First Report of <i>Fusarium cugenangense</i> Causing Leaf Blight of <i>Gentiana rhodantha</i> in China

Gentiana rhodantha Franch. ex Hemsl., a member of the Gentianaceae family, is a notable ethnomedicinal herb in Guizhou Province, China (Shen et al., 2017). In June 2024, leaf blight was observed on G. rhodantha in a cultivation field in Wudang District, Guiyang City, Guizhou Province, affecting 60% of the surveyed plants. The initial symptoms included localized chlorosis and wilting at the leaf tips, which gradually progressed toward the petiole. In cases of severe infection, complete chlorosis of the foliage occurred, culminating in plant mortality. Nine symptomatic leaves were collected from three plants, surface sterilized in 75% ethanol and 1% NaClO for one min each, and washed three times in sterile water, then incubated on potato dextrose agar (PDA) medium at 28°C in darkness for five days. Through hyphal tip culturing (Zhai et al., 2014), nine fungal isolates (constitute 75% of isolations) with identical morphological characteristics were obtained. Colonies exhibited pale purple centers with fluffy white margins and orange-red undersides. Microconidia were mainly oval and slightly curved, with 0 to 1 septum, measuring 4.4 to 15.0 × 1.7 to 3.6 μm (average 7.6 × 2.8 μm, n=50). Macroconidia were sickle-shaped, mostly 3-septate, measuring 23.7 to 50.1 × 2.9 to 5.6 μm (average 34.9 × 4.4 μm, n=50). Chlamydospores were solitary or paired, terminal or intercalary, with diameters of 5.4 to 15.6 μm (average 8.5 μm, n=50). These morphological features were consistent with those of Fusarium oxysporum species complex (Yang et al., 2024; Li et al., 2024). For molecular identification, genomic DNA was extracted from the representative strain KH19. The primers TEF1-728F/TEF1-986R (Carbone et al., 1999), RPB2-5F/RPB2-7cR (Liu et al., 1999), and T1-F/bTub-R (O’Donnell et al., 1997) were used to amplify three gene regions: translation elongation factor 1-alpha (TEF1-α), RNA polymerase II second largest subunit (RPB2), and beta-tubulin (TUB2). The sequences of strain KH19, with accession numbers PV891797 (TEF1-α), PV891796 (RPB2), and PV891798 (TUB2), were deposited in GenBank, exhibiting a homology of 99% to 100% (282/284, 956/958, 890/913) with the F. oxysporum species complex (FOSC) (accession nos. OM978704, PV690771, KP765699). Based on morphological features of the asexual morph and maximum-likelihood analyses of combined EF1-α, RPB2, and TUB2 gene sequences with sequences of representative type isolates in the FOSC, the strain was identified as F. cugenangense. Leaves on three healthy one-year-old potted G. rhodantha plants were wounded with sterile needles and inoculated with 5-mm mycelial plugs of the KH19. Non-colonized plugs of the same medium were used as the control. The inoculated plants were incubated at 27°C, 85% relative humidity, and a 12-hour photoperiod. The experiment was repeated three times. After seven days, the leaves inoculated with KH19 exhibited symptoms consistent with those observed in field, while the controls were asymptomatic. F. cugenangense was subsequently re-isolated from the inoculated leaves but not from the controls, thus fulfilling Koch's postulates. Previously, this pathogen was known to cause root rot in strawberries (Shrestha et al., 2024) and wilt in blackberries (Cho et al., 2021). This is the first report of F. cugenangense causing leaf blight in G. rhodantha in China. This finding is crucial for the early detection of the disease and the formulation of effective management strategies to protect G. rhodantha.