Location: Horticultural Crops ResearchTitle: A rapid, sensitive and field-deployable isothermal assay for the detection of Verticillium alfalfae Author
|Reyes Gaige, Andres - Monsanto Company|
|Dung, Jeremiah - Oregon State University|
Submitted to: Canadian Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2018
Publication Date: 6/13/2018
Citation: Reyes Gaige, A., Dung, J.K., Weiland, G.E. 2018. A rapid, sensitive and field-deployable isothermal assay for the detection of Verticillium alfalfae. Canadian Journal of Plant Pathology. https://doi.org/10.1080/07060661.2018.1474262.
DOI: https://doi.org/10.1080/07060661.2018.1474262 Interpretive Summary: Verticillium alfalfae is an important soilborne pathogen that causes the disease Verticillium wilt and reduces alfalfa hay yield in susceptible alfalfa varieties. Verticillium alfalfae is also a quarantine pathogen that could be spread to other countries where resistant alfalfa varieties are not available. Therefore, we developed a rapid, DNA-based detection assay that could be used to test alfalfa hay and seed for the presence of the pathogen. We tested our detection assay to confirm that it was only able to detect V. alfalfae, and not other Verticillium species that are not pathogens of alfalfa. This reduces the chance of receiving a false detection when using the assay. We also tested the assay using infected plant samples and showed that we could detect very minute amounts of V. alfalfae. The assay that we developed is easily adapted to a kit that can be used by both alfalfa farmers and by diagnostic laboratories. These results are important because they provide a tool for both growers and inspection agencies to determine whether alfalfa hay and seed is contaminated with the quarantine pathogen, V. alfalfae.
Technical Abstract: The genus Verticillium contains 10 plant pathogenic species that are responsible for billions of dollars of damage annually to > 400 plant hosts worldwide. In alfalfa, Verticillium alfalfae causes Verticillium wilt, a serious disease of susceptible cultivars with the potential to reduce yields by the second harvest year and to limit the productive stand life to < 3 years. Susceptible alfalfa cultivars exhibit disease symptoms that include v-shaped chlorosis in leaflets, discoloration, stunting and wilting of shoots. International quarantines sometimes prohibit the entry of alfalfa samples contaminated with the Verticillium pathogen. In this investigation, we developed a recombinase polymerase amplification (RPA) assay coupled with a lateral flow device for the specific and sensitive detection of V. alfalfae. The primers targeted the TEF-1a gene and were designed with mismatches to increase the specificity of the assay. This assay detected as little as 800 fg of DNA from all tested isolates of V. alfalfae. Moreover, nonspecific amplification of closely related V. nonalfalfae or other Verticillium species was not observed. Detection of V. alfalfae in inoculated alfalfa cultivars with high resistance (55V50) and low resistance (La Jolla) further demonstrated the specificity and sensitivity of the diagnostic assay. Additionally, RPA detected V. alfalfae from inoculated plants using a crude DNA extraction method. The assay is easy-to-use and will allow growers, diagnostic labs, and regulatory agencies to determine if V. alfalfae is present in alfalfa products.