2012 Annual Report 1a.Objectives (from AD-416): 1. Develop and validate molecular identification and genotyping resources for insect biocontrol fungi Beauveria spp. and Metarhizium. 2. Distinguish fungal pathogens from species with potential for the biological protection of plants through molecular systematics investigations of complex coelomycete genera, including Colletotrichum and Pestalotiopsis. 1b.Approach (from AD-416): Genomic sequences will be mined with specialized software to develop novel molecular identification and genotyping methods for the insect pathogens Beauveria and Metarhizium. Marker data obtained from strains collected by the scientists, collaborators or acquired from culture collections will form the basis for systematic and taxonomic revisions and the creation of on-line molecular and morphological identification resources. A molecular epidemiological analysis of Beauveria pathogens of coffee berry borer, an important insect pest of coffee, will be conducted in coffee farms in Puerto Rico. Molecular methods for species identification will be critically tested. Population genetic analyses combining microsatellite, mating type and SNP data will be used to analyze the genetic structure of epizootics against the environmental diversity of indigenous Beauveria. In addition, the efficacy and persistence and geographic spread of GHA, the Beauveria mycoinsecticide Mycotrol® strain that has been released for control of coffee berry borer, will be assessed. For the project on pathogenic and endophytic strains of Colletotrichum and Pestalotiopsis, strains will be obtained from a network of cooperators in the Neotropics. Initial diversity screens of Colletotrichum strains will be conducted using markers APN2 and APN2/MATigs; diversity screens for Pestalotiopsis will utilize variable intron regions of elongation factor-1 alpha (EF-1a) and beta tubulin (B-tub). Strains selected from the diversity screens will be used for multi-locus phylogenetic analyses (B-tub, EF-1a, RPB1, RPB2 and other loci) to delineate species boundaries, infer species relationships, and determine whether endophytic and pathogenic ecologies are phylogenetically associated. 3.Progress Report: This report documents progress for the Project 1275-22000-277-00D, Systematics of Biological Control Microfungi for Management of Plant Diseases and Insect Pests and continues research from Project 1275-22000-269-00D, Systematics of Microfungi used in Biological Control of Plant Diseases and Insect Pests. This project focuses on National Program 303, Component 1, Plant Diseases, Diagnostics, Etiology and Systematics. Progress was made on both objectives, which fall under Problem Statement 1, Diagnostics, Etiology and Systematics of Plant Disease and use molecular and morphological approaches to characterize fungi with potential for biological control using both molecular sequence data and microscopy. Under Objective 1, progress was made in developing improved methods for identifying species of insect pathogenic fungi. These methods are needed to conduct epidemiological studies of these organisms and to track the field behavior of strains released for insect control applications. A pipeline has been developed for the comparative analysis of genome sequences of the insect control fungi Beauveria and Metarhizium. Under Objective 2, progress was been made developing eight nuclear DNA markers for use in species discovery and diagnostics. These markers will be used to resolve the phylogeny of endophytic and pathogenic fungi in the complex genera Colletotrichum and Pestalotiopsis, the most frequently isolated pathogens and endophytes of cacao and other tropical tree crops.