**3. Howard T. Dulmage's methods and contributions on** *Bt*

Howard T. Dulmage was a microbiologist who established his line research in the study of pathogenic bacteria insects [33] and is considered one of the most important pioneers in the development of technologies for the implementation of *Bt* as a control agent of biological pests [34].

Working at the US Department of Agriculture, at the Agricultural Research Service (USDA-ARS), in Brownsville, Texas, Howard T. Dulmage from the pink worm, *Pectinophora gossypiellia*, a strain of *Bacillus thuringiensis* variety kurstaki, in 1969, which is 200 times more active in laboratory tests against the pink bollworm, *Pectinophora gossypiella*; the tobacco budworm, *Heliothis virescens*; and the cabbage looper, *Trichoplusia ni* [21], higher than that of the known strains, which is marketed as "Dipel" by the company United States of America [35–37].

Strain HD-1 is one of the best-studied strains, since it is characterized by the carrying of a variety of Cry anti-Lepidoptera genes, *Cry1Aa*, *Cry1Ab*, *Cry1Ac*, *Cry2Aa*, *Cry2Ab*, and *Cry1Ia*, and since its discovery, the outlook for *Bt*-based products has expanded and is still the most commercial success of microbial control of pest [4, 38].

H. Dulmage sets up the basis for the fermentation and formulation procedures of Bt culture extracts for their commercialization [39] and were among the most important pioneers in the development of technologies for the implementation of Bt as a biological pest control agent. He established diverse methodologies for mass production product formulation and power standardization [40].

At the beginning of the 1970s, two great advances were obtained by Dulmage, the first was based on the recovery of the spore-crystal complex by means of precipitation with lactose-acetone to produce powders and wettables, which was rapidly developed and adapted in the industry. The second was the adoption of a standardized system to calibrate the potency of the different preparations of *Bt* and the establishment of international toxicity units (ITU)/mg, which allowed the comparison of the different products developed [41, 42]. The equation proposed by Dulmage [43] is the following:

[Image [43]] is the following:

$$\text{Test extract percentage (ITU/mg)} = \frac{\text{standard LC}\_{50} [\text{standard potency (ITU/mg)}]}{\text{test extract LC}\_{50}} \tag{1}$$

Dulmage established better bioassay methods to assess the effectiveness of powders [37, 44].

In 1984, Dulmage participated in the establishment of a bioassay protocol for toxicity assessment of *Bacillus thuringiensis* var. israelenensis powders. This protocol differs from the one previously suggested by WHO in the Guidelines for Bti Production regarding the follow aspects:

**189**

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

concentrations assayed.

different times of storage [38].

**3.2 Bti strain collection**

required.

• Specifies a standard cup for larval exposure to Bti extracts.

ence standard strains and for the establishment of new ones.

be involved in studies on Bti as dipteran biocontrol agent.

World Health Organization, and was published in 1983 [45].

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

• Establishes a number of 20 larvae per cup and three replications for the

• If a minimum of six extract concentrations is tested, a repetition of the assay is

• A computational probit analysis is required for evaluating the toxicity as LC50.

• A mortality or pupation higher than 5% in the control invalidates the bioassay.

Additionally, the study suggested a variability coefficient of less than 20% for each repetition. Dulmage, together with a team of colleagues, tested the validity of this protocol and suggested some considerations for the management of the refer-

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

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

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

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 *Protecting Rice Grains in the Post-Genomic Era*

control agent of biological pests [34].

America [35–37].

control of pest [4, 38].

Dulmage [43] is the following:

Production regarding the follow aspects:

powders [37, 44].

stimulating the process of apoptosis (**Figure 3**) [1, 3, 30].

its expression in genetically modified plants [31, 32].

**3. Howard T. Dulmage's methods and contributions on** *Bt*

cabbage looper, *Trichoplusia ni* [21], higher than that of the

production product formulation and power standardization [40].

opening of these channels causes an abnormal movement of the ions in the cytosol,

The germination of the spores also contributes to the death of insect, since the vegetative cells can replicate within the host's hemolymph and cause septicemia; however, the δ-endotoxins alone are sufficient to kill some insect species if they are produced in high doses. This feature has been exploited by expressing the delta endotoxin genes in bacteria that better adapt to a particular environment, as well as

Howard T. Dulmage was a microbiologist who established his line research in the study of pathogenic bacteria insects [33] and is considered one of the most important pioneers in the development of technologies for the implementation of *Bt* as a

Working at the US Department of Agriculture, at the Agricultural Research Service (USDA-ARS), in Brownsville, Texas, Howard T. Dulmage from the pink worm, *Pectinophora gossypiellia*, a strain of *Bacillus thuringiensis* variety kurstaki, in 1969, which is 200 times more active in laboratory tests against the pink bollworm, *Pectinophora gossypiella*; the tobacco budworm, *Heliothis virescens*; and the

known strains, which is marketed as "Dipel" by the company United States of

Strain HD-1 is one of the best-studied strains, since it is characterized by the carrying of a variety of Cry anti-Lepidoptera genes, *Cry1Aa*, *Cry1Ab*, *Cry1Ac*, *Cry2Aa*, *Cry2Ab*, and *Cry1Ia*, and since its discovery, the outlook for *Bt*-based products has expanded and is still the most commercial success of microbial

H. Dulmage sets up the basis for the fermentation and formulation procedures of Bt culture extracts for their commercialization [39] and were among the most important pioneers in the development of technologies for the implementation of Bt as a biological pest control agent. He established diverse methodologies for mass

At the beginning of the 1970s, two great advances were obtained by Dulmage,

the first was based on the recovery of the spore-crystal complex by means of precipitation with lactose-acetone to produce powders and wettables, which was rapidly developed and adapted in the industry. The second was the adoption of a standardized system to calibrate the potency of the different preparations of *Bt* and the establishment of international toxicity units (ITU)/mg, which allowed the comparison of the different products developed [41, 42]. The equation proposed by

Test extract potency (ITU/mg) <sup>=</sup> standard LC50[standard potency (ITU/mg)] \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ test extract LC50

Dulmage established better bioassay methods to assess the effectiveness of

In 1984, Dulmage participated in the establishment of a bioassay protocol for toxicity assessment of *Bacillus thuringiensis* var. israelenensis powders. This protocol differs from the one previously suggested by WHO in the Guidelines for Bti

(1)

**188**


Additionally, the study suggested a variability coefficient of less than 20% for each repetition. Dulmage, together with a team of colleagues, tested the validity of this protocol and suggested some considerations for the management of the reference standard strains and for the establishment of new ones.
