Selection of strains from Bacillus thuringiensis genes containing effective in the control of Spodoptera frugiperda.  

Ana Rita Nunes Lemes , Suzana C. Marucci , Juliana R. V. Costa , Eliane C. C. Alves , Odair A. Fernandes , Manoel Victor F. Lemos , Janete A. Desidério
Univ Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 14884-900, Jaboticabal, São Paulo, Brazil
Author    Correspondence author
Bt Research, 2015, Vol. 6, No. 1   doi: 10.5376/bt.2015.06.0001
Received: 24 Oct., 2014    Accepted: 29 Jan., 2015    Published: 31 Jan., 2015
© 2015 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.
Abstract

The bacterium Bacillus thuringiensis (Berliner) is an important entomopathogen that can replace chemical pesticides in crops. In addition to the endotoxins found in B. thuringiensis, new proteins have been studied recently, such as Vip and Chitinase, which can enhance toxic effects by acting synergistically. Thus, the objectives of this work were to select new B. thuringiensis isolates carrying cry1Fa, vip3Aa, and chi; to verify polymorphisms based on PCR-RFLP  analysis; and to evaluate mortality and existence of synergistic effects of these genes on Spodoptera frugiperda larvae. A total of 114 isolates were subjected to screening via PCR to detect the vip3Aa, cry1Fa, and chi genes. Neonatal larvae of S. frugiperda were bioassayed using isolates containing one, two or all three genes and were compared to the standard strains. The PCR-RFLP analysis showed the absence of polymorphisms in these gene regions. The B. thuringiensis isolates from different regions of Brazil contained different combinations of the cry1Fa, vip3Aa, and chi genes. The presence of vip3Aa appeared to act synergistically with chi, and the combination of all three genes caused increased mortality in S. frugiperda larvae. Due to their insecticidal potential, these isolates are a prospective source of genes that can be used to genetically modify crops to increase the efficacy of pest control.

Keywords
Biological control; Vip3Aa; Cry1Fa; chitinase

Background
Cases of resistance to Bacillus thuringiensis Berliner endotoxins among insect populations have been reported as a result from selection under laboratory and field conditions. In cases where resistance has evolved, the insects have shown mutations in genes encoding proteins involved in various stages of the mode of action (Heckel et al., 2007; Tabashnik et al., 2009). In contrast to the variety of resistance mechanisms found in strains of insects selected in the laboratory, only one important mechanism of insecticide resistance, the alteration of binding sites, has been detected in insects that have evolved resistance under field or greenhouse conditions (Ferré and Van Rie 2002; Wang et al., 2007; Tabashnik et al., 2011). Thus, searching for new families of insecticidal toxins that present different modes of action compared to that of the Cry proteins, which are broadly used in the biological control of insects, is a strategy that has been employed to circumvent and/or delay the development of resistance. The discovery of new B. thuringiensis genes is crucial to develop new bioinsecticides and to provide a source of different genes for constructing genetically modified plants.
In the case of Bt maize, which contains the cry1Fa gene and is currently commercially available to control Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), new genes associated to cry1Fa gene could be utilized with the aim of obtaining pyramidized maize plants to manage the development of resistance to Bt maize in S. frugiperda populations. One new insecticidal protein family that could be employed is Vip3A, which was first described by Estruch et al. (1996). The genes encoding this protein family exhibit a high specificity for lepidopterous insects and are highly active against these insects; therefore, they are considered alternative genes for controlling the development of resistance to Cry proteins.
Other proteins that have been investigated for their potential use in the control of insects include chitinases (Chi) (Arora et al., 2003; Ding et al., 2008), which are also present in B. thuringiensis isolates. Studies have demonstrated that chitinase hydrolyzes the peritrophic membrane chitin in the insect gut, which causes the formation of pores and facilitates the binding of delta-endotoxins to their receptors located in the gut epithelium, thus increasing the toxicity of B. thuringiensis.
The objectives of this work were to select new B. thuringiensis isolates and to verify the existence of synergistic effects among the cry1Fa, vip3Aa, and chi genes in the control of S. frugiperda larvae. The gene vip3Aa appeared to act synergistically with chi and the combination of all three genes increased mortality of S. frugiperda. These results suggest potential applications for biological control as well as construction of pyramidized plants. Additionally, the different modes of action of these genes could be used to increase toxicity and to reduce the likelihood of the development of resistant insect populations.
1 Results
1.1 Detection of the cry1Fa, vip3Aa, and chi genes
The presence of the cry1Fa, vip3Aa, and chi genes in the isolates was confirmed via DNA amplification using pairs of primers specific for the genes (Table 1). It was verified that the isolates produced an amplification product of the expected size (553 bp) for the cry1Fa gene, as did the positive control, B. thuringiensis var. aizawai HD137. For the vip3Aa gene, the expected 2439-bp product was obtained from the isolates and from the positive control, B. thuringiensis var. HD125. Finally, a chi gene product with the expected size of 2031 bp was obtained from the isolates and from the positive control, B. thuringiensis var. alesti (Figure 1). Conversely, no amplicons were obtained from B. thuringiensis var. tenebrionis (the negative control), a Coleoptera -specific strain that does not carry the three studied genes.

 

 

Table 1 Specific oligonucleotide primers for the cry1Fa, vip3A, and chi genes

 

 

 

Figure 1 Agarose gel electrophoresis of the gene amplification products: (A) cry1Fa, (B) vip3Aa and(C) chi, from samples isolated from Bacillus thuringiensis; M: 1 kb DNA ladder molecular size marker; PC: positive control.

 

The PCR analysis revealed that among the 114 isolates,all three genes (cry1Fa + vip3Aa + chi) were amplified in only 12 (10.53%); 79 isolates (69.30%) amplified the cry1Fa gene only; 26 (22.81%) amplified vip3Aa only; 54 (47.37%) amplified the chi gene only; 5 (4.38%) amplified the combination of cry1Fa + vip3Aa; 33 (28.95%) amplified the combination of cry1Fa + chi; and 1 (0.88%) amplified the vip3Aa + chi combination (Figure 2). The PCR detection of the cry, vip, and chi genes revealed that cry1Fa and chi had the highest gene profile frequencies in the collection.

 

 

Figure 2  The frequencies of the cry1Fa, vip3Aa and chi genes(Lepidoptera-specific) in 114 isolates of Bacillus thuringiensis.

 

1.2 Polymorphism analysis of the cry1Fa, vip3Aa, and chi genes via PCR-RFLP
After the cry1Fa, vip3Aa, and chi genes were amplified from the isolates, we used the PCR-RFLP technique to analyze the polymorphisms in the sequences from the 12 isolates from which all three genes had been successfully amplified.
No differences in the obtained banding patterns were found among any of the isolates. This result suggests that there are no polymorphisms in the amplified fragments that could be detected using the applied restriction enzymes; therefore, it was not possible to determine any associations between polymorphisms and the mean mortality of S. frugiperda.
1.3 Bioassays
None of the B. thuringiensis isolates used in the bioassays caused 100% mortality among the S. frugiperda larvae, but several isolates exhibited efficacies greater than 70%, which can be considered efficacious (Campanini et al., 2008). The mortality rates varied among the different gene combinations throughout the evaluation period (Table 2). This finding suggests that the effectiveness in causing mortality may be related to a synergistic mechanism among the three genes, which was also found by Costa et al. (2010) for Aedes aegypti (L.) (Diptera: Culicidae).
All the treatments that contained the cry1Fa + vip3Aa + chi gene combination produced mortality rates between 60% and 94%. Isolates I_10 and I_17 caused the highest mortality (Table 2). Additionally, there were differences in mortality between the treatments with the pair-wise combinations cry1Fa + vip3Aa, cry1Fa + chi and vip3Aa + chi; these combinations resulted in 80%, 12%, and 62% mortality, respectively. In comparison, when applied individually, cry1Fa, vip3Aa, and chi caused 16%, 29%, and 2% mortality, respectively, which was significantly different compared with the isolates containing two or three genes (Table 2). Thus, it was found that vip3Aa, cry1Fa, and chi alone were not effective in controlling S. frugiperda, which was also true for the cry1Fa + chi combination. Furthermore, the synergy of the chi + vip3Aa combination appeared to produce a stronger effect compared with the treatment with vip3Aa alone.
Neither the Coleoptera-specific standard strain B. thuringiensis var. tenebrionis, which was used as a negative control, nor the blank (water) treatment caused any larval mortality (Table 2).

 

 

Table 2 Gene content of B. thuringiensisand S. frugiperda larval mortality.

 

2 Discussions
The research on B. thuringiensis conducted during the past 20 years has shown that this species is more complex than was originally thought. Rather than killing insect larvae only with the well-known insecticide crystal proteins (ICP), this bacterium also produces a series of other substances that aid in increasing mortality, including b-exotoxin, chitinase, hemolysin, phospholipase C, vegetative insecticidal proteins, and most likely other compounds that are yet to be discovered (Schnepf et al., 1998; Donovan et al., 2001; Lin and Xiong, 2004 ).
The pioneering research that characterized the chitinase protein distribution in B. thuringiensis was performed by Liu et al. (2002), who showed that chitinase was produced by B. thuringiensis isolates and that some of the chitinases exhibited the potential to increase the toxicity of the bacterium for the control of Spodoptera exigua (Hubner, 1808) (Lepidoptera: Noctuidae) larvae. Among the 70 strains tested, 38 exhibited toxicity, indicating their potential to be applied synergistically with other B. thuringiensis crystal proteins. Thus, the majority of isolates tested in this work were confirmed to produce chitinase, and the isolates containing all three genes caused the highest mortality, which suggests a possible synergy among the genes. The chitinase protein may have caused the partial digestion of the insect’s peritrophic membrane, which may have facilitated the action of the Cry1Fa and Vip3A proteins.
Multiple binding sites for Cry1 toxins are present in many insects (Ferré et al., 1991; Luo et al., 1999; Hernández-Martínez et al., 2014). The results obtained by Luo et al. (1999) demonstrated that there are at least two union sites with high affinity for the Cry1 toxin in S. frugiperda. The work performed by Sena et al. (2009) complemented the results of Luo et al. (1999) by demonstrating a union between Cry1Ab and Cry1Fa in S. frugiperda which reinforced the idea of competition for the sole union site in the gut. In relation to interactions with vip3 gene, this work evaluated the competition between the cry1 and vip3 genes in S. frugiperda, demonstrating that it is possible to combine vip with cry. These isolates could be used to isolate, to sequence, and to produce Cry1 and Vip3A proteins. Therefore, these genes may be used in the construction of pyramidized plants which could represent an alternative for combating the resistance of insects to toxins present in the currently used genetically modified (GM) plants that carry usually only one toxin. For example, S. frugiperda showed resistance to the Cry1F protein expressed in the event TC1507 of transgenic maize in Puerto Rico (Storer et al., 2012).
We suggest that vip3A possibly acts more synergistically with cry1Fa, as this combination resulted in higher mortality (80%), similar to the isolates that contained all three genes. The isolates containing individual genes caused lower larval mortality. This difference may be related to a possible synergy between the genes, in which the presence of the vip3A gene, in different combinations with the other genes, appears to increase the mortality of S. frugiperda larvae.
It is possible that chitinase, along with the Cry1Fa and Vip3A proteins, may cause mortality by partially digesting the insect’s peritrophic membrane, thus aiding in toxin penetration. However, we could not verify the action of chitinase in this work. The cry1Fa + vip3A combination resulted in 80% mortality, which was not significantly different from that caused by the isolates containing all three genes. Similar findings were reported by Costa et al. (2010), who found that the increase in the insecticidal activity of the isolated genes was not associated with the presence of chi, which was most likely due to the action of other toxins involved; i.e., the differences in mortality may also be related to the gene contents of each isolate.
The results showed that the Vip3Aa protein presented a synergistic effect with the protein Cry1Fa that probably increased the mortality of S. frugiperda through binding different union sites in this insect’s gut cells (Senna et al., 2009). Given the frequency of resistance observed, a possible strategy to manage resistance in field populations may be the use of new isolates of B. thuringiensis with different genetic profiles, such as those found in the isolates examined in this study. Thus, these proteins can be applied in the development of pyramidized maize plants, which could drastically reduce the likelihood of the development of resistance. Overall, the isolates were more efficient than the positive controls in causing mortality in S. frugiperda larvae.
The search for new isolates that contain genes with different modes of action in the insect’s gut can also aid pest management programs in avoiding or reducing the probability of the development of resistance to Bt toxins. This possibility provides the rationale for the utilization of B. thuringiensis-based products (Arantes et al., 2002).
The results of the polymorphism analysis also indicate the absence of random alterations in the studied genes that could be detected using the selected restriction enzymes and similarities between the isolates in relation to the amplified gene regions for all three proteins. However, it is important to note that the presence of a gene does not mean that it is expressed at equal levels or that it does not contain an unidentified polymorphic region.
The long-term success of crops expressing insecticidal B. thuringiensis proteins depends largely on reducing the capacity of insects to develop resistance to these proteins and, thus, to circumvent mortality. Therefore, it is important to seek new isolates with different profiles to diversify the gene expression of proteins in the available cultivars. So this work is essential to select isolates through selective bioassay with different genes. Therefore, the strategy of combining three distinct genes is promising. The relationship between toxicity and content of genes of isolates of B. thuringiensis studied suggests joint action. The synergy between genes results in an increase in the toxicity of the isolates to S. frugiperda larvae.
Hence, these genes can be used in the construction of pyramidized plants containing genes expressing proteins with different modes of action because Cry and Vip do not compete for the same binding sites. The assays performed in this study showed that isolates I_10 and I_17, which contained all three of the studied genes, can be used as sources of donor genes to be expressed in maize plants and can be used in the production of bioinsecticides for the control of this pest.
3 Materials and Methods
3.1 Insects
S. frugiperda larvae from a laboratory colony maintained for four years at the Applied Ecology Laboratory of UNESP, Jaboticabal campus, Brazil, were used. The insects were reared using an artificial diet (larva) or 5% honey (adults) and were maintained under controlled conditions (25 ± 1ºC, 70 ± 10% RH and a 14 h photophase) (Barreto et al., 1999).
3.2 Bacterial isolates and cultivation conditions
The B. thuringiensis isolates are part of the collection maintained at the Laboratory of Bacterial Genetics and Applied Biotechnology (LGBBA) of UNESP, Jaboticabal campus. The isolates were originally collected in soils from different states in the southeastern region of Brazil (São Paulo, Minas Gerais, and Paraná states).
We analyzed 114 isolates and the following four standard strains: B. thuringiensis var. alesti, B. thuringiensis var. HD125 and B. thuringiensis var. aizawai HD137, which were used as positive controls and B. thuringiensis var. tenebrionis, which was used as a negative control in the bioassays. All the isolates, including the standard strains, were multiplied in nutrient agar medium (meat extract 3 g/l, bacteriological peptone 5 g/l and agar 15 g/l) and incubated in a BOD chamber for 12 h at 30°C, followed by the extraction of total DNA.
3.3 PCR amplification of the vip3Aa, chi, and cry1Fa gene regions
Total DNA was extracted using the InstaGene Matrix kit (BioRad, Richmond, CA, USA) according to the manufacturer's recommendations. To detect the cry1Fa, vip3Aa, and chi genes from the B. thuringiensis isolates, we employed oligonucleotide primers developed by the LGBBA research group at FCAV/UNESP, Loguercio et al. (2002), and Lin and Xiong (2004), respectively.
The amplification reactions for the cry1Fa, vip3Aa, and chi genes were optimized according to the sequences of the primers and contained 1X buffer solution, 1.5-2 mM MgCl2, 200-250 µM dNTPs, 0.3-1 mM of each primer, 1 U of Taq DNA polymerase, 2-3 µl of the DNA sample and sterilized, distilled water (q.s. 20 µl). The PCR cycling conditions were optimized according to the annealing temperatures of the individual oligonucleotide primers. The following conditions were used for the cry1Fa gene: an initial denaturation step for 5 min at 95ºC followed by 30 cycles of 1 min at 95ºC for denaturation, 1 min at 50ºC for annealing and 1 min at 72ºC for extension, with a final step for 5 min at 72ºC to complete the extension. For the vip3Aa gene, the conditions consisted of an initial denaturation step for 2 min at 94ºC followed by 30 cycles of 30 s at 94ºC for denaturation, 45 s at 53ºC for primer annealing and 1 min at 72ºC for extension, with a final step for 5 min at 72ºC to complete the extension. Finally, the conditions used for the chi gene involved an initial denaturation step for 5 min at 94ºC followed by 30 cycles of 1 min at 94ºC for denaturation, 1 min at 48ºC for primer annealing and 1.5 min at 72ºC for extension, with an additional step of 10 min at 72ºC to complete the extension. At the end of the cycling program, the samples were maintained at 10ºC.
The amplified fragments were analyzed in agarose gels that contained ethidium bromide (0.5 µg/ml): 1.5% gels for the cry1Fa and vip3Aa genes and 1% gels for the chi gene. The samples were compared to a 1-kb DNA ladder molecular marker (Fermentas, Vilnius, Lithuania), then visualized under UV light and photographed using GEL DOC 2000 (BioRad) and Quantity-one software (Bio-Rad, New York, USA).
3.4 Detection of polymorphisms
The restriction enzymes used for the PCR-RFLP analyses were selected using the pDRAW32 program (http://www.acaclone.com) based on the sequences of the studied genes. Bromophenol blue (0.5%) in glycerol (50%; 4 µl) was added to the digested products. After electrophoresis using 1X TBE buffer (89 mM Tris, 89 mM boric acid and 2.5 mM EDTA, pH 8.2), the agarose gels (1%) were analyzed using the GEL DOC 2000 documentation system (Bio-Rad).
The reactions using the chi primers were performed in a total volume of 10 ml, containing 4 ml of the amplified product from each isolate, 1 ml of 10X buffer, 1 ml of enzyme (10 U), and 4 ml of Milli-Q sterilized water. The restriction reactions for the vip3Aa gene vip5/vip6 sequence were also performed in a volume of 10 ml, containing 2 ml of the amplified product from each isolate, 1 ml of 10X buffer, 1 ml of enzyme (10 U), and 6 ml of Milli-Q sterilized water. Finally, for the cry1Fa gene, the total reaction volume of 10 ml contained 2 ml of the amplified product from each isolate, 1 ml of 10X buffer, 1 ml of enzyme (10 U), and 6 ml of Milli-Q sterilized water.
The reactions were incubated at 37ºC, after which 3 ml of each reaction was added to 3 ml of sample buffer, and this mixture was subjected to electrophoresis in a 1% agarose gel. Each gel also contained a sample of the non-digested amplified product and the 1-kb DNA ladder molecular marker to allow a comparison of the results.
3.5 Bioassays
The bacterial isolates and standard strains used in this work (Table 2) were cultivated in Petri dishes containing Agar Nutrient medium and were incubated for 7 days at 30ºC. To prepare spore/crystal solutions, bacterial colonies at the surface of the medium were collected and transferred to centrifuge tubes (Corning, New York, USA) containing 9 ml of Milli-Q sterilized water and 0.005% TWEEN-20®. After complete homogenization via vortexing, the spores were diluted and quantified using a Neubauer chamber (Barreto et al., 1999), which allowed the solutions to be standardized to a concentration of 3 × 108 spores/ml.
To purify the Vip3Aa and Chi proteins, aliquots of the spore solutions from the B. thuringiensis samples were inoculated into Petri dishes containing Terrific Broth (TB) solid culture medium added to agar. The Petri dishes were incubated at 30°C for approximately 20 h. Subsequently, a pre-culture of the sample was prepared, for which one colony was collected, incubated in a 125 ml Erlenmeyer flask containing 10 ml of TB medium and placed in an incubator shaker (New Brunswick model G25) at 275 rpm and 30°C. The OD595 nm was recorded every 20 min using a spectrophotometer (Beckman DU 640B), with expected values of 0.3 for each sample.
After the spectrophotometric measurements were performed, 1 ml of the pre-culture of each sample was inoculated into 40 ml of TB culture medium and shaken at 275 rpm (30°C) for 12 h. The samples were then centrifuged (Beckman J2-21) at 6368 x g for 20 min at 4°C, and the supernatants were stored until later use in selective bioassays.
The selective bioassays were performed using S. frugiperda neonatal larvae. The larval diet was poured into a 16-cell insect-rearing tray. After the diet solidified, 300 ml of a bacterial suspension containing one of the following solutions: Cry/VipAa/Chi, Cry/Vip3Aa, Cry/Chi, or Vip3Aa/Chi was added, according to the specified treatment. After this mixture dried at room temperature (ca. 25°C), one larva was transferred directly onto the surface of the diet in each cell using a fine brush. Immediately after the larvae were transferred, the trays were sealed with plastic film and were stored in a room maintained at 26 ± 2ºC.
A randomized block design was adopted and the bioassay was replicated four times (replicates). All treatments were evaluated using 32 larvae per treatment per replicate. Sterilized water was used in the negative control. The larval mortality was assessed 1, 3, 5 and 7 days after the larvae were transferred to the diet.
3.6 Statistical analysis
The data were subject to an analysis of variance, and the means were compared using SAS PROC MIXED (SAS Institute, 2004).
Authors' Contributions
ARNL, JAD and OAF conceived and designed the experiments. ARNL, SCM, and JRVC performed the experiments.  ECCA and JRVC contributed reagents/material/analysis tools. ARNL, JAD, MVFL,and OAF wrote the paper. All authors read and approved the final manuscript.
Acknowledgments
The National Council for Scientific and Technological Development (CNPq) for granting a scholarship.
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