A rapid, sensitive and field-deployable isothermal assay for the detection of Verticillium alfalfae

Abstract

The genus Verticillium contains 10 plant pathogenic species that are responsible for billions of dollars of crop losses annually on more than 400 plant species worldwide. On alfalfa, Verticillium alfalfae causes Verticillium wilt, a disease with the potential to reduce yields of susceptible cultivars by the second harvest year and to limit the productive stand life to less than 3 years. Susceptible alfalfa cultivars exhibit disease symptoms that include chlorotic V-shaped lesions at the leaf tip that cause entire leaflets to become bleached and twisted, leading to defoliation. Stems often remain green after all leaflets have desiccated, and discolouration, stunting and wilting of shoots is visible. Internally, the taproot can show yellow to brown vascular discolouration. 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 translation elongation factor 1α (TEF-1α) 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, non-specific amplification of closely related V. nonalfalfae or other Verticillium species was not observed. Detection of V. alfalfae in inoculated alfalfa cultivars with high or low resistance 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.