First report of Fusarium triseptatum causing crown rot on Gypsophila paniculata in Pichincha, Ecuador

Gypsophila paniculata (commonly known as baby´s breath) is a key ornamental species cultivated for exports in Ecuador. In January 2023, crown rot symptoms were observed in the production fields of the province of Pichincha (0, 17133º N, 78, 41120º O). Affected plants exhibited symptoms of leaf and stem discoloration, wilting, and progressive crown and root rot, leading to plant death. Disease incidence reached approximately 60% within a 1-hectare plot (Fig. S1).The disease was observed not only in the sampled plot but also across major G. paniculata production areas in Pichincha Province. Five symptomatic plants were sampled to identify the causal agent. Stem fragments (~ 5 cm) were disinfected using 1% sodium hypochlorite, followed by 70% ethanol, then rinsed with sterile distilled water. Tissue pieces (~0.5 cm²) were aseptically transferred to potato dextrose agar (PDA) supplemented with gentamicin (160 mg/l). Isolates were purified by the hyphal tip method. Only two pure isolates (G13 and G15) were identified and exhibited white to purple mycelial growth. Microscopic examination revealed septate macroconidia with 3-5 septa (mean length 14.8 µm, width 3.9 µm), and unicellular oval microconidia (mean length 5.3 µm, width 2.9 µm) (Fig. S1). To confirm pathogenicity, ten healthy G. paniculata plants were grown in pots containing a sterile mixture of soil and sand (1:1). A conidial suspension (1 × 106 conidia/ml) was prepared in sterile distilled water. Each plant was inoculated by pouring 200 ml of suspension around the base of the stem (Chu et al., 2024). Plants were maintained at 18-25 °C, with a 12h/12h light/dark photoperiod. Symptoms similar to those observed in the field appeared after 20 days, and all inoculated plants exhibited disease symptoms with both isolates. Control plants treated with sterile distilled water remained asymptomatic. The pathogen was re-isolated from diseased tissues and exhibited identical morphological and microscopic characteristics to the original isolate. Genomic DNA was extracted using the Qiagen PowerSoil Kit. The internal transcribed spacer (ITS) region was amplified using primers ITS1 and ITS4 (White et al., 1990). Additionally, the translation elongation factor 1-alpha (TEF1-α) gene was amplified with EF1 and EF2 primers (O'Donnell et al., 1998), and the second largest subunit of RNA polymerase II (RPB2) with primers fRPB2-5F and fRPB2-7cR (Liu et al., 1999). Amplicons were sequenced and later assembled using MEGA 11 (Tamura et al., 2021). The sequences showed 100% identity among isolates and high similarity to Fusarium triseptatum sequences in GenBank. The sequences were deposited under the following GenBank accession numbers: ITS (PV704617), TEF (PV712478), RPB2 (PV726240). A Bayesian phylogenetic tree was constructed using Beast V10.5.0 and concatenated sequences from the FUSARIOID-ID database (Westerdijk Fungal Biodiversity Institute, The Netherlands). The concatenated sequences of the isolates (represented as F2) clustered the Fusarium oxysporum species complex (FOSC), specifically within the F. triseptatum clade, confirming its identity (Fig. S2). To our knowledge, this is the first report of Fusarium triseptatum causing crown and root rot on G. paniculata both in Ecuador and worldwide. This finding highlights the need for disease monitoring and integrated management strategies to prevent potential economic losses in the floriculture sector.