2004 Annual Report 4.What were the most significant accomplishments this past year? D. Progress Report: This report serves to document research conducted under a specific cooperative agreement between ARS and the Pennsylvania State University. Additional details of research can be found in the report for the parent CRIS 1920-22000-030-00D, "Evaluation and Characterization of Plant Pathogens for Biological Control of Weeds". Canada thistle (Cirsium arvense)infests millions of acres in in the U.S. including cropland, rangeland and conservation areas, and it is considered one of the ten worst weeds in the world. We are cooperating on research to utilize a fungal pathogen of Canada thistle, Puccinia punctiformis, using plant tissue culture propagation techniques to multiply the fungus. Field Plots During the summer of 2003 experiments were monitored for spread of local and systemic infections on Canada thistle. New systemically infected shoots emerged throughout the summer in close proximity to systemically infected shoots present at the beginning of the season. Locations of systemically infected shoots at the beginning and throughout the summer were recorded. More specifically, the distance and bearing from the center of the plot were recorded for each systemically infected shoot. These data points were then transformed into Cartesian coordinates so that within-season spread could be visualized. Leaf samples were taken weekly to track the development of teliospores from primary and secondary urediosori. Teliospores were found in primary urediosori from late June to October. Interestingly, teliospores were never observed within secondary urediosori (localized lesions). During the summer of 2003 areas surrounding plots were mowed in late July. There appeared to be more systemically infected shoots following mowing than in areas that were not mowed. This observation led to the hypothesis that late season mowing (following the onset of teliospore production) will allow systemically infected Canada thistle to increase dispersal of infective spores during a single season. In addition, re-growth of systemically infected shoots may help overcome the monocyclic nature of the pathogen and increase disease spread. Based on the observations made during the summer of 2003, experiments including mowing as an additional treatment were added in 2004. Observations of experiments set up in 2003 were continued in 2004. Locations of systemically infected Canada thistle shoots were recorded and compared to locations in the previous year. Graphical depictions of plots were created using a Cartesian coordinate system in order to visualize patterns of disease spread. In addition, the number of systemically infected shoots within a plot in the spring of 2004 was compared to the number of systemically infected shoots present in 2003. Regression analyses indicate a positive relationship between the numbers of systemically infected shoots present in 2003 and the numbers present in 2004. According to data collected thus far, in 77% of the plots the numbers of systemically infected shoots present in 2004 has increased or remained the same from 2003. Information regarding the numbers of systemically infected shoots at the end of the 2004 season is forthcoming and will be added to the analyses discussed above. Tissue Culture During 2003 difficulties generating sustainable callus from systemically infected Canada thistle shoots created set backs in this aspect of the project. Stem sections and callus often turned brown and eventually died in 1-3 transfers. To combat this problem ascorbic acid and sucrose were added to the medium. After two weeks, stem sections prepared as outlined in the 2003 report began developing large sections of callus. Thus far these cultures have sustained growth better than cultures grown without ascorbic acid. Furthermore, browning of stem pieces has been minimal. Currently efforts are being focused toward root and shoot formation from systemically infected callus. Biological control of Canada thistle using Puccinia punctiformi will be a useful addition to the limited options available to land managers for management of this invasive weed.