Fusarium Wilt on imperial chrysanthemum (C. morifolium) caused by Fusarium oxysporum in China

Imperial chrysanthemum (Chrysanthemum morifolium) is cultivated in China as medicinal plant. In October 2018 at Yunfu in Guangdong province, wilted stems, necrotic leaves, and root rot were observed on many imperial chrysanthemum plants. The incidence of disease was up to 50%. Infected plants were collected for further examination and fungal isolation. Small pieces of symptomatic tissues were surface sterilized with 75% alcohol for 10 s followed by 2.5% NaClO for 15 s, rinsed in sterile water three times, air dried, and transferred onto potato dextrose agar medium at 25°C for 7 days. Pure cultures were obtained by isolating single hyphae. Colonies were initially white, but after about 17 days, the center of the colony began to turn purple. The colonies produced were hyaline. Microconidia were single-celled, ellipsoid to cylindrical, and 3.1 to 5.4 × 1.3 to 2.7 μm. Fusiform macroconidia were produced on carnation leaf agar, which were mostly three-septate, 12.5 to 24.0 × 2.0 to 3.8 μm, with a prominent foot-shaped basal cell. The observed traits were identical to those reported for other Fusarium species (Leslie and Summerell 2006). Genomic DNA was extracted from isolated colonies and amplified with three pairs of primers: universal primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (Geiser et al. 2004), and FO1/FO2 (Fernandez-Ortuno et al. 2010). Isolates were identified to species by comparing the DNA sequences generated using BLAST against sequences in GenBank’s nucleotide database, Fusarium-ID, and Fusarium MLST. The 558-bp ITS sequence (GenBank MK385594) showed 100% identity to Fusarium oxysporum (GenBank MH911375 and SH1610159.08 FU JQ039192). The 712-bp TEF1α (GenBank MK404060) showed 99.85% identity to F. oxysporum (FD_00576), 100% identity to F. oxysporum (GenBank MG670365, JF430176, and SH1610159.08 FU JQ039192). PCR primers FO1/FO2 amplified a 699-bp cytochrome P450 (GenBank MK404061) that showed 100% identity to F. oxysporum (GenBank QDZ38461) by BLASTx in the NCBI. Based on the morphological characteristics and nucleotide homology, the isolate was identified as F. oxysporum. To satisfy Koch’s postulates, a pathogenicity experiment was conducted by using a spore suspension. Healthy plants’ roots were wounded with a sterile toothpick and soaked in spore suspension (∼1 × 10⁶ conidia/ml) for half an hour. The negative control was treated with distilled water. All of the plants were then placed in a greenhouse at 28°C, 60% relative humidity, and a 12-h/12-h light/dark photoperiod. After 7 days, leaves on the inoculated plants turned yellow and withered, and black necrotic areas appeared at the base of the stem. Sections of the stem revealed vascular bundle browning. The negative control plants remained healthy. The pathogen was reisolated from the lesions, and its identity was confirmed morphologically, thereby satisfying Koch’s postulates. To our knowledge, this is first report of F. oxysporum causing Fusarium wilt on imperial chrysanthemum in China.