Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions.

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

Clinical outcome of frequent exposure to Torque Teno virus (TTV) through blood transfusion in thalassemia patients with or without hepatitis C virus (HCV) infection.

As a consequence of the high prevalence of TorqueTeno virus (TTV) in blood donors, thalassemia patients frequently acquire various genotypes of this virus through therapeutic blood transfusions. At present, the clinical consequences of TTV infection remain indeterminate for these patients. Here, several hundred thalassemia patients were tested for the presence of TTV and its genotypes using a combination of PCR and clone-based DNA sequencing. Approximately 10% (12/118) of the patients aged 2-20 years remained negative for TTV including eight genotypes of SENV. Ferritin, aspartate-aminotransferase (AST) and alanine-aminotransferase (ALT) levels were invariably lower in TTV-negative patients (P = 0.02, <0.01, and 0.06, respectively) than in TTV-positive patients. Patients with TTV-HCV co-infection showed elevated ferritin and ALT levels compared with patients with TTV infection alone (P < 0.02 and P < 0.01). AST and ALT levels were within the normal range for all TTV-negative patients, whereas abnormal levels of AST and ALT were seen in a significant proportion of TTV-positive patients (30.7% and 33.6%, respectively) and patients with TTV-HCV co-infections (70.0% and 56.6%, respectively). Only TTV-positive patients (28.0%) and patients with TTV-HCV co-infections (36.3%) had hyper-ferritin levels (> or =3,000 ng/ml). The genotype(s) of TTV responsible for the liver dysfunction could not be determined. However, high levels of AST and ALT were found to be correlated with detection of a higher number of TTV genotypes in the patients. The data suggests that frequent and persistent TTV infection through blood transfusion is associated with hepatic dysfunction and/or damage in transfusion dependent thalassemia patients.