ABSTRACT
In summer 2012, bacterial blight symptoms (2) were observed on leaves of carrot plants in 7 out of 70 plots of carrot breeding lines at the Purdue University Meig Horticulture Research Farm, Lafayette, IN. Symptoms included small to large, variably shaped, water-soaked to dry, necrotic lesions, with or without chlorosis, at <5% incidence. Microscopic examination of symptomatic leaf sections revealed bacterial streaming from the cut ends of each leaf piece. For each of the seven plots, symptomatic leaf sections (each 5 to 10 mm2) were surface-sterilized in 1.2% NaOCl for 60 s, triple-rinsed in sterilized, deionized water, dried on sterilized blotter paper, macerated in sterilized water, and a loopful of the suspension was streaked onto yeast dextrose carbonate (YDC) agar medium (1). Colonies with morphology similar to that of strain Car001 of Xanthomonas hortorum pv. carotae from California (3) were obtained consistently from all seven plots, and serial dilutions streaked onto YDC agar medium to obtain pure cultures. One bacterial strain/plot was then subjected to a PCR assay for X. hortorum pv. carotae using the protocol of Meng et al. in (5), except for an annealing temperature of 60°C. All seven Indiana strains and Car001 produced a 355-bp DNA fragment indicative of X. hortorum pv. carotae. The Indiana strains and Car001 were each tested for pathogenicity on five 11-week-old carrot plants of a proprietary Nantes inbred line grown from a seed lot that tested negative for X. hortorum pv. carotae (1,3). Each strain was grown for 16 h in 523 broth (4) on a shaker (200 rpm) at 28°C, and diluted in 0.0125M phosphate buffer to 108 CFU/ml. Approximately 24 h prior to inoculation, the five plants for each strain were enclosed in a large plastic bag to create a moist chamber. The plants were inoculated by atomizing 30 ml of the appropriate bacterial suspension onto the foliage using an airbrush. Five plants inoculated with sterilized phosphate buffer served as a negative control treatment. The plants were re-sealed in plastic bags for 72 h, and placed in a randomized complete block design in a greenhouse set at 25 to 28°C. Symptoms of bacterial blight were first observed 14 days after inoculation, and developed on all inoculated plants by 21 to 28 days after inoculation, with slight variation in severity of symptoms among strains. Symptoms did not develop on negative control plants. Re-isolations were done 32 days after inoculation from symptomatic leaves of three replicate plants/strain and from three plants of the negative control treatment, using the protocol described for the original samples. Bacterial colonies typical of X. hortorum pv. carotae were obtained from symptomatic leaves for all seven Indiana strains and the control strain, but not from the negative control plants. Identity of the re-isolated strains as X. hortorum pv. carotae was confirmed by PCR assay. To our knowledge, this is the first report of bacterial blight of carrot in Indiana. References: (1) M. Asma. Detection of Xanthomonas hortorum pv. carotae on Daucus carota. 7-020. International Rules for Seed Testing, Annex to Chapter 7: Seed Health Testing Methods. Internat. Seed Testing Assoc., Bassersdorf, Switzerland, 2006. (2) R. M. Davis and R. N. Raid. Compendium of Umbelliferous Crop Diseases. The American Phytopathological Society, St. Paul, MN, 2002. (3) L. J. du Toit et al. Plant Dis. 89:896, 2005. (4) E. I. Kado and M. G. Heskett. Phytopathology 60:969, 1970. (5) X. Q. Meng et al. Plant Dis. 88:1226, 2004.
ABSTRACT
Protein C is a major regulatory protein critical to physiologic anticoagulation. When activated, it selectively degrades the activated forms of factors V and VIII, thereby, down-regulating blood coagulation. Using an activated partial thromboplastin time (APTT) assay, Dahlback et al. recently reported that some individuals with thrombophilia show a poor in vitro anticoagulant response to activated protein C (APC-Resistance). Subsequent studies identified a point mutation in the gene for factor V as the underlying cause of APC-Resistance. The incidence of APC-Resistance in patients with recurrent thromboembolic events approaches 50%. The APC-Resistance phenotype is also present in approximately 5% of normal Caucasian subjects. In an attempt to develop a more sensitive and specific test system, we evaluated an assay based on Textarin(Pentapharm, Basel, Switzerland). Textarin, a protein fraction of Pseudonaja textilis venom (Australian Eastern Brown Snake) activates prothrombin in the presence of phospholipid (PL), factor V and calcium ions. Based on Textarin's requirement for factor V, we developed a Textarin time assay to test for APC-Resistance. We evaluated this test system in normal subjects and the following patient populations: stable orally anticoagulated, previously diagnosed factor V Leiden, and therapeutically heparinized samples. We found the Textarin assay to be a sensitive and specific test system to identify APC-Resistance. The phenotypic Textarin APC-Resistance test correlated more closely with the genotypic abnormality of factor VR506Q than the APTT-APC-Resistance test.
