Introduction
Natural phytosanitary products (NPs) and entomopathogens are important tools to control insect pests in agroecological production systems, and may be used separately or in combination. The combined use may have positive effects on the biological parameters of entomopathogens, such as growth, virulence, and pathogenicity, thereby increasing control efficiency. Alternatively, the combination may have negative effects, thus compromising pathogen action. There are also situations in which biological parameters remain unchanged when entomopathogens are associated with NPs.
In a study of non-sporulating bacteria and plant extracts, the rosemary plant Rosmarinus officinalis L. (Lamiaceae) had negative effects on Staphylococcus aureus and Bacillus cereus at different times and concentrations (Genena et al., Reference Genena, Hense, Smânia and Souza2008), whereas the rue plant Ruta graveolens L. (Rutaceae) inhibited the growth of several bacteria, including species of the genus Bacillus (Pereira et al., Reference Pereira, Rodrigues, Feijo, Athayder, Lima and Sousa2006; Mendes et al., Reference Mendes, Lima, Franco, Oliveira, Aleixo, Monteiro, Mota and Coelho2008). It has been reported that nicotine and rue at various concentrations negatively affect the growth and toxicity of the entomopathogenic bacteria Bacillus thuringiensis under specific conditions (Krischik et al., Reference Krischik, Barbosa and Reichelderfer1988). On the other hand, in a study of the effects of NPs at various concentrations on the spores and crystals of B. thuringiensis subsp. kurstaki HD-1, obtained from the commercial product Dipel WP®, most products affected bacterial growth when the mixture was incubated with water, and no negative effects on crystal toxicity were observed (Silva et al., Reference Silva, Alves, Martinelo, Formentini, Marchese, Pinto, Potrich. and Neves2012).
Although NPs are safer than synthetic chemical products, little is known about the interaction between NPs and entomopathogens, particularly entomopathogenic bacteria. Thus, it is necessary to determine the interactive effects and possible selectivity of different NPs used in agroecological production systems in order to minimize the effects on non-target organisms, such as B. thuringiensis, and maximize the efficiency of insect pest control in an economical and environmentally sustainable manner. Therefore, the objective of this study was to evaluate the effect of NPs on the spores and crystals of B. thuringiensis subsp. kurstaki S-1905, which exhibits different crystal types and insecticidal activity, under laboratory conditions.
Materials and methods
Effects of NPs on Btk S-1905 spore viability
The lyophilized strain Btk S-1905 was provided by the Brazilian Agricultural Research Corporation (Embrapa), National Centre of Genetic Resources and Biotechnology, Federal District, Brazil. The NPs were obtained in agroecological production supply shops, and were used at the recommended concentrations (RCs) established by the manufacturers; assays were performed with NPs in combination and individually (table 1).
Natural phytosanitary products, use, composition, and concentrations used in the experiments.
Conc., concentration ml l−1; RI, resistance inductor; INS, insecticide; FUN, fungicide; BIO, biofertilizer.
Combined application: NPs mixed with Btk S-1905
A suspension was prepared with 2.3 × 1019 spores ml−1, by weighing 5 mg of the lyophilized material and adding 50 ml of sterile distilled water. From this suspension, successive dilutions were prepared using sterile distilled water, and a suspension of 2.3 × 106 spores ml−1 was obtained. Aliquots (300 µl) of this suspension were added to Erlenmeyer flasks with 50 ml of the NP prepared at the RC in sterile distilled water. Five Erlenmeyer flasks were prepared (replicates) for each NP assessed (treatment). The flasks were stored in a horizontal shaker and the mixtures were incubated at 30 ± 2 °C and 150 rpm for 2 h. The pH values were measured before and after incubation. The mixture of each flask was inoculated in five spots of 5 µl per spot in three Petri dishes containing nutrient agar (NA) (Alves & Moraes, Reference Alves, Moraes and Alves1998). The Petri dishes were kept open in a laminar flow hood for 5 min to evaporate the excess water. Then, the dishes were closed and placed in an incubator at 30 ± 2 °C for 18 h, followed by the quantification of colony-forming units (CFU) ml−1 for each spot. Sterile distilled water was used as a control.
Individual application: NPs and Btk S-1905
The same procedure described for the combined application was followed to prepare the spore suspension. The NP solutions were prepared in parallel in five Erlenmeyer flasks (replicates) per treatment. Aliquots (100 µl) from each treatment were applied to the surface of the NA medium, in three Petri dishes and scattered with a Drygalski loop. The Petri dishes were kept open in a laminar flow hood for 5 min to evaporate the excess water, and then the Btk S-1905 suspension was inoculated, as described for the combined application. The incubation and evaluation procedures were performed as described for the combined application. In the control, sterile distilled water was added to the culture medium and the bacterial suspension was subsequently inoculated.
The data obtained in both experiments were analyzed using an analysis of variance (F test) and means were compared with that of the control using Tukey's test (P < 0.05). The statistical analyses were implemented in Sisvar® (Ferreira, Reference Ferreira2009).
Effects of NPs on Btk S-1905
The effects of NPs on the toxicity of Btk S-1905 were evaluated in vivo using a bioindicator insect, the caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). In order to verify morphological changes and/or crystal protein degradation, scanning electron microscopy (SEM) and denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) were used, respectively.
Anticarsia gemmatalis caterpillars were laboratory-reared in containers and fed an artificial diet, according to Hoffmann-Campo et al. (Reference Hoffmann-Campo, Oliveira and Moscardi1985). They were maintained in a controlled temperature chamber at 26 ± 2 °C with a relative humidity of 70 ± 10% and a 14-h photoperiod until reaching the 2nd instar.
Determination of the lethal concentration (LC 50 ) of Btk S-1905
The concentration of Btk S-1905 for the bioassay was determined by estimating the LC50 for A. gemmatalis. Bacillus thuringiensis subsp. kurstaki HD-1, obtained from a purified sample from the entomopathogen bank at Embrapa Soja, Londrina, Paraná, Brazil, was used as the standard. A total of 0.02 g of each lyophilized sample was diluted in 20 ml of sterile distilled water (1000 µg ml−1 ≈ 4.6 × 1018 spores ml−1). From this suspension, six dilutions were prepared at concentrations of 24, 32, 41, 53, 69, and 90 µg ml−1.
The artificial diet for A. gemmatalis was prepared free of anticontaminants and solidified into cubes with sides of approximately 1.5 cm, which were cut using a spatula. The cubes were submerged for 5 s in the dilutions at various concentrations and, after drying, were placed in 50-ml containers. The experimental design was completely randomized, with 20 replicates (recipients) per concentration (treatment) and three A. gemmatalis 2nd instar caterpillars per repetition. For the control diet, the cubes were submerged in sterile distilled water. Then, the recipient containers were closed and placed in incubator at 26 ± 2 °C with a relative humidity of 70 ± 10% and a photoperiod of 14 h. After 48 h, the caterpillars were transferred to containers with the artificial diet, without the treatment, and were evaluated daily until the sixth day.
The data were analyzed using Micro Probit 3.0 (Thomas & Sparks, Reference Thomas and Sparks1987) to determine the LC50.
Btk-S1905 toxicity on A. gemmatalis
From the lyophilized material, suspensions of Btk-S1905 were prepared in Erlenmeyer flasks (50 ml) with sterile distilled water at a concentration equivalent to the previously estimated LC50. Then, the NPs were added at the RC, and the mixtures were incubated in a horizontal shaker at 30 ± 2 °C, 150 rpm for 2 h. The pH values were measured before and after incubation.
Using micropipettes, 150-μl aliquots from the mixture in each flask were added to the surfaces of the 1.5-cm cubes used in the artificial diet for A. gemmatalis (free of anticontaminants) on Petri dishes. Each plate received three diet cubes and was kept open in a laminar flow hood for 5 min to evaporate the excess water and for spore and crystal deposition into the diet. Subsequently, each plate received 25 2nd instar caterpillars of A. gemmatalis. The experimental design was completely randomized with three repetitions and 75 caterpillars per treatment. For the control, identical lots of caterpillars were prepared, which received the NP (at the RC) and the Btk-S1905 suspension. A negative control was also prepared with sterile distilled water, which was applied on the artificial diet. The plates were placed in incubator at 26 ± 2 °C, 70 10% relative humidity, and a 14-h photoperiod, and evaluations were carried out to determine the number of dead caterpillars at the following time points: 12, 24, 48, and 72 h.
The data were analyzed using the analysis of variance (F test), and the means were compared using Tukey's test (P < 0.05) implemented in Sisvar® (Ferreira, Reference Ferreira2009).
Interactive effects of NPs on the crystals were calculated using the χ2 test, and classified as synergistic, antagonistic, and additive, according to Benz (Reference Benz, Burges and Hussey1971) and Koppenhofer et al. (Reference Koppenhofer, Brown, Gaugler, Grewal, Kaya and Klein2000). In this classification, a synergistic effect describes a system with two effective components that, in combination, produce an effect that is greater than the sum of the independent effects. An additive effect occurs when the effect of two components combined equals the sum of the effects of the individual components. An antagonistic effect occurs when the combination of the elements produces a smaller effect than that observed for the individual elements.
Morphology and integrity of Btk S-1905 crystals observed by SEM
A total of 5 mg of the lyophilized Btk S-1905 sample was weighed and diluted in 50 ml of sterile distilled water in Erlenmeyer flasks along with the NPs (at the RC), resulting in a mixture of approximately 2.3 × 1019 spores ml−1. The flasks were placed in a horizontal shaker and the mixtures were incubated at 30 ± 2 °C, 150 rpm, for 2 h. Then, 1.5-ml aliquots of each mixture were removed to prepare samples for SEM. The rest of each mixture was stored in an amber glass flask in a freezer at −10 °C. The aliquots of the mixtures were transferred to microcentrifuge tubes FANEM® Model 243 and centrifuged for 5 min at 8944 g , three times, to create a pellet. The supernatant was discarded, and the sedimented materials were fixed with a 2% paraformaldehyde, 2% glutaraldehyde, and phosphate buffer (0.1 M PO4) solution for 4 h. Subsequently, the samples were washed in phosphate buffer for 15 min three times, and fixed again with 1% osmium tetroxide (OsO4) for 2 h. Later, a second washing was carried out in phosphate buffer for 15 min, three times.
Using a stereoscopic microscope, the samples were fixed with historesin on glass slides and then dehydrated using an alcoholic sequence (alcohol 70%: 3 × 15 min, alcohol 80%: 3 × 15 min, alcohol 90%: 4 × 10 min, and alcohol 100%: 4 × 10 min) and with CO2 at the critical point. The samples were then mounted in stubs with silver and coated with gold for 3 min by the sputtering process using a BAL-TEC SCD 050 sputter. Samples were observed in high-vacuum mode with a 20 kV electron beam intensity by SEM, and images were recorded by digital microphotography.
Integrity of Btk S-1905 crystals analyzed by SDS-PAGE
Samples of the suspensions prepared as described in the section ‘morphology and integrity of Btk S-1905 crystals observed by SEM’ were analyzed by SDS-PAGE to check for the presence of Cry proteins. For that purpose, the crystals were solubilized and the protein was extracted according to the methods proposed by Lecadet et al. (Reference Lecadet, Chaufaux, Ribier and Lereclus1991).
In brief, 1.5 ml of bacterial suspension was transferred to an autoclaved 2-ml microcentrifuge tube. The tubes were centrifuged at 15,115 g for 20 min, the supernatants were discarded, and the pellets were washed with 1.5 ml of 0.5 M NaCl for 20 min. The tube walls were dried with filter paper after discarding the 0.5 M NaCl solution. The sediments were washed twice with 1.5 ml of 1 mM phenylmethyl fluoride sulfonyl (PMFS) and centrifuged at 15,115 g for 20 min. After discarding the 1 mM PMFS, the pellets were resuspended in 500 µl of 1 mM PMFS and stored at −20 °C. The spore–crystal suspension and NPs were analyzed by 10% SDS–PAGE, as described by Laemmli (Reference Laemmli1970). For this purpose, 15 µl of the spore–crystal preparation was used to load the gel and electrophoresis was performed at a voltage of 25 mA for 3 h. As a control, Btk-S1905 was incubated with sterile distilled water under the same conditions used for the NPs. Bacillus thuringiensis subsp. kurstaki (HD-1) was used as the standard.
Results
Effects of NPs on Btk S-1905 spore viability
Combined application: NPs mixed with Btk S-1905
The products Biogermex and Ecolife® significantly reduced colony formation by 99.8 and 100%, respectively, and Planta Clean significantly increased colony formation by 12.2%. For the remaining products, colony formation did not differ from that of the control (table 2).
Mean (±SE) CFU ml−1 of Btk S-1905, after incubation (30 ± 2 °C, 150 rpm, 2 h) with sterile distilled water and natural phytosanitary products (at the RC), initial and final pH, inoculation of nutrient agar culture medium in Petri plates, and incubation (30 ± 2 °C, 18 h).
Means (±SE) followed by the same lowercase letter in a column do not differ significantly by Tukey's test (P < 0.05).
1 Equation: (Mean (CFU per ml) treat/Mean (CFU per ml) control) × 100 − 100 where positive values indicate an increase in CFU ml−1 and negative values indicate a decrease when compared to controls.
VC, variation of coefficient.
Individual application: NPs and Btk S-1905
Negative effects on colony formation (CFU ml−1) were observed using Biogermex and Ecolife® individually, with significant reductions of 35.46 and 100%, respectively. For the remaining products, there were no significant differences in colony formation from that of the control (table 3).
Mean (±SE) CFU ml−1 after the inoculation of Btk S-1905, with natural phytosanitary products (at the RC) in the nutrient agar medium in Petri plates (30 ± 2 °C, 18 h).
Means (±SE) followed by the same lowercase letter in a column do not differ significantly by Tukey's test (P < 0.05).
1 Equation: Mean (CFU per ml) treat/Mean (CFU per ml) control × 100 − 100, where positive values indicate an increase in CFU ml−1 and negative values indicate a decrease compared to controls.
VC, variation of coefficient.
Effects of the natural products on Btk S-1905
The LC50 (95% confidence interval) values for Btk S-1905 and Btk HD-1 were 35 (31–39) µg ml−1 (slope 3.02 ± 0.39) and 31 (22–37) µg ml−1 (slope 2.80 ± 0.68), respectively, with no significant difference between the strains.
Btk-S1905 toxicity on A. gemmatalis
In the in vivo bioassay, the effects of Biogermex, Ecolife®, and Planta Clean were antagonistic to the action of crystal toxins; when these products were combined with Btk S-1905, the average mortality rates of A. gemmatalis were 35.30, 18.4, and 23.7%, respectively, which were all lower than the mortality for Btk-S-1905 (51.3%) and higher than those for the individual products (0% Biogermex, 3.0% Ecolife®, and 1.3% Planta Clean). The pH values for the aforementioned combinations ranged from acidic (Biogermex ≈ 3.89 and Ecolife® ≈ 2.90) to alkaline (Planta Clean ≈ 9.77) (table 4).
Average percent mortality (±SE) of Anticarsia gemmatalis caterpillars by Btk S-1905 (LC50) and natural phytosanitary products at the RC, isolated and combined, after incubation (30 ± 2 °C, 150 rpm, 2 h), at 12, 24, 48, and 72 h and total, initial, and final pH values.
Means (±SE) followed by the same lowercase letter in a column and uppercase letter in a row do not differ significantly according to Tukey's test (P < 0.05).
1 Percentage of caterpillars that died in 72 h.
2 Expected mortality (EM) was calculated by the equation EM = M1 + M2(1 − M1), where M1 = mortality caused only by the entomopathogen; M2 = mortality caused only by the pesticide.
3 χ2 = (OM − EM)2/EM, where χ2 (tabulated) = 3.84, degrees of freedom = 1, P ≤ 0.05, OM, observed mortality; EM, expected mortality; VC, variation of coefficient; VC 1, treatment; VC 2, time; VC 3, total mortality.
Btk S-1905, Bacillus thuringiensis subsp. kurstaki S-1905; CE, chrysanthemum extract; BG, Biogermex; ECO, Ecolife®; PI, Pironim; PC, Planta Clean; Additv., additive; Antag., antagonistic.
The remaining products had an additive effect on crystal protein toxicity; chrysanthemum and Pironim extracts, used separately, resulted in the highest A. gemmatalis mortality rates (74.8 and 61.0%, respectively), which were significantly higher than those of the Btk S-1905 treatment (51.3%) (table 4).
Morphology and integrity of Btk S-1905 crystals observed by SEM
For all treatments, Btk S-1905 crystals did not exhibit morphological changes, indicating that in the study conditions, the products did not affect the crystal proteins (fig. 1).
Scanning electron microscopy of the mixture of Btk S-1905 spores and crystals and natural phytosanitary products (in the RC) after incubation (30 ± 2 °C, 150 rpm, 2 h). 1 – control, 2 – Pironim, 3 – Biogermex, 4 – Ecolife®, 5 – chrysanthemum extract, 6 – Planta Clean; CE, chrysanthemum extract; S, spore; BC, bipyramidal crystal; CC, cuboid crystal; SC, spherical crystal.
Integrity of Btk S-1905 crystals analyzed by SDS–PAGE
The degradation of proteins was not observed in an SDS–PAGE analysis, as evidenced by the lack of fragments showing a molecular weight below 10 kDa in any of the treatments, supporting the results of the SEM analysis (fig. 2).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Btk S-1905 proteins and natural products (in the RC) after incubation (30 ± 2 °C, 150 rpm, 2 h): 1 – molecular marker, 2 – Bacillus thuringiensis subsp. kurstaki HD-1, 3 – control –Btk S1905 (water), 4 – Biogermex, 5 – Planta Clean, 6 – Pironim, 7 – chrysanthemum extract, 8 – Ecolife®.
Discussion
For the combined application of NPs mixed with Btk S-1905, the results observed for Biogermex and Ecolife® were similar to those of Silva et al. (Reference Silva, Alves, Martinelo, Formentini, Marchese, Pinto, Potrich. and Neves2012), who examined B. thuringiensis subsp. kurstaki HD-1 spores obtained from the commercial product Dipel WP® and NPs. According to Silva et al. (Reference Silva, Alves, Martinelo, Formentini, Marchese, Pinto, Potrich. and Neves2012), these products significantly reduced the CFU ml−1, regardless of concentration. The authors also observed a significant reduction in CFUs (24.0%) using the Pironim product at the RC, in contrast with the results of this study (3.8%) (table 2).
The individual applications of NPs and Btk S-1905 spores on the surface of culture medium had a negative effect on the CFU ml−1 when using Biogermex and Ecolife® (table 3).
The negative effects observed in both experiments may be related to variation in pH, since products with acidic pH values, such as Biogermex and Ecolife®, reduced the CFU ml−1. In contrast, the CFU ml−1 increased for chrysanthemum extract and Planta Clean (which have basic pH values) (tables 2 and 3).
According to a study of the effect of pH on the germination of B. thuringiensis spores in the soil, a higher acidity results in a more substantial reduction in germination, and germination does not occur at pH values of <5 (Petras & Casida, Reference Petras and Casida1985). The influence of pH on spore germination was also studied by Wilson & Benoit (Reference Wilson and Benoit1993), who assessed the action of intestinal fluid components, alone and in combination, on B. thuringiensis subsp. kurstaki spores, and observed the cessation of germination when the alkaline pH was removed. Moreover, it is important to highlight that in a similar study, Biogermex and Ecolife® showed acid pH values and significantly reduced the CFU ml−1 of B. thuringiensis subsp. kurstaki HD-1 at three concentrations (Silva et al., Reference Silva, Alves, Martinelo, Formentini, Marchese, Pinto, Potrich. and Neves2012).
The Pironim product had an acidic pH, as did the Biogermex and Ecolife® products, but did not significantly reduce the CFU ml−1 (table 3), indicating that pH should not be considered the main factor determining spore germination and CFU ml−1. Three distinct processes are involved in the germination of bacterial spores, i.e. the presence of specific receptors on the inner membrane of the spore, the presence of ion channels, and the action of lytic enzymes in cell cortex degradation. Thus, germination may be triggered by the presence of amino acids, sugars, and nucleosides that bind to specific receptors as well as salts, high pressure, and Ca+2 ions (Setlow, Reference Setlow2003).
In addition to the effect of pH, the negative results observed for Biogermex and Ecolife® in both experiments may be related to the composition and mode of action of these products. Ecolife® consists of bioflavonoids, citrus phytoalexins, and ascorbic acid (Technical Bulletin), similar to the composition of Biogermex. Flavonoids and terpenoids act as defense mechanisms against insects and microorganisms (Dixon et al., Reference Dixon, Dey and Lamb1983; Cowan, Reference Cowan1999) and are able to bind to the cell wall, disabling it or even breaking down the cell membrane (Tsuchiya et al., Reference Tsuchiya, Sato, Miyazaki, Fujiwara, Tanigaki, Ohyama, Tanaka and Linuma1996).
Given the above, the reductions in CFU ml−1 observed for Biogermex and Ecolife® may be related to the prevention of the germination process and/or to the destruction of the bacterial membrane after germination. Such effects on cells are supported by similar results obtained for the effects of Ecolife® on cells of the Gram-negative phytopathogenic bacteria Ralstonia solanacearum and Xanthomonas axonopodis pv. manihotis, which exhibit a zone of inhibition proportional to the increase in product concentration (Motoyama et al., Reference Motoyama, Schwan-Estrada, Stangarlin, Fiore and Scarpim2003).
The effects of the NPs on the toxicity of Btk S-1905 on A. gemmatalis in this study were not in agreement with those of Silva et al. (Reference Silva, Alves, Martinelo, Formentini, Marchese, Pinto, Potrich. and Neves2012), who did not observe an effect of Ecolife® on the toxicity of Btk HD-1 in Dipel WP®. This difference may be explained by the fact that in the present study, a purified bacterium was used at a lower concentration known to be effective (LC50), while Silva et al. (Reference Silva, Alves, Martinelo, Formentini, Marchese, Pinto, Potrich. and Neves2012) used Btk HD-1 at the RC, half of the RC, and the twice the RC.
Bipyramidal, cuboid, and spherical crystals were observed, supporting the results of Medeiros et al. (Reference Medeiros, Ferreira, Martins, Gomes, Falcão, Dias and Monnerat2005) and Monnerat et al. (Reference Monnerat, Batista, Medeiros, Martins, Melatti, Praça, Dumas, Morinaga, Demo, Gomes, Falcão, Siqueira, Silva-Werneck and Berry2007). The bipyramidal crystal morphology is associated with the Cry 1 protein, which is effective against Lepidoptera and Coleoptera, and the cuboid crystals are associated with the Cry 2 protein, which is effective against Diptera and Lepidoptera (Silva et al., Reference Silva, Silva-Werneck, Falcão, Gomes, Fragoso, Quezado, Neto, Aguiar, de Sá, Bravo and Monnerat2004). These morphological types can provide important information regarding entomopathogenic activity for different orders of insects when analyzed in conjunction with biochemical and molecular analyses (Habib & Andrade, Reference Habib, Andrade and Alves1998; Who, Reference Who1999).
The solubilization of the crystal protein may occur in alkaline conditions (pH above 8) (Habib & Andrade, Reference Habib, Andrade and Alves1998), as expected for the Planta Clean product, but the mortality results for this NP indicated that the pH is not the only determinant of the solubilization of Btk S-1905 crystals (table 3).
For the treatments in this study, the proteins had two major polypeptides of 130 and 65 kDa (fig. 2). The 130-kDa polypeptides are considered characteristic of strains against Lepidoptera and Coleoptera, whereas the 65-kDa polypeptide is characteristic of strains infecting Lepidoptera and Diptera (Monnerat & Bravo, Reference Monnerat, Bravo, Melo and Azevedo2000).
Several factors can affect the insecticidal activity of B. thuringiensis, such as intestine structure and function, toxin diversity, protein structure and solubilization, interactions among toxins (Gill, Reference Gill1995), as well as the structure and process of spore germination (Liu et al., Reference Liu, Tabashnir, Moar and Smith1998).
Given the above, the antagonistic effects of Biogermex®, Ecolife®, and Planta Clean are probably associated with the binding of these products to the crystal protein, hindering solubilization in the insect intestine or preventing the binding of proteins to specific receptors in the intestinal membrane. When used separately, these products did not significantly affect insect mortality. In addition, SEM and electrophoresis analyses did not indicate morphological changes in crystal proteins. Nevertheless, faster action of the entomopathogen when mixed with a natural plant product may be related to the metabolite stressor effect, which makes insects more susceptible to bacterial toxins (Saito & Lucchini, Reference Saito and Lucchini1998).
With respect to the effect of metabolites on entomopathogenic bacteria, Lord & Undeen (Reference Lord and Undeen1990) studied the effects of tannins on B. thuringiensis subsp. israelensis toxicity and observed a reduction in the mortality of Aedes aegypti (Diptera: Culicidae) larvae when the bacteria and tannins were mixed without previous incubation, and an increase in mortality after incubation. According to the authors, the tannins can bind to intestinal proteolytic enzymes and consequently prevent the solubilization of crystals. However, after incubation, small amounts of proteins that may have been released from the crystal bind to tannins, reducing the action of tannins on the toxin.
A similar result was obtained in a study on the effects of tannins on B. thuringiensis subsp. kurstaki HD-73 toxicity to Heliothis virescens F. (Lepidoptera: Noctuidae) larvae (Navon et al., Reference Navon, Hade and Federici1993). According to the authors, the LC50 for the bacteria alone was 27.0 ng g−1 and for the bacteria mixed with tannins at 3.2 mg g−1 was 59.1 ng g−1, demonstrating that δ-endotoxin activity was antagonized or possibly inactivated.
In a study of B. thuringiensis combined with azadirachtin at known sublethal concentrations, the effects on different instars of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) were complementary, and the metabolite facilitated B. thuringiensis action (Singh et al., Reference Singh, Rup and Koul2007). Likewise, Rajguru et al. (Reference Rajguru, Sharma and Banerjee2011) reported that Btk associated with aqueous extracts of various plants causes increased mortality in Spodoptera litura (Lepidoptera: Noctuidae) larvae on the sixth day of evaluation.
Factors other than secondary compounds may be related to the reduction in larval mortality for mixtures of B. thuringiensis and other compounds, such as the presence of particulate matter (Ignoffo et al., Reference Ignoffo, Garcia, Kroha, Fukuda and Couch1981; Lord & Undeen, Reference Lord and Undeen1990) and solutes, such as phosphoric acid and citric acids, which may be stressors for insects, thereby reducing food and consequently toxin intake (Lord & Undeen, Reference Lord and Undeen1990). Cations, physicochemical and biochemical factors, such as pH, ionic and divalent forces (Tran et al., Reference Tran, Vachon, Schwartz and Laprade2001), temperature (Vachon et al., Reference Vachon, Schwartz and Laprade2006), and insect bacterial flora (Broderick et al., Reference Broderick, Raffa and Handelsman2006) are also associated with reductions in larval mortality in mixtures of B. thuringiensis.
An effect of pH on crystal proteins and, consequently, on mortality was not observed in this study for treatments with an antagonistic effect, with pH values ranging from acidic (2.90 for Ecolife® + Btk S-1905 and 3.89 for Biogermex + Btk S-1905) to alkaline (9.77 for Planta Clean + Btk S-1905) (table 4). Similar results were reported for the effect of pH and chlorine variation on Dipel SC and Thuricide 32B biopesticides, where neither product affected mortality, regardless of the presence of chlorine (Neisess, Reference Neisess1980). However, inorganic salt sodium and calcium carbonate increased the mortality of larvae by B. thuringiensis, and this may be related to the alkaline nature of these salts, which is expected to increase protein solubilization (El-Moursy et al., Reference El-Moursy, Sharaby and Awad1993).
The use of natural insecticides in combination with B. thuringiensis is a viable strategy. However, the effects on spores and crystals must be considered to obtain and optimize results. The spores play an important role in Bt pathogenicity. In addition to causing septicemia, they contribute to bacterial toxicity because cell wall constitutive proteins are similar to crystal proteins (Habib & Andrade, Reference Habib, Andrade and Alves1998). Furthermore, the association of B. thuringiensis with secondary metabolites is a key strategy for managing resistance to bacteria, since the insect intestine participates in the detoxification process and bacterial toxins may affect this process, thereby increasing susceptibility (Gill, Reference Gill1995). Field studies are recommended to confirm the results of the in vitro examinations, since in laboratory conditions, the pathogen is more highly exposed to the products than expected in the natural environment.
Acknowledgments
Dr Flavio Moscardi (In memoriam) by the co-adviser and all support spent on development of this work. Professor Dr Celia Guadalupe and the laboratory technician Osvaldo Capello's Microscopy Laboratory Electronics for all the support in the execution of scanning electron microscopy analysis.
