bacillus thuringiensis

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Rectangular and rhomboid inclusions are the typical forms of ..... endotoxin was injected intravenously into mice causing paralysis and death. The δ-endotoxin.

In: Soil Microbes and Environmental Health Editor: Mohammad Miransari, pp.

ISBN 978-1-61209-647-6 © 2011 Nova Science Publishers, Inc.

Chapter 3

BACILLUS THURINGIENSIS: SOIL MICROBIAL INSECTICIDE, DIVERSITY AND THEIR RELATIONSHIP WITH THE ENTOMOPATHOGENIC ACTIVITY J. Hernández-Fernández,1 1 and SA López-Pazos 2 1

Universidad de Bogotá Jorge Tadeo Lozano, Facultad de Ciencias Naturales e Ingeniería, Programa de Biología Ambiental, “GENBIMOL” Genética, Biología Molecular y Bioinformática, Carrera 4 No. 22-61. Bogotá, Colombia 2 Instituto de Biotecnología, Universidad Nacional de Colombia, Calle 45, Carrera 30, Bogotá, Colombia

ABSTRACT Bacillus thuringiensis is the most important biological cause for pest insect control that affects commercial crops. In the biopesticides market, this bacterium represents 100 millions of dollars/year. This bacterium is toxic to Lepidoptera, Coleoptera, Diptera, Hymenoptera and Mallophaga insect orders, among others. A great number of strains have been characterized with regard to their toxic factors, spores, genomes, habitat and biological activity. Only for Cry toxins (the most important lethal protein) at least 600 different sequences has been found and classified into 67 families groups. Some mechanism of action models have been proposed for Cry proteins that implies toxin activation and their recognition by larvae insect gut receptors. The other toxic component, the cytolitic proteins (Cyt proteins), interacts with membrane lipids in insect midgut. This technology is very useful to produce transgenic crops, which can control pests around the world. The microorganism lives in a widely spectrum of environmental places: the soil is the most important, but it has also been found in the leaves and dead insects. Our research has allowed us to find strains and proteins with an important future to protect commercial crops in Colombia, including Tuta absoluta, Tecia solanivora and Spodoptera frugiperda. In this chapter the bacterium and its diversity and how this multiplicity has important impact on their biological activity is presented. Our work in this context will integrate strain characterization, gene identification and biological tests into a lepidopteran model. The future prospects are discussed. 1 [email protected]

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Keywords: Bacillus thuringiensis, Cry protein, Cyt protein, Toxic element, Pest insect, Bioassay, Characterization.

GENERAL FEATURES OF BACILUS THURINGIENSIS Bacillus thuringiensis is the spore-forming soil bacterium widely used in agriculture as a biological pesticide. It has been used in pest insect management for more than 70 years. The Interest for microbial insecticides dates from middle of last century; research of disease Bombix mori silkworm made by Pasteur in 1849, focused the attention of bacteriologists on insect pests and the discovery of microbial disease. The important point about B. thuringiensis is that it can produce an insecticidal crystal protein, which is toxic against pest insects. Structure inclusion consists of union of many polypeptides with molecular mass between 27-140 kDa. These crystals represent 20-30% of the total protein after the processes of cellular lysis and spore liberation. These proteins are named Cry and Cyt or δ-endotoxins, representing the active ingredient for commercial preparations. A given strain will synthesize between one and five or more of these toxins packaged into a multiple crystals. However, in more cases acristalliferous strains have been described. Because Cry proteins are of high specificity and safety for the environment, they are considered alternatives to chemical pesticides, controlling pest insects in agriculture. On the other hand, many strains are able to produce Cyt proteins, which are membrane pore-forming proteins, without significant selectivity in their action mechanism. They are lethal to dipteran larvae and have shown broadly cytolytic and hemolytic activity in vitro. In addition to Cry and Cyt proteins, B. thuringiensis has other virulence factors enabling it to survive and multiply within the host, evade immune system and cause septicemia. Some of these virulence factors, such as phospholipase, enterotoxins, -exotoxins and chitinases have no specificity in the order of the insects they affect and are widely distributed within different serovars of B. thuringiensis. Others proteins produced by B. thuringiensis are vegetative insecticidal proteins (VIP) proteins with activity against pest moths.

BACKGROUND A Japanese biologist, Ishiwata Shigetane in 1901, isolated a bacterium as the cause of a disease afflicting silkworm, named Bacillus soto. This event marks the beginning to biological pest control. Berliner in 1911 (Thüringen, Germany) isolated a bacterium, which was aerobic with the ability to form spore from a diseased larva of the Mediterranean flour moth, Anagasta kuhniella, and identified it as B. thuringiensis. Berliner suggested that this pathogen could be used for insect control. The commercial production began in France in 1938, with the name Sporeiner® and in the United States in 1957, under the name Thuricide®. Hannay and Fitz-James (1955) and Angus (1956), observing that the strains of B. thuringiensis produces parasporal crystal proteins inclusions in the course of sporulation, demonstrated that these are responsible for the insecticidal action. Gonzalez and coworker in the 1980’s found that the cry genes coding for crystal proteins, were localized on plasmids. Helen Whiteley and Ernest Schnepf in 1981, at the University of

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Washington, cloned the first gene encoding for B. thuringiensis toxin: the cry1Aa gene, which was toxic to tobacco hornworm larvae. Immediately, many other cry genes were cloned quickly, and this event led to the development of B. thuringiensis transgenic plants (Schnepf et al., 1998). In the 1980s, several studies successfully demonstrated that plants can be genetically engineered. In1996, B. thuringiensis cotton hits the market (Schnepf et al., 1998). In 2010, B. thuringiensis bioinsecticides are considered as the most effective, specific and environmentally-friendly preparations for the insect pest control. Table 1. Bacillus thuringiensis serovars characterization according to the H antigen

Source: International Entomopathogenic Bacillus Centre. Pasteur Institute, Paris, France. 2010 (URL: http://wdcm.nig.ac.jp/CCINFO/CCINFO.xml?590).

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Classification of Bacillus thuringiensis strain The B. thuringiensis strains are classified into serotypes according to their H flagellar antigen determinants, proposed by De Barjac and Boneffoi in 1962. To date, up to 92 serotypes and 84 serovars have been described and defined as subspecies (International Entomopathogenic Bacillus Centre, Pasteur Institute, Paris, France, 2010) (Table 1). However, serotype is not strictly related to a specific toxicity against a given insect (Schnepf et al., 1998). The H-serotyping may not be enough to show the molecular characteristic of a strain. For example, the group strains of morrisoni serovar are toxic to lepidopteran, dipteran and coleopteran insect orders (http://www.bgsc.org/). Monoclonal antibodies can be directly used against crystal protein epitope and are correlated with a host spectrum of investigated strain. They represent the most direct way to classify new B. thuringiensis isolates and range production of insecticidal activity based on the homology in the analyzed strain (Schnepf et al., 1998). The toxins were originally classified by Höfte and Whiteley in 1989, presented in a classification with fourteen sequence of genes encoding Cry proteins into four classes, according to their amino acid sequence homology and insecticidal specificity: CryI (Lepidopteran-specific), Cry II (Lepidopteran and Dipteran specific), CryIII (Coleopteranspecific) and CryIV (Dipteran-specific). Two additional classes, CryV and CryVI (nematodeactive toxins), were added by Feitelson and coworkers in 1992. Recently, and according to the same criteria, Crickmore and coworkers (1998) established a new homology-based classification system. In their method of classification, each protein acquired a name consisting of the mnemonic Cry and four hierarchical lines consisting of numbers, capital letters, lower case letters and numbers (e.g. Cry1Aa1). Therefore, proteins with less than 45% sequence identity are placed in the primary level (Cry1, Cry2, etc.), and with 78% and 95% identity constitute the borders for the secondary and tertiary level, respectively. This system replaces the old nomenclature that used roman numerals. This information is constantly updated and available in the following URL: http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/.

Bacillus thuringiensis Crystal Proteins Biodiversity The variety of known Cry proteins is the result of a continuing effort in many countries to isolate and characterize B. thuringiensis new strains with the goal of finding novel toxins, which can control the pests with significant agronomical and medical implications. Thousands of strains have been screened and today, nearly 600 sequences of cry genes have been reported, extensively studied and classifieds into 67 Cry and 2 Cyt protein families (Table 2). B. thuringiensis does not have a history of mammalian pathogenicity and research has focused on the insecticidal nature of the crystal proteins (Schnepf et al., 1998). The characterizations of B. thuringiensis native strains, globally, allowed the recognition of gene combinations multitude, which are more common than the others. The amazing diversity of Cry toxins is believed to be due to a high degree of genetic plasticity (Schnepf et al., 1998). The genes enconding the crystal proteins are found on transmissible plasmids and flanking transposable elements that may facilitate gene spread easily, leading to the evolution of new toxins (Schnepf et al., 1998).

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Table 2. Cry protein groups and their diversity

Source: URL: http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/.

Individual Cry toxins have been studied to define the spectrum of insecticidal activity regularly resulting in the restriction of a particular order of insects and a few species (Roh et al., 2007). The high number of identified Cry proteins has permitted the comparative sequence analyses and investigation of elements important for insect functioning and specificity. The isolated strains show a wide range of specificity primarily targeting different insect orders of Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes) and Coleoptera (beetles and weevils) (Roh et al., 2007). However, some Cry toxins have been reported to inhibit the members of Hymenoptera (wasps and bees) Mallophaga, and Acari and other invertebrates such as Nemathelminthes and Platyhelminthes (Schnepf et al., 1998). A few toxins have a broader spectrum of activity that can control two or three orders of insects. For example, Cry1Ba is active against larvae of moths, flies, and beetles (van Frankenhuyzen, 2009). The combination of toxins in a given strain, therefore, defines the activity spectrum of that strain (Schnepf et al., 1998).

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Within the diversity of Cry proteins are three main groups: the three-domain Cry family, the binary-like toxins family (e.g. Cry35/36) and Mtx-like toxins type (e.g. Cry15, Cry23). Cry1 toxins are part of the three domains family. These toxins are the most diverse group of Cry proteins. Cry1 proteins are synthesized in protoxins form and must be processed by proteases to produce an active toxin, including protoxins of 70-130 KDa (Roh et al., 2007). To date, seven three-dimensional structures have been described by X-ray crystallography: Cry1Aa, Cry1Ac, Cry2Aa, Cry3Aa, Cry3Ba, Cry4Aa and Cry4Ba (López-Pazos and Cerón, 2007). These toxins had shown a low amino acid sequence homology, but have a similar structure of three-functional domains (López-Pazos and Cerón, 2007). Domain I is located at the end N-terminus and is involved with the pore formation in the membrane of the cells in the insect gut. The domain I is composed of an α-helical bundle in which six helices surround a central helix. Domain II, the most variable, is involved in the interaction with the membrane receptor and the specificity, this domain consists of three -sheets packed together to form a -prism with pseudo threefold symmetry. Domain III is also related to receptor binding and specificity and probably pore formation. The domain III forms a -sandwich, in this conformation; two antiparallel -sheets pack together with a “jelly roll” topology. Domain III indicates less structural variability than domain II (López-Pazos and Cerón, 2007). The protoxin and the active toxin sequences are mainly different due to their large carboxylterminal end highly conserved among some of the protoxin sequences. The possible function of this long carboxyl-terminal segment is to help Cry proteins as they form an ordered structured crystalline strain (Schnepf et al., 1998).

Action Mode of Bacillus thuringiensis Cry Proteins Three theories explain the toxicity mechanism of Cry proteins. In the Bravo model, Cry proteins are synthesized as a protoxin that crystallizes to form inclusions. When a susceptible insect larva (flies, moths, beetles, etc.) ingested the crystals, they are solubilized in the environment of the midgut, which is characterized by an alkaline pH, where they break the disulfide bonds (Figure 1). Host proteases are able to recognize cleavage sites on the protoxin and degrade them to produce active toxin that afterward binds to specific receptors on the midgut epithelium. In general the Cry1 N-terminal toxin cut is located in the first 25 to 30 amino acids, both Cry3A and Cry2Aa toxins in the residues 58 and 49, respectively. In the Cry protoxin higher molecular weight (130-140 kDa), also produced a cut in the second third of the protein, eliminating the last 500-600 residues of the C-terminal (Pigott and Ellar, 2007). Activated Cry toxins perform two specific functions: receptor binding and ion channel formation. The Cry protein interaction with the 210-kDa cadherin-like glycoprotein receptor (Zhang et al., 2005) or/and 120-kDa aminopeptidase N (APN) (Yaoj et al., 1999) induces conformational changes in the structure of the toxin that leads to the formation of the oligomer, which is responsible for membrane insertion and pore formation. Generally, oligomerization of toxin subunits form pore structures, which are inserted into the membrane. The pore is negligibly selective allowing the free pass of ions and water into the cells, the pass of several monovalent cations into columnar cells, causing depolarization of the cell membrane until the cell lysis and eventual death of the insect larvae.

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Figure 1. Mode of action of B. thuringiensis Cry proteins against midgut larvae. A: Toxic forms, B: Solubilization and activation, C: Receptor binding, D: Hemolymph invasion. Adapted from: http://www.inchem.org/documents/ehc/ehc/ehc217.htm.

An alternative hypothesis has been suggested, which indicates Cry toxicity is independent of toxin oligomerization (Pigott and Ellar, 2007). Yaoi and coworkers at Tokyo University cloned and purified the first Cry toxin-binding receptors from B. mori insect. This receptor was a 120-kDa aminopeptidase N (APN), which has been made since then for comprehension of action mode. The best-studied receptors are lepidopterans and Cry1A toxins, which are largely characterized. Receptors in the APNs family and a protein family of cadherin-like receptors have been identified in lepidopteran species (Bravo, 2007). Thirty eight members of APNs in five different classes have been described. In nematodes, glycolipids are assumed to be an important class of Cry toxin receptors (Pigott and Ellar, 2007). Other putative receptors are alkaline phosphatases (ALPs), a 270-kDa glycoconjugate and a 252-kDa protein (Schnepf et al., 1998; Pigott and Ellar, 2007). While several studies suggest that APNs be able to serve as Cry-binding proteins and their ability to mediate pore-formation in vitro is well established, a direct role in cytotoxicity has been yet to be decisively recognized. The APN protein apparently has a mass of 120 kDa and is anchored to the membrane through a group glicosilfosfatidil-inositol (GPI), with a mass between 175-210 kDa depending on the lepidopteran insect (Pigott and Ellar, 2007).

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Toxin interaction with the cadherin-like glycoprotein receptor promotes an additional cut of the amino terminal end, facilitating the formation of an oligomer or pre-pore formed by four monomers that is responsible for the membrane insertion and pore formation (Pigott and Ellar, 2007). To date, almost all cloned cadherin-like glycoprotein genes encode proteins that bind to toxin and have been found to confer toxin susceptibility. Cadherins play a fundamental role in the Cry1A toxin action mode, it has yet to be investigated that whether other putative receptors, are required for full toxicity (Pigott and Ellar, 2007). The theory that Cry1Ab terminates cell growth solely by osmotic lysis has been challenged. The results by Zhang and coworkers (2005) to estimate the correlation between pore formation and cytotoxicity has not been satisfactorily confirmed. They proposed a different mode of action in which monomeric Cry1Ab binds to cadherin receptor B. thuringiensis-R1 and initiates an Mg2-dependent signaling cascade that promotes cell death (Pigott and Ellar, 2007). A third hypothesis has been proposed to explain the action mode of Cry1Ac toxin. The model suggests that cytotoxicity is due to the combined effects of osmotic lysis and cell signaling. The monomeric Cry1Ac binds to the cadherin-like protein to activate it. This results in the activation of an intracellular signaling pathway regulated by phosphatases (Pigott and Ellar, 2007).

Biological Activity of B. Thuringiensis Almost 3000 insect species have been found to be susceptible to at least one of B. thuringiensis toxins (Huang et al., 2004). However, B. thuringiensis products are highly selective and environmentally safe (Schnepf et al., 1998). B. thuringiensis is widely used for the control of Lepidoptera species; also this bacterium is active against dipteran and coleopteran organisms (Roh et al., 2007). Other reports showed that B. thuringiensis is also effective on Hymenoptera, Homoptera, Orthoptera, and Mallophaga orders (van Frankenhuyzen, 2009; Schnepf et al., 1998). B. thuringiensis is active on many lepidopterans such as Bombix mori, Ephestia kuhniella sep (Mediterranean Flour Moth). The most important antilepidopteran strain was, for many years, B. thuringiensis kurstaki HD-1. HD-1 is effective on many pest lepidopterans and several toxins have been identified as useful to control this order (Table 3) (van Frankenhuyzen, 2009). In 1976 Sharpe found, B. thuringiensis strain galleriae NRRL B-4027 to be toxic against Popillia japonica larvae. Later, Krieg et al. (1983) discovered an important strain: B. thuringiensis tenebrionis BI 256-82, which is toxic to Leptinotarsa decemlineata (potato pest). In 1986, the anticoleopteran B. thuringiensis san diego M7 was isolated, containing the same plasmid profile similar to tenebrionis isolate (Herrnstadt et al., 1986). Donovan et al. (1988) were studying a B. thuringiensis strain (EG2158) toxic to coleópteran insects, with important differences of Bt tenebrionis. Many coleoptera-specific strains were isolated subsequently such as B. thuringiensis morrisoni, B. thuringiensis tolworthi, B. thuringiensis PS52A1, B. thuringiensis kumamotoensis or B. thuringiensis japonensis buibui. Today, this biological control is effective and transgenic crops have been produced around the world (Ostlie, 2001). Rectangular and rhomboid inclusions are the typical forms of anticoleopteran crystals.

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Table 3. Some active B. thuringiensis toxins against different pest-insect orders Order and insects Lepidoptera Cydia pomonella Trichoplusia ni Spodoptera exigua Artogeia rapae Bombyx mori Hyphantria cunea Plutella xylostella Manduca sexta Spodoptera frugiperda Spodoptera littoralis Helicoverpa zea Heliothis virescens Ostrinia nubilalis Spodoptera litura Lymantria dispar Galleria mellonella

Coleoptera Diabrotica undecimpunctata Leptinotarsa decemlineata Cotinis spp. Cyclocephala sp. Popillia japonica Chrysomela scripta Diptera Musca domestica Aedes aegypti

Active toxin Cry1Aa, Cry1Ab, Cry2Aa2, Cry15Aa1 Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa1, Cry2Ab2, Cry2Ac1 Cry1Ab1, Cry1Ba1, Cry1Bd1, Cry1C, Cry1Ca1, Cry1Fa1, Cry1Gb1, Cry1Ka1 Cry1Ba1, Cry1Ka1, , Cry1Ba1, Cry1Ia1, Cry1Ka1 Cry1Ba1, Cry1Ka1 Cry1Ba1, Cry1Bd1, Cry1Ea2, Cry1Gb1, Cry1Ia1, Cry1Ka1, Cry2Ab1 Cry1C, Cry1Ea1, Cry1Ea4, Cry2Ac1, Cyt1Aa2, Cyt2Aa1 Cry1C Cry1C, Cry1Ca2, Cry1D, Cry1Ea1, Cry1Fa1 Cry1Fa1, Cry2Aa1, Cry2Ab2 Cry1Fa1, Cry2Aa1, Cry2Ab2, Cry2Ac1 Cry1Fa1, Cry2Aa1, Cry2Ab2 Cry1Ia1 Cry2Aa1, Cry2Ab, Cry2Ab2 Cry9Aa1

Cry3Ba1, Cry3Bb1, Cry3Ba1, Cry3Bb1, Cry8Aa1, Cry8Ba1 Cry8Ba1 Cry8Ba1 Cyt1Aa4

Anopheles gambiae Culex quinquefasciatus Callíphora stygia Lucilia cuprina Lucilia sericata

Cry1Bd1, Cry1Gb1 Cry1C, Cry2Aa1, Cry2Ab, Cry2Ab2, Cry2Ac1, Cry4Aa, Cry4Aa1, Cry4Ba, Cry4Ba1, Cry10Aa1, Cry11Aa, Cry11Aa1, Cry17Aa1, Cry19A, Cry20Aa1, Cry27Aa1, Cyt1Aa2, Cyt1Aa4, Cyt1Ab1, Cyt2Aa1 Cry4Aa, Cry11Aa, Cry17Aa1, Cry19A, Cry27Aa1, Cyt1Ab1 Cry4Aa, Cry4Ba, Cry11Aa, Cry17Aa1, Cry19A, Cry27Aa1, Cyt1Aa2, Cyt1Ab1, Cyt2Aa1 Cry4Aa1, Cry4Ba, Cyt1Aa2, Cyt2Aa1 Cry4Aa1, Cry4Ba, Cry11Aa, Cry11Ba1, Cyt1Aa4, Cyt1Aa2, Cyt2Aa Cyt1Aa2, Cyt2Aa Cyt1Aa2, Cyt2Aa

Hymenoptera Acromyrmex Diprion pini Cephacia abietis

Cry1, Cry9 Cry5A, Cry5B Cry5A, Cry5B

Nematode Caenorhabditis elegans Pratylenchus spp. Fasciola hepatica Panagrellus redivivus Pratylenchus scribneri

Cry5Aa1, Cry5Ab1 Cry5Aa1, Cry5Ab1, Cry6Ba1, Cry12Aa1 Cry5Ab1 Cry6Aa1 Cry6Aa1

Anopheles stephensi. Culex pipiens

Coleopteran specificity Cry proteins are: Cry1Ia (81.2 kDa), Cry1Ba (139.6 kDa), Cry3 (72-75 kDa), Cry6Aa1 (54 kDa), Cry8 (130-133 kDa), Cry34 (14kDa) and Cry35 (44 kDa)

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among others (Table 3). Each anticoleopteran Cry toxins has special characteristics e.g. Cterminal is absent in the Cry3A protoxins, perhaps by coevolution due to acidic pH or type of proteases in coleopteran midgut (Schnepf et al., 1998). In 1977 Goldberg and Margalit isolated a spore forming bacterium with a great insecticidal power, from a mosquito dead larva of Negev desert. Bioassays showed that four genders are sensitive to this microorganism (Anophelesk, Uranotaenia, Culex and Aedes). The bacterium is a new B. thuringiensis serotype, named israelensis, the first isolate with Diptera activity (Barjac, 1978). Other strains with the same toxicity are: B. thuringiensis jegathesan, B. thuringiensis medellin, B. thuringiensis fukuokaensis and B. thuringiensis higo. Antidipteran crystals have cuboidal, spherical and rhomboidal shapes (Hofte and Whiteley, 1989). Cry proteins dipteral-specific are: Cry2, Cry4, Cry10, Cry11, Cry17, Cry19, Cry20, Cry27, Cyt1Aa1, Cyt1Aa4, Cyt1Ab y Cyt2Aa (Table 3). B. thuringiensis can adversely affect the activity of livestock dipterans (Gough et al., 2002). A subset of proteins was tested against species in other orders, in particular Orthopteran, Hymenopteran, Mallophagan, flatworms and nematodes (van Frankenhuyzen, 2009; Schnepf et al., 1998). Some Orthoptera members are sensitive to B. thuringiensis such as Periplaneta americana, Blattella germánica, Blatta orientalis and Dociostaurus maroccanus (QuesadaMoraga and Santiago-Alvarez, 2001; Quesada-Moraga et al., 1997; Lonc et al., 1997; Hernández-Crespo et al., 1994). First, β-exotoxina was used for its controlling effects (Latchininsky and Launois-Loung, 1992), then δ-endotoxins of 130 and 66 kDa, with relationship to Cry1 and Cry2 proteins (Beegle and Yamamoto, 1992; Quesada-Moraga and Santiago-Alvarez, 2001; Lonc et al., 1997). On the other hand, Hymenoptera is a determinant order because their potential damage is wide in commercial crops. Various cry gene products have lethality against these insects, specially Cry1, Cry5A and Cry9 toxins. Action mechanism in hymenopteran is similar to that described in lepidopteran species. Hymenopteran pests with susceptibility to B. thuringiensis are Acromyrmex, Trichogramma dendrolimi, Diprion pini, Cephacia abietis and Monomorium pharaonis (Table 3) (Pinto et al., 2003; Garcia-Robles et al., 2001; Schuler et al., 2001; Takada et al., 2001; Hill and Foster, 2000; Vobrazkova et al., 1976). In Mallophaga order, there are blood sucking species and disease causing vectors to humans and animals such as Bovicola (Damalinia) ovis, Menopon gallinae and Eomenacanthus stramineus. B. thuringiensis kurstaki HD-1 (Cry1 and Cry2), B. thuringiensis finitimus (Cry26 and Cry28), and B. thuringiensis kenyae (Cry1) have high toxicity against these organisms. It is possible that the insecticidal activity could be due to 70 kDa toxic factor, similar to Vip3A (Gough et al., 2002; Hill et al., 1998; Estruch et al., 1996; Drummond et al., 1995; Chilcott and Wigley, 1994; Drummond and Pinnock, 1992; Drummond et al., 1992; Lonc and Lachowicz, 1987; Lonc et al., 1986; Pinnock, 1994; Gingrich et al., 1974). In flatworm case, B. thuringiensis exotoxin inhibits RNA synthesis and their regeneration (Ziller, 1976). On the other hand, some δ-endotoxins (35 to 155 kDa) has been used in biological control of mites such as Tetranychus urticae (red spider mite), the house dust mite Dermatophagoides pteronyssinus and pest mites of livestock, birds and store products. Action mode depends on toxin; there is not spore germination (van deer Geest et al., 2000; Payne et al., 1994). Finally, nematode-active toxins are in the Cry5, Cry6, Cry12, Cry14, Cry21 and Cry55 groups (Table 3); many strains have been assayed, especially B. thuringiensis kurstaki, B. thuringiensis israelensis B. thuringiensis darmstadiensis PS17, B. thuringiensis PS86Q3, B.

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thuringiensis PS52A1, B. thuringiensis YBT1518, B. thuringiensis PS69D1, B. thuringiensis PS33F2, B. thuringiensis sotto PS80JJ1 and B. thuringiensis J112.. Among the sensitive nematodes are Trichostrongylys columbriformes, Caenorhabditis elegans, Panagrellus redivivus, Pratylenchus spp., Meloidogyne javanica and M. incognita, Nippostrongylus brasiliensis (rat parasite, superfamily Trichostrongylidae) and Fasciola hepática (class Trematoda) (van Frankenhuyzen, 2009; Khyami-Horani et al., 2003; Wei et al., 2003; Borgonie et al., 1996; Zuckerman et al., 1993; Meadows et al., 1989; Osman et al., 1988; Bottjer et al., 1985; Ignoffo and Dropkin,1976).

Ecological Context of Bacillus thuringiensis Very little is known about the natural ecology of B. thuringiensis. It is native in many habitats such as soil, dead insects, leaves or stored goods (Schnepf et al., 1998). Morphology and genetic composition of B. thuringiensis is highly variable among different types of soils and places. B. thuringiensis composition seems to be influenced by several factors including soil humidity, organic matter, temperature, structure and pH, macro/micro-nutrients, richness and local insect distribution (Uribe, 2004). B. thuringiensis can be found in a great number of terrestrial ecosystems such as deserts, steppes, tropical humid forest, high mountains, beaches and caves (Bravo et al., 1998; Ibarra et al., 2003; Schnepf et al., 1998). All strains are amylase and protease producers allowing the organism the use of complex and available materials such as carbon and nitrogen sources (Bernhard y Utz, 1993; Sharp et al., 1989). Synthetic and semisynthetic media have been designed for growth, crystal production and sporulation (Holmberg et al., 1980). Isolation includes hot treatment for spores, acetate enrichment and antibiotic selection (Delucca et al., 1981; Travers et al., 1987). The regulation of nitrogen and other mineral elements (magnesium, cupper, manganese, calcium and zinc) affect sporulation and crystal synthesis, besides it is necessary for an ionic balance; idoneous pH for high crystal production is between 5.5-6.5 (Içgen et al., 2002(a); Içgen et al., 2002 (b)). Taxonomically, B. thuringiensis is a member of a simple genetic group, including B. anthracis, B. cereus and B. mycoides (Priest et al., 1988; Priest et al., 1994). This group has been defined by similar phenotypic characteristics, high DNA homology and conserved regions in 16S rRNA (Kaneko et al., 1978; Nakamura, 1994; Priest et al., 1994). B. thuringiensis has a great diversity because of its genetic plasticity. Bacterium spores survive for several years, although the population declines so fast (Schnepf et al., 1998). Niches of B. thuringiensis in the environment are entomopathogen, phylloplane, and soil (Schnepf et al., 1998). Noteworthy, B. thuringiensis is able to multiply in the insect hemolymph. Many factors contribute to virulence such as δ-endotoxins, phospholipases, β-exotoxins, proteases, chitinases, and the VIPs. The Cry toxins are the most important compound allowing the development of the organism in insects. B. thuringiensis strains that have been analyzed for insecticidal activity include three major pathotypes with toxicity to lepidopteran, coleopteran and dipteran species (Roh et al., 2007). Cry toxins compose a set of proteins suitable for use as biological insecticide or for expression in transgenic plants, because it is considered one of the insecticides most harmless to humans, vertebrates, non-target insects and plants, besides being biodegradable completely (Schnepf et al., 1998).

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B. thuringiensis presence and insect population are not related at least in part due to a high degree of genetic plasticity in the bacterium (Schnepf et al., 1998). Various diversity studies have been done around the world. These studies have many important steps: isolation and morphological observations, toxin determination and bioassay tests. Firstly, samples (soil, water, leaves, dead insects, sediments, etc) are collected from locations with diverse crops, urban locations, forests, high mountains, non-cultivated areas and beaches. For bacteria isolation, heat-acetate method is used, because acetate is known to inhibit spore germination, so other spores germinate and then the growing cells and other non-spore-forming bacteria are sterilized by heat treatment (Travers et al., 1987). Putative B. thuringiensis colonies are found in flat, dry, white shapes and with uneven borders. The main character by which B. thuringiensis is differentiated from the other spore-forming microorganisms, is crystal production evaluated by microscopy (Lecadet et al., 1999). Most strains produce irregular inclusions, only 50% form typical crystals and 17% are bypiramidal (47% are toxic to lepidopteran, 1% to dipteran and 0.5% to both diptera and lepidoptera and 34% are nontoxic). The crystal morphology may not always predict activity; however abundance of heterogenous could be a source of novel biological properties. For confirmation of identity, many biochemical and molecular tests was developed for the rapid identification of different types of isolates such as protein differentiation, serotyping, plasmid content, monoclonal antibodies, known and unknown toxin genes and bioassays proofs against insect pest larvae (Figure 2). B. thuringiensis occurs naturally in many soils. It has been isolated from soil samples taken from around the world, and more than half of these isolates were undescribed strains of B. thuringiensis. It has been isolated from approximately 30% of the soil sampled (Jara et al., 2006).

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Figure 2. Characterization methodology in B. thuringiensis diversity projects. The most important objective is the identification and isolation of functional cry genes by the development of commercial products and transgenic plants.

The bacterium usually can survive only a short time in this environment. The half life of the insecticidal action of the toxins is about nine days, but a small quantity can be quite persistent. It was observed that spore number declined by one order of magnitude after two weeks (West and Burges, 1985; Petras and Casida, 1985). The profile of the crystal protein was found the most similar among B. thuringiensis strains to that of B. thuringiensis kurstaki (cry1 and cry2 genes), which is one of the most frequently obtained from nature. The most important cry gene in soil is cry1 type. For instant, in 1998 it was found that strains containing cry1 genes were the most abundant (Ben-Dov et al. 1997); Bravo group detected 49.5% of cry1 gene (Bravo et al. 1998); in Colombia 73% of cry1 gene was reported (Uribe et al. 2003); Wang et al. (2003) detected 76.5% of cry1 gene and 70% of cry2 gene; Thammasittirong and Attathom (2008) reported strains containing cry1-type genes (81.3%) at the same frequency as strains harboring cry2 gene (80.6%). Other cry genes have been discovered in a very low frequency. The most common serovar of B. thuringiensis isolated from environment is kurstaki. B. thuringiensis kurstaki has a broad spectrum of activity against a wide range of lepidopteran species attacking field crops of cotton, corn, soybeans, tomatoes, lettuce, strawberries, grapes, and peaches (Schnepf et al., 1998). B. thuringiensis does not appear to move readily in soil (Drobniewski, 1994). When two varieties of B. thuringiensis were applied in adjacent plots, no cross-contamination observed, indicating that the bacterium does not move laterally in soil. Other studies indicated that B. thuringiensis was not recovered passing a depth of six centimeters after irrigation, and that movement beyond the application plot was less than ten yards (Entwistle et al. 1993; Akiba, 1991). B. thuringiensis is occurring naturally in phylloplane, with a high percentage in tropics compared with the rest of the world, maybe due to absence of seasons, which favors the stability of the microbiota. The B. thuringiensis communities on leaves are about 102 CFU cm-2 of leaf (Collier et al., 2005; Maduell et al., 2002; Smith and Couche, 1991). B. thuringiensis may have been deposited on the plant through the followings: atmospheric deposition from distant sources, contamination from local soil populations by currents of air and precipitations, deposition of cells from insects or movement from roots to the foliage (Lindow and Andersen, 1996). cry genes composition of B. thuringiensis isolates indicates that cry1-like genes are the most abundant. B. thuringiensis phylloplane strains contain complex cry genotypes, which might have been resulted by plasmid transfer between different isolates in natural environments. Apparently, B. thuringiensis proportion did not differ with increasing altitude (1800-2900 m) (Jara et al., 2006). The phylloplane could be the ordinary home for B. thuringiensis, because it can be isolated more readily from the aerial vegetable surfaces. The most of isolates found on phylloplane correspond to B. thuringiensis kurstaki serovar (Jara et al., 2006). B. thuringiensis has been isolated more readily from dead insects or stored-product dusts. Some studies indicates that B. thuringiensis has been isolated from dead insects in a low number (approximately 2%) (Guz et al., 2009; López-Pazos et al., 2009), it is surprising because the insects are good source of food for B. thuringiensis development and maybe it is due to limiting factors (antibiotic compounds in lymph or natural competition with other

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organisms). The inclusions found in B. thuringiensis isolates from dead insects have different molecular weights relative to the environmental strains e.g. B. thuringiensis strains isolated from Premnotrypes vorax (Coleoptera) individuals have proteins of 125, 40 and 35 kDa; in the same way, B. thuringiensis strains isolated from gypsy moth (Lepidoptera) have proteins with molecular weights of 20 to 64 kDa representing new opportunities (Guz et al., 2009; López-Pazos et al., 2009). Most screening studies of susceptibility to B. thuringiensis proteins have shown a frequent insecticidal toxicity of this organism to lepidopteran species, while only very few isolates and toxins have been found active against Coleoptera (Hernandez et al. 2005). B. thuringiensis tenebrionis, isolated from larvae of beetle Tenebrio molitor (Krieg et al., 1983), produces the Cry3 protein (67 kDa), which is toxic to Coleoptera order (Federici, 1999). Other strains of B. thuringiensis producing Cry3 proteins have been isolated from soil and grain dust samples (Federici, 1999). Strains of B. thuringiensis, which are toxic to mosquitotoxic have been isolated from soil, rhizoplane, phylloplane, insects, animal feces and water. These B. thuringiensis isolates belong to israelensis, canadensis, morrisoni and darmstadiensis among others. Mosquitocidal B. thuringiensis strains possess a plasmid DNA with cry and cyt genes, so different levels of toxicity maybe due to presence or partial presence of these genes (Balaraman, 2005). B. thuringiensis has been isolated from rivers and municipal water structures after an aerial application of commercial products. Water management processes are not adequate to destroy its spores (Menom, 1985). Viable cells were recovered from the water for up to 200 days and in the sediment for up to 270 days after application (Hoti and Balaraman, 1991). The bacterium has been found in the air with the flow over 3000 meters downwind during an aerial application. The distance at which B. thuringiensis is capable of drifting, depends upon the amount and method of application, as well as the climatic conditions (Barry et al., 1993). B. thuringiensis is environmentally much more favorable than many synthetic pesticides, however environmental and health effects must be carefully tested before use. B. thuringiensis should be used only when necessary, and in the smallest quantities possible, and as part of integrated pest management program. Each strain of B. thuringiensis may exhibit different toxicity to insects, rodents and humans. B. thuringiensis and its commercial products have low oral acute toxicity to rats after feeding large doses. Other kinds of contacts have few acute consequences. Rats inhaling air spores indicated sings of respiratory depression. B. thuringiensis products are irritating to skin and eyes of rabbits (Swadener, 1994). Purified δendotoxin was injected intravenously into mice causing paralysis and death. The δ-endotoxin caused destruction of rat, mouse, sheep, horse, and human red blood cells. There have been few studies assessing the toxicity of B. thuringiensis to humans, mainly from occupational exposures. A working farmer, splashed by B. thuringiensis on his face and eyes developed skin irritation, burning, swelling, and redness. The complications were found to be related to the degree of exposure. Workers with preexisting health problems were more likely to report adverse effects (Swadener, 1994). Some of the most serious problems about the use of B. thuringiensis come from the effects it can have on the fauna, it feed on. Comparative reports showed that the environmental effects of B. thuringiensis are less than those of synthetic insecticides (Swadener, 1994).

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CONSIDERATIONS AND PERSPECTIVES The Cry toxins are close to a specific ideal insecticide against target insect, it does not contaminate the environment and the development of resistant is low in insect populations. However, there are many problems related to the use of Cry toxins for controlling both pest insects and disease vectors, to eventually replace the use of chemical insecticides. Some pest insects are not controlled effectively by Cry toxins discovered to date. It is therefore necessary to continue development of projects to isolate and characterize novel proteins that have activity against harmful pests. Otherwise using recombinant DNA and site directed mutagenesis methods to change the sequences of B. thuringiensis genes cry that have been identified and characterized may also be effective. For these reasons, there has been important progress in Colombia regarding the conformation of native strains B. thuringiensis collections. The Jorge Tadeo Lozano University, CORPOICA (the Colombian Corporation for Agricultural and Livestock Research), the National University of Colombia and the Corporation for Biological Research have been conducting research since 1992 to find novel B. thuringiensis strains that have a capacity to produce -endotoxins with a wider action spectrum. In this way, Sergio Orduz group has been evaluating the new B. thuringiensis strains with activity against insects Diptera, which could control vectors of human disease, identified a new serotype named B. thuringiensis subsp. medellin. Hernández and coworkers (2010), at the Jorge Tadeo Lozano University, Molecular Biology Laboratories, collected soil samples from various regions and different ecosystems, in five departments of Colombia. They isolated about 100 strains of B. thuringiensis and characterized them at microscopic, biochemical, molecular and biological level. The main goal of this study was to isolate and characterize new and native B. thuringiensis strains harboring cry1 genes in order to evaluate their insecticidal activity against Tuta absoluta larvae. So, it was possible to identify the genes that encode each of the strains Cry toxins, the step towards the realization of bioassays. The native B. thuringiensis strains from Colombian soils had amorphous, bi-pyramidal, square, round and triangular crystal forms. Eighteen morphological groups with different crystal combinations were established, showing high biodiversity. The Colombian native strain revealed protein bands ranging from 28 to 150 kDa, and between 1-5 protein bands were observed for individual native strains (Hernández et al., 2010). The molecular characterizations permit the identification between 1-5 specific cry1 genes in each of the native B. thuringiensis strains, producing thirteen different profiles. The characterization of novel native B. thuringiensis strains from Colombia contributes to an appreciation of the high B. thuringiensis biodiversity in Colombian agricultural areas. With respect to toxicity, ten native B. thuringiensis strains were tested against second instar larvae of T. absoluta. The two most toxic strains produced a mortality of 42 and 36%, slightly higher than the B. thuringiensis kurstaki HD1strain. The LD50 of the two native strains ZCUJTL11 and ZBUJTL39 indicated two and three fold more toxicity, respectively, than the reference Btk HD1 strain. The native B. thuringiensis strain ZCUJTL11 showed the highest potential to develop a microbiological control method against T. absoluta. The high potency of this isolate to adversely affect T. absoluta may be due to minor variations in the amino acid sequences of the proteins, higher levels of accumulation of the toxins, the presence of other

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toxin genes that were not detected by the primers used in the screening, or a combination of these factors (Hernández et al., 2010). In another study, Lopez, Orozco and Hernández (2005, 2007), standardized the LSSP methodology (Chain Reaction Polymerase with a single primer in low stringency) to identify cry1B polymorphisms in native isolates of B. thuringiensis. The isolation named BtGC120 showed a lower similarity (43%) with respect to this same gene in HD1 reference strain. The nucleotide sequence obtained from this fragment of 806 bp showed 93% identity with the sequence of the genes of B. thuringiensis morrisoni cry1Bc1 and cry1Bb1 B. thuringiensis strain EG5847. The +3 open reading frame predicted a protein of 268 amino acid residues with 88% identity with the protein Cry1Bc. This sequence revealed two domains, an endotoxin N involved in the formation of pores and another endotoxin M associated with receptor recognition. The biological assessment of native strain BtGC120 against first instar larvae Spodoptera frugiperda insect pest produced an LC50 of 1.896 ng of total protein per cm2. This study shows that the LSSP-PCR is a methodology that specifically identifies sequence variation of the cry genes of B. thuringiensis with the potential to find new genes with novel biological activities. Identification of new and more powerful Cry proteins can help in the development of new biopesticides, genetically engineered plants or have a reservoir of Cry toxins that can be used for the events of resistance emergence. Development and commercialization of agricultural varieties expressing B. thuringiensis Cry toxins (e.g. B. thuringiensis corn and B. thuringiensis cotton) is an alternative method to traditional synthetic insecticides, used for the control of important agricultural pests. Both corn and cotton, genetically engineered, has been adopted by farmers in 22 countries to control lepidopteran pests such as corn borers (mainly Ostrinia nubilalis) and the budworm-bollworm complex (Heliothis virescens, Helicoverpa spp., Pectinophora gossypiella) in cotton. Recent reports indicate the use of B. thuringiensis crops has resulted in economic benefits to growers and reduced the use of conventional insecticides; however, their potential impact affecting predators and parasitoids remain a concern. Since 2002, Colombia has adopted transgenic plants for the agricultural propose. In 2009, 18.874 hectares of cotton, 16.793 hectares of corn and 4 hectares of blue carnation were planted. Genetically produced B. thuringiensis plants represent benefits for the farmer and the environment, which is translated into reduced costs and less pollution, because possible insect resistance may decrease the efficient use of chemical pesticides. So far, ten types of cry genes have been introduced in 26 different plant species: cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1Ca, cry1H, cry2Aa, cry3A, cry9C and cry6A. The friendly strategies with environmental benefits, used for insect controlling, based on the B. thuringiensis Cry proteins, should increase in the future, in particular with the broad implementation of transgenic crops. The detection of new toxins and new ways of presenting the toxin to the susceptible insects, which includes the improvement of recombinant microorganisms and proteomic technology, could be adapted to the study of Cry B. thuringiensis proteins. Furthermore, interaction studies between Cry toxins and pest insects involving modes of action and resistance mechanisms should be carried out. Such researches are essential studies that will allow for improvement of existing B. thuringiensis application strategies and the ability to design the most efficient options.

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Jara S., Maduell P., Orduz S. 2006. Diversity of Bacillus thuringiensis strains in the maize and vean phylloplane and their respective soils in Colombia. J. Appl. Microbiol. 101: 117-124. Kaneko T., Nozaki R., Aizawa K. 1978. Deoxyribonucleic acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. Microbiol. Inmunol. 22: 639-641. Khyami-Horani H., Hajaij M., Charles J. F. 2003. Characterization of Bacillus thuringiensis ser. jordanica (serotype H71), a novel serovariety isolated in Jordan. Curr Microbiol. 47:26-31. Krieg A., Huger A.M., Langenbruch G.A., Schnetter W. 1983. Bacillus thuringiensis var. tenebrionis, a new pathotype effective against larvae of coleopteran. Z. Angew. Entomol. 96: 500-508. Latchininsky A. V., Launois-Loung M. H. 1992. “Le Criquet Maroccain, Dociostaurus maroccanus (Thunberg, 1815) dans la Partie Orientale de son Aire de Distribution. Etude Monographique Relative a l’exURSS et aux Pays Proches.” CIRAD-GERDAT PRIFAS: Montpellier/VIZR:Saint-Petersbourg. Lecadet M. M., Frachn E., Casmao V., Ripouteau H., Hamon S., Laurent P. Thiery I. 1999. Updating the H-antigen Classification of Bacillus thuringiensis. J. Appl. Microbiol., 86: 660-672. Lindow S.E., Andersen G.L. 1996. Influence of inmigration on epiphytic bacterial populations on navel orange leaves. Appl. Environ. Microbiol. 62: 2978-2987. Lonc E, Lachowicz TM. 1987. Insecticidal activity of Bacillus thuringiensis subspecies against Menopon gallinae (Mallophaga: Menoponidae). Angew Parasitol. 28:173-176. Lonc E., Lecadet M.M., Lachowicz T.M., Panek M. 1997. Description of Bacillus thuringiensis wratislaviensis (H-47), a new serotype originating from Wroclaw (Poland) and other B. thuringiensis soil isolates from the same area. Lett. Appl. Microbiol. 24: 467-473. Lonc E, Mazurkiewicz M, Szewczuk V. 1986. Susceptibility of poultry biting lice (Mallophaga) to Dipel and Bacilan (Bacillus thuringiensis). Angew Parasitol. 27:35-37. López S., Hernández J. 2005. Estandarización de LSSP-PCR para la identificación de nuevos genes cry1 en aislamientos nativos de Bacillus thuringiensis. Tesis de Maestría. Pontificia Universidad Javeriana, Bogotá-Colombia López-Pazos S. A. Cerón J. 2007. Three-dimensional structure of Bacillus thuringiensis toxins: a review. Acta Biol. Colomb. 12: 19-32. López-Pazos S.A., Martínez J.W., Castillo A.X., Cerón Salamanca J.A. 2009. Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae). Rev. Biol. Trop. 57:1235-1243. Maduell P., Callejas R., Cabrera K.R., Armengol G., Orduz S. 2002. Distribution and characterization of Bacillus thuringiensis from marine sediments of Japan. Curr. Microbiol. 40: 418-422. Meadows J., Gill S.S., Bone L.W. 1989. Factors influencing lethality of Bacillus thuringiensis kurstaki toxin for eggs and larvae of Trichostrongylus colubriformes (Nematoda). J. Parasitol. 75:191-194. Menon A.S., De Mestral J. 1985. Survival of Bacillus thuringiensis var. kurstaki. Water, Air Soil Pollut. 25:265 274. Nakamura L.K. 1994. DNA relatedness among Bacillus thuringiensis serovars. International Journal of Systematic Bacteriology. 44: 125-129.

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