Michael Rott

Application of a real-time PCR method for the detection of pine wood nematode, Bursaphelenchus xylophilus, in wood samples from lodgepole pine

A real-time PCR polymerase chain reaction (real-time PCR) method was developed to detect and differentiate Bursaphelenchus xylophilus (pine wood nematode, PWN) from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus heat shock protein 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 0.005 ng of B. xylophilus genomic DNA, as well as DNA extracted from single nematodes. The practical application of this real-time PCR diagnostic method for the detection of B. xylophilus from actual wood samples of lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees containing a heterogeneous population of nematodes, rather than pure cultures or individual nematodes, is demonstrated. This method works well in the presence of potential inhibitors associated with wood after Baermann extraction and thus eliminates the need to produce pure nematode samples through further culturing and/or isolation of nematodes with a high-power microscope.

Detection of Genetically Modified Canola Using Multiplex PCR Coupled with Oligonucleotide Microarray Hybridization

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.

An effective PCR-based diagnostic method for the detection of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in wood samples from lodgepole pine

A molecular diagnostic method has been designed for the detection and identification of Bursaphelenchus xylophilus. Heat shock protein 70 gene sequences from B. xylophilus and the closely related B. mucronatus were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by PCR. As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimisation, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than one nematode. Species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta var. latifolia) trees from British Columbia, Canada, containing an unknown nematode population, thus bypassing the need for culturing or recovering the nematode before analysis.

Microarray immunoassay for the detection of grapevine and tree fruit viruses

Development and application of DNA microarrays for plant disease diagnosis has to date been limited, and for antibody arrays even more so. In this work, an antibody microarray procedure was developed and its usefulness for the detection of plant viruses demonstrated. Using the conventional monoplex immunoassay ELISA technique as a benchmark, the procedure was used to detect several grapevine and tree fruit viruses. In a direct labelling approach, Arabis mosaic virus (ArMV), and Grapevine fanleaf virus (GFLV) were detected after incubating the antibody array with alkaline phosphatase-conjugated viral extract. Indirect detection using a double or triple antibody sandwich format also resulted in good reaction signals, using either a chromogenic or fluorescence dye. In a multiplex system, four grapevine viruses were detected without compromising sensitivity and specificity. Compared to ELISA, the antibody microarray system is similar with respect to sensitivity and specificity, and a high correlation (R(2), 0.759) was observed in regression analysis of virus concentration measurements. Receiver operating characteristic (ROC) curve analysis provided evidence of the good performance of the microarray system (AUC>0.8).

Amplification-free detection of grapevine viruses using an oligonucleotide microarray.

A single-colour microarray hybridization system was designed and evaluated for the detection of viruses infecting grapevine. Total RNA (≥0.5μg) from infected plants was converted to cDNA and labelled with Cy3 using two different strategies. While amine-modified and labelled cDNA was adequate for the detection of nepoviruses, the 3DNA technique, a post-hybridization detection method that uses intensely fluorescent dendrimer reagents, was required for the detection of closteroviruses in infected plants. Threshold detection levels were based on the ratio between viral specific and 18S rRNA positive control signal intensities. Oligonucleotides between 27 and 75 nucleotides in length were evaluated and compared. Viruses detected include eight nepoviruses, two vitiviruses, and one each of closterovirus, foveavirus, ampelovirus, maculavirus and sadwavirus. Results of this work demonstrate the potential of microarray technique to detect viral pathogens without sequence bias amplification of template RNA.

Occurrence and Detection of Globodera rostochiensis on Vancouver Island, British Columbia: An Update

The potato cyst nematode (PCN), Globodera rostochiensis, has been present in Central Saanich on Vancouver Island for at least 45 years. Eradication/control efforts have been ongoing, with regulations enacted in the early 1980s restricting the planting of host crops and movement of soil. Surveys monitoring for cyst populations have been minimal since the regulations have been in place with only one limited study in the early 1990s. In this report, a survey of eight fields was undertaken, chosen as the most likely sites that may still harbor viable PCN cysts. Conventional sampling/detection methods were considered inadequate for the detection of very low cyst populations, and an innovative bioassay was developed to improve detection while minimizing costs and labor. Viable cysts were recovered from two fields, both with past quarantine infractions. Fields with no known infractions were found free of viable cysts. Lack of viable cysts found in fields with no infractions suggests that the quarantine restrictions in place since the early 1980s have been effective in reducing or eliminating PCN from these fields. Further systematic and comprehensive retesting of all fields within the quarantine zone is now required, which could lead to the reduction or lifting of some quarantine restrictions.

Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control

The majority of plant viruses contain RNA genomes. Detection of viral RNA genomes in infected plant material by next generation sequencing (NGS) is possible through the extraction and sequencing of total RNA, total RNA devoid of ribosomal RNA, small RNA interference (RNAi) molecules, or double stranded RNA (dsRNA). Plants do not typically produce high molecular weight dsRNA, therefore the presence of dsRNA makes it an attractive target for plant virus diagnostics. The sensitivity of NGS as a diagnostic method demands an effective dsRNA protocol that is both representative of the sample and minimizes sample cross contamination. We have developed a modified dsRNA extraction protocol that is more efficient compared to traditional protocols, requiring reduced amounts of starting material, that is less prone to sample cross contamination. This was accomplished by using bead based homogenization of plant material in closed, disposable 50ml tubes. To assess the quality of extraction, we also developed an internal control by designing a real-time (quantitative) PCR (qPCR) assay that targets endornaviruses present in Phaseolus vulgaris cultivar Black Turtle Soup (BTS).

Comparative analysis of cherry virus A genome sequences assembled from deep sequencing data

Cherry virus A (CVA) is a ubiquitous graft-transmissible virus that mainly infects Prunus spp. Next-generation sequencing was applied to 39 tree fruit specimens infected with CVA, and 75 full and 16 partial-length CVA genome sequences were assembled. Phylogenetic analysis of these and 11 previously sequenced CVA genomes resulted in six major clusters with no observable relationship between the host and the assembled genome sequences. Recombination analysis detected four recombinants. Consistent single-nucleotide polymorphism (SNP) patterns were observed between the 75 full-length genomes and their sequence clouds, which supports a quasispecies model for CVA evolution.

Incidence, distribution and genetic diversity of Grapevine red blotch virus in British Columbia

Abstract Grapevine red blotch virus (GRBV) is a recently identified virus reported mainly from several grape-growing regions in the USA. To date, no information about the occurrence of GRBV is available in British Columbia (BC). Accordingly, a large-scale survey was conducted across all grape-growing regions of BC to determine the incidence and distribution of GRBV. A total of 2000 composite samples representing nine white and eight red Vitis vinifera cultivars from 128 vineyard blocks were tested for GRBV by PCR using virus-specific primers. The results showed that 32 out of 2000 (1.6%) composite samples were positive for GRBV, indicating a low incidence of this virus in BC vineyards. An analysis of the vineyard blocks positive for GRBV revealed that they were established between 2011 and 2014, suggesting a recent introduction of GRBV into BC, probably through infected plant material. Complete genome characterization and phylogenetic analysis of 29 representative GRBV isolates from BC grouped 15 isolates into clade I, sharing less than 95% identity at the nucleotide level, whereas 14 isolates grouped into clade II, sharing more than 95% identity at the nucleotide level. This result was consistent with the presence of both genetic variants in BC vineyards. Putative recombination events were detected among aligned complete genome sequences of BC isolates of GRBV and global isolates of the virus. These findings underscore the need for domestic clean plant propagation programmes and development of long-term management strategies to minimize GRBV spread in BC.

Epidemiology and Genetic Diversity of Grapevine Leafroll-Associated Viruses in British Columbia

Grapevine leafroll disease (GLD) is a complex associated with one or more virus species belonging to the family Closteroviridae. The majority of viruses in this complex are vectored by one or more species of mealybugs (Pseudococcidae) and/or scale insects (Coccidae). Grape-growing regions of British Columbia (BC), including Okanagan, Similkameen, and Fraser valleys and Kamloops (BC central interior), Vancouver, and Gulf islands, were surveyed during the 2014 and 2015 growing seasons for the presence of four major grapevine leafroll-associated viruses, including Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, and GLRaV-4. In total, 3,056 composite five-vine samples were collected from 153 Vitis vinifera and three interspecific hybrid vineyard blocks. The results showed GLRaV-3 to be the most widespread, occurring in 16.7% of the composite samples, followed by GLRaV-4 (3.9%), GLRaV-1 (3.8%), and GLRaV-2 (3.0%). Mixed infections of two or more GLRaVs were found in 4.1% of the total samples. The relative incidence of GLRaVs differed among regions and vineyard blocks of a different age. Characterization of partial CO1 region from a total of 241 insect specimens revealed the presence of Pseudococcus maritimus, Parthenolecanium corni, and other Pulvinaria sp. in BC vineyards. Spatial patterns of GLRaV-3 infected grapevines in three vineyard blocks from three different regions in the Okanagan Valley showed variable degrees of increase in disease spread ranging from 0 to 19.4% over three growing seasons. Regional differences in the relative incidence and spread of GLD underline the need for region-based management programs for BC vineyards.