Toxicity of Native Bacillus Thuringiensis Isolates on the Larval Stages of Pine Processionary Moth Thaumetopoea Wilkinsoni at Different Temperatures

Turkish Journal of Agriculture and Forestry Turk J Agric For (2013) 37: 163-172 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1201-21

Toxicity of native Bacillus thuringiensis isolates on the larval stages of pine processionary moth Thaumetopoea wilkinsoni at different temperatures

1, 2 3 Semih YILMAZ *, Salih KARABÖRKLÜ , Uğur AZİZOĞLU , 4 4 3 Abdurrahman AYVAZ , Mikail AKBULUT , Musa YILDIZ 1 Department of Agricultural Biotechnology, Seyrani Agricultural Faculty, Erciyes University, 38039 Kayseri, Turkey 2 Department of Biology, Faculty of Arts and Sciences, Osmaniye Korkut Ata University, 80000 Osmaniye, Turkey 3 Graduate School of Natural and Applied Sciences, Erciyes University, 38039 Kayseri, Turkey 4 Department of Biology, Faculty of Science, Erciyes University, 38039 Kayseri, Turkey

Received: 17.01.2012 Accepted: 13.08.2012 Published Online: 26.03.2013 Printed: 26.04.2013

Abstract: Pine processionary moth, Thaumetopoea wilkinsoni Tams, is an important defoliating lepidopteran pest of pine trees. The aim of this study was to determine the required spore–crystal concentration of local Bacillus thuringiensis (Bt) isolates, optimal ambient temperature, and larval stage to control this troublesome forest pest. The susceptibility ofT. wilkinsoni larvae decreased with older stage and lower temperatures. The optimum temperature was found to be 15 °C or higher for the control of early larval stages. At the highest spore–crystal concentration (500 µg g–1), the most effective isolate (SY49.1) caused 83% mortality for the second-stage larvae at 5 °C. However, an approximately 4-fold decrease in mortalities was observed in late-stage larvae for all isolates examined at this temperature. Nevertheless, other Bt isolates, excluding SY27.3, caused nearly complete mortality at 25 °C for early-stage larvae. Considering the distribution of seasonal temperature, Bt products should be applied at the highest ambient temperature to earlier stages for efficient control. We propose that local Bt isolates SY27.1, SY49.1, and SY62.1 could be used to develop environmentally safe bioinsecticides to control this important pest species. These results indicate that larval stage and environmental temperature should be taken into consideration for efficient control of T. wilkinsoni using the spore–crystal mixture of Bacillus thuringiensis isolates.

Key words: Bacillus thuringiensis, cry gene, larval stage, Thaumetopoea wilkinsoni, toxicity

1. Introduction Bt-based pesticides represent about 98% of the market Pine processionary moth Thaumetopoea wilkinsoni Tams for microbial pesticides (Copping and Menn 2000). is an important lepidopteran pest distributed throughout The toxicity of Bt is due to the production of crystalline temperate regions of the Middle East and Turkey protein protoxins, known as δ-endotoxins (Broderick et al. (Salvato et al. 2002; Semiz et al. 2006; Simonato et al. 2006). The synthesis of toxins as intracytoplasmic protein 2007; Kerdelhué et al. 2009). This pest causes economic crystals occurs during Bt sporulation (Höfte and Whiteley losses due to growth retardation of pine trees caused by 1989). When ingested by susceptible insects, the pathogen defoliation (Cetin et al. 2007; Erkan 2011). The life cycle of causes serious lesions or death (Oliveira et al. 2006). pine processionary moth is characterized by 2 long-lasting Cry1, Cry2, and Cry9 groups of proteins are specifically stages (larva and pupa) that are particularly susceptible toxic to lepidopteran larvae (Bravo 1997). The selective to microbial pathogens (Battisti et al. 1998). The main toxicity of Cry proteins against insects and the lack of damage to trees is caused by larvae, which live gregariously pathogenicity for mammalian cells make these molecules from autumn to spring in silk nests containing hundreds a safe biocontrol agent when used against insect pests on a of individuals (Battisti et al. 1998; Semiz et al. 2006). This commercial scale. pest can be controlled by chemical insecticides as well as Bt products are major alternative tools to chemical viruses, fungi, parasites, and Bacillus thuringiensis (Bt) pesticides and are employed in forest protection programs strains (Rausell et al. 1999). for the control of certain defoliating lepidopteran pests The gram-positive bacterium Bt is used worldwide (Frankenhuyzen 1993, 1995; Schnepf et al. 1998). Bt as a biopesticide for the control of several lepidopteran, products are effectively used against the larvae of pine coleopteran, and dipteran pests (Schnepf et al. 1998). processionary moths T. pityocampa and T. wilkinsoni * Correspondence: [email protected] 163 YILMAZ et al. / Turk J Agric For

(Battisti et al. 1998; Özçankaya and Can 2004; Gindin was added to 20 mL of Luria Bertani broth (LB) buffered et al. 2007; Cebeci et al. 2010). The isolation and use of with 0.25 M sodium acetate (pH 6.8), and the mixture new Bt strains with novel or particularly high insecticidal was incubated at 200 rpm for 4 h (30 °C). Next, 1 mL of activities could have impact for effective control of insect the sample was heated at 80 °C for 5–10 min to eliminate pests (Ibarra et al. 2003; Silva-Werneck and Ellar 2008). vegetative cells. A 50-µL aliquot was spread on nutrient agar Temperature, larval stage, spore–crystal concentration, plate and incubated overnight at 30 °C. Different colonies and application methods can influence the efficacy of were transferred to a new LB agar medium to obtain pure different strains of the microorganism (Kouassi et al. 2001; colonies for characterization. Bt subsp. kurstaki (Btk) Bauce et al. 2002; Carisey et al. 2004). Thus, determination HD1 (Institute of Biotechnology, National Autonomous of these parameters has critical importance for the control University of Mexico) was used as the reference strain for of pine processionary moth larvae (Vaňková and Švestka comparison in characterization studies of local isolates. 1976; Rausell et al. 1999). In this study, 4 Bt isolates 2.2. Polymerase chain reaction analysis previously found effective againstEphestia kuehniella, Molecular characterization of the isolates was performed Plodia interpunctella, and Thaumetopoea pityocampa by polymerase chain reaction (PCR) analysis using general larvae (Yılmaz 2010) were evaluated against T. wilkinsoni (cry2, cry5) and specific (cry1Aa/Ad, cry1Ab/Ac, cry1Ac, larvae at different temperatures in laboratory conditions. cry1Ad, cry1B, cry1C, cry9A, cry9C) primers (Table 1). DNA extraction of the isolates was carried out according to 2. Materials and methods the method of Bravo et al. (1998). PCR mixture contained

2.1. Bacillus thuringiensis isolates 1.4 µL MgCl2 (25 mM), 1.5 µL Taq buffer (10X), 0.5 µL Soil samples from depths of 2–10 cm were collected during dNTP mix (10 mM each), 0.5 µL primers ( 100 nM), 0.1 spring 2008 from 80 different locations in Adana, Turkey, µL Taq DNA polymerase (5 U µL–1), 1.3 µL bovine serum -1 to isolate Bt (Yılmaz 2010). Bt isolates were obtained by the albumin (0.08 µg µL ), 7.2 µL sterile dH2O, and 2 µL (5 method of Travers et al. (1987). One gram of soil sample ng µL–1) template DNA in a final volume of 15 µL. The

Table 1. Characteristics of the primer pairs used to identify cry genes by PCR analysis.

Primer pair Product size (bp) Tm (°C) Sequence Reference F 52 5’-TTATACTTGGTTTCAGGCCC-3’ cry1Aa/Ad 246 Ceron et al. (1994) R 53 5’-TTGGAGCTCTCAAGGTGTAA-3’ F 47 5’-AACAACTATCTGTTCTTGAC-3’ cry1Ab/Ac 216 Ceron et al. (1994) R 42 5’-CTCTTATTATACTTACACTAC-3’ F 42 5’-GTTAGATTAAATAGTAGTGG-3’ cry1Ac 180 Ceron et al. (1994) R 48 5’-TGTAGCTGGTACTGTATTG-3’ F 48 5’-CAGCCGATTTACCTTCTA-3’ cry1Ad 171 Ceron et al. (1994) R 53 5’-TTGGAGCTCTCAAGGTGTAA-3’ F 49 5’-CTTCATCACGATGGAGTAA-3’ cry1B 367 Ceron et al. (1994) R 48 5’-CATAATTTGGTCGTTCTGTT-3’ F 49 5’-AAAGATCTGGAACACCTTT-3’ cry1C 130 Ceron et al. (1994) R 46 5’-CAAACTCTAAATCCTTTCAC-3’ F 50 5’-TAAAGAAAGTGGGAGTCTT-3’ cry2 1556 Masson et al. (1998) R 47 5’-AACTCCATCGTTATTTGTAG-3’ F 55 5’-TAAGCAAAGCGCGTAACCTC-3’ cry5 322 Poojitkanont et al. (2008) R 55 5’-GCTCCCCTCGATGTCAATG-3’ F 55 5’-GTTGATACCCGAGGCACA-3’ spe-cry9A 571 Bravo et al. (1998) R 51 5’-CCGCTTCCAATAACATCTTTT-3’ F 50 5’-CTGGTCCGTTCAATCC-3’ spe-cry9C 306 Bravo et al. (1998) R 51 5’-CCGCTTCCAATAACATCTTTT-3’

F: forward; R: reverse; Tm: melting temperature; bp: base pair.

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PCR amplification was performed under the following β-mercaptoethanol, and 0.1% bromophenol blue, pH 6.8) conditions: initial denaturation at 95 °C for 2.5 min was added. This mixture was maintained for 5–10 min in followed by 34 cycles at 94 °C for 1 min, 48 °C for 1 min, boiling water. The Btk HD1 strain was used as a reference. and 72 °C for 1 min, and a final extension step at 72 °C for The gel was stained with 0.4% Coomassie Brilliant Blue 5 min (Bravo et al. 1998). Melting temperatures for each R250, as described by Temizkan and Arda (2004). primer pair are given in Table 1. Following amplification, 2.6. Bioassay the PCR products (15 µL) were electrophoresed (at 80 V T. wilkinsoni larvae were collected from their natural habitat for 2 h) on 1X Tris-acetate-EDTA (TAE with ethidium in the 2010–2011 growing season in Osmaniye, Turkey. bromide) buffer in 1% agarose gel. The specific PCR Bioassays with second, third, fourth, and fifth stages of products were excised from the gel and purified for further larvae were carried out in December, January, February, analysis using a Fermentas DNA extraction kit (K0513) and March, respectively. Larval stages were determined according to the manufacturer’s instructions. according to head capsule size and body morphology 2.3. Obtaining Bt spore–crystal mixture (EPPO/CABI 1997). Freeze-dried spore–crystal mixtures Bt isolates were grown in 150 mL T3 medium (3 g of local isolates SY27.1, SY27.3, SY49.1, and SY62.1 were tryptone, 2 g tryptose, 1.5 g yeast extract, 0.005 g MnCl2, suspended in sterile distilled water at 100, 250, and 500 –1 6 g NaH2PO4, 7.1 g Na2HPO4) and incubated for 7 days at µg g concentrations. One gram of field-collected pine 30 °C (Travers et al. 1987). Later, the cell suspensions were (Pinus brutia) needles was surface sterilized by immersion centrifuged at 15,000 × g for 10 min at 4 °C. Pellets were in 2% sodium hypochlorite solution (NaClO) for 60 s and washed twice in 20 mL sterile dH2O and centrifuged for 10 rinsing in sterile water. The needles were then soaked in min at 15,000 × g. 1 mL of spore–crystal mixture suspension for 20 min 2.4. Freeze-drying and scanning electron microscope and transferred to petri plates together with 10 larvae, view which were left in an acclimatized chamber at 5 ± 1, 15 Bt spore–crystal mixtures were freeze-dried using ± 1, and 25 ± 1 °C and 60 ± 5% relative humidity with a a Labconco-Welch freeze-dryer according to the photoperiod of 14:10 (light:dark) h for 10 days. Fresh and manufacturer’s instructions and were stored at 4 °C untreated pine needles were supplied for the larvae in each until further use. Spore–crystal samples were spread on plate after 4 days of application. The number of dead larvae a microscope slide and fixed after air-drying at room was recorded daily for 10 days. Sterile dH2O was used as temperature. Later, they were sputter-coated with 10 nm the control treatment instead of spore–crystal suspension. Au/Pd using a SC7620 mini-sputter coater (Quorum Three replicates were set up for each treatment. Technologies) and viewed using a LEO 440 scanning 2.7. Statistical analysis electron microscope at 20 kV beam current. Mortality percentages were corrected using Abbott’s 2.5. Protein electrophoresis formula (Abbott 1925) and subjected to analysis of variance Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (one-way ANOVA) for comparing the toxicity of isolates (SDS–PAGE) was conducted as described by Valicente according to concentrations. Means were separated at the et al. (2010), with some modifications. SDS–PAGE was 5% significance level by using the Tukey–Kramer honestly performed using 12% running and 5% stacking gels. The significant difference post hoc test with 2001 SPSS lyophilized spore–crystal mixtures were resuspended in 1 software. Mortality percentages were transformed using mL of 0.01% Triton X-100 solution. This step was repeated arcsine √x transformation to meet normality for probit 3 times. Pellets composed of a mixture of spore and crystal analysis (Steel and Torrie 1980). A log10 transformation were solubilized in 500 µL solubilization buffer (0.01% was used to calculate the slope values. Mortalities were Triton, 10 mM NaCl, and 50 mM Tris-HCl, pH 8.0), and subjected to probit analysis using the same statistical 1 aliquot of 100 µL was withdrawn after this step. The program to estimate the LC95 values of isolates against T. mixtures were centrifuged at 14,000 rpm for 5 min, and wilkinsoni larvae. the pellets were resuspended in 500 µL sodium bicarbonate 3. Results buffer (50 mM NaHCO3 and 10 mM β-mercaptoethanol, pH 10.5) and incubated for 3 h at 37 °C under continuous 3.1. Characterization of Bt isolates shaking. Samples were centrifuged at 14,000 rpm for Four of the total 120 Bt isolates (SY27.1, SY27.3, SY49.1, 10 min, and the supernatants were transferred to a new and SY62.1), proven to be effective against E. kuehniella, P. tube. Remaining pellets were resuspended in 250 µL interpunctella, and T. pityocampa larvae, were characterized 0.1 M Tris, pH 8.0. Equal amounts of supernatant and according to PCR and SDS–PAGE analysis. Furthermore, resuspended pellet were sampled and an equal volume of 16S rDNA and cry gene sequences of these isolates proved sample buffer (0.0625 M Tris, 2.3% SDS, 10% glycerol, 5% that they were Bt (Yılmaz 2010).

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3.2. Scanning electron micrograph of spore–crystal 3.4. SDS–PAGE analysis mixture The crystal protein profile of the isolates was determined To obtain a more detailed view of the spore–crystals of by SDS–PAGE analysis. Although each isolate produced the SY27.1, SY27.3, SY49.1, and SY62.1 isolates, samples a characteristic banding pattern, some differences were were examined under a scanning electron microscope. It observed among them (Figure 2). The purified crystals of was observed that local isolates produced bipyramidal, Btk HD1, SY27.1, SY27.3, SY62.1, and SY49.1 displayed spherical, cubic, and irregularly shaped spherical crystal major protein bands around 65, 100, 130, and 200 kDa, proteins with different sizes, similar to Btk HD1 (Figure 1). along with several smaller bands. 3.3. Screening of cry genes 3.5. Bioassay The total DNAs of Btk HD1, SY27.1, SY27.3, SY49.1, and The susceptibility of T. wilkinsoni larvae declined with SY62.1 were screened for cry genes that code toxins active older stage and lower temperatures. A concentration- against lepidopteran pests. We observed that the tested dependent increase in larval mortality was determined at isolates harbored more than one cry gene. The cry gene all stages and temperatures tested. Significant differences were observed in mortality rates among treatments for profiles of local Bt isolates are given in Table 2. both larval stage and application temperatures (Figure 3). The early larval stages were the most sensitive to the Table 2. Local Bt isolates and their cry genes. spore–crystal mixture of all isolates at 5 °C. For example, an approximately 4-fold decrease in larval mortality for all Isolates cry genes the isolates was observed at the fifth stage compared to the second. The insecticidal activity of the isolates exhibited a SY27.1 cry1Ad, cry1Ac, cry1Ab/Ac, cry1B, cry2, cry9C similar trend, with higher mortality rates at 15 °C. Nearly complete mortality was observed at 25 °C for second-stage SY27.3 cry1Ab/Ac, cry1Aa/Ad, cry5, cry9C larvae. SY49.1 and SY27.1 were the most effective isolates at all tested concentrations at 25 °C on the third, fourth, SY49.1 cry1Aa/Ad, cry1B, cry1C, cry5, cry9A, cry9C and fifth stages. However, although it was not statistically significant, the mortality trend caused by Btk HD1 at 15

SY62.1 cry1Ab/Ac, cry2, cry9C °C on third-, fourth-, and fifth-stage larvae was higher compared to that of local isolates (Figure 3).

SY27.1 (Yılmaz 2010) SY27.3 SY49.1

S I

SY62.1 (Yılmaz 2010) Btk HD1 Figure 1. Electron micrograph of local Bt isolates’ spore–crystal mixture (B: bipyramidal; C: cubic; S: spherical; I: irregular; Sp: spore).

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The effect of temperature on Bt-mediated mortality HD1 rker rker k

Y27.3 Y27.1 Y62.1 Y49.1 was more pronounced at the third, fourth, and fifth larval Ma S S S S Bt Ma stages. Even in the least sensitive stage, the increased mortality effect of higher temperature was obvious. For 200 kDa 116 kDa 120 instance, the mortality rate between 5 °C and 25 °C was 4-fold higher for SY49.1 for the fifth stage. The same trend 85 was also observed for other isolates except SY27.3, which 66.2 70 was the least effective isolate in all treatments. Although 60 this least efficient isolate (SY27.3) caused complete 50 45 mortality at the second stage with the concentration of 500 –1 40 µg g , larval mortality rates were not much different from the control at the fifth stage. 35 Lethal concentrations (LC95) of Btk HD1, SY49.1, 30 SY27.1, SY27.3, and SY62.1 were also calculated for T. wilkinsoni larvae (Table 3). The concentrations required 25 25 to kill 95% of the population (LC95) indicated a general decrease depending on increasing temperature values 20 for each larval stage. However, a general increase in LC95 values was observed depending on increasing larval stages 18.4 (Table 3). 15 14.4 4. Discussion Figure 2. SDS–PAGE (12%) analysis of spore–crystal mixture of Bt is an important entomopathogenic organism in forest standard and local B. thuringiensis isolates. protection against defoliating pests in Lepidoptera

–1 –1 –1 Control 100 µg g–1 250 µg g–1 500 µg g–1 Control 100 µg g 250 µg g 500 µg g Control 100 µg g–1 250 µg g–1 500 µg g–1

100 XY 100 B b AX ab AX b AX X stag e b BY X Y stag e Y 100 stag e A XY Y A A XY bB B

80 XY b 80 ab A °C 80 a A °C a A b X second 60 A X second 60 at 5 °C a a second 60 at 15 of at 25 a of 40 of a 40 A 40

larvae a larvae 20 α α α α α 20 larvae 20 ortalty α α α α α ortalty

ααααα ortalty M

0 M 0 M 0 % % Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 Btk HD1 SY49.1 SY27.1SY27.3SY62.1 % Btk HD1 SY49.1SY27.1SY27.3SY62.1 Isolates Isolates Isolates

–1 –1 –1 Control 100 µg g–1 250 µg g–1 500 µg g–1 Control 100 µg g–1 250 µg g–1 500 µg g–1 Control 100 µg g 250 µg g 500 µg g 100 100 100 C Y C XY XY stag e Y 80 80 BC C XY 80 X b b X XY thrd 60 60 b C b AB

BC 60 of at 15 °C b b b 40 X X X X 40 AB 40 A ab

larvae at 5 °C AX B

A larvae at 15 °C 20 a a AX AX larvae A α ααa α a α a 20 a 20 a α α α α α α α α αα % Mortalty of thrd stage Mortalty

% Mortalty of thrd stage 0 0 0 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % Btk HD1 SY49.1 SY27.1SY27.3SY62.1 Btk HD1 SY49.1SY27.1SY27.3SY62.1 Isolates Isolates Isolates

–1 –1 –1 Control 100 µg g–1 250 µg g–1 500 µg g–1 –1 –1 –1 Control 100 µg g 250 µg g 500 µg g 100 Control 100 µg g 250 µg g 500 µg g 100 100 Y 80 B Y 80 80 X c BC bc AB Y bc 60 C C 60 XY 60 b X B 40 b BC X 40 b X 40 b X BX ab

larvae at 5 °C X 20 AX A a A X larvae at 25 °C 20 a AX a AX larvae at 15 °C α a α α α a α aA 20 aA α ααα α 0 α α α α α 0 0 % Mortalty of fourth stag e % Mortalty of fourth stag e Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 % Mortalty of fourth stage Btk HD1 SY49.1SY27.1SY27.3SY62.1 Btk HD1 SY49.1 SY27.1SY27.3SY62.1 Isolates Isolates Isolates

Control 100 µg g–1 250 µg g–1 500 µg g–1 Control 100 µg g–1 250 µg g–1 500 µg g–1 Control 100 µg g–1 250 µg g–1 500 µg g–1 100 100 100 80 80 Y 80 B BY 60 60 60 b Y Y Y b Y AB B AB XY b 40 40 AB 40 B a a XY a AB b larvae at 15 °C larvae at 5 °C X

X A X larvae at 25 °C 20 A AX a X AX 20 a A a 20

Mortalty of fh stag e a X α a ααα aA α a α ααα α ααα α aA α % % Mortalty of fh stag e 0 0 % Mortalty of fh stag e 0 Btk HD1 SY49.1 SY27.1 SY27.3 SY62.1 Btk HD1 SY49.1 SY27.1SY27.3SY62.1 Btk HD1 SY49.1SY27.1SY27.3SY62.1 Isolates Isolates 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.

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Table 3. Toxicity of isolates at 5, 15, and 25 °C against different larval stages ofT. wilkinsoni.

¥ 2 Isolate LC95 95% fiducial limits Slope ± SE χ df P December, 5 °C (second stage) Btk HD1 395.24 563.68–1182.25 1.12 ± 0.52 15.91 10 0.102 SY49.1 328.02 474.73–1027.48 0.81 ± 0.56 17.06 10 0.073 SY27.1 572.93 440.19–878.24 1.39 ± 0.52 12.37 10 0.261 SY27.3 888.00 626.00–1805.19 0.93 ± 0.47 10.86 10 0.368 SY62.1 624.36 468.38–1023.87 0.93 ± 0.51 13.77 10 0.184 December, 15 °C (second stage) Btk HD1 270.66 156.80–1688.12 0.86 ± 0.62 15.43 10 0.132 SY49.1 412.13 264.85–1616.33 0.63 ± 0.23 17.07 10 0.073 SY27.1 445.54 307.14–960.30 1.03 ± 0.57 17.51 10 0.064 SY27.3 835.88 612.54–1480.06 1.20 ± 0.47 9.22 10 0.511 SY62.1 354.55 213.44–1856.41 1.11 ± 0.15 18.14 10 0.045 December, 25 °C (second stage) Btk HD1 * * * * * * SY49.1 81.78 56.65–142.27 2.41 ± 0.30 1.04 10 1.000 SY27.1 * * * * * * SY27.3 343.29 228.39–829.21 1.13 ± 0.67 18.03 10 0.054 SY62.1 238.22 178.62–377.20 1.97 ± 0.87 13.31 10 0.207 January, 5 °C (third stage) Btk HD1 1370.68 809.41–171.00 0.68 ± 0.48 14.80 10 0.140 SY49.1 1298.72 853.99–3593.32 0.40 ± 0.37 13.10 10 0.218 SY27.1 1063.91 775.57–1929.94 1.20 ± 0.49 12 10 0.285 SY27.3 1723.20 1025.85–9243.65 0.59 ± 0.49 12.50 10 0.253 SY62.1 471.059 846.45–6322.84 1.17 ± 0.53 15.87 10 0.103 January, 15 °C (third stage) Btk HD1 501.95 354.32–1009.97 0.88 ± 0.54 16.15 10 0.095 SY49.1 659.86 449.73–1628.93 0.60 ± 0.50 16.83 10 0.078 SY27.1 729.38 492.16–1925.71 0.41 ± 0.19 16.45 10 0.087 SY27.3 1017.13 736.92–1876.58 0.84 ± 0.47 8.26 10 0.603 SY62.1 1005.67 643.13–3884.34 0.46 ± 0.16 15.40 10 0.118 January, 25 °C (third stage) Btk HD1 537.10 351.99–1679.92 0.44 ± 0.12 11.85 10 0.295 SY49.1 188.10 141.76–290.63 1.97 ± 0.87 5.03 10 0.889 SY27.1 404.63 275.71–891.64 0.76 ± 0.62 16.16 10 0.095 SY27.3 769.99 585.65–1220.40 2.17 ± 0.51 11.48 10 0.321 SY62.1 508.76 387.79–791.85 1.10 ± 0.52 4.65 10 0.913

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Table 3. (Continued).

¥ 2 Isolate LC95 95% fiducial limits Slope ± SE χ df P February, 5 °C (fourth stage) Btk HD1 1335.92 914.68–3074.87 1.45 ± 0.54 14.40 10 0.155 SY49.1 1531.30 957.05–5852.94 0.47 ± 0.38 8.83 10 0.548 SY27.1 1426.90 911.66–4670.65 0.47 ± 0.37 10.68 10 0.383 SY27.3 1699.24 1039.61–7926.20 1.04 ± 0.54 14.20 10 0.164 SY62.1 1463.42 966.13–4006.29 1.34 ± 0.55 13.71 10 0.186 February, 15 °C (fourth stage) Btk HD1 615.43 432.68–1308.83 0.85 ± 0.49 17.91 10 0.056 SY49.1 966.26 566.92–5513.63 0.48 ± 0.30 17.49 10 0.064 SY27.1 951.50 614.01–3430.10 0.38 ± 0.14 15.52 10 0.114 SY27.3 929.65 645.21–2207.55 1.83 ± 0.52 17.51 10 0.064 SY62.1 1121.44 689.81–6536.63 0.46 ± 0.37 14.83 10 0.138 February, 25 °C (fourth stage) Btk HD1 821.12 547.80–2346.73 0.63 ± 0.47 16.55 10 0.85 SY49.1 482.51 336.57–1008.89 0.59 ± 0.56 16.44 10 0.088 SY27.1 610.57 418.47–1448.76 0.51 ± 0.47 16.89 10 0.077 SY27.3 1772.04 1022.85–19347.39 1.30 ± 0.47 10.38 10 0.407 SY62.1 664.30 445.29–1790.41 0.51 ± 0.33 15.71 10 0.108 March, 5 °C (fifth stage) Btk HD1 1489.12 954.85–4767.63 0.72 ± 0.49 12.11 10 0.278 SY49.1 1706.00 1013.31–10996.36 0.48 ± 0.26 8.45 10 0.585 SY27.1 1500.80 943.20–5510.70 0.40 ± 0.17 9.29 10 0.524 SY27.3 1340.65 813.84–12181.47 0.58 ± 0.51 16.49 10 0.086 SY62.1 1735.47 1039.39–10048.84 0.59 ± 0.52 11.33 10 0.332 March, 15 °C (fifth stage) Btk HD1 986.24 682.04–2162.77 0.60 ± 0.46 9.06 10 0.526 SY49.1 1055.99 713.21–2574.26 0.48 ± 0.46 9.84 10 0.454 SY27.1 1203.31 784.67–3514.23 0.49 ± 0.33 10.08 10 0.433 SY27.3 1837.36 1065.59–17343.80 0.51 ± 0.22 12.35 10 0.262 SY62.1 1356.51 875.51–4156.68 0.93 ± 0.48 13.15 10 0.215 March, 25 °C (fifth stage) Btk HD1 1047.75 721.86–2326.39 0.71 ± 0.46 8.79 10 0.552 SY49.1 568.84 394.68–1250.62 0.87 ± 0.53 15.95 10 0.101 SY27.1 637.01 482.07–1020.73 0.90 ± 0.50 12.94 10 0.227 SY27.3 1260.91 790.84–8185.61 1.08 ± 0.56 17.36 10 0.067 SY62.1 1002.98 682.13–2373.41 0.46 ± 0.36 12.67 10 0.243

*Statistics cannot be computed, possibly due to linear dependence among covariates; ¥: standard error of mean.

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(Frankenhuyzen 1993, 1995). Our local isolates were T. pityocampa larvae (Cebeci et al. 2010). In the present characterized by PCR and SDS–PAGE analysis by study, the lowest mortality rate was observed at 100 µg g–1 determining the presence of cry genes and Cry proteins. at 5 °C for the fifth-stage larvae. We also obtained lower PCR analysis revealed that SY27.1, SY27.3, SY49.1, and mortality rates at 5 °C compared to applications at 15 and SY62.1 harbor more than one type of cry gene having 25 °C for all larval stages. Our results were in accordance insecticidal activity, primarily against lepidopteran pests. with the studies reported by Kailidis et al. (1977) and Wang et al. (2003) reported that the Cry1, Cry2, and Vaňková and Švestka (1976). They reported that low Cry9 groups of proteins exhibit the strongest insecticidal temperatures have a negative effect on the effectiveness of activity against lepidopteran larvae. Thammasittirong and B. thuringiensis against insect pests. Poor feeding activity Attathom (2008) indicated that most of the cry9-carrying and consequently lower intake of toxins may also result in strains also harbored cry1 and cry2 genes. Similarly, our decreased mortality rates at lower temperatures. local isolates SY27.1 and SY62.1 had cry1, cry2, and cry9 The mortality caused by local Bt isolates on second- types of genes, and had considerably higher mortality stage larvae at 500 µg g–1 spore–crystal mixture ranged effects than the control. SY49.1 also had 3 cry1-type and between 50% and 83% at 5 °C. However, this rate averaged 2 cry9-type genes and showed very high mortality effects between 10% and 20% on fifth-stage larvae at the same against T. wilkinsoni larvae. Although SY27.3 carries temperature. Mortality of T. wilkinsoni larvae at 100 µg g–1 cry1Ab/Ac, cry1Aa/Ad, cry5, and cry9C genes and several and 25 °C averaged between 70% and 100% for second- major Cry protein bands, its mortality effect was fairly stage larvae, but this rate was between 3% and 50% for low. In other isolates, cry1 and cry9 genes might show a fifth-stage larvae at the same temperature. The early stages synergistic effect in combination with cry2 and cry1B. Local were found to be the most susceptible stages and revealed isolates produced varying-sized cubical, bipyramidal, and the highest temperature- and concentration-dependent spherical crystal proteins containing 65, 100, and 130– mortalities under laboratory conditions. The reason for 140 kDa. These are typical of lepidopteran active crystal this is that the binding affinity of Cry1Ab to brush border proteins of the Cry1, Cry2, and Cry9 groups and exhibit membrane vesicles (BBMV) varies with larval development the strongest activity against these pests (Schnepf et al. stage (Rausell et al. 2000). Rausell et al. also indicated that 1998; Wang et al. 2003; dos Santos et al. 2009). The isolates a decrease was apparent in the binding affinity to BBMV of also produced protein bands of around 40–45 kDa. SY27.3 last instar larvae compared to first instars. produced Cry proteins that are visible with electron We observed that the susceptibility of T. wilkinsoni microscopy, but it did not show a considerable level of larvae to Btk HD1 at 15 °C was higher than that to local toxicity against T. wilkinsoni larvae. Similarly, Bozlağan Bt isolates. However, the larvae were more susceptible et al. (2010) and Ammouneh et al. (2011) reported that to SY49.1 and SY27.1 isolates at 5 and 25 °C. The results their isolates revealed different levels of toxicity against showed that T. wilkinsoni larvae were highly susceptible to E. kuehniella larvae. Another researcher stated that some the Bt spore–crystal mixture, but the mortality rates were strains sharing the same cry genes showed significantly variable with larval stage and application temperature. A different insecticidal potency (Martínez et al. 2005). similar result was reported by Vargas et al. (1994). They Several studies have clearly revealed that environmental stated that T. pityocampa larvae were more susceptible factors (sunlight, temperature), target insect species to B. thuringiensis subsp. aizawai (serotype H7) than to and their larval stages, preparation and handling of the mexicanensis (H27), konkukian (H34), and andaluciensis product (concentration and distribution methods), and (H37). In this study, nearly complete mortality was the plant where the pest feeds can influence the efficacy of obtained at 25 °C for the highest concentration on different strains of a microorganism (Kouassi et al. 2001; second-stage larvae. Approximately 90% larval mortality Bauce et al. 2002; Carisey et al. 2004). Local Bt isolates was observed at the highest concentration at 15 °C for caused 60% to 100% mortalities at 500 µg g–1 on the second SY49.1, SY27.1, SY62.1, and Btk HD1 against second- stage of T. wilkinsoni larvae at 15 and 25 °C. Similarly, in a stage larvae. study carried out by Er et al. (2007), it was indicated that This study indicates that the tested local Bt isolates may 100 and 1000 µg g–1 concentrations of Btk spore–crystal have potential for the development of environmentally mixtures caused higher than 90% larval mortality on T. safe bioinsecticides against T. wilkinsoni larvae. The results solitaria larvae at 20 ± 5 °C. In another study performed suggest that Bt products should be applied to earlier in Turkey’s pine forests in December 2005 at 16 °C, 2 stages of the pest at the highest possible environmental commercial Bt products caused 97% to 99% mortality on temperature during the season of larval activity.

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