First Report of <i>Verticillium dahliae</i> Race 3 Causing Verticillium Wilt of Potato in Minnesota

Verticillium wilt (VW) of potato is mainly caused by two species of fungal pathogens called <i>Verticillium dahliae</i> and <i>Verticillium albo-atrum</i> (Johnson et al. 2010). <i>V. dahliae</i> is a broad-host range pathogen that severely affects potato production. In a recent study, three pathogenic races (1, 2, and 3) of <i>V. dahliae</i> infecting lettuce from coastal California were identified based on the presence of three distinct effectors: Ave1, VdR2e, and VdR3e (Mandal et al. 2024). Potato plants with VW symptoms, including foliar chlorosis, flagging, and vascular discoloration, were observed in July and August of 2022 and 2023 on more than 80% of potato plants grown in a naturally infested experimental disease nursery farm in Sherburne County, Minnesota (45°20'18.0"N 93°49'41.2"W), a major potato growing county. In July 2023, multiple stem samples were collected and surface sterilized. After 5-7 days of growth on water agar, 12 colonies exhibiting microsclerotia and verticillate phialides were selected. The phialides were arranged in (1-)2-3(-8) whorls below the transverse septum and (1-)2-4(-6) whorls near the apex (Inderbitzin et al. 2011). Following genomic DNA isolation, species were verified through PCR amplification and sequencing of the internal transcribed spacer regions using ITS1/ITS4 and DF/DR Vd.ITS primers (Gardes et al. 1993; Inderbitzin et al. 2013) (Genbank accession no.: ITS1/4: PQ644054, PQ644303, PQ644449, PQ644509, PQ644510, PQ644586, PQ665302, PQ644587, PQ644605, PQ665303, PQ644606, PQ644629; DF/DR Vd.ITS: PQ644632, PQ644634, PQ644896, PQ644897, PQ644903, PQ644902, PQ644907, PQ644906, PQ645019, PQ665304, PQ645052, PQ645073, PQ662628, PQ662629). BLAST searches resulted in 99% sequence identity with V. dahliae. Isolate PN15.23 (ITS1/4: PQ644587, DF/DR: PQ644906) used for potato infection study, shared the highest nucleotide identity (99%, 531/536 bp, 99.57%, 458/460 bp) with <i>V. dahliae</i> isolated from <i>Aribdopsis arenosa</i> (ON193840). Race typing of isolates was conducted using race-specific effector primers for Ave1 (race 1), VdR2e (race 2), and VdR3e (race 3), with known isolates Ls16, Ls4325, and Ca4317 as PCR positive controls (Mandal et al. 2024). Amplification and sequencing of VdR3e confirmed the presence of <i>V. dahliae</i> race 3 (PQ662628 and PQ662629) with 99.37% (111/114 amino acid) protein sequence similarity (BLASTX) to PNH37750. To fulfill Koch's postulates, the PN15.23 isolate was used to infect five, six week old Umatilla potato plants grown in Sungro soil germination mix by inoculating the soil near the root zone with <i>V. dahliae</i> conidia (10 ml of 2x106 cells/ml). Plants were maintained in greenhouse conditions at 22.5°C, with a 16/8 light/dark photoperiod and 65% relative humidity. Three weeks post-infection, watering frequency was reduced to 33% to exacerbate infection symptoms. Infected plants displayed typical VW symptoms, while control plants had no visible VW symptoms. Re-isolation based on apparent morphology was successful from four out of five infected plants. These four had identical <i>V. dahliae</i> ITS1/4 and DF/DR sequences to PN15.23. No growth was observed in five controls. To our knowledge, this is the first report of race 3 V. dahliae causing VW in Minnesota or in potato, highlighting the potential for continued yield losses until resistant cultivars for all three races are identified or developed. These findings are crucial for plant pathologists and breeders developing disease management strategies.