2007 Annual Report 1a.Objectives (from AD-416) Chalkbrood and other diseases cause significant mortality in bees, especially the alfalfa leafcutting bee, a pollinator that is key to successful production of alfalfa seed crops. Chalkbrood and other unidentified diseases are the primary reason that US seed growers are unable to produce enough of their own bees and must import them from Canada every year. A better understanding of the phylogenetic relationship between bees and the Ascosphaera (the causative agents of chalkbrood) will improve our understanding of the evolution of host/pathogen relationship. In addition, we would like to gain knowledge on the genetics of the immune response of the bees to pathogen invasion to develop effective disease control strategies to assist in breeding more resistant bee populations. 1b.Approach (from AD-416) Phylogeny Work. Fungi in the genus Ascosphaera are known to occur only in association with bees. A better understanding of the phylogeny of this unusual group will aid in identifying host specificity and pathogenicity factors. Sequence information has previously been published for the ITS gene in most known species of Ascosphaera, but phylogenies based on one gene are incomplete. We will obtain sequence information for three other genes for a number of Ascosphaera species. We will obtain ~50 kb of double-stranded sequence data using the CIBS DNA sequencing facility at Utah State University. Genetics of Immune Response. Temperature significantly affects the immune response of bees, where conditions of heat-shock make the bees less susceptible to infection from Ascosphaera infections. Microarrays will be used to identify upregulation of genes during heatshock and during pathogen invasion to look for commonalities and to identify key genes responsible for increased immunity. Once key compounds have been identified in the alfalfa leafcutting bee in response to A. aggregata and A. proliperda invasion, we will test whether a similar immune response occurs in honey bees when they are challenged with A. apis. 3.Progress Report This report serves to document research conducted under a specific cooperative agreement between ARS and Utah State University. Additional details of the research can be found in the report for the parent CRIS 5428-21000-010-00D Pollinating-Insect Biology, Management, and Systematics Research. This is the first year of the cooperative research. We sequenced the ITS and EF genes for 16 species and isolates of Ascosphaera, and the RP1 and RP2 genes for five species. We are currently in the process of completing the RP genes and assembling a phylogeny of the Ascosphaera genus. We were able to use the Ascosphaera apis genome sequence to assist us in developing primers for the new genes (EF and RP), which had not been previously been sequenced for any of the Ascosphaera. This year, we also hired a new Research Associate who has initiated building a cDNA library for both the alfalfa leafcutting bee and Ascosphaera aggregata (the pathogen that cases chalkbrood in this bee). The purpose is to develop a micro-array of the genes that are activated during the infection process so that we can identify key components of the immune response in this bee and determine both the genetics of the immune response (to determine whether it is possible to breed for a more resistant bee), and to determine what environmental factors enhance and inhibit bee immunity. ADODR monitoring activities include e-mails, conference calls, and site visits since the ARS facility is co-located with the Cooperator.