The use of synthetic organic insecticides developed during the last half of this century to control pests of agricultural and veterinary importance poses risk to human health and threatens serious environmental problems. The efforts suggesting and developing alternate control strategies for insect pest management have been gaining importance lately. One contemporary approach that has received attention is the exploitation of Bacillus thuringiensis (B.t.) toxin proteins as insecticides, as these have been - shown to be selectively fatal to some insects-and harmless to non-target organisms including man. The use of recombinant DNA technology has facilited the cloning of toxin genes in plants, in seeds and soil inhabiting bacteria, and thereby providing means for toxin delivery to insects. B.t. is a soil bacterium found almost under all environmental conditions.
The aim of present study is to search local strains of this bacteria with effective biocidal activity, to evaluate the toxicity of different strains of Bacillus sp. To work out the growth conditions of selected Bacillus with specific objective of enhancing their toxin . production, to genetically characterize the bacteria with an object to generate baseline date for future transformation experiments.
1. For this, a total of 500 soil samples 'were collected from different areas of Pakistan. The isolates were processed through Gram's staining, endospore staining and through a number of biochemical tests. The percentage for the presence of different species of Bacillus in soil samples was 33% for B. coagulans, 18% for B. megaterium, 13% for B. brevis, 12% for B. sphaericus, 11% for B. firmus, 7% for B. thuringiensis, 3% for B. stearothermophilus and 2% for B. alvei. So the most common species among soil samples were B. coagulans. The percentage occurrence of B. thuringiensis isolated from houseflies was 20% and from mosquito was 17%.
2. The antibodies were raised in domesticated rabbits against each isolate which ere used for accurate identification of the species. Different species showed different reactions. The antibodies were raised against B. alvei (isolate 1), B. brevis (isolate 7), B. thuringiensis (isolate 16), B, coagulans (isolate 23), B. firmus (isolate 53), B. megaterium (isolate 63), B. sphaericus (isolate 79) and B. stearothermophilus (isolate 90), and were used against all the 90 isolates. The clumping reaction of these isolates against their respective antibodies was, positive for the species B. firmus, B. stearothermophilus, B. sphaericus and B. alvei. For B. brevis sample CMBL7-10 and CMBL12, clumps were formed between the antibodies raised in rabbit serum and bacterial suspension, while no such agglutination reaction was observed for sample CMBL3-6, 11 13 and 14. For B. megaterium, clumping reaction was positive for sample CMBL63-66, 68-70, 72-74 and 78, while no such reaction was observed for sample CMBL 67,71 and 75-.77. For B. coagulans, production of antibodies was positive for sample CML23, 24, 27, 29-34, 37, 39, 44-50,and negative for sample CMBL 25, 26, 28, 35, 36, 38, 40-43 and 51-52. Against B. thuringiensis antibodies were raised for sample CMBLI6-19, and sample CMBL15-20 were showing no agglutination reaction
3. The growth conditions of some bacterial species i.e., B. sphaericus, B. thuringiensis, B. coagulans, B. megaterium and B.. alvei were optimized. For this purpose, optimum temperature, pH, inoculum size and growth pattern were studied. In addition to this, hydrocarbon degrading ability, salt tolerance and heavy metal resistance and antibiotic resistance were also checked. The optimum temperature was 37°C for B. thuringiensis-l, B. thuringiensis-2, B. sphaericus, B. coagulans, B. megaterium and B. alvei. pH 7 was the most suitable pH for the maximum growth of Bacillus thuringiensis-1, Bacillus thuringiensis-2; B. sphaericus and B. megaterium, while pH 7.5 was suitable for B. coagulans and B. alvei. Inoculum size 10% of the total bacterial culture was most suitable for the optimum growth of B. thuringiensis-l, B. sphaericus, B. coagulans, B. megaterium and B. alvei while 2000 volume of inoculum size was found optimum for B. thuringiensis-2.
4. Growth curves of isolates showed typical sigmoid growth pattern. 5. Bacteria were grown in the presence of different hydrocarbons (Benzoic acid, Salicylate and Phenanthrene). The maximum tolerance ability of Bacillus thuringiensis-l for benzoic acid was 195 mM, for salicylate was 80 mM and for phenanthrene was 10 mM. For Bacillus thuringiensis-2, 190 mM was the inhibitory concentration of benzoic acid, 70 mM for salicylate and 12 mM for phenanthrene. Bacillus sphaericus can degrade benzoic acid at the concentration of 195 mM, salicylate at 80 mM and phenanthrene at 12 mM. The maximum concentrations of various hydrocarbons degraded by Bacillus coagulans were: 20mM of benzoic acid, 12 mM of salicylate and 0.5 mM of phenanthrene. Bacillus megaterium was able to degrade benzoic acid upto 10 mM, salicylate upto 7 mM and phenanthrene upto 0.5 mM. For Bacillus alvei, maximum tolerant concentration for benzoic acid was 10 mM, for salicylate was 7 mM and for phenanthrene was 0.5 mM
6. Different species of Bacillus were grown in the presence of high salt concentrations, heavy metals (such as Pb2+, Cd2+, Co2+, Cu2+, Hg2+, Fe++, Zn++, Cr6+) Bacillus thuringiensis-1 showed maximum tolerance against sodium chloride and potassium dichromate. In these cases, growth was still present even in 6 M NaC1 and 3 M K2Cr2 O7. B. thuringiensis-l could tolerate 20 mM each of Pb(CH3COO)2, CuCl2 and FeSO4, 19 mM ZnSO4, 12 mM CdCl2, 10 mM HgCl2 and 8 mM CoC2. Bacillus thuringiensis-2 could tolerate 6 NaCl. The MIC of Pb(CH3COO)2 and FeSO4 was 25 mM, while for cadmium chloride and copper chloride was 20 mM. For zinc sulphate, it was 19 mM, for mercuric chloride 10 mM, for cobalt chloride 12 mM and for potassium dichromate 3 M. Bacillus sphaericus showed maximum tolerance for sodium chloride as it was 6 M. It could also tolerate lead acetate, ferrous sulphate and zinc sulphate uptill 20 mM, cadmium chloride upto 12 mM cobalt chloride upto 11 mM, copper chloride upto 21 mM, mercuric chloride upto 10 mM and potassium dichromate upto 3 M. The MICs of different salts and heavy metals against Bacillus coagulans are as follows: NaCI 0.5 M, Pb(CH3COO)2 9 m.M, CdCI2 0.4 mM, CdCI2 5 mM, CuCI2 15 mM, HgCI2 0.3 mM, FeSO4 26 mM, ZnSO4 3 mM and K:Cr2 O7 7 mM. Minimum concentrations of salts and metals to inhibit the growth of Bacillus megalerium were 3 M for NaCl. 9 mM for Pb(CH3COO)2 and K2Cr2O7. 9 mM for CoCI2, 5 mM for CoCl2, 15 mM for CuCI2. and mM for HgCI2, 25 mM for FeSO4 and 3 mM for ZnSO4. Bacillus alvei could tolerate sodium chloride uptill 3 M. The MICs of different metals against B. alvei was as follows: Pb(CH3C,OO)2 and K2Cr2 O7 9 mM, CoCI2 5 mM, CdCI2 and HgCI2 0.5 mM, ZnSO4 3 mM, CuCI2 16 mM and FeSO4 25 mM.
7. The effect of different antibiotics (ofloxcin, ceporex, ,azactum, amakin, enoxabid, urixin, doxycline and erythromycin on Bacillus isolates were also studied. Bacillus thuringiensis-l was sensitive to ofloxcin (10 μg), ceporex, (30 μg), azactum (30 μg), and amakin (30 μg), and resistant to enoxabid (30 μg), urixin (50 μg), doxycline (30 μg) and erythromycin (15 μg). Bacillus thuringiensis-2 was sensitive to enoxibid (30 μg), urixin (50 μg), ofloxin (10 μg), ceporex (30 μg), azactum (30 μg), and amakin (30 μg), and resistant to doxyc1ine (30 μg), and erythromycin (15 μg). Bacillus sphaericus was sensitive to enoxabid (30 μg), urixin (50 μg) ofloxin (10 μg), azactum (30 μg), and amakin (30 μg), while resistant to ceporex (30 μg), doxycline (30 μg) and erythromycin (15 μg). Bacillus coagulans was resistant to all antibiotics tested. Bacillus alvei was susceptible to enoxabid (30 μg), urixin (50 μg), ofloxcin (10 μg) and amakin (30 μg), while resistant to ceporex (30 μg), azactum (30 μg), doxyc1ine (30 μg) and erythromycin (15 μg). Bacillus megaterium was susceptible to enoxabid (30 μg), urixin (50 μg), ofloxcin (10 μg) and amakin (30 μg), while resistant to ceporex (30 μg) azactum (30 μg), doxycline (30 μg) and erythromycin (15 μg).
8. Bacterial strains i.e., Bacillus thuringiensis, and Bacillus sphaericus were further proceeded to optimize the medium suitable for high yield of spores and dry cell weight of these species. Four different media were used. The compositions of media varied with respect to the presence and absence of some chemicals and also in their quantities. As amount of glucose in medium 1 is 2 g, 5 g in medium 2, 15 g in medium 3 and 30 g in medium 4. Bactopeptone is present only in medium 1, while absent from rest of the media. Yeast extract is not present in medium 1, 2 and 3, while 4.5 g is present in medium 4. For Bacillus thuringiensis. medium 4 was the most suitable medium which produced 5.9xl09 spore count as it has more amount of glucose as compared to other medium and also the addition of yeast extract. For Bacillus sphaericus, the most suitable medium for the maximum yield of spores was medium 2 producing 4.14x 107 spores/ ml.
9. Different isolated species of Bacillus were evaluated for their toxicity against common household pests viz., houseflies, cockroaches and mosquitoes. Against houseflies, the most toxic strains in this study were B. thuringiensis and B. sphaericus which caused 68% and 65.3% mortality, respectively. Other strains were also toxic as B. megaterium, B. firm us, B. brevis, B. stearothermophilus, B. coagulans and B. alvei showed 59%, 55%, 53%, 52%, 32% and 29% mortality, respectively.
10. Against cockroaches, the most toxic isolate were found to be B. thuringiensis-l (77%) and B. thuringiensis-2 (70%). B. sphaericus showed 60% and B. alvei 57% mortality with vegetative cells. B. megaterium and B. coagulans showed 50% mortality, whereas B. firmus and B. brevis showed 47% mortality. B. stearothermophilus showed 43% mortality. With spores, B. thuringiensis-l showed the maximum mortality (83.3%) followed by B. thuringiensis-2 (80%), B. sphaericus (73.3%), B. alvei (70%), B. coagulans (66.7%), B. brevis (63.3%), B. megaterium (60%), B. firmus (60%) and B. stearothermophilus (50%).
11. Against mosquitoes, different doses of vegetative cells of Bacillus species were tested at a concentration lx107, 5x107, lx108, 5x108 and lx109. B. thuringiensis-2 was most effective when 5x 107 cells/ml were added in the medium containing mosquitoes as it showed 100% mortality while B. thuringiensis isolated from houseflies showed 100% mortality when 5x 107 cells/ml of the medium. B. thuringiensis-l and B. megaterium caused 100% mortality when lxl08 cells were added in the medium. B. coagulans and B. alvei showed 100% mortality when 5x 108 cells/ml were added in the medium. B. stearothermophilus and B. thuringiensis isolated from mosquitoes showed 100% and 58% mortality when lxl09 cells/ml of the medium were added. B. brevis was the least effective insecticide among all the isolated species of Bacillus as it showed 57% mortality when 1 x 109 cells/ml of the medium were added. With reference to spores, a dose of lxl07 cells/ml caused 98% and 95% mortality in B. thuringiensis-l and B sphaericus, respectively. Other Bacillus sp. as B. brevis and B. tlzuringiensis-2 showed 92° and 90% mortality, respectively. B. alvei and B. coagulans caused 87% mortality against mosquitoes and B. thuringiensis isolated from houseflies was 85% toxic. E stearothermophilus was 75% lethal, whereas both B. firmus and B. thuringiensis isolate from mosquitoes were effective with mortality rate of 52%. B.. megaterium had least mortality effect (43%) compared to other strains against mosquitoes.
12. All the strains harbor a single 23kb plasmid. The competent cells of E. coli C600 were successfully transformed with the plasmids of different strains of bacillus. The transformants could grow in the LB medium containing ampicillin (50 μg/ml) and also in medium containing streptomycin (30 μg/ml).
13. Bacterial protein was analyzed by polyacrylamide gel electrophoresis in both sporulated and non-sporulated forms of Bacillus species to verify if the toxicity of spores was due to the production of more proteins in spores than in vegetative growth.