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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

2013 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:
Substantial progress was made on Objective 1, Production, and Objective 2, Formulation, of microbial agents for controlling insects and weeds. Under Objective 1, nutritional and environmental conditions were optimized for the liquid culture production of microsclerotia (MS), a stable fungal form, of the insect-killing fungus, Metarhizium brunneum (Met). Microsclerotia of the weed-killing fungus Mycoleptodiscus terrestris were produced for use in field trials for control of the aquatic weed Eurasian water milfoil. Liquid culture production methods are under development for the insect-killing fungus Beauveria bassiana and for the fungus Trichoderma harzianum for control of plant diseases. Under Objective 2, progress continued on developing low-cost formulations of Isaria fumosorosea (Ifr) for use in controlling the Asian citrus psyllid. Substantial progress was also made on developing granular MS formulations of Met for use in controlling lesser mealworms in poultry houses, white grubs in turf, and other soil-dwelling insect pests. In collaboration with Cornell scientists, hydromulch-based MS formulations of Met are being evaluated for control of the Asian longhorned beetle. Studies were initiated to identify insect-killing fungi that could be used to control invasive wood-boring ambrosia beetles. These insects spread fungal pathogens that lead to laurel wilt disease and Fusarium die-back. In a collaborative effort with scientists at the Universities of California and Florida, we identified several fungi capable of infecting and killing ambrosia beetles. Research is underway to evaluate suitable formulations and application technologies for control of these pests. DNA analysis of more than 200 ambrosia beetles was used to determine their susceptibility to different insect-killing fungi and identified the importance and potential of co-fungal infections impact on biocontrol efficacy. Temperature optima were identified that supported high yields of MS of Met. The development of formulation and stabilization processes that improved the room temperature storage stability of Met MS granules significantly increased their potential for commercial use. Air-dried MS granules of Met produced high concentrations of spores that infected and killed white grubs, mealworms, and various beetles. Formulations of Ifr spores were tested against cucumber beetles, Asian citrus psyllids, and ambrosia beetles in collaboration with ARS, university, and industry scientists. In addition, foreign and domestic species of Met are currently being optimized for MS production under a Cooperative Research and Development Agreement (CRADA) with an industrial partner. A selective and quantitative DNA-based assay was developed for Ifr species which provides a powerful tool for evaluating population dynamic in the field after release of the bioinsecticide. This novel approach uses high resolution melting of DNA to identify Ifr species. The assay can distinguish two Ifr species in the presence of each other when obtained from environmental samples.


4.Accomplishments
1. Controlling invasive ambrosia beetles. The nation’s $322 million avocado crop is facing an emerging disease problem caused by invasive wood-boring ambrosia beetles. The insects spread fungal pathogens that lead to laurel wilt disease and Fusarium die-back. ARS Crop Bioprotection Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, Illinois, in a collaborative effort with scientists at University of California and University of Florida evaluated beneficial insect-killing fungi as an approach to controlling the invasive insects. The research project was able to identify several beneficial fungi capable of killing the insects and rank their effectiveness. This research offers an environmentally friendly option to control this emerging threat. Avocado farmers and their consumers benefit from this research through the development of a new safe pest control product.


Review Publications
Dunlap, C.A., Biresaw, G., Shearer, J.F. 2012. Captive bubble and sessile drop surface characterization of a submerged aquatic plant, Hydrilla verticillata. Current Topics in Phytochemistry. 11:53-58.

Behle, R.W., Jackson, M.A., Flor-Weiler, L.B. 2013. Efficacy of a granular formulation containing Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) microsclerotia against nymphs of Ixodes scapularis (Acari: Ixoididae). Journal of Economic Entomology. 106(1):57-63.

Jackson, M.A., Jaronski, S. 2012. Development of pilot-scale fermentation and stabilization processes for the production of microsclerotia of the entomopathogenic fungus Metarhizium brunneun strain F52. Biocontrol Science and Technology. 22(8):915-930.

Shapiro Ilan, D.I., Gardner, W., Wells, L., Cottrell, T.E., Behle, R.W., Wood, B.W. 2013. Effects of entomopathogenic fungus species, and impact of fertilizers, on biological control of pecan weevil (Coleoptera: Curculionidae). Environmental Entomology. 42:253-261.

Bharadwaj, A., Stafford III, K.C., Behle, R.W. 2012. Efficacy and environmental persistence of nootkatone for the control of the blacklegged tick (Acari: Ixodidae) in residential landscapes. Entomological Society of America. 49(5):1035-1044.

Shapiro Ilan, D.I., Cottrell, T.E., Jackson, M.A., Wood, B.W. 2013. Control of key pecan insect pests using biorational pesticides. Journal of Economic Entomology. 106:257-266.

Jaronski, S., Jackson, M.A. 2012. Mass production of entomopathogenic hypocreales. In: Lacey, L.A., editor. Manual of Techniques in Invertebrate Pathology. 2nd edition. New York, NY: Academic Press. p. 255-284.

Petzold-Maxwell, J., Jaronski, S., Clifton, E.H., Dunbar, M.W., Jackson, M.A., Gassman, A.J. 2013. Interactions among Bt maize, entomopathogens, and rootworm species (Coleoptera: Chrysomelidae) in the field: effects on survival, yield and root injury. Journal of Economic Entomology. 106(2): 622-632.

Last Modified: 11/22/2014
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