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Isolation and characterization of Bacillus thuringiensis strains active against Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae)/Selecao e caracterizacao de linhagens de Bacillus thuringiensis ativas contra Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae).

Introduction

The lesser cornstalk borer Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae) is a polyphagous pest, and larvae feed on more than 60 species of cultivated plants (Viana, 2004). These plants include crops of high economic value, such as corn, beans, wheat, soy, peanuts, and sugarcane, which suffer extensive losses by attack from this pest. The larvae damage newly germinated plants and reduces the number of seedlings per planting area. Larvae penetrate the stalk of a recently sprouted plant, make galleries toward the central core and then feed inside the stem causing new leaves to dry up and die, resulting in the so-called "dead heart" (Gallo et al., 2002).

The lesser cornstalk borer is a difficult pest to control because it can remain close to the plant stem, within the stem, or in silken web habitats and land shelters they build on the soil surface. In experiments conducted with different control methods, pest management using pheromone and light traps and soil cover with Crotalaria jucea resulted in a small reduction in the pest population (Jham, Silva, Lima, & Viana, 2007; Gill, McSorley, Goyal, & Webb, 2010). Thus, preventive chemical control and seed treatment remain the most widely used methods to control E. lignosellus. However, when chemicals are indiscriminately applied, human contamination and environmental imbalance can result, leading to an increase in the pest population.

Entomopathogenic bacteria, such as Bacillus thuringiensis, are among the alternatives to reduce the use of insecticides for pest control. The insecticidal characteristics of these bacteria are caused by the formation of parasporal crystals in the early sporulation phase. These crystals are composed of Cry proteins, which are toxic to a variety of insects that attack crops of high economic value (Vilas-Boas, Peruca, & Arantes, 2007; Vidal-Quist, Castanera, & Gonzalez-Cabrera, 2009).

The toxic activity of these proteins against insect pests led to the formulation of bioinsecticides and the selection of genes encoding insecticidal proteins to produce transgenic plants resistant to different species of insects. Several Bacillus thuringiensis isolates specific to insects of the orders Lepidoptera, Coleoptera, and Diptera have been investigated (Pardo-Lopez, Soberon, & Bravo, 2013). These isolates typically harbour one or more cry genes, and the isolates containing a wider range of genes are the most targeted. Thus, further studies are required to select these isolates, identify the cry genes, and assess isolate toxicity (Sun, Fu, Ding, & Xia, 2008).

Although the search for isolates of B. thuringiensis that are effective against E. lignosellus is of great significance for the management of this insect pest, studies remain limited. Therefore, the aim of this work was to select and characterize native isolates of B. thuringiensis toxic to E. lignosellus, with the goal to conduct further studies focused on new formulations of bioinsecticides and development of genetically modified plants.

Material and methods

Insect rearing

Larvae of E. lignosellus were reared on an artificial diet according to the methodology described by Greene, Leppla, and Dickerson (1976). The adults were maintained at 27 [+ or -] 2[degrees]C and 60 [+ or -] 10% RH with a 14h photoperiod in plastic cages (10 cm diameter, 20 cm height) coated with filter paper and closed on the upper end with tissue and on the lower end with a petri dish (14.3 cm diameter) and fed a 10% aqueous honey solution. The eggs obtained were transferred to petri dishes at 25[degrees]C for incubation; the first instar larvae were used in bioassays.

Bacterial isolates

Forty-seven native isolates of B. thuringiensis were examined from the Collection of Entomopathogenic Microorganisms of Londrina State University (Universidade Estadual de Londrina, UEL) and the Brazilian Agricultural Research Corporation, Embrapa Genetic Resources and Biotechnology (Empresa Brasileira de Pesquisa Agropecuaria Embrapa Recursos Geneticos e Biotecnologia). The HD-1 strain of B. thuringiensis subsp. kurstaki was obtained from the Collection of B. thuringiensis at the Institut Pasteur, Paris, France.

Selective bioassay to choose the most toxic isolates

Suspensions of each B. thuringiensis isolate were prepared by adding 1.0 mg of lyophilized material to 1.0 mL of sterile distilled water. The artificial diet was prepared according to Greene et al. (1976) and distributed (3 mL) when still liquid into glass tubes (2 cm diameter x 3 cm height). After the diet solidified, 50 [micro]L of a mixture of spores and crystals were applied on the diet surface. The glass tubes were kept in a laminar flow hood until the complete absorption of the suspension by the diet. Subsequently, five first-instar larvae were released inside each glass tube, which were sealed with a plastic lid. The bioassay consisted of three replicates with four glass tubes for each B. thuringiensis isolate. The standard strain B. thuringiensis subsp. kurstaki HD-1 (Btk) and water were used as positive and negative controls, respectively. The insects were maintained in an incubator (27 [+ or -] 2[degrees]C, 60 [+ or -] 10% RH and a 14h photoperiod) for six days after which mortality was assessed. The corrected mortality was calculated using Abbott's control adjusted mortality (Abbott, 1925). The data were subjected to analysis of variance (ANOVA), and the means were compared using Tukey's test at 5% probability. The most toxic isolates, those that caused a reliable mortality (above 85%) and therefore had potential for further testing, were used in dose-response bioassays and evaluated according to their molecular, protein and morphological profiles. The mortality rate was selected based on the minimum efficacy threshold (80% efficacy) required for pesticide registration in Brazil (MAPA, 1995).

Estimation of the lethal concentration ([LC.sub.50]) of B. thuringiensis isolates

Bioassays for dose estimation were performed with the 12 isolates that showed the greatest toxicity in the selective bioassays and with the HD-1 standard strain. Seven suspensions of spores and crystals of B. thuringiensis were prepared to estimate the concentration of each isolate that would cause 50% mortality in E. lignosellus larvae ([LC.sub.50]). The suspensions were prepared with 5.0 mg of lyophilized material that was diluted in 5 mL of sterile distilled water. Dilutions were performed using the initial suspension to obtain the seven concentrations used in the study (1.0, 0.2, 0.1, 0.05, 0.025, 0.008, and 0.0025 mg [mL.sup.-1] in sterile distilled water), and the number of spores per mL of water in each dilution was counted using a Neubauer chamber. The bioassay was conducted in the same way as previously described. For each concentration evaluated, three replicates with four tubes were used, for a total of 20 larvae per replicate and 60 per concentration. The mortality data were subjected to Probit analysis (Finney, 1971) to determine the lethal concentration. The [LC.sub.50] bioassay results were analysed by checking for the overlap of the 95% confidence intervals according to Probit analysis.

Protein and molecular characterization of B. thuringiensis isolates pathogenic to E. lignosellus

Genomic DNA samples of the B. thuringiensis strains were isolated according to the method described by Ricieto, Fazion, Carvalho Filho, Vilas-Boas, and Vilas-Boas (2013). The isolates were cultivated for 15h at 30[degrees]C on plates containing Luria-Bertani (LB) medium (Bertani, 1951). With the aid of a sterile toothpick, a colony of approximately 2 mm in diameter was transferred to microtubes containing 200 [micro]L of TE (10 mMTris; 1 mM EDTA; pH 8.0). The suspension was homogenized and incubated in boiling water for 10 min. Then, the suspension was centrifuged at 12,000 xg for 3 min, and the supernatant was transferred to a new tube and used as DNA template in the PCR reactions. The presence of the genes cryl, cry2, cry3, cry4A, cry4B, cry10, and cryll was analysed using specific primers and amplification conditions (Ceron, Orti, Quintero, Guereca, & Bravo, 1995, Bravo et al., 1998; Ibarra et al., 2003; Vidal-Quist et al., 2009). DNA amplification was performed using an Endurance TC412 thermocycler. For each amplification reaction, a total reaction volume of 20 [micro]L was prepared that contained 1 U Taq DNA polymerase (Invitrogen, Brazil), 2.0 [micro]L of Buffer 10 (200 mM Tris-HCl, pH 8.0, 500 mM KCl), 1.5 mM Mg[Cl.sub.2], 0.25 mM dNTP, 0.5 [micro]M each primer, 2 [micro]L of DNA and sterile Milli-Q water. The same reaction was used for all the primers. The PCR products were visualized by electrophoresis on 1.2% agarose gel in TBE buffer (89 mM Tris Borate, 2 mM EDTA, pH 8.0) stained with Syber Safe (Invitrogen, UK) using a 100 bp DNA ladder (Invitrogen, UK). After electrophoresis, the gel images were captured using a Sony Cyber-shot 8.1 digital camera.

The B. thuringiensis isolates were characterized by the protein profile of their crystals using protein electrophoresis on 10% polyacrylamide gel (SDS-PAGE). Initially, the crystals were obtained according to the protocol described by Lecadet, Chaufaux, Ribier, and Lereclus. (1992). Each isolate was cultivated in NB medium (Downes & Ito, 2001) at 30[degrees]C for 72h at 200 rpm, until complete sporulation. The B. thuringiensis subsp. kurstaki HD-1 standard strain was used as the reference.

Morphological characterization of B. thuringiensis isolates

The morphological characterization of the isolates was initially performed by optical microscopy using a microscope (Model CHS; Olympus Optical Co. Ltd., Tokyo, Japan) with a 100x phase contrast lens. For electron microscopy, the lyophilized material of each isolate used in the previous bioassays was directly deposited over metal supports and coated with gold for 180 s, using a 40 mA current under vacuum (10-1 mbar) in a BAL-TEC model SCD-050 Sputter Coater (Santos et al., 2009). Subsequently, the material was analysed using a scanning electron microscope Philips QUANTA 200 (FEI) in high vacuum under 20 kV tension with a working distance of 10.2 mm. The selected images were captured and stored for later analysis.

Results

Selective bioassay and determination of the Median Lethal Concentration ([LC.sub.50]) of Bacillus thuringiensis isolates

Among the 47 isolates of B. thuringiensis tested, 12 isolates (25.53%), in addition to B. thuringiensis subsp. kurstaki (Btk) HD-1 strain, caused mortality of E. lignosellus above 85%, for a total of 13 isolates, which were selected for all other conclusive tests. Dose-response bioassays were used to evaluate these 13 isolates. The X2 values related to the [LC.sub.50] were no significant for 12 of the isolates, which indicated that the data were homogeneous for those strains and fit the Probit analysis model (Table 1) (Finney, 1971).

The [LC.sub.50] values of the selected isolates varied from 0.038E+8 to 0.855E+8 spores mL'1. The lowest [LC.sub.50] values were obtained for the group consisting of BR83, BR145, BR09, BR78, and S1534. The [LC.sub.50] values within that group did not differ statistically according to the Probit analysis, as shown by the overlap of the 95% confidence intervals (Table 1). Only the[LC.sub.50] value of isolate BR83 was significantly lower than that of the HD-1 standard strain, with a toxicity that was three-fold greater than that of the standard. Additionally, the BR 83 isolate was approximately 22-fold more toxic than the S1269 isolate, which had the highest [LC.sub.50]. Only the S1450 isolate presented a significant [X.sup.2]; thus, the [LC.sub.50] could not be estimated (Table 1).

Protein and molecular characterization of B. thuringiensis isolates pathogenic to E. lignosellus

The PCR technique using total DNA of isolates and specific primers for the detection of cryl, cry2, cry3, cry4A, cry 4B, cry 10, and cryll resulted in the amplification of fragments of the expected sizes (Bravo et al., 1998; Ceron et al., 1995; Ibarra et al., 2003; Vidal-Quist et al., 2009) and consequently, the determination of which cry genes were in the isolates of B. thuringiensis.

The amplicons produced with the greatest frequency corresponded to cryl, cry2, and cry3 genes. The cryl gene was detected in all isolates, except BR52 and BR53, which also did not exhibit the cry2 gene. The expected fragment for the cry3 gene appeared in BR145, S1534, and S1302 isolates, whereas the fragment for the cryll gene occurred only in the BR53 isolate. Only the BR52 isolate did not show a PCR product consistent with the selected primers. The fragments of the expected size for the other primers used were not observed in all isolates, indicating the absence of cry4A, cry4B, and cryl0 genes in some isolates (Table 2). The protein profile analysis of the spore-crystal mixtures of the isolates from the selective bioassays revealed bands of 60, 65, 70, 80, and 130 kDa. The isolate used as the standard, B. thuringiensis subsp. kurstaki, had a protein profile of 65 and 130 kDa (Hofte & Whiteley, 1989; Lereclus, Delecluse, & Lecadet, 1993). All data related to the protein and molecular characterization of B. thuringiensis isolates are presented in Table 2. An image of protein electrophoresis on 10% polyacrylamide gel (SDSPAGE) illustrating the protein profile of some isolates is shown in Figure 1.

Morphological characterization of B. thuringiensis isolates

Morphological analysis using optical and scanning electron microscopy showed protein inclusions of different forms. The BR83, BR09, BR78, and S1450 isolates showed three different crystalline protein inclusions: bipyramidal, spherical, and cuboidal. Isolates BR145, S1534, S1302, and BR38 exhibited bipyramidal and spherical crystals, whereas isolates S545, S1269, BR52, and BR53 contained only bipyramidal crystals (Table 2).

Discussion

The selective and dose bioassays revealed that B. thuringiensis isolates were active against E. lignosellus. Of the isolates tested, 25.53% caused mortality above 85%; therefore, these isolates can be further tested as a possible alternative method for the management of populations of E. lignosellus. Based on dose bioassays, isolate BR83 had the lowest lethal concentration. Furthermore, BR83 had superior activity compared with that of the standard B. thuringiensis subsp. kurstaki HD-1 strain, although the activity was not significantly different from that of the BR145, BR09, BR78, and S1534 isolates. Those four isolates carried cryl and cry2 genes, which are toxic to insects of the order Lepidoptera (Ricieto et al., 2013). In addition to these genes, BR145 and S1302 isolates also carried the cry3 gene, with reported toxicity to Lepidoptera and Coleoptera (Brizzard & Whitley, 1988).

The cry1 and cry2 genes were found in almost all selected isolates, except for BR52, which did not yield an amplification product for any of the used primers, and BR53, which amplified only for the cry11 gene. The genes in the Cry1 subfamily are the most abundant and occur in approximately half of the isolates identified to date. The cry2 gene is also very common, particularly among isolates harbouring the cryl gene (Porcar & Juarez-Perez, 2003; Arrieta, Hernandez, & Espinoza, 2004).

Isolates containing cryl and/or cry2 genes were also the most abundant in the collection studied by Vidal-Quist et al. (2009), representing over 45% of the total. In studies with the BR37 isolate, Santos et al. (2009) identified eight genes of which seven were in the Cry1 group, showing the frequent occurrence of cry1 genes in isolates of B. thuringiensis (Bravo et al., 1998).

Genes in the Cry1 and Cry2 protein families have been reported in the isolates used for the control of E. lignosellus, and the effects of Cry1 proteins in transgenic peanut and soybeans plants have been examined under field conditions. In peanuts, various levels of resistance to the lesser cornstalk borer were provided by the introduction of the cry1Ac gene, from complete larval mortality to 66% reduction in larval weight (Singsit et al., 1997). For soybean, plants expressing the cry1Ac gene had four-fold more resistance to E. lignosellus than that of the wild-type isolate (Walker, All, Mcpherson, Boerma, & Parrott, 2000).

Assessments of genetically modified corn crops revealed that hybrids containing cry9C, cry1F, and cry1Ab genes did not differ in resistance to E. lignosellus; however, transgenic plants were superior to non-transgenic hybrids (Vilella, Waquil, Vilela, Siegfried, & Foster, 2002). Additionally, in laboratory bioassays, isolates containing cry2A gene and the HD-1 standard isolate proved effective against E. lignosellus (Moar, Pusztai-Carey, & Mack, 1995).

These examples demonstrate the different methodologies used for the control of E. lignosellus, but the mortality rates are different between isolates harbouring many genes and those caused by genetically modified plants carrying only the primary gene responsible for the toxicity. However, to select new isolates that carry toxic genes to be tested individually against the pest, the bioassays used in this study must be conducted. Thereby, the selected gene, after many tests, can be inserted into plants for pest control and increase the possibilities for pest management.

The protein profiles of most of the tested isolates that amplified with the cry1 and cry2 genes (BR83, BR145, BR09, BR38, S1534, and S1450) showed bands of 130 and 65/70 kDa, which confirmed their specificity for Lepidoptera. Cry1 class proteins have a molecular weight of approximately 130-140 kDa (Hofte & Whiteley, 1989), whereas proteins of the Cry2 and Cry3 groups, which are active against Lepidoptera and Coleoptera, have values of 65-70 kDa (Bravo et al., 2004).

Although the BR09 isolate did not differ from the most toxic isolate (BR83) or the HD-1 standard, the [LC.sub.50] of the BR09 isolate was approximately 1.8-fold lower than that of HD-1. Santos et al. (2009) obtained a similar value when testing the same isolate against Spodoptera eridania. Nevertheless, [LC.sub.50] values may vary among species; for example, in studies against Spodoptera cosmioides, the [LC.sub.50] of the HD-1 standard strain was two-fold lower than that of the BR09 isolate (Santos et al., 2009). Moreover, in a selection performed by Constanski et al., 2015, no differences were detected between the [LC.sub.50] of the HD-1 standard strain and that of the other isolates tested against S. eridania and S. cosmioides.

The toxicological profile of the BR09 isolate that was effective against E. lignosellus can be explained by the expression of cryl and cry2 genes, similar to the HD-1 strain, which has the crylAa, crylAb, crylAc, cry2A, and cry2B genes in its genome (Li et al., 2005). Furthermore, SDS-PAGE protein analysis of the spore and crystal mixture revealed two polypeptides with approximate molecular weights of 70-130 kDa. Santos et al. (2009) also tested the BR09 isolate and showed a gene profile with cry1Aa, cry1Ab, cry1Ac, and cry2Aa genes.

Although the S1450 isolate caused mortality of E. lignosellus larvae above 85%, in the selective bioassays, the X2 value was significant, i.e., the data did not fit the Probit model. Thus, the [LC.sub.50] could not be estimated for isolate S1450. This isolate is in the kurstaki serotype described in the literature as toxic to insects of the order Lepidoptera (Monnerat et al., 2007) and causing 100% mortality of Agrotis ipsilon (Lepidoptera: Noctuidae). Additionally, cry1Aa, cry1Ab, cry 1 Ac, cry1, and cry2 genes were revealed by the molecular analysis, which confirmed the activity of the isolate against Lepidoptera (Menezes, Fiuza, Martins, Praca, & Monnerat, 2010).

The BR78 isolate, with an [LC.sub.50] that did not differ from that of the most toxic isolates, also carried cry1 and cry2 genes. However, only two primary polypeptides of approximately 65 and 80 kDa were associated with this isolate, which are related only to the cry2 gene. The result was similar for the S1302 isolate, which despite amplifying with cry1, cry2, and cry3 genes, revealed only a band of 70 kDa. As a possible explanation, a poor performance or even the absence of the promoter led to low expression of the cryl gene, which prevented the display of a 130 kDa band.

According to Alper et al. (2014), some strains that harbour the same genes may not be as effective as other toxic strains that have those same genes, indicating that these genes could be poorly expressed because of a weak promoter in the strains. Thus, poor expression of the cry genes in the BR78 and S1302 isolates could explain the cry genes in the genetic profile but not in the protein profile.

Armengol, Escobar, Maldonado, and Orduz (2007), identified isolates toxic to S. frugiperda containing cry1Aa, cry1Ab, cry1Ac, cry1B, and cry1D genes, with protein profiles that revealed bands only at 60 kDa. According to these authors, the correlation between the identified

protein profiles and the cry genes cannot occur when the cry proteins are encoded by unknown genes or have not been amplified by the primers used. Additionally, the identified genes may encode proteins with low-level or inactive expression.

Although the [LC.sub.50] did not differ from that of the HD-1 standard isolate, only the BR52 isolate failed to obtain amplification of the expected fragment sizes with the specific primers employed. Nevertheless, the optical and electron microscope observations revealed the production of protein inclusions, suggesting that the isolate might contain genes not covered by the set of primers used in the PCR analysis or cry genes not yet described. Additionally, the analysis of the protein profile showed a polypeptide of approximately 70 kDa that corresponded to the Cry2 class, possibly explaining the insecticidal activity of isolate BR52 against Lepidoptera.

Only isolate BR53 expressed the cry11 gene. However, this gene was most likely not the cause of the toxicity of this isolate against E. lignosellus, because the gene is usually associated with activity against larvae of Diptera, such as Simulium spp., Culex spp. and Aedes aegypti (Vidal-Quist et al., 2009). Thus, isolate BR53 might contain some other gene toxic to E. lignosellus not disclosed by the set of primers used, requiring further investigation to search for new cry genes.

Based on the different shapes of protein crystals detected by optical and electron microscopy, the types of Cry proteins that composed the crystal could be inferred, which provided information on the insecticidal activity of the isolates (Lereclus, Delecluse, & Lecadet, 1993; Vilas-Boas et al., 2007).

The similarity between the morphological and molecular analyses was notable in this study. Isolates that expressed cryl and cry2 genes contained primarily bipyramidal, cuboidal, and spherical shaped crystals. These three different shapes were observed in the BR09, BR78, and BR83 isolates, which contained the cryl and cry2 genes, and in the S1450 isolate that harboured cry1, cry2, and cry3 genes. The BR38 isolate with cry1 and cry2 genes and the S1302, S1534, and BR145 isolates with cry1, cry2 and cry3 genes all contained bipyramidal and spherical crystals. The crystal protein composition was studied in some of these isolates previously, and those studies confirm the shapes observed in this study (Praca et. al., 2004; Santos et al., 2009; Ricieto et al., 2013). Only bipyramidal crystals were produced by the isolates S545 and S1269, which contained only the cryl gene, and BR52 and BR53.

The selection of isolates containing Cry1 and Cry2 proteins and harbouring more than one Cry protein is essential in the prospecting for new virulent cry genes against lepidopterans such as E. lignosellus. Once identified, these are the isolates that can be used for pest management either through bioinsecticide formulations or as a source for transgenic plant manipulation.

Bacillus thuringiensisis the most successful pathogen agent used for insect control, representing almost 2% of the total insecticide market and up to 90% of bioinsecticide formulations. Currently, formulations based on B. thuringiensis are increasing, and this market is expected to continue to grow. Most of these products are based on spore-crystal preparations derived from a few strains such as B. thuringiensis var. kurstaki HD1. Thus, the detection of B. thuringiensis strains with new genes is extremely important to develop more efficient bioinsecticides (Bravo, Likitvivatanavong, Gill, & Soberon, 2011; Lemes et al., 2015).

For some products formulated with B. thuringiensis, their use in agriculture is limited, because Cry toxins are more specific for first larval instars, are sensitive to sun radiation and have limited activity against borer insects (Bravo et al., 2011). Therefore, the internal feeding behaviour of E. lignosellus inside stalks and stems makes management with pesticide and biologic product applications alone difficult. Thus, to more effectively E. lignosellus control, plants must be genetically modified with B. thuringiensis genes.

The discovery of these cry genes is of great interest for the production of new transgenic plants and also for genes tacking or pyramidalization. For example, insertion of different cry genes into genetically modified plants that use different receptors in the insect midgut membrane can help to extend the protection against more insect pests. Additionally, the beginning of resistance can be delayed or prevented, because more than one toxic protein would be acting against the same insect species (Sanahuja, Banakar, Twyman, Capell, & Christou, 2011; Hernandez-Rodriguez, Hernandez-Martinez, Van Rie, Escriche, & Ferre, 2013).

Researchers and commercial companies have recently used the technique of gene pyramidalization. As an example, the SmartStax[R] corn developed in cooperation between Monsanto and Dow Agro Sciences companies showed that the pyramidalization of cry1A.105, cry2Ab, cry3Bb1, cry34Ab1, cry35Ab1, and cry1Fa2 genes was essential for managing pesticide-resistant pests, in addition to providing effective control against a long list of pests in Coleoptera and Lepidoptera, including E. lignosellus (Marra, Piggott, & Goodwin, 2010).

The isolates evaluated in this study have potential for biotechnological control of E. lignosellus. Additionally, the isolates can be a gene source for the production of new crops or the management of currently insect-resistant plant cultivars. The genome sequencing of the studied isolates ensured accurate quantification of cry genes for further selection and use against E. lignosellus through insertion into the genome of economically relevant crops attacked by this pest.

Conclusion

Among the 47 isolates studied, 12 caused mortality above 85%. Isolates BR145, BR09, BR78, S1534, and S1302 had the lowest [LC.sub.50] values and did not differ from the standard HD-1. The protein profiles produced bands with molecular masses of 60-130 kDa. The molecular characterization showed the presence of cry1, cry2, cry3, and cry11 genes.

The morphological analysis identified three different crystal inclusions: bipyramidal, spherical and cuboidal. As a result of these characterizations, these isolates have potential for biotechnological control of E. lignosellus and should be important candidates for more studies and the development of new biopesticides or genetically modified plants.

Doi: 10.4025/actasciagron.v39i4.32707

Acknowledgements

The authors wish to thank the National Council for Scientific and Technological Development (CNPq) for financial support.

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Received on July 13, 2016.

Accepted on December 9, 2016.

Janaina Zorzetti (1) *, Ana Paula Scaramal Ricietto (1), Fernanda Aparecida Pires Fazion (1), Ana Maria Meneguim (2), Pedro Manuel Oliveira Janeiro Neves (1) and Gislayne Trindade Vilas-Boas (1)

(1) Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 445, 86047-902, Londrina, Parana, Brazil. (2) Instituto Agronomico do Parana, Londrina, Parana, Brazil. * Author for correspondence. E-mail: zorzettijanaina@gmail.com

Caption: Figure 1. Protein profile produced by isolates toxic to Elasmopalpus lignosellus (Zeller, 1848) (Lepidoptera, Pyralidae). M, Rainbow molecular weight marker (GE); 1 - BR09; 2 - BR38; 3 BR83; 4 - BR52.
Table 1. Mortality (%) and [LC.sub.50] of Bacillus thuringiensis
isolates against first instar larvae of Elasmopalpus lignosellus
(Zeller, 1848) (Lepidoptera, Pyralidae) (n = 60) on the sixth day of
the dose-response bioassay.

Isolate      (a)        (b) [LC.sub.50]        [CI.sub.(95%)]
          Mortality   (Spores [mL.sup.-1])
             (%)                              Lower      Upper

BR83      100.00 a         0.038E+8 a        0.025E+8   0.053E+8
BR145     100.00 a        0.059E+8 ab        0.020E+8   0.122E+8
BR09       100.00a        0.063E+8 ab        0.037E+8   0.102E+8
BR78      100.00 a        0.086E+8 ab        0.030E+8   0.179E+8
S1534     100.00 a        0.090E+8 ab        0.043E+8   0.189E+8
S1302     93.33 ab         0.097E+8 b        0.071E+8   0.134E+8
HD1       100.00 a         0.114E+8 b        0.084E+8   0.151E+8
BR52      100.00 a        0.141E+8 bc        0.098E+8   0.250E+8
BR38      100.00 a         0.346E+8 c        0.233E+8   0.445E+8
BR53      100.00 a         0.378E+8 c        0.239E+8   0.593E+8
S545       91.23 b         0.392E+8 c        0.218E+8   0.767E+8
S1269     94.92 ab         0.855E+8 d        1.39E+8    0.543E+8
S1450     95.00 ab            (-)              (-)        (-)

Isolate   Slope [+ or -] SE    [X.sup.2]

BR83      1.92 [+ or -] 0.26    2.004 ns
BR145     1.25 [+ or -] 0.13   11.009 ns
BR09      1.47 [+ or -] 0.13   11.070 ns
BR78      1.19 [+ or -] 0.14   10.806 ns
S1534     1.46 [+ or -] 0.18    7.098 ns
S1302     1.30 [+ or -] 0.13    3.724 ns
HD1       1.38 [+ or -] 0.15    3.384 ns
BR52      1.38 [+ or -] 0.24    6.702 ns
BR38      2.17 [+ or -] 0.31    1.637 ns
BR53      1.69 [+ or -] 0.23    5.969 ns
S545      1.39 [+ or -] 0.20    8.546 ns
S1269     1.43 [+ or -] 0.30    3.978 ns
S1450     0.96 [+ or -] 0.16   15.880 s *

(a) Mortality (%) from selective bioassay. Means followed by the
same letter in the column are not significantly different (Tukey's
test) at p< 0.05; (b) Means followed by the same letter in the
column are not different from one another based on the overlap of
95% confidence intervals, according to Probit analysis;
(-)significant [X.sup.2]: Estimate of the [LC.sub.50] was not
possible. s * significant based on the Chi-squared test (p < 0.05).
ns- not significant based on the Chi-squared test (p < 0.05).

Table 2. Genetic protein profiles and morphological characterization
of crystals of Bacillus thuringiensis isolates.

Isolate      Genetic profile     Protein
                                 profile
                                  (kDa)

BR83           cryl, cry2        130/70
BR145 (a)    cryl, cry2, cry3    130/65-70
BR09 (b)       cryl, cry2        130/65
BR78           cryl, cry2         65/80
S1534       cryl, cry2, cry3     130/70
S1302       cryl, cry2, cry3       70
HD1            cryl, cry2        65/130
BR52               --              70
BR38           cryl, cry2        130/70
BR53              cryll            70
S545              cryl             130
S1269             cryl             130
S1450          cryl, cry2        130/65

Isolate            Morphological analysis

            Bipyramidal   Spherical   Cuboidal

BR83             +            +          +
BR145 (a)        +            +          -
BR09 (b)         +            +          +
BR78             +            +          +
S1534            +            +          -
S1302            +            +          -
HD1              +            -          +
BR52             +            -          -
BR38             +            +          -
BR53             +            -          -
S545             +            -          -
S1269            +            -          -
S1450            +            +          +

(a) Data from Ricieto et al. (2013); (b) Data from Santos
et al. (2009).
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Author:Zorzetti, Janaina; Ricietto, Ana Paula Scaramal; Fazion, Fernanda Aparecida Pires; Meneguim, Ana Mar
Publication:Acta Scientiarum. Agronomy (UEM)
Date:Oct 1, 2017
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