Egyptian Journal of Biological Pest Control, 25(3), 2015, 729-734 Proceeding of 4 th International Conference, ESPCP2015, Cairo, Egypt, 19-22 October 2015
Efficacy of Indigenous Bacillus thuringiensis Strains for Controlling Major Vegetable Pests in Bangladesh Shishir* , A.; A. A. Bhowmik* ; N. R. Akanda* ; A. Al Mamun** ; S. N. Khan* and M. M. Hoq* of Microbiology, University of Dhaka, Dhaka – 1000, Bangladesh,
[email protected]. for Advanced Research in Sciences, University of Dhaka, Dhaka – 1000, Bangladesh. (Received: October 19, 2015 and Accepted: December 30, 2015)
* Department ** Centre
ABSTRACT Integrated Pest Management (IPM) and bio-intensive pest management (BIPM) (where Bt biopesticide is an indispensible component of it) are the suggested alternatives of chemical pesticides. So, a holistic approach to the isolation and detection of potential Bt strains, production at industrial scale and administration in the field is necessary to include Bt biopesticide in the IPM and BIPM of Bangladesh. In this connection, the bioinsecticide prepared from potential indigenous Bt strain JSc1 was applied in cabbage, cauliflower and organic tea farming and was found to be efficient in controlling the target lepidopteran pests such as Helicoverpa armigera, Spodoptera litura, Plutella xylostella, Hyposidra spp. etc. Results indicated that more than 85% of the treated crops were protected from the infestation and destruction by the pests. Obtained data analyzed using ANOVA test suggested the inclusion of Bt biopesticide in the IPM of Bangladesh as no such differences were observed with the chemical pesticides currently in use.
Key words: Bacillus thuringiensis, Bt biopesticides, chemical pesticides, organic farming. INTRODUCTION The indiscriminate use of chemical pesticides in agriculture is causing serious health problems and environmental pollutions in developing countries due to their extremely hazardous and recalcitrant nature which upon mobilization through irrigation, farming and flooding results in bioaccumulation and biomagnifications accessing the food chain. It also affects soil health, microbial flora, water bodies and aquatic lives i.e. fauna and fishes (Jackson, 1991 and Ramaswamy, 1992). In addition, emergence of pest resistance to pesticides is another concurrent major problem. Integrated pest management (IPM) and Bio-intensive Pest Management (BIPM) are the efficient programs to deal this complex situation which accommodate all possible eco-friendly practices (i.e. organic farming, protecting natural enemies, reducing chemical insecticides, use of biological controls such as biopesticide, transgenic crop etc.) with greater use of economical, sustainable and environmentally safe alternatives. Bacillus thuringiensis (Bt), for its insecticidal δ- endotoxins (Cry proteins) has been the most widely used biopesticide in agriculture (Crickmore et al., 1998) and the preference is due to its high specificity as well as safety from the environmental pollutions, public health problems etc. With a view to develop efficient Bt biopesticides, so that the missing component of IPM in Bangladesh agriculture is provided, Bt strains were initially isolated from different ecosystems of Bangladesh and characterized by biochemical typing, 16S rRNA gene analysis, plasmid and cry genes profile analyses etc (Shishir et al., 2012 and Shishir et al., 2014). Screening for insecticidal genes viz. cry1, cry2, cry3, cry4A, cry8, cry9, cry10, and cry11 in the native Bt strains revealed their presence in varied proportion rendering cry1, cry2, and cry3 the most prevalent (Shishir et al., 2014) and the presumption of degree and spectrum of entomo-toxicity of the strains were made. The novel toxicity of Bt strains, harboring cry1A- type genes was then reported by us against the 3rd instar larvae of Bactrocera cucurbitae where maximum mortalities were recorded for Btk HD-73 (96%) and the indigenous Bt JSc1 (93%) (Shishir et al., 2015). Based on the findings, production of Bt biopesticide from locally available cheap raw materials including agro-industrial by products was considered to be feasible and useful. Since the expense for raw materials is one of the principal costs involved in overall Bt production, cost effective media were developed from the raw materials such as defatted mustard seed meal, defatted soybean meal, molasses, sea water etc that causing minimal costs (Hasan et al., 2011 and Mourin et al., 2015) and the sporulation as well as δ- endotoxin yield was also reported to be enhanced by regulating critical factors like C:N ratio, cystine concentration, protease activity etc (Mourin et al., 2015). Bt biopesticides are usually composed of spores and crystals protein mixtures, harvested from the production media, readily produced by aerated liquid fermentation. They are easily harvested and have a long shelf life when formulated properly (Ghribi et al., 2007). Therefore, the Bt biopesticide prepared from the indigenous Bt strain JSc1 on the cost effective media (Hasan et al., 2011 and Mourin et al., 2015) were applied in cabbage, cauliflower and tea farming and itsefficacy was compared to the conventional chemical pesticides currently in use.
730 MATERIALS AND METHODS Bacterial strains and culture conditions Reference Bt strain kurstaki HD-73, kindly provided by Okayama University, Japan and indigenous Bt strain JSc1 were used in this study. LB agar (per litre: tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 15 g) and LB broth were used for culture maintenance, propagation, subculture and spore count throughout the study and incubation temperature was maintained at 30°C for all types of cultures and the liquid cultures were incubated in an orbital shaker at 180 rpm if not otherwise stated. Inoculum preparation An isolated colony from overnight incubated LB agar medium plate at 30°C was dispensed in 3.0 ml of LB medium and incubated overnight at 30°C. Aliquots (0.2 ml) were used to inoculate 250 ml Erlenmeyer flasks containing 50 ml LB medium. After 6h of incubation at 30°C and 200 rpm, the cell density was determined at 600 nm and was used as inoculum in amounts to start with an initial OD 600nm = 0.1. Bio-insecticide production in 3.0 L bioreactor Bt bioinsecticide production was carried out at 30°C in a 3.0 L fully controlled bioreactor (New Brunswick Scientific, USA) containing 2.0 L of finally optimized medium (10% soybean extract, 0.5% molasses, 20% (v/v) sea water, 300 mg/L cystine and 4% ammonium sulfate) (Mourin et al., 2015) with continuous regulation of pH using 2N HCl and 2N NaOH. Dissolved oxygen (dO 2 ) level in the medium was automatically controlled and maintained at 30% by providing aeration at 1.0 standard liter per minute (SLPM) and agitation at 250 rpm. Fermentation was carried out up to 24 hours for maximum δ- endotoxin yield. Determination of spore and δ- endotoxin Number of spores and δ- endotoxin concentration were estimated at the end of the fermentation process. For spore count, 1.0 ml of the fermentation broth was heated at 80°C for 10 min and 100 µl of appropriate dilution after serial dilution was inoculated on LB agar medium by spread plate technique. After overnight incubation at 37°C, the colonies were counted and multiplied by the dilution factor to estimate the exact numbers. The crystal protein was partially purified following the method described by (Mourin et al., 2015) where 1.0 ml of fermentation broth was washed twice with cold sterile distilled water, once with 1.0 M NaCl and 5.0 mM EDTA and once with 5.0 mM EDTA alone. The washing steps were accomplished each time by centrifugation at 10000 rpm for 10 min. Finally, the washed pellet was resuspended in 1.0 ml of 0.1 N NaOH solution for 1 h at room temperature. Then, the partially purified crystal protein concentration in the supernatant was estimated according to Bradford method (Bradford, 1976). Field trial Bt preparation containing both Cry proteins and spores was applied in the field. The efficacy was analyzed both qualitatively and quantitatively against the caterpillars in comparison to a positive control, chemical pesticide and a negative control, water treatment usually. Bt preparations were applied at an optimum concentration of 0.3 mg/ ml of alkali soluble proteins i.e. crystal protein. Application of Bt preparation in cabbage and cauliflower farming The efficacy of Bt JSc1 spore-crystal protein preparation was tested against the pests of cabbage and cauliflower in Narshingdi. Bt preparation was applied besides control (water) and chemical pesticides (lambda cyhalothrin, emamectin benzoate) to control the common cabbage and cauliflower pests such as Helicoverpa armigera, Spodoptera litura, Plutella xylostella, Hyposidra spp. etc which belong to order Lepidoptera. Treatments were applied following randomized complete block design (RCBD) in cabbage and cauliflower farming (Fig. 1A). Treatments were both replicated and blocked along 4 blocks, each containing 3 replicates. Experiment was performed with 360 plants, 30 plants in each replicate and 90 plants in each block. Survival data of plants from pest infestation and damage from each replicate was collected and statistically analyzed.
Fig. (1): Application of different treatments: A) In cabbage farming followin g Randomized Complete Block Design (RCBD). (C: chemical pesticide; N: negative treatment, B: Biopesticide). B) Treatments in organic tea farming.
Application of Bt preparation in organic tea farming Field evaluation of Bt JSc1, garlic (+ chilli) formulation and Neem formulation was done against tea looper,
731 Hyposidra spp. twice in the year (March-April and June-July). In both cases, 60 matured tea bushes were selected for experiments and no pesticide was applied to the bushes since trimming (Fig. 1B). Treatments were applied weekly and the numbers of plants survived from pest infestation were recorded for individua l treatments. Data were analyzed statistically by ANOVA test and their efficacies were compared. RESULTS AND DISCUSSION Efficacy of Bt preparation in cabbage and cauliflower farming Qualitative results suggested that the efficacy of Bt biopesticide and chemical pesticide in protecting the cabbage and cauliflower from pest infestation were equal (Fig. 2). As cabbage and cauliflower damaging lepidopteran pests such as H. armigera, S. litura, P. xylostella, Hyposidra spp., were challenged by sporecrystal preparation from Bt JSc1 in parallel to chemical pesticide and control treatments, Bt biopesticide preparation was found to exert almost similar bioactivity against the pests with cabbage and cauliflower as did the chemical pesticide. At the final stage of the farming, it was observed that the untreated plants were completely damaged whereas chemical pesticide or Bt biopesticide treated plants were protected and the healthy cabbage and cauliflower were grown. For cabbage, more protectionwas provided by Bt biopesticide than the chemical pesticide but for cauliflower, it was opposite with slight difference. Quantitative analysis of the treatments revealed that both Bt biopesticide and chemical pesticide treatments protected about 90% of the plants whereas only 25% plants survived with negative treatment i.e. untreated plants (Fig. 3A). Average weight of cabbage was slightly higher for Bt biopesticide (1.885 kg) than that of chemical pesticides (1.855 kg) and it was opposite for cauliflower (Bt- 1.36 kg, and Chemical- 1.56 kg). The average weight of survived untreated plants was almost 38% less in both cases (cabbage- 1.165 kg and cauliflower- 0.918 kg) (Fig. 3B). The condition of untreated plants indicates the level of pest infestation which was very high in the experiment as survival of untreated plants were only 25% and their weights were almost 38% less from the treated plants. The efficiency of Bt biopesticide based on these two criteria suggests that the replacement of chemical pesticide will provide food safety as a surplus over the food security without affecting the production yield. Performance of the Bt biopesticide preparation was more effective on cabbage farming over cauliflower. This might be due to the difference in their textures which facilitated more protection in cabbage than in cauliflower. Efficacy of Bt preparation in organic tea farming Efficacy i.e. the mean survival percentage of tea plants with Bt biopesticide (53.8%) was found to be very close to the other treatments i.e. garlic (51.6%) and neem formulations (44.3%), currently in use in organic tea garden. The maximum number of survived plants (more than 75%) in the 4th week was recorded for Neem formulation treatment and was dropped as the treatment was stopped after 4th week, followed by a resume in the 6th week. On the other hand, relatively lower survival percentages were recorded for Bt biopesticide and garlic formulation initially but both of them retained their impacts with time even with an interruption (Fig. 4). Both Bt biopesticide and garlic formulation protected maximum numbers of plants after 10th week of their application but neem formulation could not regain its initial protection efficiency level. The drastic drop in protection by neem formulation might be due to its short residual activity and thus the larvae exposed to it might have survived. Adding to this, resistance might have evolved with them and transferred to the offspring. But this was not evidenced for Bt biopesticide preparation and garlic formulation. Although the variance was observed to be higher for Bt biopesticide, this does not imply random and higher fluctuation of performance rather consistent improvement of protection over time. Statistical Analysis of Variance Cabbage and cauliflower farming The statistical comparison of the efficacy of Bt biopesticide to the chemical pesticide by ANOVA (TwoWay) produced the F values which indicated that at α = 0.05, significant differences among the treatments prevailed both in cabbage (Bt biopesticide, Chemical pesticide and Control) (F= 175.658; df=11; P-value= 0.00000473) and cauliflower (Bt biopesticide, Chemical pesticide and Control) (F= 661.151; df = 11; P-value = 0.0000000922) farming (Table 1). As significant differences among the treatments were determined statistically (Table 1), pair wise difference analyses among the multiple treatments were calculated by Fisher Least Significant Difference (LSD) method. The analyzed data provides much evidences to conclude that, at α = 0.05 level of significance, Bt biopesticide and chemical pesticide did not differ significantly whereas the pairs of Bt biopesticide and control as well as chemical pesticide and control differed significantly in cabbage farming. It means that the efficacy
732
Fig. (2): Impact on pest infestation and growth of cabbage and cauliflower in different treatments. Untreated plants were found to be damaged and Bt biopesticide produced comparable results to chemical pesticides.
Fig. (4): Comparison of potentialities of Bt biopesticide, Neem formulation and Garlic formulation in protecting the tea plants from pest infestation.
Fig. (3): Comparison between the Bt preparation and chemical pesticides in terms of A) Survival rate against pest infestation, B) Productivity of cabbage and cauliflower plants.
Table (1): Statistical data obtained from the ANOVA (Two- Way) test performed on the average survival of cabbage and cauliflower plants from the pest infestation. ANOVA for Randomized Complete Block Design (RCBD) Source of Variation
Cabbage
Cauliflower
Df
SS
MS
F
P-value
df
SS
MS
F
P-value
Treatment
2
6021
3010.6
175.658
0.00000473
2
6795
3398
661.151
0.0000000922
Block
3
89
29.6
1.724
0.261
3
103
34
6.676
0.0244
Residual
6
103
17.1
6
31
5
Total
11
6213
11
6929
Table (2): Fisher Least Significant Difference (LSD) Method for Multiple Comparison Test Group vs Group (Contrast) Biopesticidevs Chemical pesticide Biopesticidevs Control Chemical pesticide vs Control
Difference (taking the absolute value) Cabbage Cauliflower 1.5 3.75 48.25 48.5 46.75 52.25
Least Significant Difference (LSD) Cabbage Cauliflower 7.154864 3.868907 7.154864 3.868907 7.154864 3.868907
733 Table (3): Statistical data obtained from the ANOVA (One- Way) test performed on the average survival of tea plants from the pest infestation Summary (Organic Tea farming)Treatments Bt biopesticide Garlic formulation Neem formulation Total
Sample size 11 11 11 33
Sum of survival percentage 592 568 487 1647
Mean 53.8181 51.6363 44.2727 49.9090
Variance 32902 30448 23221 136.5852
ANOVA (Organic Tea farming): Source of Variation Between Groups Within Groups Total
df 2 30 32
SS 550.3636 3820.3636 4370.7272
MS 275.1818 127.3454
F 2.1609
p- level 0.1328
F crit 3.31583
Omega Sqr. 0.06573
of Bt biopesticide and chemical pesticide in controlling the pests in cabbage existed highly in comparison to the no treatment. But there was insignificant difference between these two efficient treatments (Table 2). Again, in cauliflower farming, similar results were observed i.e. Bt biopesticide and chemical pesticide did not differ significantly at α = 0.05, whereas the pairs (Bt biopesticide and control and chemical pesticide and control) differed significantly at α = 0.05. These results suggest that the substitution of chemical pesticide currently in use in cabbage and cauliflower farming with the Bt biopesticide preparation will be feasible in terms of controlling pests keeping food safety and without affecting the production yield. The experiment was performed in randomized complete block design (RCBD) where treatments (Bt biopesticide, chemical pesticide and negative control) were blocked and replicated. This design is most effective if the patterns of non-uniformity (changing soil types, drainage patterns, fertility gradients, direction of insect migration into the field, etc.) in a field were identified. If the extraneous variability associated with field was not removed prior to testing for a treatment effect, it would be more difficult to detect treatment effects via F- test. In other words, the denominator in the F- test would be larger than needed because it contains variability associated with field. If the potential sources of variation were not identifiable, this design can still be used keeping the blocks as square as possible which usually keeps the plots within a block most uniform. The goal of RCBD was to maximize the differences among the blocks while minimizing the differences within the block so that statistically sound unbiased data are produced. Organic tea farming Statistical data obtained from the ANOVA (One- Way) produced an F value indicating that at α = 0.05, there was insignificant difference between the mean survival of tea plants for all the treatments throughout the field trial replicates (F= 2.1609; df= 32; P = 0.1328). This inferred that these three treatments, Bt biopesticide formulation, garlic formulation and neem formulation were of equal effects in organic tea farming (Table 3). Hence, Bt biopesticide preparation can be used in tea farming in parallel to other biopesticides in use and for its longer residual activity as well as lost cost, it will be highly potential eco-friendly biological pest control agent in organic tea farming. ACKNOWLEDGMENT This study was partly supported by the grant of USDA Agricultural Biotechnology research program coordinated by Ministry of Education, Government of Bangladesh. We also acknowledge Nurjahan Akhter Laboni, graduate student at Institute of Statistical Research and Training, University of Dhaka, for her statistical analysis and supports. REFERENCES Bradford, M. M. 1976. A Rapid and Sensitive Method for the Quantization of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254. Crickmore, N., Zeigler, D. R., Feitelson, J., Schnepf, E., Van-Rie, J., Lereclus, D., Baum, J. and Dean, D. H.
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