Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters.

AIMS: To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method. METHODS AND RESULTS: The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0.1 MPN per litre, with a 95% confidence level of 0.01-0.7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0.9 MPN per litre for pond outflow and from not detectable to 8.3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10(4) MPN per hour. CONCLUSION: This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife. SIGNIFICANCE AND IMPACT OF THE STUDY: This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.

First Report of Leaf Smut on Fescue (Festuca rubra) Caused by Urocystis agropyri in Interior Alaska.

In June and early July of 2003 and July of 2007, a smut disease was found on fescue (Festuca rubra L.) at the Fairbanks Golf and Country Club and vicinities, Interior Alaska. Diseased plants were pale green-to-slightly yellow and stunted. The lesions were long, narrow, yellowish green streaks parallel to the veins of leaves and sheaths. At a later stage, sori developed under the epidermis and the leaves and sheaths turned silvery gray. Rupture of the epidermis resulted in the release of masses of black spores that covered the leaves, especially along and within infected sheaths. Microscopic observation showed spore balls consisting of one-to-four, dark reddish brown teliospores surrounded by small, hyaline-to-light brown sterile cells. The spore balls were globose to elongate and 16 to 32 × 18 to 38 µm. The teliospores were globose, dark reddish brown, and 7.5 to 20 µm in diameter. Urocystis agropyri is recognized by R. W. Smiley et al. (2) and J. D. Smith et al. (3) as occurring on turf grasses; K. Vánky (4) restricts this species to Elymus spp. and recognizes U. ulei as the valid species infecting Festuca spp, including F. rubra. On the basis of host symptoms and morphology of spores, the smut found on F. rubra in Alaska fits better with published descriptions of U. agropyri (Preuss) Schrot (2,3), however, it likely is not conspecific with flag smut of wheat. For molecular verification of this identification, DNA was isolated from teliospores with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Primers were designed for amplification of the large subunit ribosomal gene based on the only two published sequences of U. ranunculi (Lib.) Moesz (GenBank Accession No. AF009879) and U. colchici (Schltdl.) Rabenh (GenBank Accession No. AF009878) (1). A 400-bp PCR product was amplified by the forward primer 3'-GCATTGTAAACTCAGAAGTGTTATCCG-5' and reverse primer 5'-TTCCCTAAACCTATATCCGGCG-3'. Nucleotide sequence of the PCR product (GenBank Accession No. AY547283) shared 97.7% homology with U. ranunculi and 98.2% with U. colchici. Comparison of nucleotide sequences will be more meaningful in the identification of species of Urocystis when additional sequences are published. To our knowledge, this is the first report of U. agropyri from turfgrass in Alaska. References: (1) D. Begerow et al. Can. J. Bot. 75:2045, 1997. (2) R. W. Smiley et al. Compendium of Turfgrass Diseases. 3rd ed. The American Phytopathological Society, St Paul, MN, 2005. (3) J. D. Smith et al. Page 189 in: Fungal Diseases of Amenity Turf Grassess. E. & F. N. Spon, London, 1989. (4) K. Vánky. Page 308 in: European Smut Fungi. Gustav Fischer Verlag, NY. 1994.