Effect of Bacillus thuringiensis CAB109 on the growth, development, and generation mortality of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidea)

Abstract

The efficiency of Bacillus thuringiensis (Bt) CAB109 on Spodoptera exigua (Hübner) (Lepidoptera: Noctuidea) larvae was investigated. This study introduces a novel concept of generation mortality (GM) was introduced. Bt CAB109 suspensions at sub-lethal concentrations of 0, 10², 10³, 10⁴, 10⁵, and 10⁶ cfu/ml were prepared and used to treat the second instar larvae of S. exigua. The results showed that the mortality rates of the larvae were 5.0, 8.3, 15.0, 23.3, 36.7, and 55.0% respectively after 7 days of treatment. The mean weights of treated larvae with different concentrations were 2.63, 2.19, 2.03, 1.87, 1.34, and 0.96 mg respectively after 6 days, while the developmental durations of such larvae were 16.3, 16.8, 17.5, 18.2, 19.5, and 21.2 days, respectively. Treatment with Bt affected the growth of the larvae at all instars (from the first to the fifth one).

Through the comprehensive interference index of population control (CIIPC), the GM was calculated and the percentages were 30.4, 50.3, 63.8, 77.2, and 90.6% for the six tested concentrations respectively. Thus, the GM can be used to evaluate the efficiency of biological pesticides in agricultural practices in the future.

Background

Spodoptera exigua (Hübner) (Lepidoptera: Noctuidea) is a worldwide pest (Da-yong et al. 2009), which mainly attacks vegetables and field crops. Currently, chemical pesticides are being used to control this pest; however, they are not ideal because they cause environmental pollution (Gui-lan et al. 2002). Therefore, it is necessary to use methods that will not pollute the environment (Da-yong and Yong-man 2010 and Da-yong et al. 2012). Bacillus thuringiensis (Bt) is currently the most widely used bio-pesticide (Lan-lan et al. 2008 and Qing-xian 2008). Previous studies showed that Bt can not only kill the target pests, but can also inhibit, hinder, and prolong duration of the growth and reproduction of the pests (Barker 1998 and Erb et al. 2001). Therefore, only using the concept of generation mortality to evaluate the effect of chemicals is not sufficient.

In the present study, the efficacy of sub-lethal concentrations of Bt CAB109 on the mortality rate, growth, and duration of larvae of S. exigua under laboratory conditions was evaluated.

Materials and methods

Bacillus thuringiensis (Bt CAB109) was kindly provided by the Laboratory of Pest Biological Control, College of Agriculture and Life Sciences, Chungnam National University (Korea). The Bt CAB109 strain was cultured in Nutrient Agar (NA) medium at 27 °C for 4 days (when the spore and the parasporal crystal separated from each other) (Da-yong and Yong-man 2010). Next, the culture was washed with sterile water and centrifuged at 4 °C for 10 min. The supernatant was collected and the concentration of cells in the pellet was about 10¹⁰ cfu/ml; then the pellet was stored at 4 °C until further use. S. exigua (larvae and adults) were collected and reared at 25 °C in 16:8 h (light to dark) cycles, and the relative humidity 50–60%. The second instar S. exigua larvae were selected for use at different treatments.

Effect on larval growth and development

On larval mortality

The Bt suspension was diluted to final concentrations of 0 (control), 1 × 10², 1 × 10³, 1 × 10⁴, 1 × 10⁵, and 1 × 10⁶ cfu/ml; 100 μl of the diluted suspension was drawn using a pipette and incubated into artificial feed (0.5 g) and mixed well. Twenty larvae (second instar) of S. exigua that were fasted for 3 h were placed in a plate with the artificial feed containing concentration of the Bt suspension culture and incubated for 3 h. The larvae were then transferred to a new plate containing artificial feed without Bt and reared for 7 days, after which the larval mortality rate was calculated. All the experiments were repeated four times.

On larval weight

The second instar larvae of S. exigua were divided into groups of 10 and weighed to obtain the average. These groups were treated with Bt as described before. Six days later, the groups of treated larvae were weighed and the average of larva weight was estimated. All the experiments were repeated four times.

On larval duration

Twelve of the second instar S. exigua larvae were treated with Bt for 3 h, after which they were transferred into a 12-well culture plate with the diet and reared until pupation, and the larval duration was calculated. All experiments were repeated four times.

Generation mortality (GM)

GM is the mortality or decrease of the pest numbers at various growth stages (egg, larva, pupa, and adult) in one generation after treating the second instar larvae of S. exigua with Bt CAB109, which was calculated according to the theory of interference index of population control (IIPC) defined by Xiong-fei et al. (2000).

On larvae

The Bt suspension was diluted to final concentrations of 0 (control), 10², 10³, 10⁴, 10⁵, and 10⁶ cfu/ml; then second instar larvae were treated as mentioned before and kept for 7 days, after which the number of live larvae was calculated; all experiments were repeated three times.

On pupae

The second instar larvae of S. exigua were treated as described before and reared until pupation. The pupae were then transferred into a new plate and kept until emergence of adults. The ratio of live pupae was then determined. All experiments were repeated three times.

On adults

The adults were then transferred into a plate and the numbers of live adults in each concentration were calculated. All experiments were repeated three times.

On eggs

The normal adults were grouped in 1:1 female to male ratio in a plate and provided with absorbent cotton containing 10% (w/v) glucose solution. The number of eggs laid was estimated. All experiments were repeated three times.

Formula used for the calculations

$$ \mathrm{Interference}\ \mathrm{Index}\ \mathrm{of}\ \mathrm{Population}\ \mathrm{Control}\ \left(\mathrm{IIPC}\right)\kern0.5em =\kern0.5em \frac{\mathrm{survival}\kern0.5em \mathrm{rate}\kern0.5em \mathrm{of}\kern0.5em \mathrm{treated}\kern0.5em \mathrm{group}}{\mathrm{survival}\kern0.5em \mathrm{rate}\kern0.5em \mathrm{of}\kern0.5em \mathrm{treated}\kern0.5em \mathrm{group}} $$

Comprehensive interference index of population control (CIIPC)

$$ {\displaystyle \begin{array}{l}\mathrm{CIIPC}={\mathrm{IIPC}}_1\times {\mathrm{IIPC}}_2\times {\mathrm{IIPC}}_3\times {\mathrm{IIPC}}_4\\ {}{\mathrm{where}\ \mathrm{IIPC}}_1=\frac{\mathrm{larvae}\ \mathrm{survival}\ \mathrm{rate}\ \mathrm{of}\ \mathrm{treated}\ \mathrm{group}}{\mathrm{larvae}\ \mathrm{survival}\ \mathrm{rate}\ \mathrm{of}\ \mathrm{control}\ \mathrm{group}}\\ {}\kern5em {\mathrm{IIPC}}_2=\frac{\mathrm{pupae}\ \mathrm{survival}\ \mathrm{rate}\ \mathrm{of}\ \mathrm{treated}\ \mathrm{group}}{\mathrm{pupae}\ \mathrm{survival}\ \mathrm{rate}\ \mathrm{of}\ \mathrm{control}\ \mathrm{group}}\\ {}\kern5em {\mathrm{IIPC}}_3=\frac{\mathrm{adults}\ \mathrm{survival}\ \mathrm{rate}\ \mathrm{of}\ \mathrm{treated}\ \mathrm{group}}{\mathrm{adults}\ \mathrm{survival}\ \mathrm{rate}\ \mathrm{of}\ \mathrm{control}\ \mathrm{group}}\\ {}\kern5em {\mathrm{IIPC}}_4=\frac{\mathrm{eggs}\ \mathrm{number}\ \mathrm{of}\ \mathrm{treated}\ \mathrm{group}}{\mathrm{eggs}\ \mathrm{number}\ \mathrm{of}\ \mathrm{control}\ \mathrm{group}}\end{array}} $$

Generation mortality

$$ \mathrm{GM}=\left(1-\mathrm{CIIPC}\right)\times 100\% $$

Data analysis

The data were analyzed using the OriginPro 9.0 software.

Results and discussion

Effect on larval mortality

Data of larval mortality are shown in Fig. 1. A significant difference was found between the results of the treatments when the Bt CAB109 concentration was more than 10³ cfu/ml. Furthermore, at 10³ cfu/ml, the mortality showed an obvious increase.

Fig. 1

Effect of Bt CAB109 on the mortality rate of S. exigua larvae

Effect on larval weight

The larvae were first treated with Bt CAB109 at different concentrations for 2 h then were reared for 6 days. The survived larvae were weighed. It was found that the average weight of the survived larvae was lower than that of the control and the increase of Bt concentration resulted in decrease of the larval weight (Fig. 2).

Fig. 2

Effect of Bt CAB109 on body weight of S. exigua larvae

The weight of each treated larva (in Fig. 2) was calculated using the following equation: W = W6 − W0, where W0 is the weight before treatment, and W6 is the weight of the larvae treated with Bt for 6 days.

Effect on larval duration

The duration of larvae treated with Bt increased with increase in concentration. The prolongation of treatment time caused significant differences among treatments (Fig. 3). The durations were 5, 3, and 2 days at Bt concentrations of 1 × 10⁶, 1 × 10⁵, and 1 × 10⁴ cfu/ml respectively.

Fig. 3

Effect on Bt CAB109 duration of larvae of S. exigua

Effect on the larvae, pupae, adults, and eggs

The Bt CAB109 could evidently affect all stages of S. exigua (Table 1). The CIIPC value decreased with increase in the concentration of Bt. It was found that the smallest IIPC value had the greatest in survival of all the pest stages.

Table 1 Effect of different concentrations of Bt CAB109

Effect on GM

Bt CAB109 affected not only the larvae, pupae, and adults, but also the eggs laid by the adults (Table 1). Therefore, we introduced the novel concept of GM to explain the relationship between the concentration of Bt and stages of S. exigua. The GM increased with increase in the concentration of Bt CAB109.

S. exigua was reported to have relatively less sensitivity to Bt (Yue-qiu and Xing-fu 2002 and Bao-shan et al. 2006). Our results revealed that unlike insecticides, Bt had sublethal effects on S. exigua and affected its biological aspects and development. These results are in agreement with those reported by Da-yong and Yong-man (2010) who stated that Bt affected the growth and development of S. exigua. In addition, Ming et al. (2002) and Donglin et al. (2007) obtained the same results when fed S. exigua larvae on Bt cotton. Although chemical insecticides quickly and efficiently control pests, unlike biological pesticides, the biological pesticides could have a sublethal effect, which could directly affect the weights of the larva, pupa, and the adult pests; growth and development; eclosion rate; egg count; and deformity development, which would inevitably result in the decline in crop yields; moreover, biological pesticides will cause less air pollution than the chemical ones (Shen et al., 1994, Xiao-hui et al. 1999 and Choi et al. 2008). Therefore, it is necessary to evaluate efficient methods for the biological control of pests using pesticides that have the follow-up effect (Shu-liang et al. 2006).

Most target pests would be killed by Bt, but there are some exceptions such as S. exigua, which has relatively lower sensitivity to Bt (Yue-qiu and Xing-fu 2002 and Bao-shan et al. 2006). Moreover, Bt was deterrent or feeding inhibition for insects to Bt. The amount of Bt toxin was insufficient to cause the death of insects, but it was enough to affect their normal growth and development (Da-yong and Yong-man 2013). The weight of the insects reduced, which was explained by reduction in food intake. Thus, although the pests were still alive, the degree of harm caused to the plants reduced. At the same time, the development duration of surviving larvae would be prolonged, the generation number would decrease, and the harm to the crops would be reduced.

In this study, with a CAB109 concentration of 1 × 10⁶ cfu/ml, the larval mortality was only 52.6%, but the subsequent actual GM could be up to 90.6%. Therefore, if Bt is applied manually, high mortality in a short period cannot be expected. At the time of the occurrence of pests of relatively low density, Bt can be proven effective for pest control, with little or no chemical pesticides. Thus, biological pesticides such as Bt are important for environmental protection and pollution-free agricultural production (Gay 2012 and Pretali et al. 2016).

Conclusion

The generation mortality can be a comprehensive and systematic reflection of the actual control effect of Bacillus thuringiensis for the biological pesticide in the actual control effect, which can make a reasonable assessment.