Characterization of two Bacillus thuringiensis ssp. morrisoni strains ...

Biocontrol Science and Technology

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Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae) Hatice Kati , Ikbal Agah Ince , Kazim Sezen , Serife Isci & Zihni Demirbag To cite this article: Hatice Kati , Ikbal Agah Ince , Kazim Sezen , Serife Isci & Zihni Demirbag (2009) Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae), Biocontrol Science and Technology, 19:5, 475-484, DOI: 10.1080/09583150902836377 To link to this article: http://dx.doi.org/10.1080/09583150902836377

Published online: 19 May 2009.

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Date: 02 October 2015, At: 12:37

Biocontrol Science and Technology, Vol. 19, No. 5, May 2009, 475484

RESEARCH ARTICLE Characterization of two Bacillus thuringiensis ssp. morrisoni strains isolated from Thaumetopoea pityocampa (Lep., Thaumetopoeidae) Hatice Katia*, Ikbal Agah Incea,b, Kazim Sezenb, Serife Iscib and Zihni Demirbagb

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a

Department of Biology, Giresun University, Faculty of Arts and Sciences, 28049, Giresun, Turkey, bDepartment of Biology, Karadeniz Technical University, Faculty of Arts and Sciences, 61080, Trabzon, Turkey (Received 29 July 2008; returned 11 September 2008; accepted 19 February 2009) The pine processionary moth Thaumetopoea pityocampa Den. and Schiff. (Lep., Thaumetopoeidae) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey. Two Bacillus thuringiensis isolates obtained from T. pityocampa were identified and characterized in terms of crystal shape using electron microscopy, SDSPAGE analysis, cry gene contents, Hserotype and insecticidal activity. Examination by a scanning electron microscope showed that Tp6 and Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes. On the other hand, the presence of Cry3 and Cry1 proteins were confirmed by observation of approximately 65- and 130-kDa proteins by SDSPAGE in Tp6 and Tp14 isolates, respectively. According to Hserotype results, these isolates were identified as Bacillus thuringiensis ssp. morrisoni (H8a8b). Toxicity tests were performed against six insect species belonging to Lepidoptera and Coleoptera. The highest insecticidal activity was 100% for Tp6 isolate on larvae of Agelastica alni and Leptinotarsa decemlineata and 100% for Tp14 isolate on larvae of Malacosoma neustria. Our results indicate that isolates Tp6 and Tp14 may be valuable biological control agents for various coleopteran and lepidopteran pests. Keywords: Bacillus thuringiensis ssp. morrisoni; biological control; coleopteran and lepidopteran pests; cry gene; Thaumetopoea pityocampa

Introduction Bacillus thuringiensis is a Gram-positive, spore-forming bacterium that synthesises a large diversity of crystal proteins (Cry and Cyt) during the sporulation phase. Some of these are toxic for a wide range of insects belonging to the orders Lepidoptera, Coleoptera, Diptera, Hymenoptera and Homoptera as well as being active against nematodes, mites and protozoa (Johnson, Bishop, and Turner 1998; Schnepf et al. 1998). This characteristic has resulted in B. thuringiensis being the most widely used bacterium over the last 50 years for biological control of pests and vectors of disease, and its safety to non-target insects, birds and mammals has been well demonstrated (Siegel 2001; Jensen et al. 2002). *Corresponding author. Email: [email protected] ISSN 0958-3157 print/ISSN 1360-0478 online # 2009 Taylor & Francis DOI: 10.1080/09583150902836377 http://www.informaworld.com


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Bacillus thuringiensis has been isolated from grain dust, diseased insect larvae, animal feed mills, phyloplane and aquatic environments (Meadows, Ellis, Butt, Jarret, and Burges 1992; Itoua-Apoloyo et al. 1995; Bernhard et al. 1997; Hansen, Damgaard, Eilenberg, and Pedersen 1998). Considering the aspect of biological control, the best source of B. thuringiensis isolates is target insects, however this is not always the case. So far, several B. thuringiensis have been isolated from insects and used against various pests in agriculture and forestry. The pine processionary moth Thaumetopoea pityocampa (Den. and Schiff.) is one of the most harmful insect pests for pine species in Mediterranean countries including Turkey (Ince, Demir, Demirbag˘, and Nalcacioglu 2007). Mart, Uygun, Altin, Erkilic, and Bolu (1995) suggested the use of Bacillus thuringiensis as an environmentally friendly alternative method against T. solitaria larvae. Insecticides based on this bacterium are known to be effective, with various successes, against T. processionea L. (Martin and Bonneaux 2006), a pest of oak trees, T. wilkinsoni Tams (Gindin, Navon, Protasov, Saphir, and Mendel 2007a,b) and T. pityocampa (Schiff.) (Battisti, Longo, Tiber, and Triggiani 1998), pests of pine trees. In this study, we characterized two B. thuringiensis strains (Tp6 and Tp14) isolated from T. pityocampa in detail and tested for insecticidal activity on various insect pests. Material and methods Bacillus thuringiensis isolates and strains Tp6 and Tp14 isolates used in this study were obtained from the Microbiology Laboratory, Department of Biology at Karadeniz Technical University, Trabzon, Turkey. They were isolated from healthy larvae of Thaumetopoea pityocampa (Ince, Kati, Yilmaz, Demir, and Demirbag 2008), and stored at 208C. B. thuringiensis ssp. tenebrionis (Plant Genetic Systems J. Plateaustroat 22, 9000 Gent, Belgium) and B. thuringiensis ssp. kurstaki HD-1 (Bacillus Genetic Stock Center, Columbus, OH, Dean and Zeigler 1994) were used as reference strains.

Microscopic examination In order to obtain spore-crystal mixtures, isolates were grown in nutrient agar medium at 308C for 5 days, until lyses. Spore-crystal mixtures were suspended in 1 mL ice-cold 1 M NaCl and centrifuged at 13,000 g for 5 min. The pellet was resuspended in distilled H2O. a. Light microscopy: The presence and morphology of crystals were recorded during sporulation by direct examination of the smear of spore-crystal mixtures from isolates under light microscopy (100), and confirmed by staining with Coomassie brilliant blue (0.25% solution in 50% ethanol and 7% acetic acid) as described by Sharif and Alaeddinoglu (1988). b. Scanning electron microscopy: Spore-crystal mixtures from isolates were airdried on cover-glass and coated with gold. Spores and crystals were examined with a JSM 6400 scanning electron microscopy (JEOL Ltd, Tokyo, Japan) operated at 6 and 10 kV.


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In addition to the SEM, physiological, and molecular tests, the strains were sent to the Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan for serotyping.

Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE)


Spore-crystal mixtures from isolates were re-suspended in sample buffer (60mM TrisHCl (pH 6.8), 25% glycerol, 5% 2-mercaptoethanol, 0.1% bromophenol blue), boiled 10 min, and electrophoresed in 10% SDSPAGE as described by Laemmli (1970). The gel was then stained with Coomassie brilliant blue.

Solubilization and proteolytic digestion of crystal proteins Solubilization of spore-crystal mixtures of isolates was performed as described previously with minor modifications (Iriarte et al. 2000). Spore-crystal mixtures were incubated in solubilizing buffer (Na2CO3 50 mM, 0.1 M NaCl and 10 mM dithiothreitol (DTT), pH 11.3) for 2 h under continuous shaking (Hoefer, San Francisco, CA). Protein concentration was determined using the Bradford procedure with bovine serum albumin as protein standard (Bradford 1976). Solubilized proteins were separated from the spores by centrifugation at 13,000 g for 5 min. Trypsin was then added in a ratio of 1:20 (trypsin/protein, w/w), and the suspension was inoculated at 378C for 4 h. The same ratio of trypsin was added after 1 and 2 h. The proteolytic products were analyzed in 10% SDSPAGE.

PCR analysis Polymerase chain reaction with both universal and specific primers was carried out to examine strains Tp6 and Tp14 for their cry gene content. The universal primers designed for detecting cry1, cry2, cry3 and cry4 genes (Ben-Dov et al. 1997), and specific primers for identification of cry1Ab and cry3Aa genes (cry1Ab Forward 5?-ATGGATAACAATCCGAACATC-3?, Revers 5?-TTATTCCTCCATAAGAAG TAA TTCCAC-3?; Cry3Aa Forward 5?-ATGAATCCGAACAATCGAAGTGAA3?, Revers 5?-TTAATTCACTGGAATAAATTCAAT-3?) were synthesized and used in the polymerase chain reaction (PCR). DNA templates were prepared as described by Sambrook, Fritsch, and Maniatis (1989). Reactions were routinely carried out in 25 mL: 100 ng of template DNA mixed with reaction buffer, 150 mM (each) deoxynucleoside triphosphate, 0.5 mM (each) primer, and 0.5 U of Taq DNA polymerase. Amplification was carried out in a DNA thermal cycler (Hybaid) using the stepcycle programs for identification of genes cry1, cry2, cry3 and cry4 (Ben-Dov et al. 1997). It was performed with 30-cycle program (each cycle consisting of denaturation at 948C for 60 s, annealing at 558C for 50 s, and extension at 728C for 90 s), followed by a final extension step at 728C for 10 min. Each experiment was associated with negative (without DNA template) and positive (with B. thuringiensis ssp. tenebrionis, B. thuringiensis ssp. kurstaki) controls. PCR products were analyzed by 1.3% agarose gel electrophoresis. The gel was then examined in BioDoc Analyse System (Biometra GmbH, Goettingen, Germany).


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Insect toxicity assays Insects were collected from potato and hazelnut fields in Trabzon and Giresun, and from pine forest fields in Samsun and Zonguldak, Turkey during the period of 2005 2008. Collected insects were brought to the Microbiology Laboratory at the Biology Department, Karadeniz Technical University, Trabzon, Turkey. Toxicity assays were carried out with the spore-crystal mixtures of Tp6 and Tp14 isolates and standard strains (positive controls), B. thuringiensis ssp. tenebrionis and B. thuringiensis ssp. kurstaki. In order to obtain spore-crystal mixtures, isolates were grown in nutrient agar medium at 308C for 5 days, until lyses. Spore-crystal mixtures were suspended in 1 mL of ice-cold 1 M NaCl and centrifuged at 13,000 g for 5 min. The pellet was re-suspended in distilled H2O. Tp6 and Tp14 suspensions contain a mixture of approximately 109 sporescrystals/mL adjusted using a spectrophotometer. Sterilised distilled water was used in bioassay as a negative control. Bioassays with Agelastica alni L. (Col.: Chrysomelidae) collected from hazelnut fields, Leptinotarsa decemlineata Say (Col.: Chrysomelidae) collected from potato fields, Yponomeuta malinellus Zell. (Lep.: Yponomeutidae) collected from apple fields, Galleria mellonella L. (Lep.: Pyralidae) obtained from laboratory culture, Thaumetopoea pityocampa Den. and Schiff (Lep., Thaumetopoeidae) collected from pine forest fields, and Malacosoma neustria L. second and third stadium larvae (Lep.: Lasiocampidae) collected from hazelnut fields were performed with the crystal-spore mixture applied on the diet. Diets were prepared from fresh hazelnut leaves for A. alni and M. neustria larvae, from fresh potato leaves for L. decemlineata larvae, from fresh apple leaves for Y. malinellus and from fresh pine leaves for T. pityocampa larvae. Artificial diet was used for the larvae of G. mellonella. The diets were placed into individual sterilised glass containers (80 mm in diameter). Spore-crystals mixtures (109 spores-crystals/mL) were applied to the surface of the diet. Ten second and third instar larvae were placed on the diet in containers. Containers were kept at 26928C and 60% RH on a 12:12 h photoregime, with the fresh unamended diet reintroduced after consumption of the treated diets. Larval mortalities were recorded daily and all dead larvae were removed from containers. Ten tested insects were transferred to dishes. Mortality was recorded 10 days after initiation of the treatment. Infectivity tests were carried out with the positive and negative controls. All bioassays were repeated three times on different occasions. Means were analyzed using one-way analysis of variance (ANOVA) and compared by least significant difference (LSD) test (Minitab 1997). Results and discussion There has recently been an increased interest in finding new and better B. thuringiensis strains with higher insecticidal activity for control of pest insects. In this study, we report detailed identification and insecticidal activity of B. thuringiensis Tp6 and Tp14 isolates from T. pityocampa. The crystal morphologies of these isolates were observed under light (data not shown) and scanning electron microscopy (data not shown). The isolates contained both spores and crystals that were released from the rod-shaped sporulated bacteria. The crystals are the most distinguishing characteristics of B. thuringiensis strains (Gonzalez, Dulmage, and Carlton 1981). Examination by a scanning electron microscope showed that Tp6 and



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Tp14 isolates have flat square and bipyramidal crystal shapes, respectively. Morphological characteristics of Tp6 and Tp14 isolates were similar to those of other B. thuringiensis strains previously described (Ho¨fte and Whiteley 1989). B. thuringiensis Tp6 and Tp14 isolates, in contrast to the reference strain, B. thuringiensis ssp. morrisoni PG-14, which produced ovoidal inclusions, produced flat-square and bipyramidal inclusions, respectively (Choi et al. 2004). Serological studies indicated that H antigenic structure of the isolates Tp6 and Tp14 is identical to B. thuringiensis ssp. morrisoni (H8a8b). The classification of B. thuringiensis into serovars has been very useful, but has limitations (De Barjac and Frachon 1990). There is no correlation between serotype classification and spectrum of insecticidal activity. The morrisoni serotype (H8a8b) harbors three strains that are active against Lepidoptera (strain HD-12), Diptera (strain PG-14), and Coleoptera (B. thuringiensis ssp. tenebrionis) (Krieg, Schnetter, Huger, and Langenbruch 1987). The SDSPAGE protein components of B. thuringiensis ssp. morrisoni, B. thuringiensis ssp. kurstaki HD-1, Tp6 and Tp14 were examined using SDS PAGE (Figure 1). Crystals of the Tp6 isolate produce about 65-kDa protein bands similar to those of B. thuringiensis ssp. morrissoni. This protein is similar in size to those reported for Cry3 protein (Honigman et al. 1986). Crystals of Tp6 isolate were solubilized after 2 h incubation and then treated with trypsin. This yielded a trypsinresistant peptide of about 50 kDa (Figure 2). On the other hand, crystals of Tp14 isolate produced about 130-kDa protein bands similar to those of B. thuringiensis ssp. kurstaki. This protein is similar in size to those reported for the Cry1 protein. Crystals of this isolate were completely solubilized after 2 h incubation and trypsinization. It yielded a trypsin-resistant peptide about 45 kDa (Figure 2).

Figure 1. Purified crystals of the Tp6 and Tp14 isolates were separated in 10% SDS polyacrylamide gel and then stained with Coomassie blue. Lanes: M, Molecular mass standards; 1. B. thuringiensis ssp. kurstaki HD-1; 2. B. thuringiensis ssp. tenebrionis; 3. Isolate Tp6; 4. Isolate Tp14.


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Figure 2. SDSpolyacrylamide gel of undigested (Lane 1, 3, 5 and 7) and trypsin digested (Lane 2, 4, 6 and 8) purified crystal proteins. Gel then stained with Coomassie blue. B. thuringiensis ssp. kurstaki HD-1 (Lane 1 and 2); B. thuringiensis ssp. tenebrionis (lane 3 and 4); Isolate Tp6 (Lane 5 and 6); Isolate Tp14 (Lane 7 and 8); M, Molecular mass standards.

Figure 3. Agorose gel electrophoresis analysis of PCR products obtained by using the cry1, cry2 and cry3 general primers pairs. Lanes: M. Marker (100 bp DNA Ladder); 1. B. thuringiensis ssp. kurstaki HD-1 (cry1); 2. Isolate Tp14 (cry1); 3. B. thuringiensis ssp. kurstaki HD-1 (cry2); 4. Isolate Tp14 (cry2); 5. B. thuringiensis ssp. tenebrionis (cry3); 6. Isolate Tp6 (cry3).



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We have used a method based on PCR to allow rapid and highly sensitive determination of the cry gene contents of Tp6 and Tp14 isolates. DNA amplification was carried out using universal primers (cy1, cry2, cry3, and cry4). PCR analysis showed that Tp6 contains cry3 gene and Tp14 isolate contains cry1 and cry2 genes (Figure 3). Fragments with the expected sizes corresponding to cry1, cry2 and cry3 genes were amplified with DNA from Tp6 and Tp14 isolates (Ben-Dov et al. 1997). Fragments with the expected sizes of about 272 and 725 bp corresponding to cry1 and cry2 genes, respectively, were amplified with DNA from Tp14 isolate (Ben-Dov et al. 1997). Fragments with the expected size of about 589 bp corresponding to cry3 gene were amplified with DNA from the Tp6 isolate (Ben-Dov et al. 1997). Although B. thuringiensis Tp6 and Tp14 isolates were serotyped as ssp. morrisoni, Tp14 had cry genes different from the reference strain. B. thuringiensis ssp. morrisoni PG-14 showed products for cry4A, cry4B, cry10A, and cry11A (Choi et al. 2004). However, B. thuringiensis Tp6 and Tp14 isolates, unlike its reference strain, had cry3 and cry1, cry2 genes, respectively. The RFLP pattern of the PCR product from B. thuringiensis ssp. morrisoni HD-12 showed that this strain should contain cry1A(a), cry1, cry1C, cry1C(b), and cry1F genes (Kuo and Chak 1996). Also, Tp6 and Tp14 isolates were detected that contained cry3Aa and cry1Ab genes, respectively (Figure 4). In conclusion, we have provided evidence of the potential of new B. thuringiensis strains for insect pest control. The crystal-spore mixtures of Tp6 and Tp14 isolates were tested against A. alni L., L. decemlineata Say, Y. malinellus Zell., G. mellonella L.,

Figure 4. Agorose gel electrophoresis analysis of PCR products obtained by using the cry1Ab and cry3Aa specific primers pairs. Lanes: M, Marker; 1. Isolate Tp14 (cry1Ab); 2. Isolate Tp6 (cry3Aa).

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Table 1. The insecticidal effects of spore-crystal mixtures (109 spore-crystal/mL) of two B. thuringiensis isolates obtained from T. pityocampa on some coleopteran and lepidopteran pests.


Mortality (%) Isolates

A. alni

L. decemlineata

Y. malinellus

G. mellonella

M. neustria

Tp6 Tp14 Negative control1 Positive control2 Positive control3

10095 ND 0 ND 10090

10095 ND 40910 ND 80910

ND 80915 20910 90910 ND

ND 20910 0 2095 ND

ND 10090 0 10090 ND

1 Sterilised water. 2B. thuringiensis ssp. kurstaki HD-1, 3B. thuringiensis ssp. tenebrionis BTS-1, ND: not determined. ANOVA LSD test (F52.678; PB0.05).

T. pityocampa Den. and Schiff, and M. neustria L. (Table 1). The recorded mortality was 100% for Tp6 on larvae of A. alni and L. decemlineata (ANOVA LSD, P B0.05). On the other hand, this value was 20% higher than that of the crystal-spore mixture of the reference strain, B. thuringiensis ssp. tenebrionis on the larvae of L. decemlineata. The insecticidal activities are 80, 20 and 100% for Tp14 on the larvae of Y. malinellus, G. mellonella and M. neustria, respectively. Tp6 and Tp14 isolates did not show any insecticidal activity on larvae of T. pityocampa. In another study, PG-14 isolate of B. thuringiensis ssp. morrisoni was discovered in the Philippines which is as toxic as B. thuringiensis ssp. israelensis and produces the same complement of endotoxin proteins (Cyt1A, Cry4A, Cry4B and Cry11A) plus an additional 144-kDa Cry1 protein toxic to lepidopterans (Federici, Park, Bideshi, Wirth, and Johnson 2003). There is no correlation between serotype classification and spectrum of insecticidal activity. The morrisoni serotype (H8a8b) harbors three strains that are active against Lepidoptera (strain HD-12), Diptera (strain PG-14), and Coleoptera (B. thuringiensis ssp. tenebrionis) (Norris 1964; Padua, Ohba, and Aizawa 1984; Krieg et al. 1987). Our results indicate that Tp6 and Tp14 isolates obtained from T. pityocampa are B. thuringiensis ssp. morrisoni with potential use for the control of some coleopteran and lepidopteran pests. Further study will involve the characterization of novel B. thuringiensis strains and provide good sources for developing microbial pesticides against pests. Acknowledgements This work was supported by the Turkish Republic Prime Ministry State Planning Organization (21.111.004.1). The authors would like to thank Dr Michio Ohba and Dr Kumiko Kagoshima for providing the H-Serotyping, to Dr Dan Zeigler for providing the reference B. thuringiensis ssp. kurstaki HD-1, and to Dr Stefan Jansens for providing B. thuringiensis ssp. tenebrionis.

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