2008 Annual Report 1a.Objectives (from AD-416) The objectives of this agreement are (1) to optimize and transfer production and stabilization processes for the fungal bioherbicide Mycoleptodiscus terrestris (MT) for commercial-scale production, and (2) use MT formulations produced at pilot- and commercial-scale for control of hydrilla (Hydrilla verticillata) in large-scale field trials in Florida. 1b.Approach (from AD-416) In collaboration with the U.S. Army Corps of Engineers and SePRO Corporation, dry formulations of the fungal bioherbicide Mycoleptodiscus terrestris (MT) will be used to demonstrate the effectiveness of this agent in controlling the invasive aquatic weed in Florida ponds and lakes. Microsclerotia of MT will be produced and stabilized using patented ARS technology. Small-scale production of MT microsclerotia for pond studies will be produced by ARS, and the production and stabilization process for this herbicide will be optimized as field trials proceed. For large-scale hydrilla control studies, MT microsclerotia will be produced by a contract fermentor following transfer of the production and stabilization technology. 3.Progress Report Studies focused on determining optimized nutritional and environmental conditions for scale-up of the production of microsclerotia of Mycoleptodiscus terrestris (MT) using deep-tank fermentation and assessing the compatibility of MT relative to application and production temperatures. The impact of temperature, pH, aeration, agitation, and the use of low-cost sources of nitrogen such as corn steep liquor powder and cottonseed meal was examined. We conducted twelve 100-liter, sixteen 5-liter, and numerous shake-flask fermentations during these studies. Fermentation studies showed that cultures grown with corn steep liquor powder produced lower yields of MT microsclerotia compared to cultures grown with Pharmamedia®. An industrial fermentation consultant determined that the optimization of aeration and agitation parameters were critical for the commercial scale-up of our fermentation process. Aeration and agitation studies still in progress suggest that higher aeration rates will reduce fermentation times and increase yields of MT microsclerotia. The MT biomass produced in eight of the 100-liter fermentations was formulated and dried. Four of these dried MT products were used in hydrilla field trials in Florida. Temperature studies with liquid MT fermentations showed that growth temperature has a significant impact on biomass accumulation and on the number of discrete colony forming units produced within this biomass. These studies suggest that growth temperatures between 22-26ºC are optimal for inoculum development where maximal colony forming units is desired and that 26-30ºC is the optimal for the formation of microsclerotia. Studies with air-dried MT microsclerotia showed that temperatures of 24-28ºC were optimal for both hyphal germination and conidia production. Neither MT biomass nor air-dried microsclerotia germinated or grew at temperatures above 32ºC. The lack of growth by MT at higher temperatures increases the safety of the biocontrol agent for humans and other mammals while restricting its effectiveness when water temperatures exceed 32ºC. Research is monitored by occasional meetings, frequent phone calls, and written reports. This research is conducted under NP 304, Component 5, Problem Statement 2.