Disease Progress and Detection of a California Resistance-Breaking Strain of Tomato Spotted Wilt Virus in Tomato with LAMP and CRISPR-Cas12a Assays

Use of tomato cultivars with the Sw-5 resistance gene cluster has led to the occurrence of resistance-breaking (RB) tomato spotted wilt virus (TSWV) strains globally, including California and, recently, North Carolina and Texas. We documented disease on tomato infected with either an RB strain from California (CA-RB) or a wild type (CA-WT) strain of TSWV on tomato with (cultivar Mountain Merit) or without (cultivar Mountain Fresh Plus) the Sw-5b resistance gene and detected virus incidence over time using microneedle RNA extractions and LAMP. We developed a LAMP/Cas12a assay for detection of the CA-C118Y mutation in a CA-RB strain and tested the assay with field samples. Disease in the susceptible cultivar was less severe with CA-RB than with the CA-WT strain. In contrast, the resistant cultivar had little disease when inoculated with the CA-WT strain but exhibited stunting of greater than 50% when inoculated with the CA-RB strain. In the susceptible tomatoes, the detection rates over time by LAMP reaction were higher in CA-WT than in CA-RB-inoculated plants. In resistant tomato, CA-RB remained detectable by TSWV LAMP over 14 days, whereas the WT strain was undetectable. A two-step LAMP/Cas12a assay differentiated the two strains in 1 h. Our methods were validated with samples from TSWV-infected North Carolina fields. A phylogeny of NSm gene sequences that included North Carolina field samples revealed two independent origins of the North Carolina RB isolates. The LAMP/Cas12 assay showed excellent detection of the CA-C118Y mutation. The TSWV LAMP/Cas12a assay is adaptable for in-field applications on either a smart phone platform or heat block. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .