First report of fusarium wilt disease caused by Fusarium equiseti on grafted watermelon in Korea.

Abstract

In Korea, most of the grafted watermelons are a fusion of bottle gourd (Lagenaria siceraria) as a rootstock and watermelon (Citrullus lanatus) as a scionstock (Lee et al., 2010). Currently, we have collected several samples from grafted watermelon displaying symptoms of yellowing, withered and wilting leaves. When the symptomatic stem was excised, browning vascular tissues were observed due to the colonization of fungal pathogen. From the samples obtained, 25 fungal isolates were identified as species of Fusarium. Among 25 isolates, 18 were identified as Fusarium oxysporum, four as Fusarium solani, and three as Fusarium equiseti (F. equiseti) . Initial assessment showed that one of the F. equiseti isolates (NIHHS 16-126) was highly virulent to rootstock. Interestingly, this is the first time F. equiseti has been identified pathogenic to grafted watermelon. NIHHS 16-126 isolate was collected from watermelon cultivation field around Buyeo-gun (36.25951°N, 126.92044°E) county. Disease incident was estimated to infect approximately 10% of the watermelon plants cultivated in this area. NIHHS 16-126 isolate was examined to confirm its identity. On potato dextrose agar, colonies appeared yellowish-brown while the aerial mycelium was whitish to peach in color. Macroconidia were relatively long (20.21 - 51.13 × 2.30 - 4.5 μm, n=50), comprise of 3-6 septa, curvature shape and its conidiophores were with monophialides. However, microconidia formation was not observed. These morphological characteristics resemble F. equiseti characters as described by Hyun (2019). For molecular identification, an internal transcribed spacer of ribosomal DNA (ITS-rDNA), elongation factor-1α (EF-1α), and beta-tubulin (β-tub) genes were sequenced using primer pairs of ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Glass and Donaldson 1995), and Bt2a/Bt2b (Carbone and Kohn 1999). BLASTN analysis revealed that ITS-rDNA (LC648248), EF-1α (LC648250), and β-tub (LC648249) sequences were 99-100% identical to F. equiseti reference sequences (KF515650, KF747331, and KF747330) infected Avicennia marina in China (Lu 2014). Phylogenetic analysis of concatenated ITS-rDNA, EF-1α and β-tub sequences showed that this isolate clustered in the same clade as F. equiseti, confirming its identity as F. equiseti. For the inoculation, roots of 12-days-old seedlings (watermelon and bottle gourd, n=10 each) were dipped in the conidia suspension (1x106 conidia/µL) for 30 min. Inoculated seedlings were planted in the soil before being transferred to the greenhouse (temperature; 30°C, daylight; 14 hours). Control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated bottle gourd seedlings (n=10) wilted and eventually died. In contrast, none of the inoculated watermelons or control seedlings were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch's postulates. Bottle gourd is the most preferred rootstock for grafted watermelons among Korean farmers due to its ability to resist Fusarium spp. infection. Therefore, the identification of F. equiseti as a fungal that is pathogenic to rootstock is crucial information to manage fusarium wilt disease among grafted watermelon. To our knowledge, this is the first report confirming F. equiseti infection in grafted watermelon plants in Korea.