First Report of Daylily Flower Rot Caused by Fusarium proliferatum in China

Daylily (Hemerocallis citrina Baroni) is an important plant whose flowers are commonly used as a vegetable and in medicine in China. From June 2015 to July 2016, brown and rotted flowers, some of which were covered with white mycelia, were observed in a 0.8-acre commercial daylily field in Yanqing District, Beijing, China. Disease incidence was ∼40%. A total of 24 diseased flowers were collected, cut into 0.5 cm diameter sections from the edge of the rot/healthy tissues, surface-sterilized with 1% NaClO for 1 min followed by 75% ethanol for 30 s, and then rinsed twice with sterilized distilled water. Five surface-sterilized sections per flower were placed on potato sucrose agar (PSA) and carnation leaf agar (CLA) (Leslie and Summerell 2006) and incubated for 5 days at 25°C with a 12-h light-dark cycle in a chamber. Seventy-eight singe-spore isolates with similar morphological features as determined by a microscope were obtained (Song et al. 2017), and 24 were used as the research materials and incubated on PSA and CLA. The colonies on PSA were initially white, becoming purple, with rose to dark purple pigment on the surface. The macroconidia were abundant on CLA, hyaline, straight to slightly curved, 3 to 5 septate, 21.4 to 49.3 × 2.3 to 4.9 µm. Microconidia were oval, obovoid, or ellipsoidal, 0 to 1 septate, 4.5 to 15.1 × 2.2 to 4.3 µm. The cultural and morphological characteristics of the fungus were similar with previous reports of Fusarium proliferatum (Ritieni et al. 1995). Genomic DNA of all 24 isolates were extracted using the plant genomic DNA kit (Tiangen, China). The small subunit ribosomal RNA (mtSSU), elongation factor 1 alpha (EF-1α), and beta-tubulin (Tub) regions were amplified using the primers NSM1/NSM2 (Li et al. 1994), EF-1Ha/EF-2Tb (Alastruey-Izquierdo et al. 2008), and Beta1/Beta2 (Geiser et al. 2004). The amplicon sizes were ∼534, 691, and 745 bp, respectively. Three sequences of isolate HYC1410080201 were submitted to GenBank (accessions MF442355, MF448528, and MF442354, respectively). Isolates had 100% similarity with sequences of several isolates of F. proliferatum in GenBank (KX218406, KY081546, and KR861512, respectively). Pathogenicity was tested and Koch’s postulates satisfied in a growth chamber by inoculating daylily flowers. Briefly, spores were collected from 7-day-old PDA cultures of each isolate (n = 24) maintained at 25°C and suspended in sterilized water at 1 × 10⁶ conidia/ml. Spore suspension, 5 ml from each isolate, was sprayed onto the flowers of a plant with 10 plants inoculated per fungal isolate. Control plants were sprayed with 5 ml of sterile water. Each isolate was replicated three times. Five days after inoculation, small, pale yellow, water-soaked spots were observed on flowers of all inoculated plants. Under high humidity conditions, inoculated flowers were covered with a layer of white mycelia. Infected flowers wilted and dropped off, similar to symptoms observed on plants in the field. No symptoms were observed on control plants. Isolates obtained from inoculated plants had identical morphological features to the original isolates. Thus, Koch’s postulates were fulfilled and showed that F. proliferatum was the causal agent. To our knowledge, this is the first report of F. proliferatum causing daylily flower rot worldwide. Since this disease occurs during flowering and seriously affects edible and medicinal value, its presence has economic implications for daylily production.