First Report of Fusarium fujikuroi Causing Black Rot of Bletilla striata (Baiji) in China

Bletilla striata Rchb. f. (Orchidaceae family) is a traditional Chinese herb (known as baiji in Chinese) that is distributed widely in China, Japan, and other regions of Southeast Asia. Baiji is used in Chinese medicine for homoeostasis and detumescence and is also used as an ornamental plant (Shi et al. 2017). In March 2017 and 2018, a black rot disease was observed on B. striata in many nurseries in the Hunan Province of China. Symptoms first displayed as black rot from the base upward to the apex of young leaves and then occurred as a rapid wilting of all leaves resulting in death of the aboveground plant parts. Symptomatic leaves were excised near the margin of necrotic lesions, surface disinfested with 70% ethanol and 0.1% HgCl₂, rinsed with sterile distilled water, and then incubated on potato dextrose agar (PDA) at 26°C in the dark. Colonies with similar cultural morphology were consistently isolated. Colonies showed thick white aerial mycelium with a growth rate of 1 cm/day on PDA. When cultured on carnation leaf agar for 10 days at 26°C, microconidia were abundantly produced, 6.5 to 11.5 × 3.3 to 5.2 μm, clavate, and oval to kidney shaped, with zero to one transverse septa. Macroconidia were abundant, sickle shaped, 27.5 to 62 × 2.5 to 4.0 μm, slender, almost straight, slightly incurved in the apical cell and foot shaped in the basal cell, and had three to five septa. These morphological characteristics were consistent with those of Fusarium spp. (Booth 1971). The rDNA internal transcribed spacer (ITS), a portion of the translation elongation factor 1-α (EF-1α) gene, and β-tubulin gene were amplified and sequenced using the paired primers ITS4/ITS5, EF-1/EF-2, and Bt-1/Bt-2, respectively (Wang et al. 2014). Sequences were deposited in GenBank (MF426033, MH263736, and MH263737 for ITS, EF-1α, and β-tubulin, respectively). BLAST searches showed that the ITS sequence had 99% identity to strains of Fusarium sp., such as F. proliferatum (KJ528883.1) or F. verticillioides (KR183784.1), and the TEF-1α and β-tubulin showed 99 to 100% identities to the strains of F. fujikuroi (HF679028.1 and HF679024.1). In addition, the sequences of TEF-1α and β-tubulin showed 100% identities to the sequences of the Gibberella fujikuroi species complex (ID FD_01764) in the Fusarium-ID database (Geiser et al. 2004). According to the morphological characteristics and molecular identification, the pathogen was putatively determined to be F. fujikuroi (Leslie and Summerell 2006). Pathogenicity tests were conducted by placing mycelial discs (0.5 mm in diameter), obtained from colonies of representative isolate BJ-1 actively growing on PDA, on leaves of 2-month-old B. striata plants. Control leaves were mock inoculated with only sterile agar plugs. In addition, another two sets of detached and living leaves were inoculated separately using aliquots of 50 μl of conidial suspension (10⁵ conidia/ml), and the control leaves were inoculated with the same volume of sterile water. All treatments were placed in humid chambers at 26°C, and the assays were performed twice with three replicates. After 4 days, all inoculated leaves showed black rot symptoms, whereas the control leaves remained asymptomatic. F. fujikuroi was reisolated from the inoculated leaves, thus completing Koch’s postulates. Because this disease developed rapidly in B. striata, its occurrence may seriously affect production of this medicinal plant and should be taken into consideration by breeders. To the best of our knowledge, this is the first report of F. fujikuroi causing black rot on B. striata in China, as well as in the world.