First Report of <i>Fusarium asiaticum</i> and <i>F</i>. <i>meridionale</i> Causing Leaf Spot on <i>Hemerocallis citrina</i> in Sichuan Province, China

Daylily (Hemerocallis citrina) is widely cultivated in China as vegetable and medicine. Its flower buds are nutritious vegetables rich in ascorbic acid, mineral elements and et al (Wang et al. 2024). Whereas, little is known about the pathogen identification in daylily. In April 2023, leaf spot symptoms were observed on 20% daylily seedlings in Dazhou city (30.91° N, 106.87° E), Sichuan province, China. The symptomatic leaves were superficially disinfected with 70% ethanol for 20 s, rinsed twice in sterile dH2O and subsequently disinfected with 1% NaClO for 40 s, and rinsed twice in sterile dH2O again. The disinfected leaves were cut into pieces (5 × 5 mm), placed on PDA amended with streptomycin sulfate (50 mg/L), and incubated in dark at 25 ℃ for two days. The fungal isolates, displaying morphological features of Fusarium species (Leslie and Summerell 2006), were purified through transferring single spores. Consequently, two distinct type fungal isolates were obtained. Cultured on PDA, Type Ⅰ (Q22, Q26, Q27) and Type Ⅱ (Q29, Q29-1) isolates showed similar growth phenotypes. Type Ⅰ and Ⅱ isolates' cultures were initially white but gradually became yellow, and scarlet diffusible pigments were produced with time (Fig. S1). On SNA medium, Type Ⅰ isolates produced macroconidia with 3 to 6 septa, which measured 5.56±0.54 × 48.65±6.12 µm (n = 50). While, Type Ⅱ isolates' macroconidia contained 3 to 11 septa that measured 4.08±0.93 × 51.40±12.83 µm (n = 50) (Fig. S1). All the isolates were properly preserved in our lab. To further identify these isolates, DNA fragments of Beta-tubulin (TUB2) (Glass and Donaldson 1995), translation elongation factor-1 alpha (TEF1) (Rehner and Buckley 2005), and DNA-directed RNA polymerase II largest (RPB1) (Hofstetter et al. 2007) and second largest subunit (RPB2) (O'Donnell et al. 2012) were amplified and sequenced. BLASTN analyses of TUB2 (PP266396 ~ PP266398), TEF1 (PP404039 ~ PP404041), RPB1 (PP404042 ~ PP404044) and RPB2 (PP408000 ~ PP408002) of Type Ⅰ isolates showed 99.35 ~ 100% identity to those of F. asiaticum NRRL 13818 (AF212745.1, MW233069.1, MW233240.1, JX171573.1). Type Ⅱ isolates' sequences (PP408003 ~ PP408010) showed their homology with those of F. meridionale NRRL 28436 (AF212730.1, MW233092.1, MW233263.1, MW233435.1) at 100% identity. The phylogenetic tree based on combined datasets of TUB2, TEF1, RPB1 and RPB2 of Fusarium species confirmed that Type Ⅰ and Ⅱ isolates were F. asiaticum and F. meridionale, respectively (Fig. S2). Seedlings of cultivar "chuanhuanghua No.1" (n = 5) in a greenhouse (25°C, relative humidity 90%) were inoculated with conidial suspension (3 × 105 conidia / mL). Controls were treated with sterile dH2O. Fifteen days post-inoculation, natural symptoms appeared on inoculated leaves (Fig. S3). Whereas, non-inoculated controls were disease-free. The pathogenicity assay was repeated three times. F. asiaticum and F. meridionale were successfully re-isolated from diseased leaves and verified using morphological and molecular methods described above, fulfilling Koch's postulates. F. ussurianum, a Fusarium graminearum species complex (FGSC) member, and F. proliferatum were identified as causal agents of leaf spot on daylily in China (Chen et al. 2024; Li et al. 2018). To our knowledge, this is the first report of F. asiaticum and F. meridionale causing leaf spot on daylily worldwide. Our studies demonstrate that FGSC members are the common pathogens causing leaf spot on daylily in Sichuan, China.