Bacillus Thuringiensis Berliner: Isolation to Application: a Review

Bacillus thuringiensis (Bt) is a ubiquitous soil bacterium that produces an array of insecticidal crystal proteins with varied specificities. Recently a lot of effort is being made to collect, characterize, utilize novel and more potent strains of Bt. The crystal proteins (Cry proteins) produced by Bt are important in agricultural, horticultural and public health pest management. In addition to the above recently identified non-insecticidal Cry toxins are being used in cancer research. There is continuing debate among researchers on the species status for Bt and its ecology. The various toxins particularly Cry toxins are important in ecofriendly pest management in organic farming and exported oriented crops also. Many cry genes have been engineered into wide variety of crop plants and microbes, offering efficient and effective pest management strategy. Increased efforts to isolate novel strains of Bt from wide variety habitats have yielded valuable strains with new genes. Intensive use of various Bt formulations and high level of expression of various cry genes in Bt transgenic plants have resulted in resistance development in various pest species. But with the judicious use of gene pyramiding and refugia, continued successful pest management is ensured. e-mail: [email protected] Bacillus thuringiensis (Bt) is a ubiquitous, gram positive soil bacterium, which was first identified by the Japanese microbiologist, Ishiwata in 1901 from the diseased silk worm, Bombyx mori (Beegle and Yamamoto, 1992). Bt produces the insecticidally important crystalline inclusions, popularly called d-endotoxins, mainly during the late exponential phase and stationary phase of growth. The various crystal (Cry) toxins have been proved to be very specific to insects and pose no threat to the environment and nontarget animals including human beings. In addition to Cry proteins Bt also produces aexotoxin, ß-exotoxin, vegetative insecticidal proteins (Vip), louse factor, mouse factor, cytolytic toxins (cyt) etc, production of which is subspecies/strain specific (Konecka et al., 2007). Until late 1970s most of the subspecies of Bt isolated were lepidopteran active, later many Bt strains active on coloeptera, diptera, acarina, protozoa, hymenoptra, trematode, nematodes etc were discovered (Crickmore et al., 1998) In majority of the cases, Bt has been isolated from the dead insects and occurrence of Bt in soil has been reported to be very less. Later researchers isolated Bt from various sources (Bernhard et al., 1997, Morris et al., 1998, Gough et al., 2002). The above efforts yielded many novel isolates of Bt with altogether new genes widening the application of Bt in various fields. Advent of recombinant DNA techniques in early 1980s has accelerated cloning and characterization of new cry genes from the already identified subspecies as well as from uncharacterized strains of Bt. At present there are about 300 different cry genes have been characterized and the sequences are available. Additionally the interest on cry genes was due to the monogenic nature of the toxin as against other complex insecticidal genes like tc genes from Photorhadus luminescens, which are difficult to engineer into crop plants. In addition to the above, Agrobacterium mediated transformation of various crops was successful, which was instrumental in the development of Bt transgenic plants. 236 AGRICUTURAL REVIEWS In this communication the following aspects of Bt have been reviewed 1) ecology, isolation and occurrence 2) classification 3) Nature of insecticidal proteins & other toxins and their mode of action 4) field application and 5) resistance management. 1. Ecology, Isolation and Occurrence The true ecological role of Bt has not been established completely till date since it invests > 50% of its resources to produce crystal proteins (Ohba, 1986). Additionally Bt has been isolated from places where there was no apparent insect activity at all. More over Bt rarely causes epizootics in insect population, which is untypical of any obligate insect pathogens. There are many hypotheses on the possible role of the crystal proteins. These are (a) the crystalline inclusions are vestigial (b) crystal proteins have some unknown physiological role in the bacterium (c) crystalline inclusions serve as a reservoir of nutrients and (d) it is a weapon to invade insects. Since many Cry proteins have been found to be toxic to insects, it has been considered as true insect pathogen (Agaisse and Lereclus., 1995) Usual method of isolation of Bt is from diseased or dead insects or in places with high levels of insect infestation. Saleh et al. (1969) used polymyxin B sulfate and penicillin G which reduced the number of non-Bacillus organisms while isolating Bt. Later, Travers et al. (1987) found that Bt spores did not germinate in the presence of 0.25 M sodium acetate in the medium. Employing this method they detected one spore of Bt in the background of 10 bacteria/ gram of soil. Presently different strains of Bt has been recorded from Lepidoptera, Diptera, Coleoptera, Hymenoptera, Hemiptera, Orthoptera, Mallophaga and from Nematoda, Acarina and Protozoa, Trematoda etc. (de Maagd et al., 2003). Researchers have isolated Bt from different habitats such as soil (Ohba and Aizawa, 1986, 1986a; Carozzi et al., 1991; Hastowo et al., 1992; Landen et al., 1994; Vasquez et al., 1995; Bernhard et al., 1997; Hossain et al. 1997; Theunis et al., 1998; Kaur and Singh, 2000), fresh water (Ichimatsu et al., 1998), saw dust (Bravo et al., 1998; Helgason et al., 1998; Morris et al., 1998; De Lucca et al., 1982), cured tobacco leaves (Kaelin, 2000; Kaelin and Gadani, 2000), warehouse (Hongyu et al., 2000), dead insects (Chilcott and Wigley, 1993; Bernhard et al., 1997; Strongman et al., 1997, Schnepf et al., 1998), trematode (Horak et al., 1996), rice bran (Jung et al., 1998), stored products (Bernhard et al., 1997; Chaufaux et al., 1997), compost (Bernhard et al., 1997), phylloplane (Bernhard et al., 1997; Bora et al., 1994; Damgaard et al., 1998), feces samples of green house workers (Jensen et al., 2002), in gastroenteritis outbreak (Jackson et al., 1995), marine sediments (Maeda et al., 2000), in animal feed mill (Meadows et al., 1992), sericultural farms (Ohba,1984; Kim et al., 1998), on livestock ecto parasites (Gough et al., 2002), periodontitis, dairy, human pus, human nose, eye, urine (Helgason et al., 2000), Antarctica (Forsyth and Logan, 2000), in ancient glacial ice (Christner et al., 2003) etc. Many of the earlier workers have not taken sibling strains into their considerations, Bravo et al. (1998) has emphasized such grouping on siblings. Prabagaran et al. (2002) have isolated 583 strains from different agro climatic regions in India and classified the same based on crystal morphology, SDS-PAGE profile and PCR panning. Out of 583 isolates screened only 18 isolates produced crystals and were subjected to detailed investigation. Bernhard et al., (1997) and Hongyu et al. (2000 b) have grouped huge populations of Bt based on the above methods. Lopez-Meza and Ibarra (1996) characterized novel strains of Bt. It is worth to investigate in depth on limited Bt 237 Vol. 29, No. 4, 2008 isolate than characterizing large population with no proven potential. Statistical analysis with thousands of Bt strains regarding potency generally does not have any practical value. On the contrary, studies with selected elite strains with novel gene/s or high potency will have commercial value. 2. Classification Bt is evolutionarily very close to B. cereus except that the former produces crystal inclusions while the later does not. Some classical bacterial taxonomists still opine that Bt should be a subspecies of B. cereus, based on many experimental evidences. Bt and B. cereus are one species The various experiments that proved the similarities between Bt and B. cereus are as follows (a) certain strains of Bt and B. cereus killed mice, when injected intra peritoneally (Lamanna and Jones, 1963) (b) the spores of both share common antigens and also exhibited cross-sensitivity to bacteriophages (c) some B. anthracis strains were sensitive to bacteriophage from Bt and the fatty acid patterns were similar (Kaneda, 1968) (d) flagellar antigens of some Bt and B. cereus isolates over lapped (e) close enzyme serological relationship, thin section spores appeared alike in fine structure, DNA homology is 80-100% (Krieg, 1975). According to Singer (1980) strains of bacteria having greater than 70% DNA homology should be considered as single species. Bt and B. cereus are two different species The various experimental evidences that support this argument are as follows (a) DNA competition studies, immuno fluorescence and phase-microscopy, pyrograms based on pyrolysis gas-liquid chromatography, biochemical characteristics (Jones,1983) (b) Morphological and biochemical characteristics (Heimpel and Angus, 1960) (c) Later de Barjac (1990) developed identification and classification method based on serotyping and biochemical characteristics (d) Norris (1963) advanced the identification and classification of Bt by esterase patterns of vegetative cells by starch gel electrophoresis (e) various heat stable somatic O-antigens, crystal serology; phages have been examined for their usefulness in the identification and classification (de Barjac, 1990) (f) reaction to different monoclonal antibodies (Hoffte and Whiteley, 1989) (g) phospholipid and fatty acid analyses (Hathout et al., 2000) (h) electrophoresis of PCR products (Perez et al., 1994) (i) 16S rRNA sequence comparison (Joung et al., 2001) (j) AFLP (Kim et al., 1997; Ticknor et al., 2001) (k) genomic restriction digestion (Schnepf et al., 1998) (l) SDS-PAGE analysis of total proteins (Helgason et al., 1998) (m) Southern blot analysis (Kronstad and Whiteley, 1986) (n) bioassay (Martin and Travers (1989) (o) RFLP (Joung and Cote, 2001) (p) ribosomal RNA gene restriction patterns (Joung and Cote, 2002). (q) Beattie et al. (1998) employed Fourier transformation infrared spectroscopy to different B. cereus, B. mycoides and Bt (r) VilasBoas et al. (2002) identified the genetic differences between sympatric populations of B. cereus and Bt by allozyme electrophoresis and hemolytic activity (s) Radnedge et al. (2003) worked on the genomic differences that distinguished Bt from the closely related pathogens like B. cereus and B. anthracis. Serotyping based on H-flagellar antigen is regularly employed to classify Bt subspecies (Lacadet et al., 1999., Joung and Cote, 2002). 3. Nature and mode of action of insecticidal proteins and other toxins