Search Results (8)

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ⁹-tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures. Full article

Peach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis. Full article

High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21–22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ. Full article

Metagenomic approaches used for virus diagnostics allow for rapid and accurate detection of all viral pathogens in the plants. In order to investigate the occurrence of viruses and virus-like organisms infecting grapevine from the Ampelographic collection Kromberk in Slovenia, we used Ion Torrent small RNA sequencing (sRNA-seq) and the VirusDetect pipeline to analyze the sRNA-seq data. The used method revealed the presence of: Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine fanleaf virus (GFLV) and its satellite RNA (satGFLV), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Pinot gris virus (GPGV), Grapevine satellite virus (GV-Sat), Hop stunt viroid (HSVd), and Grapevine yellow speckle viroid 1 (GYSVd-1). Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for validation of sRNA-seq predicted infections, including various combinations of viruses or viroids and satellite RNA. mRT-PCR could further be used for rapid and cost-effective routine molecular diagnosis, including widespread, emerging, and seemingly rare viruses, as well as viroids which testing is usually overlooked. Full article

Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay. Full article

Gene amplification techniques such as polymerase chain reaction (PCR) are widely used for the diagnosis of plant diseases caused by viruses and viroids. It is preferable that sample preparation methods for PCR or reverse transcription (RT) PCR are rapid, straightforward, and inexpensive. We previously reported a method for the extraction of nucleic acids without mechanical tissue grinding using a buffer containing potassium ethyl xanthogenate (PEX) to detect viroid RNAs. In the present report, the previous PEX method was improved and simplified. In the simplified PEX (SPEX) method, the process of PEX buffer treatment for plant cell wall disruption is improved to one step of incubation at 80 °C for 10 min, instead of three steps that took more than 26 min at 65 °C in the previous method. Total nucleic acids could be extracted from fresh, frozen, or dried leaves of a cultivar or wild species of tobacco, tomato, citron, hop plants, and pericarps of persimmon fruits by the SPEX method. Several RNA viruses and viroids were successfully detected from the extracted nucleic acids together with an internal mRNA by RT-PCR. The SPEX method may be useful for detecting not only viruses and viroids, but also other plant pathogens. Full article

Chrysanthemum stunt viroid (CSVd) is one of the most severe threats in Chrysanthemum morifolium production. Over the last decade, several studies have reported the natural occurrence of CSVd resistance in chrysanthemum germplasms. Such CSVd-resistant germplasms are desirable for the stable production of chrysanthemum plants. Current surveys include finding new resistant chrysanthemum cultivars, breeding, and revealing resistant mechanisms. We review the progress, from discovery to current status, of CSVd-resistance studies, while introducing information on the improvement of associated inoculation and diagnostic techniques. Full article

Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future. Full article