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United States Department of Agriculture

Agricultural Research Service

Research Project: Production, Stabilization and Formulation of Microbial Agents and Their Natural Products

Location: Crop Bioprotection Research

2011 Annual Report

1a.Objectives (from AD-416)
Objective 1: Develop liquid culture methods for producing fungal biocontrol agents by optimizing the nutritional and environmental conditions during growth for the production of an appropriate fungal propagule with optimal efficacy and storage stability. Initially, research focus will include the fungi, Metarhizium anisopliae and Mycoleptodiscus terrestris important in agricultural, urban and natural ecosystems.

Objective 2: Develop novel formulation technologies for microbial biocontrol agents through the selection and application of innovative processes and ingredients that lead to improved storage stability, product delivery, field stability, and efficacy. Initially, focus will be on the fungi Isaria fumosorosea, Metarhizium anisopliae, and Mycoleptodiscus terrestris important in agricultural, urban, and natural ecosystems.

1b.Approach (from AD-416)
Our approach to the development of production methods for fungal biocontrol agents will focus on the use of deep-tank, liquid fermentation techniques. Propagule form, yield, storage stability, and biocontrol efficacy are critical “fitness” factors that must be considered during medium optimization since all are required if the biocontrol agent is to become a commercial product. Assays will be performed in our laboratory to evaluate propagule “fitness” include microscopic evaluation for propagule form, measurement of the rate of biomass accumulation and propagule formation, propagule survival after drying, and propagule viability following formulation and storage. Insect or weed biocontrol assays of our fermentation products will be performed in collaboration with plant or insect pathologists. Nutritional environments that promote the rapid development (short fermentation times) of stable fungal propagules with adequate shelf-life, and consistent biocontrol efficacy will be optimized by identifying critical nutritional components in the composition of the liquid fermentation medium. In addition to nutritional parameters, environmental conditions during culture growth will be evaluated and optimized. Production and stabilization processes for promising fungal biocontrol agents will be scaled from shake-flask studies to fermentation systems as large as 100 liters depending on field trial requirements and on commercial interest. Formulation-based solutions to critical problems related to biocontrol agent stability, efficacy, and application will be addressed using a multifaceted research approach. We will evaluate the impact of culture harvest techniques, stabilization processes, and formulation ingredients on the physical characteristics, biological activity, storage stability, and field efficacy of selected biocontrol agents. Appropriate support matrices and drying processes will be evaluated for the stabilization of the microbial control agent. Support matrices to be evaluated during drying will include various diatomaceous earths, clays, and vermiculites. Processing equipment and conditions for sizing, mixing, drying, encapsulating, and granulating microbial biopesticide formulations will be tested with the ultimate goal of producing a microbial biocontrol agent in a form suitable for use against the target pest. Selective nutrients or amendments as formulations or adjuvants will be evaluated to improve biocontrol agent performance.

3.Progress Report
This report documents progress for project 3620-22410-014-00D which started October 2010 and continues research from project 3620-22410-010-00D Production, Stabilization, and Formulation of Microbial Biocontrol Agents and Natural Products. Substantial progress was made on both Objectives 1 and 2. Under Objective 1, progress was made on optimizing liquid culture production systems for the fungal bioinsecticides Metarhizium anisopliae (Ma) and Isaria fumosorosea (Ifr) and the bioherbicide Mycoleptodiscus terrestris (Mt). Under Objective 2, significant progress was made in developing stabilization and formulation processes for microbial biocontrol agents such as Ma and Mt and other pest control compounds. Nutritional and environmental conditions were optimized during Ma culture growth to reduce fermentation times and increase yields of stable Ma propagules (microsclerotia). The stability and conidia production potential of Ma microsclerotia were evaluated following testing using various formulation, granulation, drying, and storage conditions. Promising formulations of Ma microsclerotia granules showed excellent shelf-life; and when rewetted, produced spores that infected and killed white grubs, mealworms, and sugar beet root maggots. These granular formulations of Ma microsclerotia have been tested for control of various insect pests in collaboration with ARS, university, and industry scientists. Experiments were conducted to reduce the cost of producing Ifr spores for use in spray applications for insect control. Low-cost nitrogen sources (soy flour and cottonseed meal) were shown to reduce production medium costs by 80%, and maintaining high oxygen concentrations in the liquid production medium significantly increased spore yields and reduced unwanted filamentous growth. Spores of Ifr were tested in collaboration with ARS and university scientists for control of subterranean termites, cucumber beetles, citrus psyllids, and other beetle pests. Studies were also conducted to evaluate the UV stability of Ifr spores under a variety conditions. Temperature optima were identified for the production and use of Mt in controlling the aquatic weed hydrilla. Formulations of Mt only and Mt with low-dose chemical herbicide combinations were tested in collaboration with the U.S. Army Corps of Engineers and an industrial partner for control of hydrilla. Additional formulation studies were conducted in collaboration with ARS scientists at the University of Nebraska to develop a second generation controlled release formulation of catnip oil to repel stable flies from cattle feedlots. In addition, formulations of short-chain fatty acids were developed as repellents of biting flies. In collaboration with university scientists, various natural formulations of nootkatone, an essential oil from grapefruit, were tested with the goal of reducing volatility and damage to plants by the oil. Spray-dried lignin formulations of nootkatone were developed that reduced volatility and provided safe, effective tick control with minimal plant damage.

1. Effective granular formulation of a fungal bioinsecticide. The fungus Metarhizium anisopliae (Ma) infects and kills many important soil-dwelling insect pests including meal worms, root weevils, soil grubs, rootworms, wireworms, fruit flies, and root maggots. Spores of Ma are the infectious form of the fungus but their application into soil has proven to be difficult. USDA, ARS scientists in the Crop Bioprotection Research Unit at the National Center for Agricultural Utilization Research, Peoria, IL, have discovered a very stable form of Ma, a microsclerotium, that can be produced using liquid culture fermentation and stabilized as a dry granular formulations. Dry granular formulations of Ma can be easily applied to the soil; and when rehydrated, they grow and produce infective spores of Ma in the soil environment. Successful production of this stable form of Ma by liquid fermentation has provided the active ingredient for developing granule formulations to target insect pests in soil. A prototype granule formulation containing this stable form of Ma (without nutrients) was applied to potting soil and showed insecticidal activity against the lesser meal worm. This research resulted in collaborative work with a university to evaluate granule formulations of Ma as a biopesticide for control of soil dwelling beetles that damage melons and in license applications by companies interested in the patent-pending technology. These studies have demonstrated the potential for the commercial production and formulation of dried granules of Ma for use by homeowners, farmers, and land managers in controlling soil-dwelling insect pests.

2. Improved fermentation process for biological insecticide. For many serious insect pests, chemical pesticides are not a control option. Some insects have developed resistance to chemical insecticides while others are problems in environments where chemicals are not recommended such as urban and aquatic environments or in organic farming. Isaria fumosorosea (Ifr) is a naturally-occurring fungus that produces spores that can infect and kill soft-bodied insects such as whiteflies, aphids, and subterranean termites. Liquid fermentation processes for the production of spores of Ifr has been known for many years, but high production costs remain a major constraint to the use of Ifr as an insect control agent. USDA, ARS scientists in the Crop Bioprotection Research Unit at the National Center for Agricultural Utilization Research Center, Peoria, IL, have developed a lower-cost production medium for spores of Ifr and identified environmental conditions during fermentation that promote the production of spores rather than the filamentous form of the fungus. These improvements resulted in an 80% reduction in the cost of the production nutrients and a significant reduction in product processing requirements. The improved production process results in yields of over one trillion spores per liter of fermentation broth after a short fermentation time of 40 hours. Discussions are under way with an industrial partner on the commercialization of this and other Agricultural Research Service technology related to the production and use of Ifr for insect control. This lower-cost, Ifr production process has potential to expand the use of this non-chemical insect control agent by farmers, greenhouse operators, and homeowners.

Review Publications
Dunlap, C.A., Jackson, M.A., Saha, B.C. 2010. Compatible solutes of sclerotia of Mycoleptodiscus terrestris under different culture and drying conditions. Biocontrol Science and Technology. 21(1):113-123.

Behle, R.W., Compton, D.L., Kenar, J.A., Shapiro Ilan, D.I. 2011. Improving formulations for biopesticides: Enhanced ultraviolet protection for beneficial microbes. Journal of ASTM International. 8(1):137-157.

Jackson, M.A., Dunlap, C.A., Sherer, J.F., Heilman, M.A., Palmquist, D.E. 2011. The impact of temperature on the production and fitness of microscleotia of the fungal bioherbicide Mycoleptodiscus terrestris. Biocontrol Science and Technology. 21(5):547-562. DOI: 10.1080/09583157.2011.564728

Weiler, L.F., Behle, R.W., Stafford, K. 2011. Susceptibility of four tick species Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus (Acari: Ixodidae) to nootkatone. Journal of Medical Entomology. 48(2):322-326.

Zhu, J.J., Dunlap, C.A., Behle, R.W., Berkebile, D.R., Wienhold, B.J. 2010. Repellency of a wax-based catnip-oil formulation against stable flies. Journal of Agricultural and Food Chemistry. 58:12320-12326. DOI: 10.1021/jf102811k.

Last Modified: 4/19/2014
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