Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Postenucleation orbital sialocele in a dog associated with prior parotid duct transposition

An orbital sialocele developed in a dog following enucleation for protracted glaucoma. This eye had historically been treated for keratoconjunctivitis sicca by parotid duct transposition approximately 5 years previously, and the duct was ligated distally at the time of enucleation. One month following enucleation, the dog presented with a fluctuant conical-shaped swelling ventrolateral to the orbital socket. Surgical exploration revealed a dilated, fibrotic distal portion of the previously transposed parotid duct, and saliva, within the enucleated orbit. The distal portion of the duct and saliva-containing tissues from within the orbit were excised. The remaining proximal normal portion of the parotid duct was re-routed into the oral cavity. Clinicopathologic and histologic examination of the excised orbital contents and dilated portion of duct revealed a sterile sample of saliva and moderate chronic periductal fibrosis. At a 6-month re-evaluation there was no evidence of recurrence of the sialocele, and the parotid duct was functional.