2007 Annual Report 1a.Objectives (from AD-416) Continue exploration and evaluation of microbial control agents with the long-term goal of developing innovative biological control methods against the coffee berry borer. Focus will be on studies aimed at determining whether the fungal entomopathogen Beauveria bassiana can be used as a fungal endophyte for systemic control of the coffee berry borer and in the development of molecular techniques for detection of B. bassiana in plant tissues. A search for new coffee berry borer biocontrol agents via foreign exploration for parasitoids, fungi, and nematodes will be conducted in Latin America and Africa. 1b.Approach (from AD-416) Various techniques will be tested in order to optimize inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana: (1) spraying flowers; (2) spraying seedlings; (3) injecting spore suspensions on the stem; and (4) inoculation of radicles. To determine if B. bassiana has become established in the inoculated plants, polymerase chain reaction-based identification methods and specific molecular markers will be used. The program of exploration for new biological control agents of the coffee berry borer will be accomplished through collaborations with cooperators, such as ECOSUR (Mexico) and ICIPE (Kenya). Coffee plantations will be visited and coffee berries infected with the coffee berry borer will be sampled. Standard protocols will be used to survey for parasitoids, fungi, and nematodes attacking the insect. 3.Progress Report - Spraying of Beauveria bassiana on coffee flowers in the field. The objective was to spray coffee flowers with Beauveria bassiana to determine if this would result in the establishment of the fungal insect pathogen in the coffee berry. The goal is to develop a sustainable biological control method against the coffee berry borer. Plants were sprayed in Puerto Rico in April of 2007; experiment is on-going. - Foreign exploration for natural enemies. The goal is to search for new natural enemies of the coffee berry borer in Kenya. Previously unreported natural enemies could become important biological control agents in other coffee-producing countries where these organisms could be introduced. A research collaboration has been established with Juliana Jaramillo and Christian Borgemeister (ICIPE, Nairobi, Kenya), whereby insects are collected in the field and sent to USDA for subsequent identification. Insects are being shipped periodically from Kenya and identifications are in progress. - Molecular detection of B. bassiana in sprayed plants. The goal is to develop PCR-based assays to detect Beauveria bassiana infections of leaves and fruits of coffee. This assay would complement the existing method for detecting B. bassiana endophytes by culturing methods. Initial studies have shown that detection of B. bassiana by PCR is comparable in sensitivity to assays developed for other fungi. We are currently assessing whether the level at which B. bassiana occurs within plants is within this detection. 4.Accomplishments Development of genetic markers for the specific detection of Beauveria bassiana in coffee plants and other environmental samples. The entomopathogenic fungus Beauveria bassiana naturally forms asymptomatic endophytic infections of live plant leaves, stems and possibly immature fruits of coffee and other plants. This association raises the possibility that coffee plants can serve as reservoirs of insect fungal pathogens against their insect pests, such as the coffee berry borer. We have developed multiple genetic markers for the specific and quantitative detection of Beauveria in plants and other environmental samples. These markers will enable a more rapid diagnosis of Beauveria in natural and experimentally infected plants and will facilitate understanding the utility of this phenomenon as it relates to insect biological control. This accomplishment addresses the following National Program 304 (Crop Protection and Quarantine) component: development of new and improved pest control technologies. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 7 Number of newspaper articles and other presentations for non-science audiences 1 Review Publications Posada, F.J., Aime, M.C., Peterson, S.W., Rehner, S.A., Vega, F.E. 2007. Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales). Mycological Research. 111(6):748-757. Posada, F.J., Vega, F.E. 2006. Inoculation of cocoa seedlings (theobroma cacao) with the fungal entomopathogen beauveria bassiana (ascomycota: hypocreales) and its establishment as an endophyte. Mycologia. 97:208-213. Vega, F.E., Posada, F.J., Peterson, S.W., Gianfagna, T.J., Chaves, F. 2006. Penicillium species endophytic in coffee plants and ochratoxin a production. Mycologia. 98:31-42. Vega, F.E., Posada, F.J., Infante, F. 2006. Coffee insects, ecology and control. Encyclopedia of Pest Management. http://www.dekker.com/sdek/issues~content=t713172972 Posada, F.J., Vega, F.E. 2006. inoculation and colonization of coffee seedlings (coffea arabica l.) with the fungal entomopathogen beauveria bassiana (ascomycota: hypocreales). Mycoscience. 47:284-289. Vega, F.E., Posada, F.J., Gianfagna, T.J., Chaves, F.C., Peterson, S.W. 2006. An insect parasitoid carrying an ochratoxin producing fungus. Naturwissenschaften. 93:297-299.