Upendra Thapa Shreshtha1, Gyan Sunder Shahukhal1, Kiran Babu Tiwari1, Subarna Pokhrel3, Anjana Singh2, Vishwanath Prasad Agrawal1*
1Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal;
2Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal
3School of Biological Chemistry and Engineering, Seoul National University, S. Korea
*Address for Correspondence: Prof. Dr. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775,
E-mail: vpa@wlink.com.np
ABSTRACT
Bacillus thuringiensis strains were isolated from soil samples collected from Khumbu Base Camp of the Everest region and characterized by standard microbiological techniques viz. colonial and morphological characteristics, and biochemical tests. Insect bioassay of each isolate was performed by standard method using mosquito larva. Among ten randomly selected isolates, one isolate showed the highest insecticidal activity against Dipteron insects.
Keywords: Insect-bioassay, Isolates, Khumbu region, Mosquitocidal, Mosquito larva
INTRODUCTION
Microbial insecticides are especially valuable as their toxicity to non-target animals and humans is extremely low compared to other commonly used chemical insecticides. They are safe for both the pesticide user and consumers of pesticide treated crops (Neppl, 2000). The soil bacterium, Bacillus thuringiensis, fulfills the requisites of a microbiological control agent against agricultural pest and vectors that cause massive crop destruction (Ben-Dov et al, 1999). The main target pest of B. thuringiensis insecticides include various Lepidoptera (butterfly), Diptera (flies and mosquitoes), and individual Coleopteran (Beatle) species and some strains kill off nematodes (Schnepf et al, 1998) where as B. thuringiensis var. kurstaki HD1 is highly potent strain due to its wide spread insecticidal properties (Dulmage, 1970).
METHODOLOGY
Soil sampling, isolation and biochemical characterization: Soil samples were collected from Sagarmatha National Park (SNP) and Phereche of Khumbu Base Camp of Everest region and were transported to RLABB, where the study was carried out from March 2005 to December 2005. B. thuringiensis were isolated by acetate selection method (Travers et al, 1987). The isolated organisms were identified by standard microbiological techniques including colonial and morphological characteristics, and biochemical tests (Bergey’s Manual, 1986).
Collection of Mosquito larvae: Mosquito (Lepidoptera) larvae were collected from the ditches in local area of Bode, Bhaktapur, Nepal for insect bioassay. The larvae were identified as Culex spp. by zoologists at the Central Department of Zoology, Tribhuvan University, Kirtipur and bioassay was performed as described by Pang (1994).
Insect bioassay: Larvae, collected from the ditches in local area of Bode, Bhaktapur, Nepal, were reared in a jar containing 100 ml of sterilized water containing 0.3 ml of 5% Brewer's Yeast and 5 ml of B. thuringiensis stationary phase culture and allowed to stand for 3 days. The number of deaths was recorded for one, two and three days. The crystal protein from the stationary culture of the selected individual strain was partially purified by Alkanine method (Dulmage, 1970) followed by Native-PAGE (Blackshear, 1984). The purified crystal protein was bio-assayed (30µg/ml per assay) from each band (Pang, 1994).
RESULTS
Out of 86 δ-endotoxin positive isolates, 10 randomly selected ones were used for insect bioassay. Although all isolates tested were effective against the larvae, isolate S6 was the most effective of all (Fig. 1).
1Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal;
2Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal
3School of Biological Chemistry and Engineering, Seoul National University, S. Korea
*Address for Correspondence: Prof. Dr. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775,
E-mail: vpa@wlink.com.np
ABSTRACT
Bacillus thuringiensis strains were isolated from soil samples collected from Khumbu Base Camp of the Everest region and characterized by standard microbiological techniques viz. colonial and morphological characteristics, and biochemical tests. Insect bioassay of each isolate was performed by standard method using mosquito larva. Among ten randomly selected isolates, one isolate showed the highest insecticidal activity against Dipteron insects.
Keywords: Insect-bioassay, Isolates, Khumbu region, Mosquitocidal, Mosquito larva
INTRODUCTION
Microbial insecticides are especially valuable as their toxicity to non-target animals and humans is extremely low compared to other commonly used chemical insecticides. They are safe for both the pesticide user and consumers of pesticide treated crops (Neppl, 2000). The soil bacterium, Bacillus thuringiensis, fulfills the requisites of a microbiological control agent against agricultural pest and vectors that cause massive crop destruction (Ben-Dov et al, 1999). The main target pest of B. thuringiensis insecticides include various Lepidoptera (butterfly), Diptera (flies and mosquitoes), and individual Coleopteran (Beatle) species and some strains kill off nematodes (Schnepf et al, 1998) where as B. thuringiensis var. kurstaki HD1 is highly potent strain due to its wide spread insecticidal properties (Dulmage, 1970).
METHODOLOGY
Soil sampling, isolation and biochemical characterization: Soil samples were collected from Sagarmatha National Park (SNP) and Phereche of Khumbu Base Camp of Everest region and were transported to RLABB, where the study was carried out from March 2005 to December 2005. B. thuringiensis were isolated by acetate selection method (Travers et al, 1987). The isolated organisms were identified by standard microbiological techniques including colonial and morphological characteristics, and biochemical tests (Bergey’s Manual, 1986).
Collection of Mosquito larvae: Mosquito (Lepidoptera) larvae were collected from the ditches in local area of Bode, Bhaktapur, Nepal for insect bioassay. The larvae were identified as Culex spp. by zoologists at the Central Department of Zoology, Tribhuvan University, Kirtipur and bioassay was performed as described by Pang (1994).
Insect bioassay: Larvae, collected from the ditches in local area of Bode, Bhaktapur, Nepal, were reared in a jar containing 100 ml of sterilized water containing 0.3 ml of 5% Brewer's Yeast and 5 ml of B. thuringiensis stationary phase culture and allowed to stand for 3 days. The number of deaths was recorded for one, two and three days. The crystal protein from the stationary culture of the selected individual strain was partially purified by Alkanine method (Dulmage, 1970) followed by Native-PAGE (Blackshear, 1984). The purified crystal protein was bio-assayed (30µg/ml per assay) from each band (Pang, 1994).
RESULTS
Out of 86 δ-endotoxin positive isolates, 10 randomly selected ones were used for insect bioassay. Although all isolates tested were effective against the larvae, isolate S6 was the most effective of all (Fig. 1).
DISCUSSION
Due to high toxicity of chemical pesticide to human beings, animals and beneficial insects, the use of chemical pesticides is being replaced by environment friendly bio-pesticides. The crystal proteins of B. thuringiensis, as bio-control agent, have been extensively studied worldwide. Under the present investigation, most of the bacterial strains were highly mosquitocidal. As the bacterial strains were collected from high altitude (above 4000m), where mosquitoes are not expected, the crystal proteins from the bacteria may be novel ones. In Nepalese context, though isolation and characterization of B. thuringiensis from different soil samples and their insect toxicity have been studied and tested from elsewhere, the characterization of crystal proteins for mosquitocidal properties hasn’t yet been explored from extreme environment. In an attempt to find novel crystal protein residing B. thuringiensis in high altitude, the bacteria were isolated from soil samples collected from Khumbu region of Mt. Everest base camp. The isolates showing potent insecticidal property tested against dipterans need to be studied further in larger trials so that it can have applicability to reduce the mosquitoes and different diseases caused by these vectors (Malaria, Filaria, Kala-azar etc).
ACKNOWLEDGEMENTWe express our full gratitude to CNR (Italy’s National Research Council) for supporting this work and especially thank to Mr. Deepak Singh, Mr. Yogan Khatri and Rajindra Aryal for collecting soil samples from Mount Everest region.
References:
Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y (1999). Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol; 65: 3714-6.
Blackshear PJ (1984). Systems for polyacrylamide gel electrophoresis. In Methods in enzymology (Jakoby WB eds.) vol 104: 237-255.
Claus D and Berkeley RCW (1986) ‘Genus bacillus. in: sneath PHA, ed. Bergey’s Manual of Systematic Bacteriology’, Baltimore: Williams & Wilkins, Volume. 2
Dulmage HT (1970). Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J Invertebr Pathol; 16: 385-9.
Neppl CC (2000). Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago.
Pang AS (1994). Production of antibodies against Bacillus thuringiensis delta-endotoxin by injecting its plasmids. Biochem Biophys Res Commun ; 202: 1227-34.
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev; 62: 775-806.
Shrestha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP (2006). Delta- endotoxin immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of Mount Everest region. J Food Sci Technol Nepal; 2: 128-131.
Travers RS, Martin PA and Reichelderfer CF (1987). Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol; 53: 1263-6.
Due to high toxicity of chemical pesticide to human beings, animals and beneficial insects, the use of chemical pesticides is being replaced by environment friendly bio-pesticides. The crystal proteins of B. thuringiensis, as bio-control agent, have been extensively studied worldwide. Under the present investigation, most of the bacterial strains were highly mosquitocidal. As the bacterial strains were collected from high altitude (above 4000m), where mosquitoes are not expected, the crystal proteins from the bacteria may be novel ones. In Nepalese context, though isolation and characterization of B. thuringiensis from different soil samples and their insect toxicity have been studied and tested from elsewhere, the characterization of crystal proteins for mosquitocidal properties hasn’t yet been explored from extreme environment. In an attempt to find novel crystal protein residing B. thuringiensis in high altitude, the bacteria were isolated from soil samples collected from Khumbu region of Mt. Everest base camp. The isolates showing potent insecticidal property tested against dipterans need to be studied further in larger trials so that it can have applicability to reduce the mosquitoes and different diseases caused by these vectors (Malaria, Filaria, Kala-azar etc).
ACKNOWLEDGEMENTWe express our full gratitude to CNR (Italy’s National Research Council) for supporting this work and especially thank to Mr. Deepak Singh, Mr. Yogan Khatri and Rajindra Aryal for collecting soil samples from Mount Everest region.
References:
Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y (1999). Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol; 65: 3714-6.
Blackshear PJ (1984). Systems for polyacrylamide gel electrophoresis. In Methods in enzymology (Jakoby WB eds.) vol 104: 237-255.
Claus D and Berkeley RCW (1986) ‘Genus bacillus. in: sneath PHA, ed. Bergey’s Manual of Systematic Bacteriology’, Baltimore: Williams & Wilkins, Volume. 2
Dulmage HT (1970). Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J Invertebr Pathol; 16: 385-9.
Neppl CC (2000). Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago.
Pang AS (1994). Production of antibodies against Bacillus thuringiensis delta-endotoxin by injecting its plasmids. Biochem Biophys Res Commun ; 202: 1227-34.
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev; 62: 775-806.
Shrestha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP (2006). Delta- endotoxin immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of Mount Everest region. J Food Sci Technol Nepal; 2: 128-131.
Travers RS, Martin PA and Reichelderfer CF (1987). Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol; 53: 1263-6.
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