First Report of Fusarium proliferatum Causing Bulb Rot on Lilium davidii var. willmottiae in China.

Abstract

Lilium davidii var. willmottiae, known as Lanzhou lily, is widely cultivated in China for its edible bulbs. In July 2023, symptoms of bulb rot were observed on Lanzhou lilies harvested from Lanzhou, Gansu Province, during storage at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China), at an incidence of nearly 70%. The surface of the lily scales had dark water-stained spots, after the development of which the color gradually darkened, the bulbs became soft, accompanied by a pungent smell. Finally, the whole bulb became ruined and rotten, and there were thick mycelium layers on the bulbs. The infected bulbs were washed with clean water, sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 5 min, and then rinsed three times with sterile distilled water. The 5 mm×5 mm tissue pieces from the junction of the diseased part and the healthy part were clipped, placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 fungal isolates were obtained, all of which exhibited typical Fusarium characteristics. The colonies were white to pink with white to cream-colored aerial mycelia. After 10 to 15 days of incubation, the macroconidia (n = 50) were hyaline, relatively slender with a curve, three to five septate, and 8.73 to 33.24 × 2.16 to 4.19 μm in length. The microconidia (n = 50) were hyaline and pyriform, without septa, and measured 4.04 to 8.48 × 1.24 to 2.65 μm. These morphological characteristics were similar to those described for Fusarium proliferatum (Leslie and Summerell 2006). For molecular identification, a cetyltrimethylammonium bromide (CTAB) protocol was used to extract total genomic DNA (O'Donnell et al., 1998), after which the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymerase Ⅱ subunit 2 (RPB2) genes were amplified using the universal primers ITS1/ITS4, EF1/EF2 and RPB2-5f2/fRPB2-7cr, respectively, and subsequently sequenced (White et al., 1990; O'Donnell et al., 1998; Liu et al., 1999; Reeb et al., 2004; O'Donnell et al., 2007; Jiang et al., 2018). The sequences of a representative isolate (CAAS01) were analyzed and submitted to GenBank under accession numbers OR554007 (ITS), OR594233 (TEF1-α) and OR603932 (RPB2). A BLAST analysis revealed that the sequences of the ITS, TEF1-α, and RPB2 genes shared 100%, 100%, and 100% identity, respectively, with those of Fusarium proliferatum (MT466521.1, MK952792.1, and LT841266.1) in GenBank. In addition, the ITS, TEF1-α and RPB2 sequences shared 100%, 100%, and 100% identity with those of Fusarium annulatum (LC13675, the Fusarium fujikuroi species complex; previously known as the Gibberella fujikuroi species complex) in the Fusarium-ID database. Fusarium proliferatum, whose common synonyms are Gibberella fujikuroi mating population D and Gibberella fujikuroi var. intermedia, is the anamorphic form of the Gibberella fujikuroi complex that belongs to the Nectriaceae family. A phylogenetic tree was constructed based on the combined TEF1-α and RPB2 sequences of CAAS01 and other Fusarium isolates, revealing that CAAS01 was grouped with Fusarium proliferatum. Based on sequence alignment and phylogenetic analysis, the isolate was identified as Fusarium proliferatum. To determine the pathogenicity of the isolated fungi, healthy bulbs were punctured with disposable sterilized needles and soaked in equal amounts of sterile water and conidial suspension (1×107 conidia/mL) for 30 min respectively. The pathogenicity experiment was repeated three times. After 7 days of inoculation at 25 °C and 80% relative humidity, the surface of the inoculated bulbs produced water-stained spots and mycelium layers consistent with the symptoms exhibited by Lilium davidii var. willmottiae bulbs during storage, while the uninoculated lily bulbs remained symptomless. Fusarium proliferatum was reisolated from the infected bulbs and identified based on morphological and molecular characteristics, fulfilling Koch's postulates. To our knowledge, this is the first report of bulb rot on Lilium davidii var. willmottiae caused by Fusarium proliferatum in China. This study will contribute to the development of management strategies for this postharvest disease in Lilium davidii var. willmottiae.