Pathogenicity of indigenous soil isolate of Bacillus thuringiensis to Helicoverpa armigera Hübner 1809 (Lepidoptera: Noctuidae)

  • Muhammad Zeeshan Majeed
  • Muhammad Asam Riaz
  • Muneeba Arif Khan
  • Chun-Sen Ma
  • Salman Ahmad
Open Access


This study evaluated the pathogenicity of indigenous soil isolate of Bacillus thuringiensis (Bt) strains, applied without and along with 1.0% MgCl2 salt, to Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). Toxicity and effect of Bt isolate on larval development (weight) were assessed using in vitro bioassays. Six concentrations of the tested Bt with salt (i.e., 1.0 × 107, 0.5 ×  105, 1.0 × 105, 1.5 × 105, and 2.0 × 105 cfu/ml), five without salt (i.e., 0.5 × 105, 1.0 × 105, 1.5 × 105, and 2.0 × 105 cfu/ml), and control were bioassayed against third-instar larvae of H. armigera under a complete randomized design (CRD), with four replications. Results revealed that both larval mortality and weight changes were significantly affected by time (F5, 19 = 35.98; P <  0.001 and F5, 19 = 11.01; P < 0.001, respectively) and treatments (F5, 19 = 27.45; P < 0.001 and F5, 19 = 25.07; P < 0.001). The highest larval mortality (91.1%) was exhibited by the highest concentration (1.0 × 107 cfu/ml), followed by 2.0 × 105 cfu/ml concentration, without salt (88.9%) and with salt (66.7%). Median lethal concentration (LC50) values of isolated Bt strain were 1.7 and 1.27 × 105 cfu/ml, without salt, and 1.80 and 1.13 × 105 cfu/ml, with salt, at 96 and 120 h, respectively. Regarding the impact of Bt isolate on larval development, treatment with the highest concentration (1.0 × 107 cfu/ml) had the most significant and negative impact on larval weight change (R2 = 0.53), followed by 2.0 × 105 cfu/ml Bt concentration (R2 = 0.40). There was no obvious synergistic or additive effect, but rather an inhibition was observed on the pathogenicity potential or larvicidal effect of Bt isolate.


Bacillus thuringiensis Microbial insecticides Helicoverpa armigera Toxicity evaluation 


Lepidopterous species are among the most devastating and economically important insect pests causing quantitative and qualitative losses of the yields all over the world (Ndemah et al. 2001; Mazzi and Dorn 2012). Helicoverpa spp. and Spodoptera spp. are the most damaging and cosmopolitan pest species. These species have well-adapted different agro-ecological zones around the globe from Asia to Africa to Europe (Zhang et al. 2015). They are polyphagous pests with a wide range of host plants including: cotton, maize, gram, sesame, okra, tomato, potato, and many other fruit and vegetable crops (Aggarwal et al. 2006; Nagoshi et al. 2011). Nevertheless, due to blind and extensive use of pesticides, these and other lepidopterous pest species have developed resistance to almost all groups of pesticides including organochlorines, organophosphates, carbamates, and pyrethroids (Kranthi et al. 2001).

Microbial bio-pesticides are eco-friendly and target specific alternates to hazardous synthetic pesticides (Kumar and Singh 2015; Majeed et al. 2017). Worldwide, an annual increase of 10% in the use of bio-pesticides has been estimated and among them, approximately 90% formulations are derived from Bacillus thuringiensis (Bt) (Kumar and Singh 2015; Osman et al. 2015). However, there is a great potential to look for the indigenous strains and isolates of Bt and characterize their pathogenicity and toxicity against insect pest species (Sree and Varma 2015).

The present study assessed the toxicity of local soil isolates of Bt against Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), a notorious pest of cotton, maize, gram, okra, sesame, and other crops in Indo-Pak region (Bibi et al. 2013; Qayyum et al. 2015). Moreover, as the sporulation of Bt has been found enhanced in the presence of certain inorganic ions particularly chlorides of Ca and Mg (Tabbene et al. 2009; Bibi et al. 2013), isolates of Bt toxins and spores were treated with MgCl2 salt. Apart from determination of lethal concentrations (mortality), the effect of Bt strain on the larval development (larval weight) was also assessed.


Collection and preparation of samples

Composite soil samples were collected from cultivated and non-cultivated fields from different locations of the district Sargodha (Punjab, Pakistan). Sampling sites were confirmed to have received no application of pesticide including Bt formulation. Samples were brought to the laboratory and homogenized. Five grams of aliquot samples was taken from this large composite soil sample for further processing and isolation of Bt strains.

Preparation of soil solutions

Five grams of each soil sample was taken and dissolved in 10 ml of sterilized water and was thoroughly homogenized on orbital shaker. One milliliter of this solution was put in 9 ml sterilized water in a sterilized test tube and was homogenized by shaking. From this stock solution, serial dilutions were prepared using double distilled autoclaved water.

Preparation of culture media

Nutrient agar is widely used as a general purpose medium for culturing a large number of micro-organisms. Nutrient broth and nutrient agar powder (Merck KGaA, Darmstadt, Germany), 5 g each, was dissolved in 250 ml distilled water and autoclaved at 120 °C for 15–20 min under a pressure of 16–20 psi. Then, this medium was moderately cooled at 28 °C for 20 min and poured in sterilized Petri dishes and left for 20–30 min to be solidified for further utilization in downstream process.

Sample inoculation

In order to get the bacteria present in soil samples, five culture media plates were inoculated with each soil solution by streaking soil sample dilutions starting from lower to higher dilutions. Immediately after inoculation of plates, Petri plates were wrapped with paraffin film and aluminum foil for avoiding foreign contamination and were placed in the incubator for 24 h at 30 °C, after which bacterial colonies were grown on media plates. These bacterial colonies were picked with the help of a wire lope and examined under a compound microscope for confirmation of Bacillus spp.

Furthermore, peptone agar medium (Merck) was used for the re-streaking of bacterial colonies already grown on culture media. This medium was poured on sterilized Petri dishes (3 mm layer) and upon solidification at room temperature for 30 min; it was inoculated by the bacterial colonies. Inoculated Petri dishes were wrapped with paraffin film and aluminum foil and incubated at 30 °C. After 36 h, pure colonies (whitish colonial growth) of Bt were verified under a microscope and further grown in liquid media for preparation of pure bacterial cultures containing enough bacterial cells to prepare microbial formulation of Bt.

Bacterial proliferation in liquid medium

To prepare a liquid medium for bacterial cells multiplication, 100 ml of UG medium (containing 7.5 g peptone and 6.8 g KH2PO4; pH 7.4) and 10 ml of stock solution 1 (containing 12.3 g L−1 MgSO4·7H2O, 0.17 g L−1 MnSO4·7H2O, and 1.4 g L−1 ZnSO4·7H2O), stock solution 2 (containing 2.0 g L−1 Fe2(SO4)3 and 3 g L−1 H2SO4), and stock solution 3 (containing 14.7 g L−1 CaCl2·2H2O) were added in a sterilized 250-ml conical flask, following inoculation and incubation on orbital shaker (220 rpm) at 30 °C for 48 h.

Preparation of B. thuringiensis suspensions

A liquid medium with purified bacterial colonies was centrifuged at 250 rpm for 20 min, and pure whitish colonies of bacteria gathered in the form of pellet in Eppendorf tube base were further suspended in 0.5 M sodium chloride (NaCl) solution and centrifuged again at 250 rpm for 10 min. Whitish colonies collected at the base of Eppendorf tube were re-suspended in double distilled autoclaved water and filtered through filter paper. These filtered out bacterial colonies, containing bacterial toxins, spores, and cells, were used to prepare treatment solutions either directly or by adding 1.0% MgCl2 salt. Five different Bt concentrations, i.e., 1.0 × 107, 0.5 × 105, 1.0 × 105, 1.5 × 105, and 2.0 × 105 colony-forming units (cfu) per milliliter, were prepared, using double distilled autoclaved water as solvent.

Pathogenicity bioassays

Standard leaf-dip bioassays were conducted, using sterilized glass Petri dishes (9 cm). Test solutions of Bt isolate were made in double distilled autoclaved water. Fresh unsprayed gram leaves were cut, washed thoroughly with distilled water, and shade-dried at room temperature (28 °C). These leaves were dipped for 10 s in test solutions and then were placed for 15 min on autoclaved towel paper for drying with their adaxial surface upward. Then, treated leaves were placed in Petri dishes with five laboratory reared third-instar larvae of H. armigera. Experimental design was completely randomized (CRD), with four replications for each treatment including control (water). Mortality of larvae was assessed at 24, 48, 72, 96, and 120 h post treatments. Moribund individuals were considered dead. A separate independent bioassay was carried out to assess the impact of isolated Bacillus strain on larval development (body weight), following the same experimental protocol as described above.

Statistical analysis

Statistix® (V8.1 for Windows® (Analytical Software 2005), Statistix, Tallahassee, FL) was used for statistical manipulation of data. Larval mortality data was corrected according to Abbott’s procedure (Abbott 1925) before further statistical analyses. Data normality was checked, using Shapiro-Wilk tests at α = 0.05, and data were log transformed (Log10(X + 1)) in case of abnormally distributed data. Toxicity of Bacillus strain was determined by working out median lethal concentrations (LC50) values separately for 72, 96, 120 h post exposure data, using probit analysis with 95% confidence limits (Finey 1971). The effect of Bt treatments on larval mortality and weight change was assessed by one-way and two-factor (factorial) analysis of variance, and Tukey’s highest significant different (HSD) tests at 5% level of significance were used for comparison of treatment means.

Results and discussion

According to the overall factorial analysis (Tables 1 and 2), when Bt strain with salt was tested, there was a significant effect of time (F4, 15 = 8.62; P < 0.001) and Bt treatments (F4, 15 = 8.18; P < 0.001) on larval mortality but not of their interaction (F16, 47 = 1.19; P < 0.311), while larval weight change was only affected by time factor (F4, 15 = 2.75; P < 0.038) (Table 1). Similarly, both larval mortality and larval weight changes were significantly affected by time (F5, 19 = 35.98; P < 0.001 and F5, 19 = 11.01; P < 0.001, respectively), Bt treatments (F5, 19 = 27.45; P < 0.001 and F5, 19 = 25.07; P < 0.001), and their interaction (F20, 79 = 3.98; P < 0.001 and F20, 79 = 2.85; P = 0.001) (Table 2).
Table 1

Analysis of variance comparison of mortality of the third-instar larvae of Helicoverpa armigera Hübner bioassayed with soil isolated strain of Bacillus thuringiensis applied with 1.0% MgCl2 salt



Larval mortality (%)

Larval weight change (%)


F value

P value


F value

P value











< 0.001*








< 0.001*




Time × treatments























*P <  0.001 (highly significant) and P <  0.05 (significant); two-way factorial ANOVA at α = 0.05

Table 2

Analysis of variance comparison of mortality of the third-instar larvae of Helicoverpa armigera Hübner bioassayed with soil isolated strain of Bacillus thuringiensis applied without 1.0% MgCl2 salt



Larval mortality (%)

Larval weight change (%)


F value

P value


F value

P value











< 0.001*



< 0.001*





< 0.001*



< 0.001*

Time × treatments




< 0.001*



















*P < 0.001 (highly significant) and P < 0.05 (significant); two-way factorial ANOVA at α = 0.05

Toxicity of isolated Bt strain to H. armigera larvae

There was no larval mortality at 24-h exposure to all Bt concentrations either with or without salt. This is in line with the mode of action of Bt strains as their protoxins upon ingestion by the target insect pests take about 48 to 72 h to be converted into toxic protein crystals which further bind with midgut epithelial cells and cause mortality (septicemia) symptoms (Bravo et al. 2017).

However, there was a significant mortality at 48 h (F5,19 = 212; P <  0.001), 72 h (F5,19 = 197; P <  0.001), 96 h (F5,19 = 306; P <  0.001), and 120 h (F5,19 = 478; P <  0.001) post infection without salt. At 48-h exposure, the highest mortality (22.2%) was observed for 1.0 × 107 cfu/ml Bt treatment, followed by 2.0 × 105 cfu/ml concentration, while the minimum mortality (0.0%) was recorded for 0.5 and 1.0 × 105 cfu/ml concentrations without significant difference (Fig. 1). At 72 h post exposure, maximum larval mortality (44.4%) was exhibited by 1.0 × 107 and 2.0 × 105 cfu/ml concentrations, while there was no mortality observed for 0.5 × 105 cfu/ml concentration. The highest and significant mortality at 96 h was recorded for 1.0 × 107 cfu/ml (66.7%) and by 2.0 × 105 cfu/ml (55.6%), and both were significantly different from other lower concentrations of Bt (Fig. 1). Similarly, at 120 h, minimum larval mortality was recorded for 0.5 × 105 cfu/ml (11.1%), statistically significant from mortalities at 1.0 × 105 cfu/ml (33.3%), and 1.5 × 105 cfu/ml (44.4%). The highest larval mortality was exhibited by 1.0 × 107 and 2.0 × 105 cfu/ml concentration (88.9%), without a significant difference (Fig. 1). Median lethal concentrations (LC50) at 72, 96, and 120 h were 2.2 × 105 cfu/ml, 1.7 × 105 cfu/ml, and 1.27 × 105 cfu/ml, respectively (Table 3).
Fig. 1

Mean mortality of the third-instar larvae of Helicoverpa armigera Hübner exposed to different concentrations of Bacillus thuringiensis isolate with or without 1.0% MgCl2 salt. Columns represent mean percent mortality ± standard error (n = 4). For each time interval, bars with similar alphabetic letters are not significantly different (ANOVA; P ≤ 0.05)

Table 3

Median lethal concentration (LC50) values of Bacillus thuringiensis strain isolated from soil bioassayed against the third-instar larvae of Helicoverpa armigera Hübner


Observation time (h)

LC50 (× 105 cfu/ml)

95% FL


χ2 (df = 10)

P value

Without salt




4.005 ± 0.343






2.218 ± 0.188






3.571 ± 0.206



With salt



1.804 ± 0.192






2.397 ± 0.194






3.633 ± 0.203



LC 50 lethal concentration (× 105 cfu/ml) of tested Bt treatment that killed 50% of exposed larvae, FL 95% Fiducial (confidence) limits, df degree of freedom, – incalculable

Similarly, when the Bt was tested by 1.0% MgCl2 salt, there was a significant mortality at 48 h (F4,15 = 318; P <  0.001), 72 h (F4,15 = 503; P <  0.001), 96 h (F4,15 = 209; P <  0.001), and 120 h (F4,15 = 597; P <  0.001) of exposure. At 48-h exposure, the highest mortality (22.2%) was observed for 2.0 × 105 cfu/ml Bt concentration, followed by 1.5 × 105 cfu/ml concentration (11.2%), while no mortality was recorded for 0.5 × 105 cfu/ml and 1.0 × 105 cfu/ml Bt concentrations (Fig. 1). At 72 h post exposure, maximum larval mortality (33.6%) was exhibited by 2.0 × 105 cfu/ml concentration, and it was significantly different from larval mortalities at 1.5 × 105 cfu/ml and 1.0 × 105 cfu/ml. At 96 h post exposure, the highest mortality was recorded for 2.0 × 105 cfu/ml (44.5%) followed by 1.5 × 105 cfu/ml (33.3%), and both were significantly different from lower concentrations of Bt (Fig. 1). Likewise, minimum larval mortality was recorded for 0.5 × 105 cfu/ml (11.1%) at 120-h exposure and was significantly different from mortalities at higher Bt concentrations. LC50 values for Bt with salt were 2.5 × 105 cfu/ml, 1.8 × 105 cfu/ml, and 1.1 × 105 cfu/ml for 72, 96, and 120 h, respectively (Table 3). Estela et al. (2004) and Makhlouf et al. (2016) demonstrated the same trend of larval mortality, i.e., 72 and 85%, respectively, at 120 h of exposure to many soil isolates of Bt.

Effect on larval development (body weight) of H. armigera

Weight of H. armigera third-instar larvae was also determined at each treatment during the entire Bt bioassay and is represented in Fig. 2. Average weight of third-instar larva of H. armigera was 72.5 ± 8.7 Mg. At 48 h post exposure, average larval weight increased almost three times for all treatments except for treatment with the highest Bt concentration (1.0 × 107 cfu/ml) for which only 1.5 time increase was observed but without significant difference (Fig. 2). However, a significant difference was observed in average larval weight change after exposure of 72 h and upwards. At 96-h exposure, a significant reduction in average larval weight was recorded for 1.0 × 107 and 2.0 × 105 cfu/ml Bt concentration treatments, while the maximum weight gain was exhibited by control (363%) and at 0.5 × 105 cfu/ml Bt concentration (264%). The same trend was recorded for larval weight change at 120-h exposure (Fig. 2). Regarding individual treatment impact, 1.0 × 107 cfu/ml Bt concentration had the most significant and negative impact on average larval weight change (R2 = 0.53), followed by 2.0 × 105 cfu/ml (R2 = 0.40), while the most significant and positive effect was exhibited by the control (R2 = 0.88), followed by 0.5 × 105 cfu/ml Bt concentration (R2 = 0.61) (Fig. 2).
Fig. 2

Mean weight change of the third-instar larvae of Helicoverpa armigera Hübner exposed to different concentrations of Bacillus thuringiensis isolate with or without 1.0% MgCl2 salt. Data points represent mean percent weight change ± standard error (n = 4)

A similar trend was observed for bioassay of Bt with MgCl2 salt (Fig. 2). Obtained results are in agreement with those of Huang et al. (2005) and Sellami et al. (2013) who revealed a significant loss of body weight in the third-instar larvae of Ostrinia nubilalis, Spodoptera littoralis, and Ephestia kuehniella after 3 to 4 days post exposure to Bt Cry1Ac andVip3 toxins.

Impact of MgCl2 mineral salt on pathogenicity of Bacillus spp. against H. armigera

Regarding the effect of mineral salt (MgCl2) on the pathogenicity potential of Bt isolate, results showed no obvious synergistic or additive effect but there was an antagonistic effect on the larval mortality and weight change (Figs. 1 and 2). The highest mortalities of larvae (55 and 65%) were observed at 96 and 120 h post exposure, respectively. One reason of this negative or antagonistic effect of MgCl2 salt on toxicity of Bt isolate would be very high amount of salt resulted in delayed toxicity of Bt toxins as evidenced by Estela et al. (2004).


In conclusion, the results of present study corroborate the biocontrol potential of different entomopathogenic agents such as B. thuringiensis found in indigenous soils for the non-chemical and eco-friendly management of economically important insect pests such as H. armigera and manifest the significance of taking into consideration the pathogenic characterization of native soil strains of B. thuringiensis against exotic insect pests as emphasized by different studies (Bravo et al., 2017).



The authors are thankful to Dr. Umar Farooq (Department of Food Science and Nutrition, University of Sargodha) for assisting in the identification and culturing of isolated Bt strain.


There is no funding source to be declared for this study.

Availability of data and materials

All datasets on which conclusions of the study have been drawn are presented in the main manuscript.

Authors’ contributions

MZM, MAR, and SA conceived and designed the experimental protocols. MAK and MAR performed the experiments. CSM and MAR provided the technical assistance in the experimentation. MZM and SA performed the statistical analyses. MZM, MAR, and MAK prepared the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Muhammad Zeeshan Majeed
    • 1
    • 2
  • Muhammad Asam Riaz
    • 2
    • 4
  • Muneeba Arif Khan
    • 2
  • Chun-Sen Ma
    • 1
  • Salman Ahmad
    • 3
  1. 1.State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingPeople’s Republic of China
  2. 2.Department of Entomology, College of AgricultureUniversity of SargodhaSargodhaPakistan
  3. 3.Department of Plant Pathology, College of AgricultureUniversity of SargodhaSargodhaPakistan
  4. 4.Department of EntomologyUniversity of GeorgiaAthensUSA

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