**3.1 Howard T. Dulmage's fermentation extracts**

From 1970 to 1988, Dulmage established the largest Bt collection in the Americas, and he collected more than 800 isolates that were named using his HD code, belonging to 21 serovarieties. From these 800 isolates, 17 belonged to the H-14 serovariety, corresponding to Bti. He conducted a series of fermentation experiments with Bt in order to optimize the production and to assess the effectiveness of powder; hundreds of fermentation extracts were generated, and some of them were donated by the US Department of Agriculture in 1989 to the International Collection of Entomopathogenic Bacillus of the Faculty of Biological Sciences of the University of Nuevo León, Mexico, which has approximately 4000 stored fermentation extracts of which 3000 of them correspond to HD strains, and currently extracts are found in the form of dry powder, with different times of storage [38].

### **3.2 Bti strain collection**

In the 1970s, Dulmage continued to the control of disease-transmitting mosquito larvae using lepidopteran-active isolates having some reported dipteran activity. When Dulmage became aware of the discovery of a new Bt subspecies capable of attacking dipteran larvae, especially simuliids (*Bacillus thuringiensis* subsp. *israelensis*) (Bti), he quickly perceived the great value of this discovery, because of the possibility to control dangerous human disease vectors, and began to be involved in studies on Bti as dipteran biocontrol agent.

One of the greatest contributions of Dulmage to Bti research was the compilation of a protocol guide for Bt H-14 serovariety local production. This guide was an extension of the procedures developed by him for the production, formulation, and standardization of lepidopteran-specific serovarieties. These guidelines were presented and discussed in the informal consultation on local H-14 Bt production, in Geneva, Switzerland, in October 1982. The 128-page booklet was prepared by Dr. Dulmage, at the request of the Scientific Working Group on biological control of vectors of the Special Program for Research and Training in Tropical Diseases of the World Health Organization, and was published in 1983 [45].

In 1985, Dulmage and a research group proved the tested strain was Bti HD-968-S-1983, which resulted to be 4.74 times more potent than the standard use (IPS-78); the potency assigned to it was 4740 ± 398 ITU/mg. They recommended the use of this strain as the potency reference standard for comparison with any Bti formulation.

Twenty samples of the strain HD-500 and HD-567 of Bti fermentation extracts from the collection of Dulmage et al. [44] recovered by lactose-acetone coprecipitation during the period from 1978 to 1983 maintained their residual toxic activity against the mosquito *Aedes aegypti*. All extracts evaluated presented toxicity at the highest tested doses (1000 ppm), and two of the stored extracts (3260 and 3501) showed LD50 of 0.12 and 1.16 ppm, respectively [40].

Bti protein crystals from fermentation extracts showed persistence of toxic activity of fermentation extracts after more than three decades. This opens the possibility of improving the use of special strains and improved formulations to control insect vectors of diseases.

### **4. New Cry toxins**

Despite the success of the application of Bt crystal proteins for the biological control of pests, at present it is still necessary to identify new Cry toxins with greater toxicity; this approach is considered one of the best ways to counter the potential resistance evolved by insects as well as in developing products against a wider spectrum of insect pests. Traditionally, Bt isolates were screened for their insecticidal spectrum by the time-consuming and laborious insect bioassays [22, 46]. Since only a limited number of cry genes have been used for insect control either in sprays or transgenic crops so far, novel insecticidal genes are required [31].

The most common technique used to predict toxicity is the polymerase chain reaction (PCR), through the identification of new cry genes [47], but high-throughput sequencing technology has also been used in the discovery of toxins [20]. Seventy-two antigenic groups (serovariety) have been distinguished for *Bacillus thuringiensis* [48]. Crickmore et al. [19] have designed an especial database for Bt toxins with links to information on host insects, based on the last update (www. lifesci.sussex.ac.uk/Home/Neil\_Crickmore/Bt/). About 952 toxin genes, encoding different entomopathogenic proteinaceous toxins, have been identified and characterized in the Bt strains isolated all around the world; however, only a small proportion of these proteins are highly toxic and therefore used in the production of bioinsecticides. This can be accomplished by either finding new wild-type strains or engineering Cry proteins with enhanced activity or altered insecticidal spectrum by swapping domains and site-directed mutagenesis; nevertheless a thorough knowledge of Cry protein structure and binding interactions with target receptors is a must [49].

Additionally, the construction of *Bt* DNA libraries in *Escherichia coli*, followed by screening by Western blotting or a hybridization-based method, or the development of DNA libraries in an acrystaliferous mutant of *Bt* followed by microscopic observation and/or SDS-polyacrylamide gel (SDS-PAGE) detection of expressed genes has also been used to detect novel Cry protein genes [44].

Moreover, a combination of genomics, transcriptomics, proteomics, and metabolomics could be used to study *Bt* toxin proteins with different characteristics and activities [21]. However, due to the interaction between different toxins produced by a strain in insect midgut, bioassays provide complementary and necessary characterization information. Due to the money, time, and material costs associated with insect rearing and time-consuming characteristics of insect bioassays, cell-based assays have been employed for toxicity characterization of *Bt* strains or toxins [50].

**191**

10.6-fold [68].

submerged root zone [71].

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

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

microorganisms [55].

Furthermore, recent studies have confirmed more new potentials of different Bt strains. These new features are including plant growth promotion [51], bioremediation of heavy metals and other chemicals [1, 52], anticancer activities [53], polymer production [54], and antagonistic effects against plant and animal pathogenic

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].

Cry1Ac, Cry1B, Cry1C, Cry1Ca1, Cry2A, and Cry9C proteins [61–63].

cantly reducing the incidence of *Chilo suppressalis* [67, 72].

(Walker) (Lepidoptera: Noctuidae) [61].

and good grain quality [65].

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*

Bt rice lines resistant to rice lepidopteran pests mainly express Cry1Aa, Cry1Ab,

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

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 signifi-

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

Other *B. thuringiensis* proteins that present high affinity are Cry9Aa and Vip3Aa.

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

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 *Protecting Rice Grains in the Post-Genomic Era*

showed LD50 of 0.12 and 1.16 ppm, respectively [40].

formulation.

insect vectors of diseases.

**4. New Cry toxins**

required [31].

is a must [49].

(IPS-78); the potency assigned to it was 4740 ± 398 ITU/mg. They recommended the use of this strain as the potency reference standard for comparison with any Bti

Bti protein crystals from fermentation extracts showed persistence of toxic activity of fermentation extracts after more than three decades. This opens the possibility of improving the use of special strains and improved formulations to control

Despite the success of the application of Bt crystal proteins for the biological control of pests, at present it is still necessary to identify new Cry toxins with greater toxicity; this approach is considered one of the best ways to counter the potential resistance evolved by insects as well as in developing products against a wider spectrum of insect pests. Traditionally, Bt isolates were screened for their insecticidal spectrum by the time-consuming and laborious insect bioassays [22, 46]. Since only a limited number of cry genes have been used for insect control either in sprays or transgenic crops so far, novel insecticidal genes are

The most common technique used to predict toxicity is the polymerase chain reaction (PCR), through the identification of new cry genes [47], but high-throughput sequencing technology has also been used in the discovery of toxins [20]. Seventy-two antigenic groups (serovariety) have been distinguished for *Bacillus thuringiensis* [48]. Crickmore et al. [19] have designed an especial database for Bt toxins with links to information on host insects, based on the last update (www. lifesci.sussex.ac.uk/Home/Neil\_Crickmore/Bt/). About 952 toxin genes, encoding different entomopathogenic proteinaceous toxins, have been identified and characterized in the Bt strains isolated all around the world; however, only a small proportion of these proteins are highly toxic and therefore used in the production of bioinsecticides. This can be accomplished by either finding new wild-type strains or engineering Cry proteins with enhanced activity or altered insecticidal spectrum by swapping domains and site-directed mutagenesis; nevertheless a thorough knowledge of Cry protein structure and binding interactions with target receptors

Additionally, the construction of *Bt* DNA libraries in *Escherichia coli*, followed by screening by Western blotting or a hybridization-based method, or the development of DNA libraries in an acrystaliferous mutant of *Bt* followed by microscopic observation and/or SDS-polyacrylamide gel (SDS-PAGE) detection of expressed

Moreover, a combination of genomics, transcriptomics, proteomics, and metabolomics could be used to study *Bt* toxin proteins with different characteristics and activities [21]. However, due to the interaction between different toxins produced by a strain in insect midgut, bioassays provide complementary and necessary characterization information. Due to the money, time, and material costs associated with insect rearing and time-consuming characteristics of insect bioassays, cell-based assays have been employed for toxicity characterization of *Bt* strains or toxins [50].

genes has also been used to detect novel Cry protein genes [44].

Twenty samples of the strain HD-500 and HD-567 of Bti fermentation extracts from the collection of Dulmage et al. [44] recovered by lactose-acetone coprecipitation during the period from 1978 to 1983 maintained their residual toxic activity against the mosquito *Aedes aegypti*. All extracts evaluated presented toxicity at the highest tested doses (1000 ppm), and two of the stored extracts (3260 and 3501)

**190**

Furthermore, recent studies have confirmed more new potentials of different Bt strains. These new features are including plant growth promotion [51], bioremediation of heavy metals and other chemicals [1, 52], anticancer activities [53], polymer production [54], and antagonistic effects against plant and animal pathogenic microorganisms [55].
