ABSTRACT
In early May 2008 and 2009, peony samples (Paeonia spp.) with symptoms of leaf spot and blight were submitted to the Virginia Tech Plant Disease Clinic. The 2008 peony was an unknown cultivar from a northern Virginia landscape. The three cultivars (Dr. Alexander Fleming, Felix Crousse, and Karl Rosenfield) submitted in 2009 were from a commercial nursery in southwestern Virginia that was reporting leaf spot progressing to severe blight, which rendered plants unsalable, on 75% of a 1,219 m2 block during a 10-day period of heavy rainfall. Bacterial streaming from spots was observed. On the basis of phenotypic and biochemical tests, the isolates were determined to be xanthomonads. Two isolates (one recovered from the 2008 sample and one from the 2009 sample) were used in the following work. Isolates were characterized by multilocus sequencing (MLST) (4). PCR reactions were prepared and cycled using 2X ImmoMix (Bioline, Tauton, MA) according to manufacturer's recommendations with an annealing temperature of 58°C. Template DNA was added by touching a single colony with a 20-µl pipette tip and placing the tip into the reaction mix for 1 min. Four bands of the expected size were visualized on an electrophoresis gel and cleaned products were sequenced in forward and reverse directions at the University of Chicago, Cancer Research Center DNA Sequencing Facility. Corresponding gene fragments of each isolate were identical. A consensus sequence (PAMDB Isolate ID No. 936) for each of the four gene fragments was constructed and compared with sequences in NCBI ( http://www.ncbi.nlm.nih.gov/nuccore/ ) and PAMDB ( http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl ) (1) databases using Blastn (2). No perfect match was found. Genetic distances between the peony isolates and all strains in PAMDB were determined by MegAlign (Lasergene; DNAStar, Madison, WI). The Xanthomonas strain most similar to the isolates recovered from the peony samples was Xanthomonas hortorum pv. hederae ICMP 1661 with a genetic distance of 0.023; this strongly suggests that the peony isolates belong to X. hortorum. For Koch's postulates, six surface-disinfested young leaflets from Paeonia lactiflora 'Karl Rosenfield' were inoculated by forcefully spraying a phosphate-buffered saline suspension of each bacterial isolate (~4.3 × 109 CFU/ml) into the underside of the leaf until leaf tissue appeared water soaked. Controls were inoculated similarly with phosphate-buffered saline solution. Moist chambers with inoculated leaves were incubated at ambient temperature under two 48W fluorescent grow lights with 12 h of light and dark. Circular spots were observed on leaves inoculated with the 2009 and 2008 isolates in 18 and 20 days, respectively. No symptoms were observed on controls. Bacterial streaming from leaf spots was observed by phase-contrast microscopy; bacteria were isolated and confirmed to be identical to the original isolates by the methods described above. To our knowledge, this is the first report of a Xanthomonas sp. causing leaf spot and blight on peony. Although bacterial blight of peony has been attributed to a xanthomonad in recent years, the pathogen had not been further characterized (3). References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) D. J. Altschul et al. J. Mol. Biol. 215:403, 1990. (3) M. L. Gleason et al. Diseases of Herbaceous Perennials. The American Phytopathological Society, St. Paul, MN. 2009. (4) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.
ABSTRACT
Recycled irrigation water is a primary source of inoculum for Phytophthora spp. and is capable of spreading propagules throughout nursery production. Chlorination commonly is used by the industry to disinfest recycled irrigation water; however, chlorine has not been fully researched as a disinfestant for this purpose. In this study, zoospores of seven species and eight isolates of Phytophthora were exposed for 2 min to free available chlorine at 0.25, 0.5, 1.0, 2.0, and 4.0 mg/liter. Zoospores, mycelial fragments, and culture plugs of P. nicotianae also were exposed to chlorine concentrations ranging from 0.25 to 8.0 mg/liter for periods ranging from 15 s to 8 min. In addition, chlorinated water was assayed monthly in 2000 and 2001 at two commercial nurseries, and quarterly in the first year at four other nurseries in Virginia, for chlorine and survival of pythiaceous species using a selective medium. No zoospores of any species tested survived endpoint free chlorine at 2 mg/liter, while limited mycelial fragments of P. nicotianae survived at 8 mg/liter, and mycelial plugs treated at the same level of chlorine were able to produce few sporangia. Phytophthora spp. were recovered only from nursery irrigation water with levels of free chlorine at 0.77 mg/liter or lower. The results of this study are essential for improving current chlorination protocols.
