First Report of Fusarium falciforme Causing Fusarium Wilt on pepper in Hainan, China.

Pepper (Capsicum annuum L.) is a significant vegetable crop, valued for its nutritional and economic importance (Pang et al. 2023). Pepper cultivation in China accounts for about 8-10% of the total vegetable planting area, contributing an output value of approximately 250 billion yuan. This makes pepper the leading vegetable in terms of both planting area and economic value. In December 2023, a total of 70% disease incidence of Fusarium wilt was observed in a 1200 m² pepper seed breeding base in Sanya City, Hainan Province, China (18°38'60″ N, 109°16'51″ E). Symptoms initially appeared as wilting on upper leaves. Subsequently, the base of the stem started to necrosis, browning, and gradually spreading upward along the stem. As the lesions expanded, the whole plant gradually wilted and died. Ten diseased plants were randomly selected from the most severely affected area (667 m²). Diseased tissues (5 mm²) were subsequently removed from the lesion edges of these plants, surface sterilized in 75% ethanol for 30 s, and rinsed with sterile distilled water three times, finally cultured on potato dextrose agar (PDA) at 25 °C. Six fungal isolates were obtained using the single-spore isolation method (HN-01 to HN-06). Colonies produced white aerial mycelia with apricot pigments in the PDA medium. The spore morphology and size were observed and measured using synthetic nutrient-poor agar (SNA) medium. Macroconidia were hyaline, slightly curved in shape with 3 or 4 septa, measuring 28.6 to 41.4 × 3.2 to 6.2 μm (av. = 34.8 ± 3.32 × 4.6 ± 0.85 um, n = 20). Microconidia were elongated, oval with 0 or 1 septum, and measured 11.2 to 16.8 × 2.6 to 5.8 μm (av. = 13.5 ± 1.47 × 4.12 ± 1.03 um, n = 20). Chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, with diameters ranging from 2.8 to 10.5 um (av. = 5.8 ± 2.31 um, n = 20). For molecular identification, genomic DNA from all six isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) method, and the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α), and RNA polymerase II beta subunit (RPB2) regions were amplified and sequenced using the primers ITS1/ITS4, EF-1/EF-2, and RPB2-5F/7cR (White et al. 1990; O'Donnell et al. 2010). The sequences were deposited in GenBank (ITS: PP779839, PP779840, PP779841, PP779842, PP779843, PP779844; EF1-α: PP797138, PP797139, PP797140, PP797141, PP797142, PP797143; RPB2: PP797144, PP797145, PP797146, PP797147, PP797148, PP797149). The sequences of all three genes showed 99 to 100% similarity with Fusarium falciforme and other closely related Fusarium species (ITS: PP735125, EF1-α: OP163897 and RPB2: MF467484). A maximum likelihood phylogenetic tree was constructed based on the ITS, EF1-α, and RPB2 sequences of all isolates, along with other closely related Fusarium species. Based on morphological and phylogenetic characteristics, all isolates were identified as F. falciforme (Xu et al. 2023; Wang et al. 2023). Ten Pepper inbred line A23-41 (5 for the inoculation treatment and 5 for the control) were tested for pathogenicity using three representative isolates: HN-01, HN-02, and HN-03. A total of 20 ul of spore suspension with concentration 10⁶ spores/ml was injected near the soil surface of the stem, while the control treatment was inoculated with 20 μl of sterile water. The plants were placed in a climatic chamber at RH80-90% and 25/20 °C (day/night) after inoculation. The experiment was repeated three times. After 10 days, Inoculated plants stem developed necrosis, browning, while the control group remained asymptomatic. The fungus reisolated from the artificially infected stems was identified through EF1-α and RPB2 sequence analysis as F. falciforme, thus fulfilling Koch's postulates. Fusarium falciforme has been previoulsy reported as causing various diseases on different hosts in several countries, including Korea (Kang et al., 2024), Malaysia (Balasubramaniam et al., 2023), and Mexico (Payán-Arzapalo et al., 2024). To the best of our knowledge, this is the initial report demonstrating that F. falciforme causes Fusarium wilt on peppers in China. The study results can provide the basis for future research on the occurrence, prevention, and management of this disease.