. (2010), with some modifications. SDS–PAGE was performed using 12% running and 5% stacking gels. The lyophilized spore–crystal mixtures were resuspended in 1 mL of 0.01% Triton X-100 solution. This step was repeated 3 times. Pellets composed of a mixture of spore and crystal were solubilized in 500 µL solubilization buffer (0.01%
• Triton, 10 mM NaCl, and 50 mM Tris-HCl, pH 8.0), and 1 aliquot of 100 µL was withdrawn after this step. The mixtures were centrifuged at 14,000 rpm for 5 min, and the pellets were resuspended in 500 µL sodium bicarbonate buffer (50 mM NaHCO 3 and 10 mM β-mercaptoethanol, pH 5) and incubated for 3 h at 37 °C under continuous shaking. Samples were centrifuged at 14,000 rpm for 10 min, and the supernatants were transferred to a new tube. Remaining pellets were resuspended in 250 µL 0.1 M Tris, pH 8.0. Equal amounts of supernatant and resuspended pellet were sampled and an equal volume of sample buffer (0.0625 M Tris, 2.3% SDS, 10% glycerol, 5%
• β-mercaptoethanol, and 0.1% bromophenol blue, pH 6.8) was added. This mixture was maintained for 5–10 min in boiling water. The Btk HD1 strain was used as a reference. The gel was stained with 0.4% Coomassie Brilliant Blue R250, as described by Temizkan and Arda (2004). Bioassay
• T. wilkinsoni larvae were collected from their natural habitat in the 2010–2011 growing season in Osmaniye, Turkey.
• Bioassays with second, third, fourth, and fifth stages of larvae were carried out in December, January, February, and March, respectively. Larval stages were determined according to head capsule size and body morphology (EPPO/CABI 1997). Freeze-dried spore–crystal mixtures of local isolates SY27.1, SY27.3, SY49.1, and SY62.1 were suspended in sterile distilled water at 100, 250, and 500 µg g –1 concentrations. One gram of field-collected pine (Pinus brutia) needles was surface sterilized by immersion in 2% sodium hypochlorite solution (NaClO) for 60 s and rinsing in sterile water. The needles were then soaked in 1 mL of spore–crystal mixture suspension for 20 min and transferred to petri plates together with 10 larvae, which were left in an acclimatized chamber at 5 ± 1, 15
• ± 1, and 25 ± 1 °C and 60 ± 5% relative humidity with a photoperiod of 14:10 (light:dark) h for 10 days. Fresh and untreated pine needles were supplied for the larvae in each plate after 4 days of application. The number of dead larvae was recorded daily for 10 days. Sterile dH 2 O was used as the control treatment instead of spore–crystal suspension. Three replicates were set up for each treatment. Statistical analysis Mortality percentages were corrected using Abbott’s formula (Abbott 1925) and subjected to analysis of variance
• (one-way ANOVA) for comparing the toxicity of isolates according to concentrations. Means were separated at the 5% significance level by using the Tukey–Kramer honestly significant difference post hoc test with 2001 SPSS software. Mortality percentages were transformed using arcsine √x transformation to meet normality for probit analysis (Steel and Torrie 1980). A log 10 transformation was used to calculate the slope values. Mortalities were subjected to probit analysis using the same statistical program to estimate the LC 95 values of isolates against T. wilkinsoni larvae. Results
• Characterization of Bt isolates Four of the total 120 Bt isolates (SY27.1, SY27.3, SY49.1, and SY62.1), proven to be effective against E. kuehniella, P. interpunctella, and T. pityocampa larvae, were characterized according to PCR and SDS–PAGE analysis. Furthermore, 16S rDNA and cry gene sequences of these isolates proved that they were Bt (Yılmaz 2010).
• Scanning electron micrograph of spore–crystal mixture To obtain a more detailed view of the spore–crystals of the SY27.1, SY27.3, SY49.1, and SY62.1 isolates, samples were examined under a scanning electron microscope. It was observed that local isolates produced bipyramidal, spherical, cubic, and irregularly shaped spherical crystal proteins with different sizes, similar to Btk HD1 (Figure 1). Screening of cry genes The total DNAs of Btk HD1, SY27.1, SY27.3, SY49.1, and SY1 were screened for cry genes that code toxins active against lepidopteran pests. We observed that the tested isolates harbored more than one cry gene. The cry gene profiles of local Bt isolates are given in Table 2. SDS–PAGE analysis The crystal protein profile of the isolates was determined by SDS–PAGE analysis. Although each isolate produced a characteristic banding pattern, some differences were observed among them (Figure 2). The purified crystals of Btk HD1, SY27.1, SY27.3, SY62.1, and SY49.1 displayed major protein bands around 65, 100, 130, and 200 kDa, along with several smaller bands. Bioassay The susceptibility of T. wilkinsoni larvae declined with older stage and lower temperatures. A concentrationdependent increase in larval mortality was determined at all stages and temperatures tested. Significant differences were observed in mortality rates among treatments for both larval stage and application temperatures (Figure 3). The early larval stages were the most sensitive to the spore–crystal mixture of all isolates at 5 °C. For example, an approximately 4-fold decrease in larval mortality for all the isolates was observed at the fifth stage compared to the second. The insecticidal activity of the isolates exhibited a similar trend, with higher mortality rates at 15 °C. Nearly complete mortality was observed at 25 °C for second-stage larvae. SY49.1 and SY27.1 were the most effective isolates at all tested concentrations at 25 °C on the third, fourth, and fifth stages. However, although it was not statistically significant, the mortality trend caused by Btk HD1 at 15 °C on third-, fourth-, and fifth-stage larvae was higher compared to that of local isolates (Figure 3). Figure 1. Electron micrograph of local Bt isolates’ spore–crystal mixture (B: bipyramidal; C: cubic; S: spherical; I: irregular; Sp: spore). S Sp The effect of temperature on Bt-mediated mortality was more pronounced at the third, fourth, and fifth larval stages. Even in the least sensitive stage, the increased mortality effect of higher temperature was obvious. For instance, the mortality rate between 5 °C and 25 °C was 4-fold higher for SY49.1 for the fifth stage. The same trend was also observed for other isolates except SY27.3, which was the least effective isolate in all treatments. Although this least efficient isolate (SY27.3) caused complete mortality at the second stage with the concentration of 500 µg g –1 , larval mortality rates were not much different from the control at the fifth stage. Lethal concentrations (LC 95 ) of Btk HD1, SY49.1, SY1, SY27.3, and SY62.1 were also calculated for T. wilkinsoni larvae (Table 3). The concentrations required to kill 95% of the population (LC 95 ) indicated a general decrease depending on increasing temperature values for each larval stage. However, a general increase in LC 95 values was observed depending on increasing larval stages (Table 3). Discussion Bt is an important entomopathogenic organism in forest protection against defoliating pests in Lepidoptera SY3 SY1 SY1 SY1 Bt Ma 85 70 60 50 40 30 25 20 15 Figure 2. SDS–PAGE (12%) analysis of spore–crystal mixture of standard and local B. thuringiensis isolates. Figure 3. Percentage mortalities of different larval stages of T. wilkinsoni after exposure to B. thuringiensis isolates at 5, 15, and 25 °C. The same letters on bars of the same color show lack of significant difference for each concentration. α α α α α 20 40 60 80 100 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % M ortality of second stag e larvae at 25 °C Isolates Control 100 µg g –1 250 µg g –1 500 µg g –1 b b A X X X A A a X A X A α α α α α Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 larvae Isolates A a a a a α α α α α Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % larvae Isolates b b A B B B Y Y Y X α α α α α Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 Isolates AX a a a X a α α α α α Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 larvae Isolates aA X X α α α α α 20 40 60 80 100 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % Mortality of third stage larvae at 15 °C Isolates Control 100 µg g –1 250 µg g –1 500 µg g –1 B b a b b C C X Y X b α α α α α Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % Mortality of fourth stag A a X a a A A a α α α α α b a A B 20 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 Isolates b c a B B A Y Y X Y 20 40 60 80 100 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % Mortality of fourth stag e larvae at 25 °C Isolates Control 100 µg g –1 250 µg g –1 500 µg g –1 α α α α α a a a a a A A A X X X 20 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 α α BC BC AB AB ab ab ab XY XY XY XY XY BC XY AB bc ab BC bc A X 20 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 b b b a b B B B A B Y Y Y X Y 20 40 60 80 100 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % Mortality of fifth stag e larvae at 25 °C Isolates Control 100 µg g –1 250 µg g –1 500 µg g –1 Table Toxicity of isolates at 5, 15, and 25 °C against different larval stages of T. wilkinsoni. Isolate LC 95 95% fiducial limits Slope ± SE ¥ χ 2 df P December, 5 °C (second stage) Btk HD1 324 568–1182.25 12 ± 0.52 91 10 0.102 SY1 302 473–1027.48 0.81 ± 0.56 06 10 0.073 SY1 593 419–878.24 39 ± 0.52 37 10 0.261 SY3 800 600–1805.19 0.93 ± 0.47 86 10 0.368
• SY1 636 438–1023.87 0.93 ± 0.51 77 10 0.184 December, 15 °C (second stage) Btk HD1 266 180–1688.12 0.86 ± 0.62 43 10 0.132 SY1 413 285–1616.33 0.63 ± 0.23 07 10 0.073 SY1 454 3014–960.30 03 ± 0.57 51 10 0.064 SY3 888 654–1480.06 20 ± 0.47 22 10 0.511
• SY1 355 244–1856.41 11 ± 0.15 14 10 0.045 December, 25 °C (second stage) Btk HD1 * * * * * * SY1 78 65–142.27 41 ± 0.30 04 10 000 SY1 * * * * * * SY3 329 239–829.21 13 ± 0.67 03 10 0.054
• SY1 222 162–377.20 97 ± 0.87 31 10 0.207 January, 5 °C (third stage) Btk HD1 1368 8041–171.00 0.68 ± 0.48 80 10 0.140 SY1 1272 899–3593.32 0.40 ± 0.37 10 10 0.218 SY1 1091 757–1929.94 20 ± 0.49 12 10 0.285 SY3 1720 1085–9243.65 0.59 ± 0.49 50 10 0.253
• SY1 4059 845–6322.84 17 ± 0.53 87 10 0.103 January, 15 °C (third stage) Btk HD1 5095 15 SY1 686 473–1628.93 0.60 ± 0.50 83 10 0.078 SY1 738 416–1925.71 0.41 ± 0.19 45 10 0.087 SY3 1013 792–1876.58 0.84 ± 0.47 26 10 0.603
• SY1 10067 613–3884.34 0.46 ± 0.16 40 10 0.118 January, 25 °C (third stage) Btk HD1 510 399–1679.92 0.44 ± 0.12 85 10 0.295 SY1 110 176–290.63 97 ± 0.87 03 10 0.889 SY1 4063 271–891.64 0.76 ± 0.62 16 10 0.095 SY3 799 565–1220.40 17 ± 0.51 48 10 0.321 SY1 5076 379–791.85 10 ± 0.52 65 10 0.913
• Isolate LC 95 95% fiducial limits Slope ± SE ¥ χ 2 df P February, 5 °C (fourth stage) Btk HD1 1392 968–3074.87 45 ± 0.54 40 10 0.155 SY1 1530 905–5852.94 0.47 ± 0.38 83 10 0.548 SY1 1490 966–4670.65 0.47 ± 0.37 68 10 0.383 SY3 1624 1061–7926.20 04 ± 0.54 20 10 0.164
• SY1 1442 913–4006.29 34 ± 0.55 71 10 0.186 February, 15 °C (fourth stage) Btk HD1 643 468–1308.83 0.85 ± 0.49 91 10 0.056 SY1 926 592–5513.63 0.48 ± 0.30 49 10 0.064 SY1 950 601–3430.10 0.38 ± 0.14 52 10 0.114 SY3 965 621–2207.55 83 ± 0.52 51 10 0.064
• SY1 1144 681–6536.63 0.46 ± 0.37 83 10 0.138 February, 25 °C (fourth stage) Btk HD1 812 580–2346.73 0.63 ± 0.47 55 10 0.85 SY1 451 357–1008.89 0.59 ± 0.56 44 10 0.088 SY1 657 447–1448.76 0.51 ± 0.47 89 10 0.077 SY3 1704 1085–19347.39 30 ± 0.47 38 10 0.407
• SY1 630 429–1790.41 0.51 ± 0.33 71 10 0.108 March, 5 °C (fifth stage) Btk HD1 1412 985–4767.63 0.72 ± 0.49 11 10 0.278 SY1 17000 1031–10996.36 0.48 ± 0.26 45 10 0.585 SY1 1500.80 920–5510.70 0.40 ± 0.17 29 10 0.524 SY3 1365 884–12181.47 0.58 ± 0.51 49 10 0.086
• SY1 1747 1039–10048.84 0.59 ± 0.52 33 10 0.332 March, 15 °C (fifth stage) Btk HD1 924 604–2162.77 0.60 ± 0.46 06 10 0.526 SY1 1099 84 SY1 12031 767–3514.23 0.49 ± 0.33 08 10 0.433 SY3 1836 1059–17343.80 0.51 ± 0.22 35 10 0.262
• SY1 1351 851–4156.68 0.93 ± 0.48 15 10 0.215 March, 25 °C (fifth stage) Btk HD1 1075 786–2326.39 0.71 ± 0.46 79 10 0.552 SY1 584 368–1250.62 0.87 ± 0.53 95 10 0.101 SY1 601 407–1020.73 0.90 ± 0.50 94 10 0.227 SY3 1291 784–8185.61 08 ± 0.56 36 10 0.067 SY1 10098 613–2373.41 0.46 ± 0.36 67 10 0.243
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