Colombian datura virus (CDV) was first described in 1968 (3) and has since been reported in Europe (4), Japan (see 4 for additional references), and the United States (1,2). CDV is a member of the family Potyviridae with flexuous, filamentous nucleocapsids that can be transmitted by mechanical inoculation and grafting and is known to be vectored by the common aphid Myzus persicae. In the fall of 2007, five Brugmansia plants of unknown species from a Parks Board Collection in a Lower Mainland nursery, British Columbia, Canada, were found to be displaying symptoms typical of a viral infection: chlorotic flecking and mottling on leaves, leaf shrivel, and vein banding. Symptomatic leaves from these five plants were tested by ELISA (Immuno Strip Test, Agdia, Elkhart, IN) for several common viruses including Impatiens necrotic spot, Tobacco mosaic, Cucumber mosaic, and Tomato spotted wilt viruses and found to be negative for all. However, rub inoculations onto the herbaceous indicators Nicotiana occidentalis and N. benthamiana resulted in severe symptom formation including necrosis, wilting, shriveling, stunted growth, petiole and stem tip collapse, as well as collapse from the base of the plants, and plant death within 2 weeks after inoculation. A leaf dip assay of the original infected Brugmansia sample and infected N. benthamiana tissue revealed flexuous, potyvirus-like particles with the electron microscope (EM). On the basis of the Brugmansia leaf symptoms and the EM results, a possible infection with CDV was suspected. Primers CDV-3 and CDV-NIb5, specific to CDV (4), were used in a reverse transcription (RT)-PCR assay that amplified an approximate 1,600-bp fragment from the original Brugmansia sample and inoculated N. bentamiana and N. occidentalis plants. The amplified portion of the genome is the extreme 3′ terminus and includes the 3′ noncoding sequence, the viral coat protein gene, and part of the viral replicase gene. Fragments were cloned into pCR2.1-TOPO (Invitrogen, San Diego, CA) and two clones from each plant (total of six clones) were sequenced in both directions. Sequences of all clones were essentially identical, with only three nucleotide differences among the clones (GenBank Accession No. EU571230). BLASTn analysis revealed the highest match to several CDV isolates ranging from 98.7 to 99.5% nucleotide sequence identity. BLASTp analysis of the 451 amino acid viral polyprotein translation product gave a similarly high match with CDV isolates, with the highest match to a Hungarian isolate of CDV (GenBank Accession No. CAD26690) of 99.8% identity, or only one mismatch out of 451 amino acids. An additional group of 15 large symptomless Brugmansia plants, located approximately 6 m from the five symptomatic plants, were also tested by RT-PCR and found to be positive. These 15 plants were of a different but also unknown species of Brugmansia. In conclusion, analysis of symptomatic Brugmansia from a Canadian collection by transfer of disease to herbaceous indicators, EM, RT-PCR, and genomic sequence comparisons, are consistent with the detection and identification of the potyvirus Colombian datura virus. To our knowledge, this is the first report of this viral pathogen in Canada. References: (1) S. Adkins et al. Phytopathology (Abstr.) 95(suppl.):S2, 2005. (2) C. R. Fry et al. J. Phytopathol. 152:200, 2004. (3) R. P. Kahn and R. Bartels. Phytopathology 58:58, 1968. (4) J. Schubert et al. J. Phytopathol. 154:343, 2006.

A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

Simple but reliable PCR techniques were developed for the detection and identification of several food crops, including crops known to contain allergens. A single pair of oligonucleotide primers (PL-1C and PL-2D), that target the trnL region of the chloroplast tRNA gene in polymerase chain reaction (PCR) analysis, was used to amplify crop specific fragments. The specific DNA fragments were of the following sizes; 387 bp (canola), 532 bp (corn), 571 bp (potato), 584 bp (soybean), 615 bp (white and red rice), 642 bp (peanut), and 662 bp (wheat). Each amplified fragment was reliably identified using 3% agarose gel electrophoresis. The amplified fragments were cloned, sequenced, and a variable region was used to design specific sense primers for identity confirmation of some selected crops. When combined with the antisense primer PL-2D, specific fragments of 403, 397, 343, and 304 bp were amplified for peanut, wheat, soybean, and rice, respectively. These are common crops known to contain allergens. The PCR techniques described may be easily adapted for the detection of other crops and may be modified for use in multiplex PCR detection techniques, or micro-/macro-array analysis.

An application of stable carbon isotope analysis to the mechanistic interpretation of ultraviolet‐B (UV‐B) effects on growth inhibition is described that is particularly useful for small plants such as Arabidopsis thaliana that are not well suited for gas exchange studies. Many investigators use tissue δ13C, relative abundance of 13C and 12C, as a proxy for water use efficiency and as an indicator of environmental effects on stomatal behaviour and on photosynthesis during growth. Discrimination against 13C is enhanced by both high stomatal conductance and damage to photosynthetic machinery. Because the thinning of the stratospheric ozone layer is permitting more UV‐B to enter the biosphere, the mechanisms of action of UV‐B radiation on plants are of particular current interest. Arabidopsis thaliana wild‐type Landsberg erecta (Ler) and the UV‐B‐sensitive mutant fah I, deficient in UV‐absorbing sinapate esters, were grown in a controlled environment and exposed to UV‐BBE doses of 0 or 6–7 kJ m−2 day−1. UV‐B exposure decreased dry matter production and δ13C in both genotypes, but growth inhibition was generally greater in fah I than in Ler. The fah I mutant also had less leaf greenness than Ler. Changes in leaf tissue δ13C were detected before growth inhibition and were evident in treatments of both genotypes that did not cause marked growth effects. This suggests that the effects of UV‐B contributing to increased carbon isotope discrimination in Ler may have been primarily associated with high stomatal conductance, and in fah I with both high stomatal conductance and damage to photosynthetic machinery.