Bulletin of Entomological Research (2018) 108, 223–231 doi:10.

1017/S0007485317000670
© Cambridge University Press 2017

Action of natural phytosanitary products

on Bacillus thuringiensis subsp. kurstaki
S-1905
E.R. Lozano*, P.M.O.J. Neves, L.F.A. Alves, M. Potrich,
G.F.L.T. Vilas-Bôas and R.G. Monnerat
Technological University Federal of Parana, Câmpus Dois Vizinhos, Brazil

Abstract

The objective of this study was to evaluate the effects of natural phytosanitary pro-
ducts (NPs) on spores and crystals of Bacillus thuringiensis subsp. kurstaki S-1905 (Btk
S-1905). For the spore assay, NPs and bacteria were applied in combination and indi-
vidually. For the combined application, Btk S-1905 + NP mixtures were inoculated on
nutrient agar (NA), and for the separate applications, the NPs were spread on NA
plates, which were later inoculated with the pathogen. The number of colony-forming
units (CFU) per milliliter was quantified after 18 h of incubation. For the crystal protein
degradation assay, the Btk S-1905 + NP mixtures were added to the diet of Anticarsia
gemmatalis (Lepidoptera: Erebidae), and mortality was evaluated at the following time
points: 12, 24, 48, and 72 h. Scanning electron microscopy and agarose gel electrophor-
esis were carried out. Biogermex and Ecolife® reduced the CFU ml−1 in both combined
and separate applications. Biogermex, Ecolife®, and Planta Clean were antagonistic to
the action of bacterial toxins, and no product affected the morphology or resulted in the
degradation of the crystal proteins. The remaining products evaluated did not reduce
the CFU ml−1 and had additive effect when combined with the crystal toxin.

Keywords: entomopathogenic bacteria, associated control, alternative control,

selectivity

(Accepted 25 June 2017; First published online 26 July 2017)

Introduction different times and concentrations (Genena et al., 2008), where-

as the rue plant Ruta graveolens L. (Rutaceae) inhibited the
Natural phytosanitary products (NPs) and entomopatho-
growth of several bacteria, including species of the
gens are important tools to control insect pests in agroecologi-
genus Bacillus (Pereira et al., 2006; Mendes et al., 2008). It has
cal production systems, and may be used separately or in
been reported that nicotine and rue at various concentrations
combination. The combined use may have positive effects on
negatively affect the growth and toxicity of the entomopatho-
the biological parameters of entomopathogens, such as
genic bacteria Bacillus thuringiensis under specific conditions
growth, virulence, and pathogenicity, thereby increasing con-
(Krischik et al., 1988). On the other hand, in a study of the ef-
trol efficiency. Alternatively, the combination may have nega-
fects of NPs at various concentrations on the spores and crys-
tive effects, thus compromising pathogen action. There are
tals of B. thuringiensis subsp. kurstaki HD-1, obtained from the
also situations in which biological parameters remain un-
commercial product Dipel WP®, most products affected bac-
changed when entomopathogens are associated with NPs.
terial growth when the mixture was incubated with water,
In a study of non-sporulating bacteria and plant extracts,
and no negative effects on crystal toxicity were observed
the rosemary plant Rosmarinus officinalis L. (Lamiaceae) had
(Silva et al., 2012).
negative effects on Staphylococcus aureus and Bacillus cereus at
Although NPs are safer than synthetic chemical products,
little is known about the interaction between NPs and entomo-
pathogens, particularly entomopathogenic bacteria. Thus, it is
*Author for correspondence necessary to determine the interactive effects and possible se-
Phone: (55)46-35368901 lectivity of different NPs used in agroecological production
Fax: (55)46-35368905 systems in order to minimize the effects on non-target organ-
Email: [email protected] isms, such as B. thuringiensis, and maximize the efficiency of
224 E.R. Lozano et al.

insect pest control in an economical and environmentally sus- Effects of NPs on Btk S-1905
tainable manner. Therefore, the objective of this study was to
The effects of NPs on the toxicity of Btk S-1905 were evalu-
evaluate the effect of NPs on the spores and crystals of B. thur-
ated in vivo using a bioindicator insect, the caterpillar
ingiensis subsp. kurstaki S-1905, which exhibits different crystal
Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). In
types and insecticidal activity, under laboratory conditions.
order to verify morphological changes and/or crystal protein
degradation, scanning electron microscopy (SEM) and de-
naturing sodium dodecyl sulfate-polyacrylamide gel electro-
Materials and methods phoresis (SDS–PAGE) were used, respectively.
Effects of NPs on Btk S-1905 spore viability Anticarsia gemmatalis caterpillars were laboratory-reared in
containers and fed an artificial diet, according to
The lyophilized strain Btk S-1905 was provided by the Hoffmann-Campo et al. (1985). They were maintained in a con-
Brazilian Agricultural Research Corporation (Embrapa), trolled temperature chamber at 26 ± 2 °C with a relative hu-
National Centre of Genetic Resources and Biotechnology, midity of 70 ± 10% and a 14-h photoperiod until reaching the
Federal District, Brazil. The NPs were obtained in agroecologi- 2nd instar.
cal production supply shops, and were used at the recom-
mended concentrations (RCs) established by the
manufacturers; assays were performed with NPs in combin- Determination of the lethal concentration (LC50) of Btk S-1905
ation and individually (table 1).
The concentration of Btk S-1905 for the bioassay was deter-
mined by estimating the LC50 for A. gemmatalis. Bacillus thur-
ingiensis subsp. kurstaki HD-1, obtained from a purified
Combined application: NPs mixed with Btk S-1905 sample from the entomopathogen bank at Embrapa Soja,
A suspension was prepared with 2.3 × 1019 spores ml−1, by Londrina, Paraná, Brazil, was used as the standard. A total
weighing 5 mg of the lyophilized material and adding 50 ml of of 0.02 g of each lyophilized sample was diluted in 20 ml of
sterile distilled water. From this suspension, successive dilu- sterile distilled water (1000 µg ml−1 & 4.6 × 1018 spores
tions were prepared using sterile distilled water, and a suspen- ml−1). From this suspension, six dilutions were prepared at
sion of 2.3 × 106 spores ml−1 was obtained. Aliquots (300 µl) of concentrations of 24, 32, 41, 53, 69, and 90 µg ml−1.
this suspension were added to Erlenmeyer flasks with 50 ml of The artificial diet for A. gemmatalis was prepared free of an-
the NP prepared at the RC in sterile distilled water. Five ticontaminants and solidified into cubes with sides of approxi-
Erlenmeyer flasks were prepared (replicates) for each NP as- mately 1.5 cm, which were cut using a spatula. The cubes were
sessed (treatment). The flasks were stored in a horizontal sha- submerged for 5 s in the dilutions at various concentrations
ker and the mixtures were incubated at 30 ± 2 °C and 150 rpm and, after drying, were placed in 50-ml containers. The experi-
for 2 h. The pH values were measured before and after incuba- mental design was completely randomized, with 20 replicates
tion. The mixture of each flask was inoculated in five spots of (recipients) per concentration (treatment) and three A. gemma-
5 µl per spot in three Petri dishes containing nutrient agar talis 2nd instar caterpillars per repetition. For the control diet,
(NA) (Alves & Moraes, 1998). The Petri dishes were kept the cubes were submerged in sterile distilled water. Then,
open in a laminar flow hood for 5 min to evaporate the excess the recipient containers were closed and placed in incubator
water. Then, the dishes were closed and placed in an incubator at 26 ± 2 °C with a relative humidity of 70 ± 10% and a photo-
at 30 ± 2 °C for 18 h, followed by the quantification of colony- period of 14 h. After 48 h, the caterpillars were transferred to
forming units (CFU) ml−1 for each spot. Sterile distilled water containers with the artificial diet, without the treatment, and
was used as a control. were evaluated daily until the sixth day.
The data were analyzed using Micro Probit 3.0 (Thomas &
Sparks, 1987) to determine the LC50.

Individual application: NPs and Btk S-1905

Btk-S1905 toxicity on A. gemmatalis
The same procedure described for the combined applica-
tion was followed to prepare the spore suspension. The NP so- From the lyophilized material, suspensions of Btk-S1905
lutions were prepared in parallel in five Erlenmeyer flasks were prepared in Erlenmeyer flasks (50 ml) with sterile dis-
(replicates) per treatment. Aliquots (100 µl) from each treat- tilled water at a concentration equivalent to the previously es-
ment were applied to the surface of the NA medium, in timated LC50. Then, the NPs were added at the RC, and the
three Petri dishes and scattered with a Drygalski loop. The mixtures were incubated in a horizontal shaker at 30 ± 2 °C,
Petri dishes were kept open in a laminar flow hood for 5 min 150 rpm for 2 h. The pH values were measured before and
to evaporate the excess water, and then the Btk S-1905 suspen- after incubation.
sion was inoculated, as described for the combined applica- Using micropipettes, 150-μl aliquots from the mixture in
tion. The incubation and evaluation procedures were each flask were added to the surfaces of the 1.5-cm cubes
performed as described for the combined application. In the used in the artificial diet for A. gemmatalis (free of anticontami-
control, sterile distilled water was added to the culture me- nants) on Petri dishes. Each plate received three diet cubes and
dium and the bacterial suspension was subsequently was kept open in a laminar flow hood for 5 min to evaporate
inoculated. the excess water and for spore and crystal deposition into the
The data obtained in both experiments were analyzed diet. Subsequently, each plate received 25 2nd instar caterpil-
using an analysis of variance (F test) and means were com- lars of A. gemmatalis. The experimental design was completely
pared with that of the control using Tukey’s test (P < 0.05). randomized with three repetitions and 75 caterpillars per
The statistical analyses were implemented in Sisvar® treatment. For the control, identical lots of caterpillars were
(Ferreira, 2009). prepared, which received the NP (at the RC) and the
 Effects of natural products on Bacillus thuringiensis 225

Table 1. Natural phytosanitary products, use, composition, and concentrations used in the experiments.

Product Use Composition Conc. (ml l−1)*

Biogermex RI and BIO Citrus bioflavonoids (vitamin P), citrus phytoalexins, ascorbic acid 200 ml
(vitamin C), citric acid, citrus polypeptides, palmitic acid,
several fatty acids, sugars, glycerides, and tocopherols
Ecolife® RI and BIO Bioflavonoids, phytoalexins, ascorbic, lactic, and citric acids 750 ml
Chrysanthemum extract INS Pyrethrins and jasmolinas 1000 ml
Planta Clean FUN Plant extracts, fatty acids, and minerals 2500 ml
Pironim INS Neem (leaves, seeds cake, and oil); natural pyrethrum; 600 ml
pyroligneous extract of Eucalyptus sp.

Conc., concentration ml l−1; RI, resistance inductor; INS, insecticide; FUN, fungicide; BIO, biofertilizer.

Btk-S1905 suspension. A negative control was also prepared a 20 kV electron beam intensity by SEM, and images were re-
with sterile distilled water, which was applied on the artificial corded by digital microphotography.
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
Integrity of Btk S-1905 crystals analyzed by SDS-PAGE
at the following time points: 12, 24, 48, and 72 h.
The data were analyzed using the analysis of variance Samples of the suspensions prepared as described in the
(F test), and the means were compared using Tukey’s test section ‘morphology and integrity of Btk S-1905 crystals observed
(P < 0.05) implemented in Sisvar® (Ferreira, 2009). by SEM’ were analyzed by SDS-PAGE to check for the pres-
Interactive effects of NPs on the crystals were calculated ence of Cry proteins. For that purpose, the crystals were solu-
using the χ2 test, and classified as synergistic, antagonistic, bilized and the protein was extracted according to the
and additive, according to Benz (1971) and Koppenhofer methods proposed by Lecadet et al. (1991).
et al. (2000). In this classification, a synergistic effect describes In brief, 1.5 ml of bacterial suspension was transferred to an
a system with two effective components that, in combination, autoclaved 2-ml microcentrifuge tube. The tubes were centri-
produce an effect that is greater than the sum of the independ- fuged at 15,115 g for 20 min, the supernatants were discarded,
ent effects. An additive effect occurs when the effect of two and the pellets were washed with 1.5 ml of 0.5 M NaCl for 20
components combined equals the sum of the effects of the in- min. The tube walls were dried with filter paper after discard-
dividual components. An antagonistic effect occurs when the ing the 0.5 M NaCl solution. The sediments were washed
combination of the elements produces a smaller effect than twice with 1.5 ml of 1 mM phenylmethyl fluoride sulfonyl
that observed for the individual elements. (PMFS) and centrifuged at 15,115 g for 20 min. After discard-
ing the 1 mM PMFS, the pellets were resuspended in 500 µl of
1 mM PMFS and stored at −20 °C. The spore–crystal suspen-
Morphology and integrity of Btk S-1905 crystals observed by SEM sion and NPs were analyzed by 10% SDS–PAGE, as described
by Laemmli (1970). For this purpose, 15 µl of the spore–crystal
A total of 5 mg of the lyophilized Btk S-1905 sample was preparation was used to load the gel and electrophoresis was
weighed and diluted in 50 ml of sterile distilled water in performed at a voltage of 25 mA for 3 h. As a control,
Erlenmeyer flasks along with the NPs (at the RC), resulting Btk-S1905 was incubated with sterile distilled water under
in a mixture of approximately 2.3 × 1019 spores ml−1. The the same conditions used for the NPs. Bacillus thuringiensis
flasks were placed in a horizontal shaker and the mixtures subsp. kurstaki (HD-1) was used as the standard.
were incubated at 30 ± 2 °C, 150 rpm, for 2 h. Then, 1.5-ml ali-
quots of each mixture were removed to prepare samples for
SEM. The rest of each mixture was stored in an amber glass
Results
flask in a freezer at −10 °C. The aliquots of the mixtures
were transferred to microcentrifuge tubes FANEM® Model Effects of NPs on Btk S-1905 spore viability
243 and centrifuged for 5 min at 8944 g, three times, to create
Combined application: NPs mixed with Btk S-1905
a pellet. The supernatant was discarded, and the sedimented
materials were fixed with a 2% paraformaldehyde, 2% glutar- The products Biogermex and Ecolife® significantly reduced
aldehyde, and phosphate buffer (0.1 M PO4) solution for 4 h. colony formation by 99.8 and 100%, respectively, and Planta
Subsequently, the samples were washed in phosphate buffer Clean significantly increased colony formation by 12.2%. For
for 15 min three times, and fixed again with 1% osmium tet- the remaining products, colony formation did not differ
roxide (OsO4) for 2 h. Later, a second washing was carried from that of the control (table 2).
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
Individual application: NPs and Btk S-1905
an alcoholic sequence (alcohol 70%: 3 × 15 min, alcohol 80%:
3 × 15 min, alcohol 90%: 4 × 10 min, and alcohol 100%: 4 × 10 Negative effects on colony formation (CFU ml−1) were ob-
min) and with CO2 at the critical point. The samples were served using Biogermex and Ecolife® individually, with sig-
then mounted in stubs with silver and coated with gold for nificant reductions of 35.46 and 100%, respectively. For the
3 min by the sputtering process using a BAL-TEC SCD 050 remaining products, there were no significant differences in
sputter. Samples were observed in high-vacuum mode with colony formation from that of the control (table 3).
226 E.R. Lozano et al.

Table 2. Mean (±SE) CFU ml−1 of Btk S-1905, after incubation Table 3. Mean (±SE) CFU ml−1 after the inoculation of Btk S-1905,
(30 ± 2 °C, 150 rpm, 2 h) with sterile distilled water and natural with natural phytosanitary products (at the RC) in the nutrient
phytosanitary products (at the RC), initial and final pH, inocula- agar medium in Petri plates (30 ± 2 °C, 18 h).
tion of nutrient agar culture medium in Petri plates, and incuba-
tion (30 ± 2 °C, 18 h). Mean CFU CFU rel.
Treatment ml−1 (×105) test (%)1
Treatment Mean CFU CFU rel. pH
ml−1 (×105) test (%)1 Control 508.7 ± 5.9a –
0h 2h Pironim 521.5 ± 7.3a +2.5
Biogermex 328.3 ± 24.7b −35.5
Control 349.8 ± 5.8a – 7.3 5.9 Ecolife® 0.0 ± 0.00c −100.0
Pironim 336.5 ± 5.0a −3.8 3.5 3.5 VC (%)= 8.7
Biogermex 0.8 ± 0.8b −99.8 3.8 3.7 Control 394.8 ± 14.1a –
Ecolife® 0.0 ± 0.0b −100.0 3.3 3.2 Chrysanthemum extract 496.8 ± 50.1a +25.8
VC (%)= 5.1 Planta Clean 459.7 ± 66.5a +16.4
Control 369.6 ± 9.4b – 7.3 5.9 VC (%)= 21.5
Chrysanthemum extract 389.5 ± 8.5ab +5.4 9.0 9.0
Planta Clean 416.8 ± 6.4a +12.8 9.6 9.6 Means (±SE) followed by the same lowercase letter in a column do
VC (%)= 5.4 not differ significantly by Tukey’s test (P < 0.05).
1
Equation: Mean (CFU per ml) treat/Mean (CFU per ml) control ×
Means (±SE) followed by the same lowercase letter in a column do 100 − 100, where positive values indicate an increase in CFU ml−1
not differ significantly by Tukey’s test (P < 0.05). and negative values indicate a decrease compared to controls.
1
Equation: (Mean (CFU per ml) treat/Mean (CFU per ml) control) VC, variation of coefficient.
× 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. Discussion
For the combined application of NPs mixed with Btk S-1905,
the results observed for Biogermex and Ecolife® were similar to
Effects of the natural products on Btk S-1905
those of Silva et al. (2012), who examined B. thuringiensis subsp.
The LC50 (95% confidence interval) values for Btk S-1905 kurstaki HD-1 spores obtained from the commercial product
and Btk HD-1 were 35 (31–39) µg ml−1 (slope 3.02 ± 0.39) Dipel WP® and NPs. According to Silva et al. (2012), these pro-
and 31 (22–37) µg ml−1 (slope 2.80 ± 0.68), respectively, with ducts significantly reduced the CFU ml−1, regardless of concen-
no significant difference between the strains. tration. 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).
Btk-S1905 toxicity on A. gemmatalis The individual applications of NPs and Btk S-1905 spores
on the surface of culture medium had a negative effect on the
In the in vivo bioassay, the effects of Biogermex, Ecolife®, CFU ml−1 when using Biogermex and Ecolife® (table 3).
and Planta Clean were antagonistic to the action of crystal tox- The negative effects observed in both experiments may be
ins; when these products were combined with Btk S-1905, the related to variation in pH, since products with acidic pH
average mortality rates of A. gemmatalis were 35.30, 18.4, and values, such as Biogermex and Ecolife®, reduced the
23.7%, respectively, which were all lower than the mortality CFU ml−1. In contrast, the CFU ml−1 increased for chrysanthe-
for Btk-S-1905 (51.3%) and higher than those for the individual mum extract and Planta Clean (which have basic pH values)
products (0% Biogermex, 3.0% Ecolife®, and 1.3% Planta (tables 2 and 3).
Clean). The pH values for the aforementioned combinations According to a study of the effect of pH on the germination
ranged from acidic (Biogermex & 3.89 and Ecolife® & 2.90) of B. thuringiensis spores in the soil, a higher acidity results in a
to alkaline (Planta Clean & 9.77) (table 4). more substantial reduction in germination, and germination
The remaining products had an additive effect on crystal does not occur at pH values of <5 (Petras & Casida, 1985).
protein toxicity; chrysanthemum and Pironim extracts, used The influence of pH on spore germination was also studied
separately, resulted in the highest A. gemmatalis mortality by Wilson & Benoit (1993), who assessed the action of intes-
rates (74.8 and 61.0%, respectively), which were significantly tinal fluid components, alone and in combination, on B. thur-
higher than those of the Btk S-1905 treatment (51.3%) (table 4). ingiensis 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
Morphology and integrity of Btk S-1905 crystals observed by SEM and Ecolife® showed acid pH values and significantly reduced
For all treatments, Btk S-1905 crystals did not exhibit mor- the CFU ml−1 of B. thuringiensis subsp. kurstaki HD-1 at three
phological changes, indicating that in the study conditions, the concentrations (Silva et al., 2012).
products did not affect the crystal proteins (fig. 1). The Pironim product had an acidic pH, as did the
Biogermex and Ecolife® products, but did not significantly re-
duce the CFU ml−1 (table 3), indicating that pH should not be
considered the main factor determining spore germination
Integrity of Btk S-1905 crystals analyzed by SDS–PAGE
and CFU ml−1. Three distinct processes are involved in the
The degradation of proteins was not observed in an SDS– germination of bacterial spores, i.e. the presence of specific re-
PAGE analysis, as evidenced by the lack of fragments showing ceptors on the inner membrane of the spore, the presence of
a molecular weight below 10 kDa in any of the treatments, ion channels, and the action of lytic enzymes in cell cortex deg-
supporting the results of the SEM analysis (fig. 2). radation. Thus, germination may be triggered by the presence
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.

Treat Times % Mortality pH

12 h 24 h 48 h 72 h Observed1 Expected2 χ2 3 Interaction 0h 2h
Water 0.0 ± 0.0Ab 0.0 ± 0.0Ac 0.0 ± 0.0Ab 0.0 ± 0.0Ab 0.0 ± 0.0c – – – 8.20 6.93
CE 6.6 ± 1.1Ba 10.6 ± 1.0Bab 40.5 ± 5.9Aa 17.2 ± 2.1Ba 74.8 ± 2.3a – – – 9.27 9.05
Btk S-1905 1.6 ± 1.6Cab 3.1 ± 1.5Cbc 32.8 ± 11.7Aa 13.7 ± 2.7Ba 51.3 ± 10.1b – – – 7.38 7.42

Effects of natural products on Bacillus thuringiensis

CE + Btk S-1905 8.10 ± 1.9Ba 12.90 ± 2.2Ba 47.50 ± 2.7Aa 20.70 ± 4.1Ba 89.60 ± 6.3a 87.72 0.04 Additiv. 9.15 8.99
VC 1 (%)= 15.0 VC 2 (%)= 26.3 VC 3 (%)= 10.7
Water 0.0 ± 0.0Aa 0.0 ± 0.0Aa 0.0 ± 0.0Ac 0.0 ± 0.0Ab 0.0 ± 0.0b – – – 8.20 6.93
BG 0.0 ± 0.0Aa 0.0 ± 0.0Aa 0.0 ± 0.0Ac 0.0 ± 0.0Ab 0.0 ± 0.0b – – – 3.58 3.57
Btk S-1905 1.6 ± 1.6Ba 3.1 ± 1.5Ba 32.8 ± 11.7Aa 13.7 ± 2.7Ba 51.3 ± 10.1a – – – 7.38 7.42
BG + Btk S-1905 0.0 ± 0.0Ba 0.0 ± 0.0Ba 9.7 ± 5.5Ab 7.9 ± 4.2ABab 35.3 ± 15.2a 51.8 5.2 Antag. 3.7 3.9
VC 1 (%)= 54.73 VC 2 (%)= 58.45 VC 3 (%)= 37.47
Water 0.0 ± 0.0Aa 0.0 ± 0.0Aa 0.0 ± 0.0Ab 0.0 ± 0.0Ab 0.0 ± 0.0c – – – 8.2 6.9
ECO 0.00 ± 0.0Aa 1.5 ± 1.5Aa 1.5 ± 1.5Ab 0.0 ± 0.0Ab 3.0 ± 3.0bc – – – 2.9 2.8
Btk S-1905 1.60 ± 1.6Ca 3.1 ± 1.6BCa 32.8 ± 11.6Aa 13.7 ± 2.7Aba 51.3 ± 10.1a – – – 7.3 7.4
ECO + Btk S-1905 4.9 ± 3.0ABa 0.0 ± 0.0Ba 5.1 ± 3.0ABb 13.6 ± 8.9Aa 23.7 ± 9.4ab 52.8 16.0 Antag. 2.9 2.9
VC 1 (%)= 52.6 2 (%)= 64.0 VCCV 3 (%)= 38.9
Water 0.0 ± 0.0Ab 0.0 ± 0.0Aa 0.0 ± 0.0Ac 0.0 ± 0.0Ab 0.0 ± 0.0c – – – 8.2 6.9
PI 48.7 ± 9.0Aa 2.5 ± 1.3BCa 9.8 ± 4.3Bb 0.0 ± 0.0Cb 61.0 ± 9.2ab – – – 3.3 3.2
Btk S-1905 1.6 ± 1.6Bb 3.1 ± 1.5Ba 32.8 ± 11.7Aa 13.7 ± 2.7Aa 51.3 ± 10.1b – – – 7.4 7.42
PI+ Btk S-1905 60.4 ± 6.3Aa 6.5 ± 1.6Ba 11.1 ± 3.6Bb 10.1 ± 3.5Ba 88.0 ± 6.4a 81.00 0.60 Additiv 3.3 3.3
VC 1 (%)= 20.4 VC 2 (%)= 34.1 VC 3 (%)= 13.6
Water 0.0 ± 0.0Ab 0.0 ± 0.0Aa 0.0 ± 0.0Ab 0.00 ± 0.0Ab 0.0 ± 0.0c – – – 8.2 6.9
PC 0.0 ± 0.0Ab 0.0 ± 0.0Aa 1.3 ± 1.3Ab 0.0 ± 0.0Ab 1.3 ± 1.3c – – – 10.0 9.9
Btk S-1905 1.6 ± 1.6Cab 3.1 ± 1.5BCa 32.8 ± 11.7Aa 13.7 ± 2.7Aba 51.3 ± 10.1a – – – 7.4 7.4
PC + Btk S-1905 12.5 ± 9.2Aa 0.0 ± 0.0Ba 1.2 ± 1.2ABb 4.7 ± 0.8ABab 18.4 ± 8.4b 51.9 21.6 Antag. 9.6 9.8
VC 1 (%)= 60.8 VC 2 (%)= 56.6 VC 3 (%)= 34.0

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.
χ = (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,
3 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.

227
228 E.R. Lozano et al.

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.

of amino acids, sugars, and nucleosides that bind to specific disabling it or even breaking down the cell membrane
receptors as well as salts, high pressure, and Ca+2 ions (Tsuchiya et al., 1996).
(Setlow, 2003). Given the above, the reductions in CFU ml−1 observed for
In addition to the effect of pH, the negative results ob- Biogermex and Ecolife® may be related to the prevention of the
served for Biogermex and Ecolife® in both experiments may germination process and/or to the destruction of the bacterial
be related to the composition and mode of action of these pro- membrane after germination. Such effects on cells are sup-
ducts. Ecolife® consists of bioflavonoids, citrus phytoalexins, ported by similar results obtained for the effects of Ecolife®
and ascorbic acid (Technical Bulletin), similar to the compos- on cells of the Gram-negative phytopathogenic bacteria
ition of Biogermex. Flavonoids and terpenoids act as defense Ralstonia solanacearum and Xanthomonas axonopodis pv. maniho-
mechanisms against insects and microorganisms (Dixon et al., tis, which exhibit a zone of inhibition proportional to the in-
1983; Cowan, 1999) and are able to bind to the cell wall, crease in product concentration (Motoyama et al., 2003).
 Effects of natural products on Bacillus thuringiensis 229

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®.

The effects of the NPs on the toxicity of Btk S-1905 on A. toxins (Gill, 1995), as well as the structure and process of
gemmatalis in this study were not in agreement with those of spore germination (Liu et al., 1998).
Silva et al. (2012), who did not observe an effect of Ecolife® Given the above, the antagonistic effects of Biogermex®,
on the toxicity of Btk HD-1 in Dipel WP®. This difference Ecolife®, and Planta Clean are probably associated with the
may be explained by the fact that in the present study, a puri- binding of these products to the crystal protein, hindering
fied bacterium was used at a lower concentration known to be solubilization in the insect intestine or preventing the binding
effective (LC50), while Silva et al. (2012) used Btk HD-1 at the of proteins to specific receptors in the intestinal membrane.
RC, half of the RC, and the twice the RC. When used separately, these products did not significantly af-
Bipyramidal, cuboid, and spherical crystals were observed, fect insect mortality. In addition, SEM and electrophoresis
supporting the results of Medeiros et al. (2005) and Monnerat analyses did not indicate morphological changes in crystal
et al. (2007). The bipyramidal crystal morphology is associated proteins. Nevertheless, faster action of the entomopathogen
with the Cry 1 protein, which is effective against Lepidoptera when mixed with a natural plant product may be related to
and Coleoptera, and the cuboid crystals are associated with the metabolite stressor effect, which makes insects more sus-
the Cry 2 protein, which is effective against Diptera and ceptible to bacterial toxins (Saito & Lucchini, 1998).
Lepidoptera (Silva et al., 2004). These morphological types With respect to the effect of metabolites on entomopatho-
can provide important information regarding entomopatho- genic bacteria, Lord & Undeen (1990) studied the effects of tan-
genic activity for different orders of insects when analyzed nins on B. thuringiensis subsp. israelensis toxicity and observed
in conjunction with biochemical and molecular analyses a reduction in the mortality of Aedes aegypti (Diptera:
(Habib & Andrade, 1998; Who, 1999). Culicidae) larvae when the bacteria and tannins were mixed
The solubilization of the crystal protein may occur in alka- without previous incubation, and an increase in mortality
line conditions (pH above 8) (Habib & Andrade, 1998), as ex- after incubation. According to the authors, the tannins can
pected for the Planta Clean product, but the mortality results bind to intestinal proteolytic enzymes and consequently pre-
for this NP indicated that the pH is not the only determinant of vent the solubilization of crystals. However, after incubation,
the solubilization of Btk S-1905 crystals (table 3). small amounts of proteins that may have been released from
For the treatments in this study, the proteins had two major the crystal bind to tannins, reducing the action of tannins on
polypeptides of 130 and 65 kDa (fig. 2). The 130-kDa polypep- the toxin.
tides are considered characteristic of strains against A similar result was obtained in a study on the effects of
Lepidoptera and Coleoptera, whereas the 65-kDa polypeptide tannins on B. thuringiensis subsp. kurstaki HD-73 toxicity to
is characteristic of strains infecting Lepidoptera and Diptera Heliothis virescens F. (Lepidoptera: Noctuidae) larvae (Navon
(Monnerat & Bravo, 2000). et al., 1993). According to the authors, the LC50 for the bacteria
Several factors can affect the insecticidal activity of B. thur- alone was 27.0 ng g−1 and for the bacteria mixed with tannins
ingiensis, such as intestine structure and function, toxin diver- at 3.2 mg g−1 was 59.1 ng g−1, demonstrating that δ-endotoxin
sity, protein structure and solubilization, interactions among activity was antagonized or possibly inactivated.
230 E.R. Lozano et al.

In a study of B. thuringiensis combined with azadirachtin at Broderick, N.A., Raffa, K.F. & Handelsman, J. (2006) Midgut
known sublethal concentrations, the effects on different instars bacteria required for Bacillus thuringiensis insecticidal activ-
of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) were ity. Proceedings of the National Academy of Sciences of the United
complementary, and the metabolite facilitated B. thuringiensis States of America 103(41), 15196–15199.
action (Singh et al., 2007). Likewise, Rajguru et al. (2011) re- Cowan, M.M. (1999) Plant products as antimicrobial agents.
ported that Btk associated with aqueous extracts of various Clinical Microbiology Reviews 12(4), 564–582.
plants causes increased mortality in Spodoptera litura Dixon, R.A., Dey, P.M. & Lamb, C.J. (1983) Phytoalexins: en-
(Lepidoptera: Noctuidae) larvae on the sixth day of evaluation. zymology and molecular biology. Advances Enzymology and
Factors other than secondary compounds may be related to Related Areas Molecular Biology 55, 1–136.
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and other compounds, such as the presence of particulate mat- ical additives to increase the activity spectrum of Bacillus
ter (Ignoffo et al., 1981; Lord & Undeen, 1990) and solutes, such thuringiensis var. kurstaki (Dipel 2X) against the rice moth
as phosphoric acid and citric acids, which may be stressors for Corcyra cephalonica. Journal of Islamic Academy of Sciences. 6(2),
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(Lord & Undeen, 1990). Cations, physicochemical and bio- Ferreira, D.F. (2009) Sistema Sisvar para análises estatísticas.
chemical factors, such as pH, ionic and divalent forces (Tran Available at http://www.dex.ufla.br/~danielff/programas/
et al., 2001), temperature (Vachon et al., 2006), and insect bac- sisvar.html (acessado em 02 de jun., 2009).
terial flora (Broderick et al., 2006) are also associated with re- Genena, A. K., Hense, H., Smânia, J. A. & Souza, S.M.R. (2008)
ductions in larval mortality in mixtures of B. thuringiensis. Rosemary (Rosmarinus officinalis) – a study of the compos-
An effect of pH on crystal proteins and, consequently, on ition, antioxidant and antimicrobial activities of extracts ob-
mortality was not observed in this study for treatments with tained with supercritical carbon dioxide. Ciência e Tecnologia
an antagonistic effect, with pH values ranging from acidic de Alimentos. 28(2), 463–469.
(2.90 for Ecolife® + Btk S-1905 and 3.89 for Biogermex + Btk Gill, S.S. (1995) Mechanism of action of Bacillus thuringiensis tox-
S-1905) to alkaline (9.77 for Planta Clean + Btk S-1905) ins. Memórias do Instituto. Oswaldo Cruz. 90, 69–74.
(table 4). Similar results were reported for the effect of pH Habib, M.E.M. & Andrade, C.F.S. (1998) Bactérias
and chlorine variation on Dipel SC and Thuricide 32B biopes- entomopatogênicas. pp. 383–427 in Alves, S.B. (Ed.) Controle
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salt sodium and calcium carbonate increased the mortality Criação massal da lagarta da soja (Anticarsia gemmatalis),
of larvae by B. thuringiensis, and this may be related to the al- EMBRAPA – Centro Nacional de Pesquisa da Soja, Londrina,
kaline nature of these salts, which is expected to increase pro- Documentos 10, 21.
tein solubilization (El-Moursy et al., 1993). Ignoffo, C.M., Garcia, C., Kroha, M.J., Fukuda, T. & Couch, T.L.
The use of natural insecticides in combination with B. thur- (1981) Laboratory tests to evaluate the potential efficacy of
ingiensis is a viable strategy. However, the effects on spores Bacillus thuringiensis var. israelensis for use against mosqui-
and crystals must be considered to obtain and optimize re- toes. Mosquito News 41, 85–93.
sults. The spores play an important role in Bt pathogenicity. Koppenhofer, A.M., Brown, I.M.Gaugler, R.Grewal, P.S., Kaya,
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toxicity because cell wall constitutive proteins are similar to nematodes and imidacloprid against white grubs: green-
crystal proteins (Habib & Andrade, 1998). Furthermore, the as- house and field evaluation. Biological Control 19, 245–251.
sociation of B. thuringiensis with secondary metabolites is a key Krischik, V.A., Barbosa, P., Reichelderfer, A.F. (1988) Three
strategy for managing resistance to bacteria, since the insect in- trophic level interactions: allelochemicals, Manduca sexta (L.),
testine participates in the detoxification process and bacterial and Bacillus thuringiensis var. kurstaki Berliner. Environmental
toxins may affect this process, thereby increasing susceptibil- Entomology 17, 476–482.
ity (Gill, 1995). Field studies are recommended to confirm the Laemmli, U.K. (1970) Cleavage of structural proteins during the
results of the in vitro examinations, since in laboratory condi- assembly of the head of bacteriophage T4. Nature 227, 680–
tions, the pathogen is more highly exposed to the products 685.
than expected in the natural environment. Lecadet, M.M., Chaufaux, J., Ribier, J.E., & Lereclus, D. (1991)
Construction of novel Bacillus thuringiensis strains with dif-
ferent insecticidal activities by transduction and transform-
Acknowledgments
ation. Applied and Environmental Microbiology 58, 840–849.
Dr Flavio Moscardi (In memoriam) by the co-adviser and all Liu, Y.B., Tabashnir, R.E., Moar, W.J. & Smith, R.A. (1998)
support spent on development of this work. Professor Dr Celia Synergism between Bacillus thuringiensis spores and toxins
Guadalupe and the laboratory technician Osvaldo Capello’s against resistant and susceptible diamond moths (Plutella xy-
Microscopy Laboratory Electronics for all the support in the lostella). Applied and Environmental Microbiology 64, 1385–1389.
execution of scanning electron microscopy analysis. Lord, J.C. & Undeen, A.H. (1990) Inhibition of the Bacillus thur-
ingiensis var. israelensis toxin by dissolved tannins.
Entomological Society of America 19, 1547–1551.
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