Development and Application of Rapidly Deployable In-Field Molecular Diagnostics for Plant Diseases
Date: May 2016
Term: 3 years
Status: Completed
Researcher(s): Tim Dumonceaux, Chrystel Olivier, Matthew Links and Hossein Borhan, Agriculture and Agri-Food Canada, Saskatoon SK
SaskCanola Investment: n/a
Total Project Cost: n/a
Funding Partners: ADF
Project Summary
Crop diseases can be devastating to the profitability of producers, and certain diseases are particularly problematic, such as clubroot, blackleg and aster yellows. Researchers at Agriculture and Agri-Food Canada initiated a project in 2013 to develop rapid diagnostic methods for a range of pathogens along with methods for rapid DNA extraction in a non-laboratory setting and apply these methods to the detection of crop pathogen DNA in agricultural products. This project provides tools for both producers and regulators to obtain crucial, timely information on plant disease prevalence and spread, which will aid efforts to contain and control these diseases.
Crop diseases can be devastating to the profitability of producers, and certain diseases are particularly problematic. For some diseases, there is an immediate need of diagnostics development, which makes the development of rapid and accurate detection methods important and timely. Although there are DNA sequencebased methods readily translatable to a variety of pathogens, they normally require complex DNA extraction and PCR amplification steps, along with DNA sequencing, which in total can require several days to get final results. Molecular diagnostics can play an important role by providing relatively rapid, accurate data that often includes strain information. Such information can be important in terms of identifying pathotypes for the purposes of disease surveillance and can also in some cases distinguish strains of variable virulence.
Researchers at Agriculture and Agri-Food Canada in Saskatoon, Saskatchewan initiated a three-year study in 2013 to develop rapid diagnostic methods for a range of pathogens along with methods for rapid DNA extraction in a non-laboratory setting and apply these methods to the detection of crop pathogen DNA in agricultural products. The objectives of the project were the development of molecular assays that are capable of rapidly and specifically detecting various strains of Plasmodiophora brassicae (clubroot); Phytoplasma spp. (e.g. Aster Yellows); and Leptosphaeria maculans (blackleg). Researchers also wanted to develop and test in-field DNA extraction and testing methodologies.
For this project, researchers focused on exploiting the unique features of loop-mediated isothermal DNA amplification (LAMP), which is a simple, rapid, inexpensive diagnostic tool that can yield results in as little as one hour. Furthermore, a positive reaction can yield strain-level data, reducing the need for DNA sequencing to confirm strain identity. The benefit is that DNA prepared for LAMP does not need to be extensively purified, which lends itself to simple, rapid, inexpensive DNA extraction. These features of LAMP make it an ideal diagnostic tool to bring into the agricultural field.
Researchers developed a panel of LAMP assays that would specifically detect problematic strains of P. brassicae, L. maculans, and phytoplasma. The panels were based on available sequence data, including complete genomes determined for P. brassicae and L. maculans, They also identified one readily available LAMP target for all strains, the chaperonln-60 (cpn60) gene, and demonstrated that the cpn60 universal target (cpn60 UT) is an appropriate molecular barcode for bacteria, and an effective target for LAMP, including for phytoplasma. Researchers also had access to a wide variety of infected material from many different strains of each of the targeted pathogens, and will be able to exploit genomic or barcode sequence data to develop effective LAMP assays for many strains of interest.
Photo:
Canola plant symptomatic with aster yellows. (top)
Results of LAMP molecular Diagnostic Assay on the plant, with the positive result (yellow) observed within 30 minutes. The pink tubes are controls and healthy plants. (Bottom)
Photo Credit: Dr. Tyler Wist. AAFC.
As a result of the project, field-deployable assays based on LAMP have been developed and validated for all of the targeted pathogens. The expertise developed while designing and validating these assays led the researchers to develop rapid diagnostic assays for other pathogens, such as the ergot pathogen (Claviceps purpurea) and Verticillium longisporum, as well as dutch elm disease, fruit tree diseases and others. As a result of this project, researchers now have the capacity to respond rapidly to producer needs with regards to the detection of pathogen DNA in agricultural samples. This includes unexpected emerging pathogens (eg. Verticillium), which can create a need for the rapid development of molecular diagnostics targeting pathogens that may not have been anticipated in advance.
A second aspect of this project involved the development of in-field DNA extraction for application of the diagnostic tools developed. Researchers have begun to perfect field-based DNA extraction methods for rapid and mobile pathogen detection. This project provides tools for both producers and regulators to obtain crucial, timely information on plant disease prevalence and spread, which will aid efforts to contain and control these diseases. They have also applied this method to the rapid extraction of DNA from insects and have a functional protocol in place.
Scientific Publications
Pérez-López, E., D. Rodríguez-Martínez, C.Y. Olivier, M. Luna-Rodríguez, and T.J. Dumonceaux. 2017. Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico. Scientific reports 7:950.
Comte, A., T. Gräfenhan, M.G. Links, S.M. Hemmingsen, and T.J. Dumonceaux. 2017. Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination. PLOS ONE 12:e0173495.
Dumonceaux TJ, Green M, Hammond C, Perez E, Olivier C. 2014. Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences In Host Plant Infection Patterns. PLoS ONE 9:e116039.
Links MG, Demeke T, Grafenhan T, Hill JE, Hemmingsen SM, Dumonceaux TJ. 2014. Simultaneous profiling of seedassociated bacteria and fungi reveals antagonistic interactions between microorganisms within a shared epiphytic microbiome on Triticum and Brassica seeds. New Phytol. 202:542-553.
Full Report PDF: n/a