Fusarium falciforme, a pathogen causing wilt disease of chrysanthemum in Vietnam

Abstract

Chrysanthemums are flowering plants of the genus Chrysanthemum in the family Asteraceae, and are widely cultivated in Vietnam. The plants may be infected by a variety of pathogens, including Alternaria spp., Ascochyta chrysanthemi, Botrytis cinerea, Fusarium spp., Puccinia spp., Septoria spp. and Verticillium albo-atrum (Trolinger et al., 2017). Of these, Fusarium species are devastating pathogens causing stem, crown or root rot and wilt on chrysanthemum and leading to significant losses, especially in warm climates (Locke et al., 1985; Mun et al., 2012). In September 2020, more than 10% of infected plants showing root rot, stem rot and wilt were observed on chrysanthemums in growing areas of Northern Vietnam. Thirty-five diseased samples were taken from three different fields in suburban areas of Hanoi. Diseased roots and stems were cut into small pieces, dipped in sodium hypochlorite solution (1%) for 45 seconds then ethanol solution (70%) for 30 seconds. These pieces were rinsed three times with sterile distilled water, dried and placed onto potato dextrose agar (PDA) containing 0.02 g/l streptomycin and 0.02 g/l penicillin. Pure cultures were obtained by single spore isolation. On PDA, the colonies were white, covering a 9 cm petri-dish after 7 days at 25°C. Yellowish pigmentation was observed (reverse view) in the centre of old cultures (Figure 1). On carnation leaf-piece agar, the microconidia formed in false heads on monophialides and were hyaline, oval, elongated oval or reniform, 0–1 septate, 8.4 to 18 (mean 13.2) × 3.2 to 5.0 (mean 4.1) μm (n = 40). Macroconidia were hyaline, curved, 2–4 septate, 26.4 to 32.8 (mean 29.6) × 4.9 to 6.3 (mean 5.6) μm (n = 40) (Figure 2). Chlamydospores were globose to subglobose, produced singly, in pairs, chains and clusters, 8.4 to 9.0 (mean 8.7) μm diameter (n = 40) (Figure 3). A representative isolate (PPRI20101) was chosen for further study and stored in the culture collection of the Plant Protection Research Institute of the Vietnam Academy of Agricultural Sciences. Fungal genomic DNA of PPRI20101 was extracted using the E.Z.N.A Fungal DNA Mini Kit (OMEGA BioTek, USA), according to the manufacturer's instructions. The internal transcribed spacer (ITS) region was amplified and sequenced using the ITS1/ITS4 primers (White et al., 1990). An assembled sequence was deposited in GenBank (Accession No. MW493136). BLAST analysis showed that the ITS sequence of PPRI20101 had 100% identity to the type strain of Fusarium falciforme (NR_164424). Isolate PPRI20101 was ultimately identified as F. falciforme (syn. Neocosmospora falciformis) based on the combination of the ITS sequence analysis and morphological characteristics. Healthy chrysanthemum plants grown in sterile soil in pots (25 cm diameter, 25 cm height) were used to confirm the pathogenicity of F. falciforme PPRI20101. One hundred millilitres of a spore suspension of F. falciforme (1×106 spores/ml) was added to the soil in each pot. The same volume of sterile water was used for controls. The chrysanthemum plants were placed in a glasshouse at 25°C for 10 days. The experiment was repeated three times. The symptoms of root rot, stem rot and wilt were visually observed seven days post-inoculation and the same pathogen was re-isolated from symptomatic roots and stems. No pathogens were isolated from the roots and stems of the control plants. To our knowledge, this is the first report of Fusarium falciforme affecting chrysanthemum in Vietnam. Further studies are essential to develop successful disease management strategies. The authors are thankful to the Syngenta Company, Vietnam for sampling activities and funding support in this study.