First Report of Root and Hypocotyl Rot of Dry Bean Caused by Aphanomyces euteiches in Alberta, Canada.

Dry bean (Phaseolus vulgaris L.) is a high-value crop grown under irrigation on 45,000 ha in southern Alberta, Canada. In 2019, one field of red beans showed premature yellowing and stunting of shoots in mid-July near Bow Island, AB. Roots had brown lesions along the length of hypocotyl and tap root, and decay and sloughing of the root cortex of lateral roots (Fig. 1). Roots were washed for 10 min under running tap water, and portions of the lesions were excised for DNA extractions using the Plant DNeasy kit (Qiagen) and multiplex PCR assays for an array of pathogens, which included Aphanomyces euteiches (Chatterton et al. 2019). Lesion pieces were also surface sterilized and plated onto PDA or cornmeal agar amended with metalaxyl, benomyl and vancomycin (CMBV) and visualized microscopically for oospores. The roots were PCR positive for A. euteiches, Rhizoctonia solani, and Pythium ultimum. Isolations yielded R. solani, and Fusarium spp. on PDA, but cultures on CMBV were over-grown with Rhizopus. Oospores measuring 24.7 +/- 1.08 m, consistent with the expected size of A. euteiches oospores of 18 - 25 m (Papavizas and Ayers, 1974), were visible in some of the root pieces. To obtain an A. euteiches isolate, soil was collected from three areas of the red bean field and used in a bait assay. Tests were performed using pea (cv. CDC Meadow) and dry bean (cv. Redbond) seeds treated with metalaxyl. Five seeds per pot were planted into field soils in 10-cm pots with 4 replicate pots/field. Soils were watered as needed until the 2nd node stage and then kept at saturation for 14 days, at which time roots were washed. Roots from the dry bean, but not the pea, plants showed severe root rot, including honey-brown discolouration of the lateral roots and extending to the hypocotyl, degradation of the root cortex, and presence of oospores. Diseased root pieces were plated onto CMBV without surface sterilization. Cultures with fast growing, white, aerial mycelia characteristic of A. euteiches on CMBV were recovered from dry bean roots from the three soil samples, considered as three isolates, and were transferred to PDATo confirm identity, total DNA was extracted from 7-day old cultures of the three isolates growing on PDA using the Qiagen DNeasy Plant Kit. The ribosomal DNA internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 and ITS4, and sequenced (White et al. 1990). The sequences, deposited in GenBank with accession numbers OM976770, OM976771, and OM976772, were 100% identical to the ITS rDNA sequence of several isolates of A. euteiches (e.g. KM486066.1; 669/669 bp; 670/670 bp; 670/670 bp) using a BLASTn query and 99.7% identical to the CBS15773 voucher specimen (HQ643116.1, 661/663 bp; 662/664 bp; 662/664 bp). To confirm pathogenicity of these three isolates, cultures were used to produce zoospores for inoculating 14-day old red bean and pea seedlings (Chatterton et al. 2015). Control plants received minimal salt media solution and did not develop symptoms. Dry bean, but not pea, seedlings displayed root rot symptoms (Fig. 2). A. euteiches was re-isolated from dry bean seedlings by plating onto CMBV, and identity confirmed as described above. Although a previous survey detected A. euteiches in a small number of dry bean fields in southern Alberta (Chatterton et al. 2019), tests to confirm Koch's postulates were not performed. Aphanomyces root rot can be a devastating pathogen of pulse crops. Precautionary measures should be taken to prevent spread of this pathogen in the small dry bean growing area of southern Alberta by encouraging producers to use rotation intervals, monitoring movement of the pathogen, and evaluating cultivars for resistance to this pathogen.

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform.

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot-and-mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user-developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user-intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT-PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn-around time of approximately 6 hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross-reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.

Microarray for Identification of the Chiropteran Host Species of Rabies Virus in Canada.

Species identification through genetic barcoding can augment traditional taxonomic methods, which rely on morphological features of the specimen. Such approaches are especially valuable when specimens are in poor condition or comprise very limited material, a situation that often applies to chiropteran (bat) specimens submitted to the Canadian Food Inspection Agency for rabies diagnosis. Coupled with phenotypic plasticity of many species and inconclusive taxonomic keys, species identification using only morphological traits can be challenging. In this study, a microarray assay with associated PCR of the mitochondrial cytochrome c oxidase subunit I (COI) gene was developed for differentiation of 14 bat species submitted to the Canadian Food Inspection Agency from 1985-2012 for rabies diagnosis. The assay was validated with a reference collection of DNA from 153 field samples, all of which had been barcoded previously. The COI gene from 152 samples which included multiple specimens of each target species were successfully amplified by PCR and accurately identified by the microarray. One sample that was severely decomposed failed to amplify with PCR primers developed in this study, but amplified weakly after switching to alternate primers and was accurately typed by the microarray. Thus, the chiropteran microarray was able to accurately differentiate between the 14 species of Canadian bats targeted. This PCR and microarray assay would allow unequivocal identification to species of most, if not all, bat specimens submitted for rabies diagnosis in Canada.