Starch Encapsulation of Microbial Pesticides - American Chemical

Chapter 17

Starch Encapsulation of Microbial Pesticides M. R. McGuire and B. S. Shasha

Downloaded by EAST CAROLINA UNIV on September 30, 2015 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0595.ch017

Plant Polymer Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604

Microbial insecticides, when formulated within starch or flour matrices are more efficacious and have longer residual activity than commercial formulations. Three starch formulations have been developed: a sprayable formulation and two granular baits. The sprayable formulation is composed of a premixed combination of sucrose and commercially available pregelatinized cornstarch or pregelatinized corn flour that can be tank-mixed at solids rates of 2-6%. Bioassays of cotton or cabbage leaf tissue treated with the sprayable formulations demonstrated increased residual activity of Bacillus thuringiensis (Bt) after simulated (greenhouse) or actual (field) rainfall. The two types of granular formulations are: (1) a conventional granule which remains discrete through wet and dry periods, and (2) an adherent granule which, upon contact with water, will partially swell and remain stuck to leaf tissue after drying. Field and laboratory tests have demonstrated that Bt will remain active longer when encapsulated in starch or flour under rainy conditions than non-encapsulated Bt. This technology has also been used for a wide variety of microbial pesticides including bacteria, viruses, fungi, protozoa, and nematodes.

Due to increased perceptions of the hazards associated with chemical pesticides, there is a need to develop alternative non-chemical pest control tools. The recent establishment of guidelines aimed at reducing pesticide application and increasing the use of integrated pest management techniques further fuels this need. One option is to use microbial insecticides typified by the bacterium, Bacillus thuringiensis (Bt). Bt is very effective at controlling certain insect species. Strains have been comercially available for use against insects in the orders Lepidoptera, Coleoptera, and Diptera. At this time many additional strains are under commercial development to control a wider variety of insects. Despite this success, the use of Bt has encountered certain problems with acceptance by growers and has lagged behind market expectations over This chapter not subject to U.S. copyright Published 1995 American Chemical Society

In Biorational Pest Control Agents; Hall, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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the past few years. Acceptance has been hindered, in part, by the short residual activity of Bt and by reduced efficacy of some of the commercial formulations available to the grower. Residual activity is affected by several environmental factors including degradation by sunlight (7) and washoff by rainfall (2). In addition, some insects find Bt unpalatable and may not consume a lethal dose of the active agent (5). The purpose of this paper is to summarize our work with novel Bt formulations and to asses the feasibility of providing new formulations for Bt and other microbial insecticides. During the last several years, we have examined formulating ingredients that may increase residual activity or enhance feeding on Bt laced substrates. Starch and flour derived from corn can be modified into film-forming materials that will entrap or encapsulate agents. These materials are inexpensive, in surplus and milling companies are eager to work towards creating new markets for their products. Besides Bt, other microbial control agents have been formulated using similar techniques. Preparation of Soluble Starch and Flour When dry starch granules are exposed to water at 0-40° C, they undergo limited, reversible swelling. Excess water and high temperature (80-100° C) causes hydration accompanied by irreversible swelling, a process known as gelatinization. Upon cooling and especially at high starch concentration, a three-dimensional gel network results, a process known as retrogradation. During this process, the reassociated molecules become insoluble in water. A water-soluble starch powder, called pregelatinized starch, can be obtained i f the water is removed before the retrogradation occurs. The pregelatinized starch, when added in high concentrations to water, will entrap other agents present in the water upon retrogradation. Because the resulting product is insoluble, the matrix holds the active agent inside and prevents leaching. Desirable characteristics of the active agent can be enhanced by the addition of sunscreens or other additives. If flour is substituted for the starch, a similar reaction occurs, except the protein content of the flour does not become soluble upon heating. The benefits of flour over starch include lower cost and the protein may act as a feeding stimulant (3) and/or a sunlight screen. Granular Formulations In a process described by Dunkle and Shasha (4), a ratio of 1 part pregelatinized starch was added to 2 parts water containing Bt and corn oil to form a single large mass. After several hours at 4° C, the mass was chopped in a Waring blendor with the addition of pearl (unmodified) cornstarch to obtain granules of a desired size. We have since learned that equal amounts of starch and water can be used to form the initial mass. Besides lowering the overall water content, this ratio hastens retrogradation and the resulting mass can be chopped into granules without the addition of pearl starch. Granules made with this process have been extensively studied as formulations for control of the European corn borer, Ostrinia nubilalis Hiibner, in field corn. Preliminary tests demonstrated that feeding stimulants could be added to the formulation that would enhance the acceptance of granules containing



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Bt to corn borer larvae (5). These authors also demonstrated the utility of using relatively inert starch granules to test the usefulness of additives that may enhance the efficacy of the formulation. In essence, Bartelt et al. (5), showed that starch by itself was not readily accepted by corn borer larvae. However, i f a combination of feeding stimulants, composed of a lipid, a protein and a sugar is added, larvae would feed on the granules in preference to plant tissue. Wheat germ, for example, contains all three ingredients. If one or more of the components of wheat germ was omitted, larvae did not respond as well to the granule. Coax (CCT Corporation, Litchfield Park, A Z ) , a commercial product, was also highly preferred by corn borer larvae in these tests. In greenhouse tests, using corn as a test plant and corn borer larvae as the test insect, Bartelt et al. (5) demonstrated that i f Coax was present in the granule, Bt content could be reduced by 75% without loss of insecticidal activity. Subsequent work reported by McGuire et al. (6) documented a similar response by corn borer larvae under field conditions (Table I and Field Evaluation, below). However, these field

Table I. Efficacy of Bacillus thuringiensis in granular formulations for control of Ostrinia nubilalis in whorl-stage field corn

Formulation

Dose

Starch Starch Starch + Congo Red Starch + Congo Red Starch + 1% Coax Starch + 1% Coax Starch + 10% Coax Starch + 10% Coax Flour + CaCl Flour + CaCl Flour + CaCl + 10% Coax Flour + CaCl + 10% Coax Flour + Molasses Dipel 10G Furadan 15G

400 1600 400 1600 400 1600 400 1600 400 1600 400 1600 1600 1600

2

2

2

2

a

Avg. % Control

15

Sites Tested

75 90 77 88 85 89 88 97 76 63 80 66 75 77 57

2 2 2 2 2 2 2 2 3 4 3 4 1 7 1

a

International Units of Bacillus thuringiensis per mg formulation. A l l formulations applied at 11.2 kg/ha. Tunneling in treated plants / tunneling in untreated control plants x 100.

b

tests were strictly efficacy tests and no measure of residual activity was taken. To determine residual activity of starch formulations, Bt preparations were exposed to direct sunlight (7). Starch alone was not sufficient to protect activity of the Bt but



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the addition of sunscreens could effectively maintain the potency over a 12 day period (7). To measure residual activity under more realistic conditions, granules were placed in the whorl of corn plants in the field and then collected over time. Assays were done in the laboratory to measure relative activity of the granules and results were reported as percent original activity remaining (8). From this study, it was apparent that sunlight alone was not an important factor in degradation of Bt activity in the whorl of a corn plant. Sunlight intensity, measured with a LiCor Spectroradiometer (LiCor, Inc. Lincoln, NE), demonstrated the lack of ultraviolet light in the whorl or within a leaf axil of a corn plant. These sites are where granules come to rest after application and where corn borer larvae feed. Instead, rainfall was the predominant mechanism that acted to remove Bt from commercial granules and the feeding site and render it ineffective. After 70 mm rain, a commercial granule lost 40% of its insecticidal activity while starch formulations lost 100 mm rainfall, all starch formulations tested maintained significantly higher activity than the commercial formulation (8). While the effectiveness of these granules has been verified, the processing demands to make large quantities of the granules reduces the feasibility of this technology being used extensively. Therefore, we began developing methods to produce the granules with smaller amounts of water. If water simply is added in less than equal amounts to the pregelatinized starch or flour, the result is a mixture of a large mass and a lot of dust; clearly an unacceptable product. However, we found we could reduce the water by mixing it with substances that would not gel the starch or at least delay the water from starting the gelling reaction. This allowed time for complete dispersion of the water throughout the starch and resulted, upon mixing, in discrete granules being formed. One example of a substance that does not gel the starch is alcohol. Therefore, by adding 25 ml of a 30% isopropanol solution to 25 g pregelatinized starch, we have reduced the amount of water by 30% and eliminated the need for grinding and sieving. By altering the percentage of alcohol, granule size can be controlled (9). We have since found that high concentrations of a salt, such as CaCl , or sugar solution, such as molasses, will allow a bigger reduction of water while maintaining adequate granule production (10). Testing of these formulations led to the discovery that granules made with this process would adhere to surfaces i f the surfaces were wet upon application. Following drying, the granules would remain stuck to the surface and resist washoff by simulated rain (9). 2

Field Evaluation. Both types of granules have been extensively tested in field corn over the last several years. These tests were aimed at determining efficacy of Bt against the European corn borer in field corn with different formulations. For these tests, newly hatched European corn borer larvae were applied to the whorl of each plant using the procedures of Guthrie (77). Approximately 7 days later, granules were applied over the row using a high clearance vehicle equipped with metering applicators calibrated to deliver 11.2 kg/ha. Approximately 6 weeks later, after the larvae had pupated, damage was assessed by splitting each corn stalk from base to


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tassel and measuring vertical tunnelling. For a more detailed description of these general procedures see McGuire et al. (6). Results of these tests (Table I) strongly indicated that each of the formulations tested with the exception of some of the granules made with CaCl performed as well as or better than a commercial formulation. A l l starch formulations were made with the first process (i.e., large amounts of water) while the flour granules were made with less water. Granules formulated at the same Bt concentration as a commercial product (1600 IU/mg) provided control equal to the commercial formulation. Granules with 400 IU/mg Bt containing Coax, however, also provided control similar to formulations with more Bt. Because information pertaining to the cost of manufacturing Bt is unavailable, it is unclear i f it is economical to replace part of the Bt with a feeding stimulant such as Coax. However, these data, coupled with the extreme versatility of the granule formation process which allows incorporation of sunlight screens, feeding stimulants, attractants, etc. suggest that this process could have utility for a wide range of approaches to pest control.


2

Sprayable Formulations When pregelatinized starch or flour is added to water in proportions of less than 10% solids (i.e.,

Starch Encapsulation of Microbial Pesticides - American Chemical

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