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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

2012 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:
Under Objective 1, pilot-scale, 100-liter liquid culture production systems were optimized for two fungal bioinsecticides, Metarhizium brunneum (Met) and Isaria fumosorosea (Ifr), and for the fungal bioherbicide Mycoleptodiscus terrestris (Mt). Under Objective 2, significant progress was made in developing low-cost formulations of Ifr used in controlling the Asian citrus psyllid. Substantial progress was also made on developing granular formulations of Met for use in controlling the lesser mealworm (a poultry litter pest) and for controlling various soil-dwelling insect pests on turf and on melon crops. In addition, gene-based diagnostic tools were developed to measure populations of Ifr and Met after field application in order to monitor their growth, persistence, and effectiveness in the field. Research on production processes for Met identified low-cost nitrogen sources that can be used for growing large amounts of Met at high concentrations. Ten different strains of Met were evaluated and shown to be capable of being used for commercial production. In addition, processes to formulate Met and to put it into a stable for commercial application were identified; these findings significantly increased the production potential of Met. Moreover, ARS researchers at Peoria, IL, found that air-dried granules of Met produced with these methods could kill white grubs, mealworms, and beetle pests on turf and on melon crops. Finally, a gene-based diagnostic tool was developed for tracking populations of Met in the soil. This tool will allow scientists and regulators to follow the fate of Met when it is applied to soil as a biocontrol agent. Substantial progress was made to reduce the cost of producing Ifr spores for insect pest control. Pilot-scale fermentation studies identified growth media that can be used to produce high numbers of Ifr spores that are needed for commercial production. Spores of Ifr tested in collaboration with ARS and university scientists proved successful for controlling cucumber beetles, Asian citrus psyllids, and insect pests of melons in the field. In addition, unique genetic markers were developed to track and monitor the persistence and effectiveness of Ifr populations in the field subsequent to application; this tool will be useful to scientists and regulators that wish to track and monitor Ifr biocontrol applications in the field.


4.Accomplishments
1. New methods to kill insect pests. Root weevils, meal worms, soil grubs, rootworms, wireworms, fruit flies, and root maggots are soil insect pests that affect a wide variety of agricultural crops, home lawns, and landscape plants, and turf. ARS researchers in the Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, developed new methods to grow and commercially produce the bioinsecticidal fungus Metarhizium (Met) to infect and killed these pests. In addition, the researchers were able to successfully use these methods to kill lesser meal worm larvae and adults (which are pests in commercial poultry operations). Koppert BV has licensed this technology from ARS through a patent on these processes filed by the Peoria, IL, researchers (USDA License No. 1474-001). The dried granular formulations of Met that the researchers at Peoria have developed will provide homeowners, farmers, and land managers an effective non-chemical control for numerous soil-dwelling insect pests.

2. Novel diagnostic tool to track and monitor beneficial biocontrol bacteria in soil. ARS researchers in the Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, developed and validated a new gene-based diagnostic tool to determine the concentration of beneficial insect-killing fungi in soil. The new assay replaced older, more labor-intensive, assays for measuring the levels of these fungi in the soil. Soils, in particular, are difficult environment to quantify microbes, due to the presence of unique chemical inhibitors; this new tool overcomes these obstacles. As a result, this gene-based diagnostic tool will further the development of these beneficial fungi as biopesticides for controlling soil-dwelling insect pests both in commercial and home settings.


Review Publications
Behle, R.W., Weiler, L.F., Bharadwaj, A., Stafford Iii, K.D. 2011. A formulation to encapusulate nootkatone oil for tick control. Journal of Medical Entomology. 48(6):1120-1127. DOI: http://dx.doi.org/10.1603/ME10282.

Jackson, M.A. 2012. Dissolved oxygen levels affect dimorphic growth by the entomopathogenic fungus Isaria fumosorosea. Biocontrol Science and Technology. 22(1):67-79. DOI: 10.1080/09583157.2011.642339.

Behle, R.W., Popham, H.J. 2012. Laboratory and field evaluations for efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda. Journal of Invertebrate Pathology. 109:194-200.

Moran, P.J., Patt, J.M., Cabanillas, H.E., Adamczyk Jr, J.J., Jackson, M.A., Dunlap, C.A., Hunter, W.B., Avery, P.B. 2011. Localized autoinoculation and dissemination of Isaria fumosorosea for control of the Asian citrus psyllid in South Texas. Subtropical Plant Science. 63:23-35.

Price, N.P., Ray, K.J., Vermillion, K., Dunlap, C.A., Kurtzman, C.P. 2011. Structural characterization of novel sophorolipid biosurfactants from a newly-identified species of Candida yeast. Carbohydrate Research. 348:33-41. DOI: 10.1016/j.carres.2011.07.016.

Last Modified: 9/22/2014
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