Watermelon Fusarium Wilt Pathogenicity Assay

To prepare inoculum, colonized agar plugs from a 7-day-old PDA culture were transferred into a liquid MBL and grown for 4 days on a rotary shaker at 170 rpm at room temperature. Spores were collected by filtering through four layers of sterile cheesecloth and the spore suspension was adjusted to approximately 1 × 10⁷ spores/ml. Pathogenicity was tested by root-dip method using watermelon (Citrullus lanatus L.) cultivar Zaojia (a susceptible cultivar to race 1). Seedlings were grown in a vermiculite: plant ash: perlite (6:2:1) mixture in a growth room at 24°C with a cycle of 16 h light/8 h dark. Two-week-old seedlings were carefully uprooted and washed in tap water to remove soil particles. Roots of the seedlings were dipped for 30 s in spore suspensions prepared from WT, targeted disruption ΔFonNot2 and complementation ΔFonNot2-C strains. The inoculated seedlings were carefully replanted in the same growth medium and allowed for disease development. Disease scores were assessed 3 weeks after inoculation using the following rating scales: 0 = no symptom, 1 = yellowing, 2 = wilting and 3 = death. Fungal biomass measurement was performed as described previously (Thatcher et al., 2009 (link)). Root and stem samples were collected at 3, 6, and 9 days post-inoculation and the transcript levels of Fon FonOpm12 and watermelon ClRps10 genes were determined by qRT-PCR using a pair of FonOpm12-specific primers FonOpm12-F and FonOpm12-R and a pair of watermelon ClRps10-specific primers ClRps10-F and ClRps10-R, respectively (Supplementary Table S1). Relative fungal biomass was calculated by normalizing FonOpm12 to watermelon ClRps10 (Lin et al., 2010 (link)).