**5.** *Bacillus thuringiensis* **development on rice crops**

Genetically engineered or transgenic crops producing Cry proteins from *Bacillus thuringiensis* are key management tools against several important insect pests. GE plants expressing Bt insecticidal proteins selectively target insect pests while having little impact on beneficial insects. Bt toxins have been widely adopted worldwide; it was calculated that over 100 million hectares of crops contained Bt genes by 2017 [56].

Bt crops produce either a single toxin or more than one Bt toxin; these are called pyramided crops. Bt pyramided crops delay evolution of resistance to target pests, insects resistant to one toxin are killed by other toxins in the pyramid [57, 58]. Nevertheless, pyramided Bt crops are vulnerable to the development of cross-resistance. The use of Bt pyramids and the simultaneous planting of non-Bt crops are the main strategies applied to produce susceptible pest insects (known as the "refuge strategy") [59].

Rice is a primary food source for more than half of the world's population making it one of the most fundamental crops. Since 1989 multiple insect-resistant genetically engineered (IRGE) rice lines expressing *Bacillus thuringiensis* insecticidal proteins had been developed [60], controlling lepidopteran pests. There are four major lepidopteran pest rice such as the rice stem borers *Chilo suppressalis* (Walker) (Lepidoptera: Crambidae), *Scirpophaga incertulas* (Walker) (Lepidoptera: Crambidae), *Cnaphalocrocis medinalis* (family Crambidae), and *Sesamia inferens* (Walker) (Lepidoptera: Noctuidae) [61].

Bt rice lines resistant to rice lepidopteran pests mainly express Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1Ca1, Cry2A, and Cry9C proteins [61–63].

Since Cry1Ab was first introduced into a japonica rice variety, many Bt genes have been found, and only a few of them were selected for developing transgenic crops [60]. Because deploying two or more Bt genes in one rice variety can delay the emergence of pest resistance [64, 74], scientists started to develop Bt hybrid rice lines with Cry1Ab/Cry1Ac into various rice plants which have both high grain yield and good grain quality [65].

Some advantages of expressing fusion proteins like Cry1Ab/Cry1Ac and Cry1Ab/Vip3A are the equalization of the expression level of the two proteins, trait integration in different crops, and highly efficient expression strains [66]. Studies on Cry1Ab/Cry1Ac fusion protein have demonstrated great effectiveness significantly reducing the incidence of *Chilo suppressalis* [67, 72].

Other *B. thuringiensis* proteins that present high affinity are Cry9Aa and Vip3Aa. These two proteins bind specifically to brush border membrane vesicles of the Asiatic rice borer *Chilo suppressalis*, which do not share binding sites [68]. Cry9Aa and Vip3Aa toxins have shown potent toxic synergy based on a specific interaction between them against *C. suppressalis* larvae with a synergism factor (SF) value of 10.6-fold [68].

The rice water weevil (*Lissorhoptrus oryzophilus* Kuschel) is another of the most destructive insect pests of cultivated rice (*Oryza sativa*) in the United States [69, 70]. This pest causes low yields in rice by damaging the roots from larval feeding in the submerged root zone [71].

Some of the strategies to control this insect pest are the use of pyrethroids, which are toxic to aquatic organisms [72], synthetic insecticides, and weed control around fields to reduce habitat for rice water weevil adults. *Bacillus thuringiensis* spp. *galleriae* (Btg) have proven to be an environmentally friendly alternative against rice water weevil larvae. Studies indicate that Btg granular formulation has biological activity against this target pest and performs as well as the pyrethroids insecticides [73], showing promising potential for rice water weevil control.

## **6. Resistance to** *Bacillus thuringiensis*

*Bt* insecticides consist of several types of insecticidal crystal proteins; hence, the development of insecticidal pesticide resistance is difficult or slow [47]. However, resistance has already been observed in laboratory, and the first case was a population of Indian meal moths, *Plodia interpunctella*, in 1985, and since then different insect species have been reported to be resistant to one or more *Bt* toxins under laboratory conditions. However, the situation in the field remains very different. To date, the only natural populations that have really developed resistance following *Bt*-based treatments have been populations of diamondback moth, *Plutella xylostella* [27, 74].

The use of transgenic plants has greatly increased the selection pressure on target pest populations and is likely to become much more acute in natural conditions if *Bt* use in agriculture and for human health applications spreads or in cases of the nonrational use of large-scale transgenic crops expressing *cry* genes [32].

In agriculture worldwide, repeated applications of Bt sprays and widespread adoption of Bt crops (transgenic crops protected from insects by the expression of *Cry* and/or *Vip3* genes) have led to resistance [75, 76].

Field populations of *Diabrotica virgifera* have shown resistance to eCry3.1Ab maize and cross-resistance among Cry3Bb1, mCry3A, and eCry3.1Ab, which are the Bt toxins most commercialized for management of western corn rootworm [77].

Resistance to Cry toxins can be developed by mutations in the insect pests that affect any of the steps of the mode of action of Cry toxins [78]. "Field populations" refers to insects on the field, since the conditions are distinct in vitro, can be developed by different mechanisms, such as altered activation of Cry toxins by midgut proteases sequestering the toxin by glycolipid moieties or esterases, by inducing an elevated immune response, and by alteration resulting in reduced binding to insect gut membrane; among all these mechanisms of resistance, the most common mechanism of toxin resistance is the reduction in toxin binding to midgut cells, which in different resistant insect species include mutations in Cry toxin receptors such as cadherin (CAD)-like proteins, alkaline phosphatase (ALP), or aminopeptidase N (APN) or mutations in the ABCC2 transporter [78].

The emergence of resistant insects is a problem that both *Bt* sprays and plant products are likely to face in the future [32]. Several strategies, such as the use of spatial or temporal refugia, high or ultrahigh doses, and gene pyramiding to express two toxins, or two insect control approaches, such that the possibility of evolution of resistance to two toxins/approaches, independent of each other, is greatly diminished, can be a promising approach to prolong the efficacy of products based on *Bt* [36, 46].

There are different methods to counteract the resistance of insects to Bt toxins, for example, assisted mutation with UV light; the combination of Bt toxins with other toxins, such as *Bacillus sphaericus* proteins; and formulations with plant extracts.

Nevertheless, a new method has been used to combat resistance to Bt toxins, the phage-assisted continuous evolution (PACE), which rapidly evolves Bt toxins to bind a new receptor with high affinity and specificity, expressed on the surface of insect midgut cells. The PACE system enhances the insecticidal activity against both sensitive and Bt-resistant insect larvae up to 335-fold, through more than 500

**193**

*Toxic Potential of Bacillus thuringiensis: An Overview DOI: http://dx.doi.org/10.5772/intechopen.85756*

**7. Formulations based on** *Bacillus thuringiensis*

insects, without harming humans or the environment [80].

There are several types of formulations, among the most used are:

protectors, and adhesive materials to improve adsorption.

fertilizers, and residues of ground plants) [81].

• Granular particles are larger and heavier than powder formulations.

• Particle size coarse of 100–1000 μm for granules and 100–600 μm for

• Once applied, the granules slowly release their active ingredient.

• Made of mineral materials (kaolin, attapulgite, silica, starch, polymers, dry

• Concentration of the active ingredient (organisms) in granules ranges from 5 to 20%.

• Some granules require soil moisture to release their active ingredient [3, 82].

with other microorganisms [79].

**Powder (DP)**

**Granules (GR)**

microgranules.

powder (talc, clay, etc.).

• Particle size of 50–100 μm.

generations of mutation, selection, and replication to bind a new receptor [23]. Collectively, these methods establish an approach to overcoming Bt toxin resistance.

The production of toxic proteins has given *Bacillus thuringiensis* enormous interest in its inclusion in phytosanitary formulations. The efficiency of products based on *Bt* depends on the type of formulation, as well as various environmental factors. Formulation depends on the persistence of toxicity and the choice of application method; other important factors are UV radiation, agitation, sedimentation, water quality, contaminants, pH, temperature, susceptibility of insects, and competition

The wide variety of formulations based on spores and crystals intended for being ingested by the white insect are the result of many years of research. The development of a large variety of spore-crystal complex matrices allows for improvements, such as increased toxic activity, increased palatability to insects, or longer storage times. These matrices use chemical, vegetable, or animal products, which are constituted in such a way that they favor contact between crystals and

Proper formulation can help to overcome several of the factors that limit or reduce its larvicidal activity and improve control performance by enabling greater contact with target larvae, ensuring stability under storage and field conditions, providing a variety of application options, and increasing the ease of handling.

• Formulated by sorption of an active ingredient on finely ground mineral

• Powders can be applied directly to the target, either mechanically or manually.

• Concentration of the active ingredient (organism) in the powder is usually 10%.

• The inert ingredients for this formulation are anticaking agents, ultraviolet

*Protecting Rice Grains in the Post-Genomic Era*

**6. Resistance to** *Bacillus thuringiensis*

around fields to reduce habitat for rice water weevil adults. *Bacillus thuringiensis* spp. *galleriae* (Btg) have proven to be an environmentally friendly alternative against rice water weevil larvae. Studies indicate that Btg granular formulation has biological activity against this target pest and performs as well as the pyrethroids insecticides [73], showing promising potential for rice water weevil control.

*Bt* insecticides consist of several types of insecticidal crystal proteins; hence, the development of insecticidal pesticide resistance is difficult or slow [47]. However, resistance has already been observed in laboratory, and the first case was a population of Indian meal moths, *Plodia interpunctella*, in 1985, and since then different insect species have been reported to be resistant to one or more *Bt* toxins under laboratory conditions. However, the situation in the field remains very different. To date, the only natural populations that have really developed resistance following *Bt*-based treatments have been populations of diamondback moth, *Plutella xylostella* [27, 74]. The use of transgenic plants has greatly increased the selection pressure on target pest populations and is likely to become much more acute in natural conditions if *Bt* use in agriculture and for human health applications spreads or in cases of the

nonrational use of large-scale transgenic crops expressing *cry* genes [32].

*Cry* and/or *Vip3* genes) have led to resistance [75, 76].

dase N (APN) or mutations in the ABCC2 transporter [78].

In agriculture worldwide, repeated applications of Bt sprays and widespread adoption of Bt crops (transgenic crops protected from insects by the expression of

Field populations of *Diabrotica virgifera* have shown resistance to eCry3.1Ab maize and cross-resistance among Cry3Bb1, mCry3A, and eCry3.1Ab, which are the Bt toxins most commercialized for management of western corn rootworm [77]. Resistance to Cry toxins can be developed by mutations in the insect pests that affect any of the steps of the mode of action of Cry toxins [78]. "Field populations" refers to insects on the field, since the conditions are distinct in vitro, can be developed by different mechanisms, such as altered activation of Cry toxins by midgut proteases sequestering the toxin by glycolipid moieties or esterases, by inducing an elevated immune response, and by alteration resulting in reduced binding to insect gut membrane; among all these mechanisms of resistance, the most common mechanism of toxin resistance is the reduction in toxin binding to midgut cells, which in different resistant insect species include mutations in Cry toxin receptors such as cadherin (CAD)-like proteins, alkaline phosphatase (ALP), or aminopepti-

The emergence of resistant insects is a problem that both *Bt* sprays and plant products are likely to face in the future [32]. Several strategies, such as the use of spatial or temporal refugia, high or ultrahigh doses, and gene pyramiding to express two toxins, or two insect control approaches, such that the possibility of evolution of resistance to two toxins/approaches, independent of each other, is greatly diminished, can be a promising approach to prolong the efficacy of products

There are different methods to counteract the resistance of insects to Bt toxins, for example, assisted mutation with UV light; the combination of Bt toxins with other toxins, such as *Bacillus sphaericus* proteins; and formulations with plant extracts. Nevertheless, a new method has been used to combat resistance to Bt toxins, the phage-assisted continuous evolution (PACE), which rapidly evolves Bt toxins to bind a new receptor with high affinity and specificity, expressed on the surface of insect midgut cells. The PACE system enhances the insecticidal activity against both sensitive and Bt-resistant insect larvae up to 335-fold, through more than 500

**192**

based on *Bt* [36, 46].

generations of mutation, selection, and replication to bind a new receptor [23]. Collectively, these methods establish an approach to overcoming Bt toxin resistance.
