(Prepared for Publication)
Upendra Thapa Shrestha1, Sarobar Ghimire1, Jay Bhandari1, Kiran Babu Tiwari1, 2, Hemant Raj Joshi1, Vishwanath Prasad Agrawal2
1:
2: Research Laboratory for Biotechnology and Biochemistry (RLABB), Sanepa, Ktm.
ABSTRACT
Bacillus thuringiensis strains were isolated from soil samples collected from
INTRODUCTION
Bacillus thuringiensis is a ubiquitous gram positive, spore forming bacterium that forms a parasporal crystal during the stationary phase of its growth cycle. B. thuringiensis was initially characterized as insect pathogen, and its insecticidal activity was attributed largely or completely (depending upon the insects) to the parasporal crystals. This observation lead to the development of bioinsecticides based on B. thuringiensis for the control of certain insect species among the order Lepidoptera, Diptera, and Coleoptera. There are more recent reports of B. thuringiensis isolates active against other insects of order (Hymenoptera, Homoptera, Orthoptera, and Mallophaga) and against Nematodes, mites and protozoa. B. thuringiensis is already a useful alternative or supplement to the synthetic chemical pesticide application in commercial agriculture for pest management and mosquito control. It is also a key source of genes for transgenic expression to provide pest resistant in plants (Schnepf et al., 1998).
Shrestha et al. (2006 and 2007) isolated more than hundred B. thuringiensis isolates from the soil samples of Khumbu region including
Open drainage system and stagnant water body are the main sites for mosquito breeding. All mosquitoes have one common requirement--they need water to complete their life cycle. Mosquitoes lay individual eggs on the stagnant water bodies. These eggs can lay dormant for several years. These eggs hatch in 24-48 hours releasing larvae that are commonly called "wrigglers". Generally, the larvae feed on microorganisms and organic material in the water, but some mosquitoes prey on the larvae of other mosquito species too. These larvae change into the pupal or "tumbler" stage within 7-10 days in preparation for adult life which are vectors for many diseases. This larva stage of mosquitoes can be completely removed from those habitats by spraying potent B. thuringiensis strains (http://en.wikipedia.org/wiki/Mosquito-borne_disease).
Therefore the main target of this research was to search of potent B. thuringiensis strains which can survive and efficiently multiply in waste water so that it can be used in biological control of mosquito breeding.
METHODOLOGY
Isolation and revival of Bacillus thuringiensis: One potent strain of B. thuringiensis isolate (S6) was revived from the previous master culture preserved in RLABB and three soil samples from (Sagarmatha National Part-SNP, S2, S7 and S11) were reprocessed to isolate B. thuringiensis following Acetate selection method to avoid any contamination (Travers et al., 1987). All isolates were subcultured on Nutrient Agar for further processing.
Characterization of B. thuringiensis for their crystal protein formation: Identification was again carried out by gram staining, spore staining and crystal staining, and biochemical tests, Sugar utilization tests and Hydrolysis tests as described in Bergey’s Manual of Systematic Bacteriology, Volume 2, 1986 (Claus & Berkeley 1986).
Insect bioassay for the selection of some more potent strains: Mosquito larvae were collected from the local areas (
Test for survivability and efficiency of b. Thuringiensis in waste water: Water samples from Mosquito breeding sites were collected. Five milliliter broth of 3 days old culture of B. thuringiensis (broth density greater than McFarland Standard No.4 =12 x 108/ml) isolates were inoculated in it. Quantitative measurement of B. thuringiensis isolates was done by Plate count method after its isolation following the Acetate selection method (Travers et al., 1987). The survivability time was recorded till 30 days or more. After survivability test, the bacteria were again inoculated in waste water containing mosquito larva and the mortality of mosquito was recorded in 1, 2 and 3 days and were recorded for up to 7 days when needed. Then dead mosquitoes ware analyzed for whether it was caused due to B. thuringiensis or not.
RESULTS
Revival and identification of B. thuringiensis: Altogether seven potent isolates were further proceeded for morphological, cultural and biochemical characterization for reidentification. All the characteristics were compared with the standards described by Bergey’s Manual of Systematic Bacteriology, Volume 2 (Claus & Berkeley 1986). All isolates used were Gram positive rods, spore forming and crystal protein producing. These potent strains were once again assayed for insect bioassay.
Insect bioassay: Insecticidal activities of sample S6 were found to be highest among seven SNP isolates. All isolates were found to be very potent but due to use of old mosquito larva few changed into adult mosquito within our experiment period. All these isolates were able to kill the mosquito larva even at their natural breeding place, waste water and were further used for survivability test in the waste water (Table 1).
Table 1: Insecticidal activities of B. thuringiensis endotoxins using Stationary phase culture
S. No. | Samples | Sets | No. of Deaths out of 10 mosquito larva | Total deaths out of 20 larva | Percent a | ||
24 hrs | 48 hrs | 72 hrs | |||||
1 | Control | I | 0 | 0 | 0 | 0 | 0 |
II | 0 | 0 | 0 | ||||
2 | S2a | I | 2 | 5 | 8 | 17 | 85 |
II | 2 | 4 | 9 | ||||
3 | S2b | I | 2 | 4 | 8 | 15 | 75 |
II | 3 | 4 | 7 | ||||
4 | S6 | I | 8 | 9 | 10 | 20 | 100 |
II | 9 | 10 | 10 | ||||
5 | S7a | I | 3 | 5 | 7 | 15 | 75 |
II | 2 | 3 | 8 | ||||
6 | S7b | I | 1 | 3 | 8 | 15 | 75 |
II | 1 | 4 | 7 | ||||
7 | S11a | I | 3 | 5 | 9 | 18 | 90 |
II | 3 | 6 | 9 | ||||
8 | S11b | I | 2 | 4 | 8 | 14 | 70 |
II | 2 | 5 | 9 | ||||
Note: a =final percentage of death of larva after 72 hours
Survivability test: The initial concentration of bacteria in waste water after inoculation was 6 x 107 per ml of waste water (5 x 12 x108 per 100 ml = 6 x 107 per ml).The growth of B. thuringiensis in waste water was found to increase for seven days and plunged gradually in subsequent days till 30 days. The survivability of B. thuringiensis isolates was recorded till 30 days and found to be alive in the waste water. The number of B. thuringiensis was found to be higher in control in compare with the test, unautoclaved waste water (all data are shown in a single table; Table 2).
Table 2: Combined data of all day's enumeration
S. no. | Bacterial count in | Isolates code (No of Bacteria per ml) | |||||
S2a | S2b | S7a | S7b | S11a | S11b | ||
1 | 0 day | 6 x 107 | 6 x 107 | 6 x 107 | 6 x 107 | 6 x 107 | 6 x 107 |
2 | 3 days | 8.35 x 109 | 7.2 x 109 | 1.10 x 1010 | 7.1 x 109 | 2.33 x 1010 | 8.9 x 109 |
3 | 7 days | 1.47 x 1011 | 1.21 x 1011 | 0.94 x 1011 | 1.38 x 1011 | 4.49 x 1011 | 4.19 x 1011 |
4 | 14 days | 1.12 x 108 | 0.99 x 108 | 0.87 x 108 | 0.66 x 108 | 1.38 x 108 | 1.19 x 108 |
5 | 21 days | 1.00 x 107 | 1.1 x 107 | 0.8 x 107 | 0.85 x 107 | 1.55 x 107 | 1.50 x 107 |
6 | 30 days | 3.0 x 106 | 5.0 x 106 | 5.0 x 106 | 4.0 x 106 | 6.0 x 106 | 4.50 x 106 |
DISSCUSSION
Altogether seven potent isolates were further proce eded for colonial morphological, cultural and biochemical characterization for reidentification. All the characteristics were compared with the standards described by Bergey’s Manual of Systematic Bacteriology, Volume 2 (Claus & Berkeley 1986). All isolates used were Gram positive rods, spore forming and crystal protein producing (Table 1). These potent strains were once again assayed for insect bioassay.
The most prominent characteristic of B. thuringiensis is the intracellular production of an insecticidal d-endotoxin in the form of a proteinaceous crystal during sporulation. The majority of B. thuringiensis strains are toxic to larvae of the order Lepidoptera. (Huber-Lukac et al., 1986). Of the total seven isolates, one potent strain S6 was revived from master culture and remaining other six strains were reisolated and recharacterized from previous soil samples in RLABB. These strains were then assayed for preliminary bioassay using mosquito larva of Culex species as target species. Cultures were grown to the stationary phase, the suitable phase for the sporulation and production of d endotoxin (Dulmage, 1970). The assay was set in duplicate to read reproducible results. Each set was set with 10 larva and 100 ml of sterilized water with 0.3 ml of 5% brewer's yeast. Five milliliter of B. thuringiensis culture of stationary phase was added in each and allowed to stand for up to three days (Bhattarai, 2002 and Subedi, 1999) .The number of deaths in each set was recorded for one, two and three days each so as to know the efficiency of individual activities of the respective isolates. Of the 7 isolates tested against the mosquito larva, S6 was the most effective one (100% efficient within three days). Most of them were highly efficient to kill the larva, ranging 70% to 100% (Table 1). Few of mosquito larva changed into adult mosquito during our experiment period as the mosquito larva used were too old. All these isolates were able to kill the mosquito larva even at their natural breeding place, waste water and were further used for survivability test in the waste water. The isolate S6 was found to be potent as well as be able to survive in waste water for many days up to 30 days (Bhandari and Khanal, 2009). Hence the isolate was not further used in this research work. Some more isolates to be tested for survivability were only focused this work.
The growth of B. thuringiensis in waste water was found to increase rapidly for seven days and plunged gradually in subsequent days till 30 days. The initial concentration of bacteria in waste water after inoculation was 6 x 107 per 100 ml of waste water (5 x 12 x108 per 100 ml = 6 x 107 per ml). The number gradually increased to about 109-1010 within 3 days and soon rose up to 1011 in 7 days per 100 ml in waste water. In spite of the presence of many grazers and phages, the bacteria were found to overcome and multiple in waste water. Soon after the seventh day, the number of bacteria started to decrease down gradually. The bacteria, B. thuringiensis previously inoculated were hardly isolated after 30 days. The number of B. thuringiensis was found to be higher in control in compare with the test, unautoclaved waste water (Table 2). The survivability of B. thuringiensis isolates was recorded till 30 days and found to be alive in the waste water. During enumeration, each colony was identified as the colony of B. thuringiensis following standard procedures.
The reasons behind the fluctuation of bacteria density with incubation period are described below. We all know that waste water is full of organic and inorganic chemicals as well as toxic chemicals. Besides, waste water also contains enormous groups of microbes (protozoa, bacteria, phages etc.) and higher animals including insects and smaller plants as well. From the above data, the bacteria seemed to multiple in the waste water however the decreased in number after 7 days indicates that certain extrinsic factors played role. The reasons might be due to continuous grazing of B. thuringiensis by certain grazers like ciliates and flagellate already present in the waste water and might be due to phage attack on those bacteria. Some toxic compounds can also influence on their growth which was observed in our experiments. The number of bacteria in control was slightly higher than that of test in which the growth suppressing factors might only be toxic inorganic compounds as there were no any biological lives in control.
The available nutrients might be one more reasonable fact in decreasing bacterial number. The nutrients in the waste water were continuously used by all organisms present there. Therefore as long as the bacteria were there, no more foods would be available for bacteria to support their growth. It is very difficult to describe the actual chemical and physical niche of the waste water. So this complex niche should be studied in detail by chemical analysis of waste water and biological density of the sample.
CONCLUSION
B. thuringiensis, one of the soil bacteria can survive and multiply in waste water for many days. This property of B. thuringiensis adds to their prominent characteristics as biopesticides even in different niches including soil and water.
ACKNOWLEDGEMENT
The authors express their sincere gratitude to UGC (University Grants Commission, Sanothimi, Bhaktapur) for supporting this work.
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http://en.wikipedia.org/wiki/Mosquito-borne_disease
