The formulations of Bacillus thuringiensis (B. t.) have been used for several decades since its discovery as an effective means of controlling Lepidoteran pests in agriculture because of the its many properties that are different from the synthetic chemical formulations. It is not toxic to mammals; it is environmental friendly, earlier it was believed to have immunity towards the pesticide resistance phenomenon, good combination with other pest control methods and the possibility of being mass produced at low cost at farm level, are the factors that make B. thuringiensis the much-needed means for IPM programmes in developing countries. It has been revealed by almost 85 years of research that Bacillus spp., especially B. thuringiensis and Bacillus sphaericus are the most effective biopesticides (Boucias & Pendland, 1998). B. thuringiensis is a bacterial species having insecticidal attributes and a specific range of insect orders are affected by it. B. thuringiensis has atleas 34 subspecies also called
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serotypes or varieties. It has over 800 strain isolates (Swadener, 1994). B. thuringiensis comprises about 5-8% of Bacillus spp. population in the environment (Hastowo et al., 1992).it has been found that more than 130 species of lepidopteran, dipteran and coleopteran insects are controlled by
B. thuringiensis till date (Dean, 1984).
Historical Background of B. thuringiensis
B. thuringiensis play an important role in the biological control of insect pest which form toxic crystal proteins at the time of sporulation. B. thuringiensis, a bacterium that produces insecticidal proteins during its sporulation is the source of well known and generally used biopesticide. This widespread soil bacterium, was discovered first in Japan in 1901 by Ishawata and then in 1911 in Germany by Berliner. It is found in grain dust from soil in abundance and other grain storage facilities, (Baum et al., 1999). Later it was found that thousands of strains of B. thuringiensis are present (Lereclus, 1993). The isolation of this bacterium from diseased larvae of Anagasta kuehniella, led to the establishment of B. thuringiensis as microbial insecticide.
The first record of its use to control insects can be traced back to Hungary at the end of 1920, and in Yugoslavia at the beginning of 1930s; it was used to control the European corn borer (Lords, 2005). The first commercial product of B. thuringiensis was Sporine it was available in 1938 in France (Waiser, 1986) for the controling flour moth (Jacobs, 1951). World War II resulted in the use of the product for very short time (Nester et al., 2002). Development of transgenic plant was also noticed. In 1987 first reports of insertion of genes encoding for B. thuringiensis delta-endotoxins into plants came. Tobacco and tomato plants were first transgenic plants to express B. thuringiensis toxins (van Frankenhuyzen, 1993). "Thuricide" was first commercialized strain on B. thuringiensis. It was commercialized in 1957 because of the increasing concern of biopesticide over the use of chemical insecticides.
During its stationary phase of growth B. thuringiensis, a gram-positive spore-forming bacterium produces crystalline proteins called deltaendotoxins (Schnepf et al., 1998). The crystal is released to the environment after analysis of the cell wall at the end of sporulation, and it comprises 20 to 30% of the dry weight of the sporulated cells (Schnepf et al., 1998)
Distribution & Habitat of B. thuringiensis
This bacterium is universal distribution (Martin & Travers, 1989). Its main habitat is believed to be soil; however it has been isolated from foliage, water, storage grains, and dead insects as well (Iriarte & Caballero, 2001). Strains isolated from dead insects has been the main source for commercially used varieties, including kurstaki, isolated from A. kuehniella; israelensis, isolated from mosquitoes, and tenebrionis, isolated from Tenebrio monitor larvae (Ninfa & Rosas, 2009; Iriarte & Caballero, 2001).. The spores of B. thuringiensis persist in soil, and vegetative growth being subjected to the availability of nutrients (DeLucca et al., 1981; Akiba, 1986; Ohba & Aizawa, 1986; Travers et al., 1987; Martin & Travers, 1989).
In USA, B. thuringiensis represented between 0.5% and 0.005% of all Bacillus species isolated from soil sample (DeLucca et al., 1981). B. thuringiensis were recovered globally from soils by (Martin & Travers, 1989). Meadows (1993) recovered B. thuringiensis from 785 of 1115 soil samples, and the percentage of samples that contained B. thuringiensis ranged from 56% in New Zealand to 94% in samples from Asia and central and southern Africa. Ohba & Aizawa (1986) isolated B. thuringiensis from 136 out of 189 soil samples in Japan.
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There are several theories regarding the ecological niche filled by B. thuringiensis. B. thuringiensis generally recycle poorly and rarely unlike most insect pathogenic microbes. The reason being natural epizootics in insects, leading to assumption that B. thuringiensis is essentially a soil micro-organism having incidental insecticidal activity (Martin & Travers 1989). are commonly reported in the The environmental occurrence of B. thuringiensis is independent of insects and the lack of association between occurrence and insect activity are the evidence supporting this idea (van Frankenhuyzen 1993). Meadows (1993) suggested four possibilities for the presence of B. thuringiensis in soil: 1) seldom grows in soil but is deposited there by insects; 2) may be infective to soil-inhabiting insects (as yet undiscovered); 3) may grow in soil when nutrients are accessible; and 4) a resemblance with B. cereus.
B. thuringiensis has been found widely in the phylloplane. Various
B. thuringiensis subspecies have been isolated from coniferous trees, deciduous trees and vegetables, as well as from other herbs (Smith & Couche, 1991; Damgaard et al., 1997). B. thuringiensis being deposited on the upper side of leaves (exposed to the sun) may remain effectual for only 1-2 days, but the one deposited on the underside of leaves (i.e. protected from the sun) may stay active for 7-10 days (Swadner, 1994).
After an aerial application of Thuricide 16B B. thuringiensis kurstaki has been isolatedfrom rivers and public water distribution systems (Ohana, 1987).
Crystal Composition and Morphology
in 1915 for the first time the existence of parasporal inclusions in B. thuringiensis was noted (Berliner, 1915), but it was not until 1950s that their protein composition was delineated (Angus, 1954). Crystalline fine structure that is a property of most of the parasporal inclusions was detected by Hannay (1953). B. thuringiensis subspecies can produce more than one inclusion, which may have different ICPs (Hannay, 1953). crystalline inclusions of B. thuringiensis ranging from 27 to 140 KDa are protoxins that are proteolytically transformed into smaller toxic polypeptides in themed gut of larva (Peferoen, 1997). Crystal protein genes are present as satellite chromosome (Kronstad et al., 1983).
On the basis of their ICP composition, the crystals have different forms (bipyramidal, cuboidal, flat rhomboid, or a composite with two or more crystal types) (Bulla et al., 1977; Höfte & Whiteley, 1989). A partial correlation between crystal morphology, ICP composition, and bioactivity against target insects has been recognized (Bulla et al., 1977; Höfte & Whiteley, 1989; Lynch & Baumann, 1985).
Classification of B. thuringiensis subspecies
In early 1960s the classification of B. thuringiensis subspecies on the basis of serological analysis of the flagella (H) antigens was introduced (de Barjac & Bonnefoi, 1962). Morphological and biochemical criteria further add-on to this classification by serotype (de Barjac, 1981). Only 13 subspecies of B. thuringiensis had been described until 1977, and all subspecies were toxic only to Lepidopteran larvae at that time. The finding of other subspecies toxic to Diptera (Goldberg & Margalit, 1977) and Coleoptera (Krieg et al., 1983) distended the host range and remarkably increased the number of subspecies. Over 67 subspecies based on flagellar
H-serovars had been identified up to the end of 1998,
Genetics of ICP
In the early 1980s, it was recognized that most genes coding for the ICPs exist on large transmissible plasmids, most of which are readily exchanged between strains by conjugation (González & Carlton, 1980; González et al., 1981). Since these early studies, many ICP genes have been cloned, sequenced and used to construct
B. thuringiensis strains having novel insecticidal spectra (Höfte & Whiteley, 1989).
The presently known crystal (cry) gene types encode ICPs that are particular to either Lepidoptera (cryI), Diptera and Lepidoptera (cryII), Coleoptera (cryIII), Diptera (cryIV), or Coleoptera and Lepidoptera (cryV) (Höfte & Whiteley, 1989). All ICPs described to date upon ingestion attack the insect gut. To date, each of the proteolytically activated ICP molecules having insecticidal activity has a variable C-terminal domain, which is accounts for receptrecognition (host susceptibility), and a conserved
N-terminal domain, which triggers pore formation (toxicity) (Li et al., 1991).
Naturally occurring B. thuringiensis strains generally contain ICPs which are active against a single order of insects. However, new strains with various plasmid contents can be formed by conjugative transfer between B. thuringiensis strains or related species (González & Carlton, 1980). Thus the complex and diverse activity spectra observed in B. thuringiensis can be explained by mobility of the cry genes and the exchange of plasmids (González & Carlton, 1980; González et al., 1981; González et al., 1982; Reddy et al., 1987; Jarrett & Stephenson, 1990). By conjugation new B. thuringiensis strains have been developed that is toxic to two insect orders.
Nutritional status of B. thuringiensis
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Availability of nutrients is the factor affecting sporulation and germination in bacilli (Hardwick & Foster, 1952), in order to delineate the mechanisms regulating spore and parasporal crystal formation it is important to study nutritional requirement of
B. thuringiensis var. thuringiensis. Growth, sporulation and crystal formation of B. thuringiensis var. thuringiensis are supported by certain amino acids, while others restrain the growth (Singer et al., 1966; Singer & Rogoff, 1968; Bulla et al., 1975; Nickerson & Bulla, 1975; Rajalakshmi & Shethna, 1977). Growth, sporulation and crystal formation in Î’. thuringiensis is promoted by a lower concentration of cystine (Nickerson & Bulla, 1975) or cysteine (Rajalakshmi & Shethna, 1977), while only vegetative growth was observed at a elevated concentration of cys/cysSH (Rajalakshmi & Shethna, 1977).
Classification of B. thuringiensis
The classification of B. thuringiensis subspecies based on the serological analysis of the flagella (H) antigens was introduced in the early 1960s (de Barjac & Bonnefoi, 1962). This classification by serotype has been supplemented by morphological and biochemical criteria (de Barjac, 1981)these are repeated lines. By serological techniques many strains of B. thuringiensis have been isolated and classified within more than 20 different varieties by serological techniques. These varieties have been grouped into five pathotypes on the basis of their effectiveness or potency for insects (Hoftey & Whiteley, 1989):
Lepidopteran-Specific (e.g. B. thuringiensis .var Kurstaki)
Dipteran-Specific (e.g. B. thuringiensis . var israelensis)
Coleopteran-Specific (e.g. B. thuringiensis .var. tenebrionis)
Those active against Lepidoptera and Dipter(e.g. B. thuringiensis . var. aizawai)
Those with no toxicity recorded in insects (e.g. B. thuringiensis . var. Dakota)
Mode of Action
Schnepf et al., (1998) have reviewed the ICP structure and function in detail. A major determinant of ICP specificity is the binding of the ICP to putative receptors and the major mechanism of toxicity is the formation of pores in the midgut epithelial cells (Van Frankenhuyzen, 1993). The crystal is dissolved in the insect's alkaline gut after ingestion of B. thuringiensis by insect. After that the digestive enzymes being present in insect's body break down the crystal structure and activate B. thuringiensis's insecticidal component, namely the delta-endotoxin (Swadner, 1994). The delta-endotoxin binds to the cells lining the midgut membrane and pores are created in the membrane, upsetting the ion balance of the gut. The insect soon stops feeding and is starved to death (Gill et al., 1992).
The classification of B. thuringiensis Cry toxins was done in the past decade on the basis of the targeted pest attacked by them (Hofte & Whiteley, 1998); however, as a result of dual toxic activity shown by some cry genes and the inconsistencies in the original proposed classification by Höfte and Whiteley (1989), Crickmore et al., (1998) suggested a modification of the nomenclature for insecticidal crystal proteins, based on the capability of a crystal protein to show some experimentally demonstrable toxic effect in a target organism (Crickmore et al., 1998; Höfte & Whiteley, 1989). Almost 70 serotypes and the 92 subspecies described to date describe the diversity of B. thuringiensis (Galan-Wong et al., 2006).
It is an established fact that many insects are vulnerable to the toxic activity of
B. thuringiensis; they include, lepidopterans which have been remarkably well studied, and activity is shown against them by many toxins (Jarret & Stephens., 1990; Sefinejad et al., 2008). A bulk of susceptible species belonging to agriculturally important families such as Cossidae, Gelechiidae, Lymantriidae, Noctuidae, Pieridae, Pyralidae, Thaumetopoetidae, Tortricidae, and Yponomeutidae belong to order Lepidoptera (Iriarte & Caballero, 2001).
General patterns of use:
The major commercial uses of B. thuringiensis generally aims against pest of agricultural and forest crops belonging to order lepidoptera; however, in current years strains active against pests belonging to order coleopteran have also been marketed (Tomlin, 1997). In public health programmes strains of B. thuringiensis kurstaki active against dipteran vectors of parasitic disease organisms have been employed (Tomlin, 1997).
Applications in agriculture and forestry
After B. thuringiensis became available in France for about 30 years it is used commercially on agricultural and forest crops (Van Frankenhuyzen, 1993). Use of
B. thuringiensis has increased to a great extent in recent years and the number of companies with a commercial interest in B. thuringiensis products has been raised from four in 1980 to at least 18 (Van Frankenhuyzen, 1993). Employing conventional spraying technology several commercial B. thuringiensis products including
B. thuringiensis aizawai, B. thuringiensis kuehniella or B. thuringiensis tenebrionise have been applied to crops. A variety of formulations have been used on major crops such as cotton, maize, soybeans, potatoes, tomatoes, various crop trees and stored grains. Formulations have ranged from ultralow-volume oil to high-volume, wettable powder and aqueous suspensions (Tomlin, 1997). Naturally occurring B. thuringiensis strains have been used mainly, but B. thuringiensis toxins expressed by transgenic microorganism using the technique of conjugation and genetic manipulation have reached the commercial market in some cases (Carlton et al., 1990). The reason for developing these modified organisms is to increase host range, lengthen field activity or improve deliverance of toxins to target organisms. For example, the transfer of coleopteran-active cryIIIA gene to a lepidopteran-active B. thuringiensis kuehniella (Carlton et al., 1990). A plasmid carrying an ICP gene has been transferred from B. thuringiensis to a non-pathogenic leaf-colonizing isolate of Pseudomonas fluorescens; fixation of the transgenic cells produces ICP contained within a membrane prolonging the persistence (Gelernter, 1990).
Applications in vector control
B. thuringiensis Kurstaki has been used in large scale programmes to control both mosquitos and blackflies (Lacey et al., 1982; Chilcott et al., 1983; Car, 1984; Car & de Moor, 1984; Cibulsky & Fusco, 1987; Becker & Margalit, 1993; Bernhard & Utz, 1993). In Germany for example to control mosqiutos 23 tonnes of B. thuringiensis Kurstaki wettable powder and 19 000 litres of liquid concentrate were used (Anopheles and Culex species) between 1981 and 1991 in the Upper Rhine Valley (Becker & Margalit, 1993). Approximately 10 tonnes of B. thuringiensis Kurstaki have been used in China to control the malarial vector, Anopheles sinensis in recent years.
Resistance of Insect Populations
During the last 15 years numerous insect populations belonging to several different species with different levels of resistance to B. thuringiensis have been obtained by laboratory selection experiments (Schnepf et al., 1998). The species comprise of Plodia interpunctella, Cadra cautella, Leptinotarsa decemlineata, Chrysomela scripta, Tricholplusia ni, Spodoptera littoralis, Spodoptera exigua, Heliothis virescens, Ostrinia nubilalis and Culex quinquefasciatus (Schnepf et al., 1998). The Indian meal moth was the first insect to develop resistance to B. thuringiensis. Kurstaki. It is a pest of grain storage areas (Swadner, 1994).
The progression of resistance is more quick in laboratory experiments than under field conditions because of higher selection pressure in the laboratory (Tabashnik, 1991). In the field no indications of insect resistance to B .thuringiensis were noticed, until the development of resistance was ob-served in the diamondback moth in crops where B. thuringiensis had been repeatedly used. Since then, in laboratory resistance has been observed in tobacco budworm, the Colorado potato beetle and other insect species (McGaughey, 1992)
Transgenic Bacillus thuringiensis plants
Transgenic plants are the plants which have their DNA being modified by genetic engineering. B. thuringiensis transgenic plants possess gene from B. thuringiensis. This gene helps these plants to produce insecticide within their cells. Agriculture has been revolutionized by transgenic plants expressing insecticidal proteins from the bacterium, Bacillus thuringiensis. B. thuringiensis has become a key insecticide because genes producing B. thuringiensis toxins have been engineered into major crops grown on 11.4 million ha worldwide? in 2000 (Shelton et al., 2002).
Transgenic Bacillus thuringiensis plants were intended to control the European corn borer (Ostrinia nubilalis) and other Lepidoptera that feed on corn tissue. The B. thuringiensis toxins produced by transgenic corn are specific to Lepidoptera and kill specifically insects that consume the plant tissue. Owing to this specificity, the impact of B. thuringiensis corn on no target organisms was believed to be insignificant (Orr & Landis, 1997; Pilcher & Rice, 1998). The majority of commercial B. thuringiensis hybrids express the endotoxin in their pollen to varying degrees and thus may pose a risks to non target Lepidoptera consuming pollen deposited on their host plants (Losey et al., 1999; Jesse & Obrycki, 2000).