Selection of Brazilian Bacillus thuringiensis Strains for Controlling Diamondback Moth on Cabbage in a Systemic Way  

Li­lian Botelho Praca , Carla Ferreira Caixeta , Ana Cristina Menezes Mendes Gomes , Rose Gomes Monnerat
Embrapa Recursos Geneticos e Biotecnologia, Parque Estacao Biologica-PqEB-Av. W5 Norte (final) Caixa Postal 02372, 70770-917, Brasilia, DF , Brasil
Author    Correspondence author
Bt Research, 2013, Vol. 4, No. 1   doi: 10.5376/bt.2013.04.0001
Received: 23 Jan., 2013    Accepted: 07 Feb., 2013    Published: 20 Mar., 2013
© 2013 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Praça et al., 2012, Selection of Brazilian Bacillus thuringiensis Strains for Controlling Diamondback Moth on Cabbage in a Systemic Way, Bt Research, Vol.4, No.1 1-7 (doi: 10.5376/bt.2013.04.0001)

Abstract

Plutella xylostella (L.) (Lepidoptera: Plutellidae) is a pest of great economic importance, due to losses caused in brassica crops worldwide. The control of this pest is hindered by the selection of insects resistant to various chemicals and by its cryptic habit. The aim of this work was to select a new B. thuringiensis strain with potential to control this insect pest in a systemic way because of its cryptic habit between the leaves of the cabbage, as a new control strategy. The strains were characterized by morphological, entomophatogenic, biochemical and molecular methods. No significant differences were observed in the CL50 values of S1905, S2122 and S2124 strains when compared to Btk HD-1 standard. The strains S1905 and S2124 had presented two main proteins with 130 and 65 kDa, while S2122 presented only 130 kDa protein. The strains presented PCR products with expected sizes for detection of some genes cry1 and cry2 and bipyramidal, spherical, and cuboidal crystals. The strains show some different cry1 genes that it's very important to develop new products with different toxins to be used in management of P. xylostella resistance to B. thuringiensis products and as a systemic bioinsecticide.

Keywords
Plutella xylostella; Biological control; Toxicity; cry genes

Introduction
Plutella xylostella L. (Lepidoptera: Plutellidae), Diamondback Moth, is a pest that causes serious damages in plants of Brassicaceae family, particularly in cabbage culture (Brassica oleraceae var. capitata) (Castelo Branco et al., 1996; França and Medeiros, 1998) in Brazil and other countries. The damage in the production is around 60% (Imenes et al., 2002).

In the central region of Brazil, the attack of P. xylostella in the field occurs along the year, although its biggest occurrence is from July till September. The critical period of attack occurs from the fourth till seventh week after transplant, and either in the head formation (Castelo Branco et al., 2003).

The main method of its control is the use of chemical insecticides. There are a lot of reports showing four insecticide applications per week increasing the control cost and creating the potential for insect resistance, principally where the cultivation is uninterrupted (Villas Boas et al., 2004). This indicates the necessity of reducing the consuming of insecticides and the importance of using safe alternatives of control.

The use of entomopathogenic bacteria Bacillus thuringiensis (Bt) is a very important alternative for insect control. During the sporulation process, B. thuringiensis produces one or more proteins that are toxic to diamondback moth that are specific, innocuous to mammals, vertebrates and plants, and are not toxic to environment (Monnerat et al., 1999; Broderick et al., 2006).

The Laboratory of Entomopathogenic Bacteria at Embrapa Genetic Resources and Biotechnology has been studying, since 2003, a new way of controlling P. xylostella using B. thuringiensis in its endophytic form, in order to protect cabbage crops and other brassicas from this important endophagous pest (Monnerat et al., 2003; 2009).

The aim of this present study was to select and characterize new Bt strains toxic to P. xylostella to be used as a systemic bioinsecticide.

1 Results and Analysis
1.1 Bioassay of the B. thuringiensis strains against
P. xylostella
The three strains S1905, S2122 and S2124 were toxic to P. xylostella. S1905, S2122 and HD-1 caused 100% of mortality after 48 hs, although S2124 caused 58.33% of mortality after 48 hours (Kruskal-Wallis: H3 =10.8, P =0.013) and 98.33% of mortality after five days (Kruskal-Wallis: H3 = 3.0, P = 0.392) (Table 1).
 

 
Table 1 Perceptual of mortality of second instar larvae of P. xylostella (media ± standard deviation) caused by Brazilian B. thuringiensis strains


After selective bioassays, the insects were submitted to lethal concentration bioassays. The CL50 of the tree B. thuringiensis strains varied between 2.336 to 4.842 µg/mL (Table 2) and all of them were similar to HD-1 (ANOVA: F = 0,673, P = 0,595).
 

 
Table 2 Estimation of Lethal Concentration (LC50) of B. thuringiensis strains toxic to second instar larvae of P. xylostella after five days


1.2 Characterization of B. thuringiensis strains toxic to P. xylostella
The analysis of the spore-crystals mixtures by SDS-PAGE showed the presence of two proteins of 130 and 65 kDa in S1905 and S2124 strains and in S2122 showed only the 65 kDa protein (Figure 1).
 

 
Figure 1 Protein profile of B. thuringiensis strains HD1, S1905, S2193 e S2124


S1905 produced amplicons for genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry2Aa and cry2Ab (Table 3). S2122 produced amplicons for seven genes cry1Aa, cry1Ab, cry1Ad, cry1C, cry1D, cry1F and cry2Ab. S2124 strain produced amplicons for cry1Ab, cry1E, cry2Aa and cry2Ab (Table 3).
 

 
Table 3 Protein profile, cry genes and types of crystal present in B. thuringiensis strains


1.3 Scanning and transmission electron microscopy of B. thuringiensis strains
The morphological characterization through scanning and transmission microscopy showed three different types of crystal protein inclusions. The strains produced bipyramidal, cuboidal and spherical crystals (Figure 2; Figure 3).
 

 
Figure 2 Scanning Electronic Microscopy of spore-crystals of Bacillus thuringiensis strains

 

 
Figure 3 Transmission of electronic microscopy of spores-crystals mixtures of B. thuringiensis strains


2 Discussion
The three strains S1905, S2122 and S2124 were tested against P. xylostella in selective bioassay showing toxicity. In the first 48 hours, S1905 and S2122 strains killed the insects faster than S2124 when compared with Btk HD-1 standard. Although with 96 hours, these strains didn’t present significative differences in the mortality. These results were similar to others researches with B. thuringiensis for the control of P. xylostella that showed high toxicity (Medeiros et al., 2005; Monnerat et al., 2007; Viana et al., 2009; Xie et al., 2010).

The three strains S1905, S2122, S2124 exhibited LC50 values similar to the standard strain Btk HD-1. All of them presented a high toxicity against P. xylostella showed results similar to the others previously described (Monnerat et al., 2007).

The result of protein profile showed in SDS-PAGE is typical to lepidopteran active crystal protein from the Cry1 and Cry2 classes (Schenpf et al., 1998; Praça et al., 2004) and it is consistent with the results of the bioassays presented in this work.

In the cause of cry genes, the three strains produced PCR amplicons for cry1 and cry2 genes that are effective for lepidopteran pests. The strain S1905 presented six cry genes like Btk HD-1 and S997 strains that were toxic to Spodoptera frugiperda and Anticarsia gemmatalis (Praça et al., 2004). The strains S2122 presented seven genes and three of them (cry1Ab, cry1C and cry1D) presented in this strain showed a similarity to B. thuringiensis aizawai, a standard strains toxic to P. xylostella. The strain S2122 and S2124 present cry1E and cryF genes respectively and it is important to comment that these genes were less abundant. Some researchers say that they are found in the rains tropic (Bravo et al., 1998).

The genes cry1C and cry1D present in S2122 encoded toxins active against S. frugiperda and S. exigua (Bravo et al., 1998; Monnerat et al., 2006). All the strains presented cry2A genes that encoded proteins toxic to lepidopteran and dipteran species (Cárdenas et al., 2001). The genes that were found most commonly in the strains reported here were cry1Ab and cry2Ab. The presence of cry1 and cry2 genes in S1905 and S2124 strains was consistent with their production of the above proteins of 130 and 65 kDa (Figure 1), respectively.

It’s important to comment that Cry1 and Cry2 toxins are the most studied toxins and that the cry1 gene is the most abundant in the nature (Bravo et al., 1998; Rosa-Garcia et al., 2008).

In the cause of S2122 strain, it didn’t present the 65 kDa protein because it probably could have silent genes and this characteristic will be studied with more details in the future. The high toxicity of the strains to P. xylostella suggests that the Cry1Ab, Cry1Ac and Cry1C toxins present in the strains can be responsible to toxicity against this insect (Monnerat et al., 1999).

This crystals morphology of the strains was similar to B. thuringiensis kurstaki HD-1 as described previously (Praça et al., 2004; Monnerat et al., 2007; Santos et al., 2009; Sezen et al., 2010). The bypiramidal crystals may be related to the presence of Cry1 proteins and show the activity against lepidopteran and coleopteran insects, the cuboidal are associated to Cry2 proteins, which show the activity to lepidopterans and dipterans (Dankocsik et al., 1990; Wu et al., 1991; Bravo et al., 1998; Praça et al., 2004; Monnerat et al., 2007; Xie et al., 2010). Cuboidal crystals appear in the studied strains, although this crystal is known as toxicity to coleopteran insects B. thuringiensis strains characterized in this study can be used in the formulation of new bioinsecticide to manage the insect’s resistance and it’s also possible to be used endophytically for controlling P. xylostella.

3 Materials and Methods
3.1 Strains and culture conditions
Three strains of B. thuringiensis from the Collection of Invertebrate Bacteria of Embrapa Genetic Resources and Biotechnology were used in this work. These strains were obtained from soil and water samples from different parts of Brazil and were previously identified as pathogenic to Lepidopteran species of insects (http://plataformarg.cenargen. embrapa.br/rede-microbiana/colecoes-de-ulturas/colecao-e-bacterias-de-invertebrados/colecao-de-bacterias-de-invertebrados). B. thuringiensis subspecie kurstaki (Btk) HD-1, obtained from the Collection of Bacillus thuringiensis and Bacillus sphaericus of the Institute Pasteur, Paris, was used as standard.

All strains and HD-1 were grown in Embrapa medium (Monnerat et al., 2007), for 72 h., 200 rpm, 30℃ Then, they were centrifuged at 12,800 xg for 30 min, at 4℃ (BR4i centrifuge Jouan). The pellets, containing spores and crystals were frozen for 16 h and lyophilized for 18 h in Labconco model Lyphlock18 freeze-dryer.

3.2 Bioassays against P. xylostella
Bioassays were carried out using second instar larvae of P. xylostella reared in the laboratory at (26 ± 2)℃, (60 ± 10)% humidity and photophase of 12 h (Medeiros et al., 2003). Two kinds of bioassays were carried out, the selective one to confirm the toxicity of the strains and the quantitative one to determine the lethal concentration required to kill 50% of the tested insects (LC50).

The selective bioassays were performed by the procedure described by Tabashnik et al (1990) with some modifications. Pieces of cabbage (Brassica oleracea L.) leaves (6 cm × 3 cm) were dipped vertically for 10 minutes in spore/crystal powder diluted 10% in distilled water. Leaves were then air dried vertically for 1 h at 25℃ then placed in a Petri dish (80 mm× 15 mm) with 10 larvae, in a total of tree replicates. The procedure of the selective bioassay was the same for each strain. After 48 h the surviving larvae were transferred to a new leaf and the mortality was assessed. Larval mortality was assessed again at day five (Monnerat et al., 1999) and the rate of mortality was determined and compared by variance analyses using Sigmastat analyses statistic (Kuo et al, 1992).

In order to determine the LC50, 6 concentrations (between 100 µg/mL and 0.01 µg/mL) of the lyophilized mixture of spores and crystals were diluted in sterile water. Then, the cabbage leaves were dipped in each concentration as described for the previous bioassay. The bioassays were repeated three times. The mortality data were evaluated on the fifth day analyzed by Probit analyses and LC50 (Robertson et al., 2002) was determined and analyzed by variance analyses (Kruskal-Wallis) using Sigma Stat analyses statistic (Kuo et al., 1992).

3.3 Characterization of the B. thuringiensis strains toxic to P. xylostella
3.3.1 SDS-PAGE (Polyacrylamide gel electrophoresis)
The proteins were extracted according to Lecadet et al (1991). The molecular mass and integrity of the proteins were determined by SDS-PAGE 10% (Laemmli, 1970). The electrophoresis was realized in Hoefer miniVE vertical electrophoresis system – Amersham Pharmacia in voltage of 120 V three hours and 30 minutes. The gel was stained and fixed in 40% methanol, 10% acetic acid and Coomassie blue (0.1%) for about 16 h, under slight shaking and it was distained in 40% methanol and 10% acetic acid for 2 h, with agitation. The HD-1 strain of B. thuringiensis subsp. kurstaki was used as control.

3.3.2 DNA sample preparation and PCR
The strains were grown on Embrapa agar medium (Monnerat et al., 2007) for 14~16 h, at 30℃ and DNA was extracted as described by Bravo et al. (1998). Molecular characterization through PCR was performed to identify the toxin-coding genes, by using a variety of oligonucleotide pairs specific for the following genes/gene families: cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry13, cry14, cry17, cry19, cry21, cry24, cry25, cry27, cry29, cry30, cry32, cry39, cry40, cyt1 and cyt2 (Ceron et al., 1994; Ceron et al., 1995; Ben-Dov et al., 1997; Bravo et al., 1998; Ibarra et al., 2003). 15 µL of the supernatant was used as DNA sample in the PCR mixture containing 2.5 U Taq DNA polymerase enzymes (5.0 U), 0.5 µM of each primer used in the reactions and 0.2 mM of deoxynucleoside triphosphates mix in a total volume of 50 µL. The amplification reactions were carried out in PTC-100 (Programmable thermal Controller – MJ Research, inc). The PCR products were visualized in 1% agarose gel in tris-borate buffer at 150 V for 1 h and stained with ethidium bromide (10 µg/mL).

3.3.3 Scanning and Transmission electron microscopy
In order to observe the spores and crystals produced by the different strains in scanning electronic microscopy, the strains were grown in sporulation medium (Lereclus et al., 1995) at 28℃ for 72 h. Crystals were isolated by centrifugation in sucrose gradients (Chang et al., 1993). These preparations were washed and lyophilized before being deposited on a metallic support. The samples were covered with gold for 180 seconds, using a sputter coater (EMITECH model K550) and observed in a ZEISS model DSM 962 scanning electron microscope.

To observe the spores and crystals of B. thuringiensis by transmission electron microscopy, the culture was centrifuged at 8 000 rpm and fixed in 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer at pH 7.0 for 4 h and maintained under agitation overnight. Fixed bacteria were washed three times for 10 minutes with cacodylate buffer and twice with water. The bacteria were post-fixed in 2% osmium tetroxide and 0.2 M cacodylate buffer for 1 h in dark conditions. After washed with cacodylate buffer for three times and twice with water for 10 minutes, the pellets were dehydrated in a 10 to 100% ethanol series for 20 minutes for each step and twice in pure ethanol for completely dehydration. The bacteria were embedded in Epon resin at 4℃ under agitation and incubated at 70℃. The ultrathin sections (60 µm) were stained in 2% uranyl acetate at dark conditions for 1 hour. The ultrathin sections were washed in water and observed in a JEOL1011C Transmission Electron Microscope. Between changes of solution, the samples were centrifuged at 8000 rpm for few seconds to form the pellets.

4 Conclusion
The strains S1905, S2122 and S2124 were toxic to P. xylostella and present different genes. These strains are important to the development of new bioinsecticides that can be used in the control of P. xylostella and the management of insect resistance in the field.

Authors’ contribution
LP conducted all the research for this paper and prepared the manuscript. CC participated in bioassays and in biochemistry and molecular characterization. AG conducted the scanning and transmission electron microscopy. RM reviewed the manuscript and coordinated the project.

Acknowledgement
This work was financially supported by FAP-DF and Embrapa.

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