Identification of Neofusicoccum parvum as the causative agent of leaf spot disease on Bletilla striata in China

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is an essential traditional Chinese medicinal plant used to treat hemorrhage, swelling, inflammation, ulcers, and pulmonary diseases (Xu et al. 2019). In April of 2020, an unknown leaf spot disease was observed on B. striata in a plantation (~ 0.2 ha) in Nanning, Guangxi province, China. Disease incidence was estimated at approximately 25% (n = 150 plants). The initial symptoms were small brown circular spots, which then expanded into reddish to brown, circular to irregular lesions 5-10 mm in diameter. As the disease developed, the whole leaf became densely covered with lesions. Finally, the lesions coalesced, killing the leaf and resulting in defoliation. To isolate the causal agent, six symptomatic leaves were collected from individual plants. Small pieces (~ 5 mm²) were cut from the margin of the necrotic lesions (n = 18), disinfected in 1% NaOCl for 2 min before rinsing three times in sterile water, and placed on potato dextrose agar (PDA) at 26°C for 3 days. Hyphal tips from the resulting cultures were transferred to PDA to obtain pure cultures. Fifteen isolates were obtained, of which twelve isolates exhibited similar morphology. Colonies on PDA were initially white, then turned dark gray after 7 days. Pycnidia were produced on the surface of PDA after 50 days. Conidia were hyaline, aseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and measuring 11.5 to 15.2 × 4.9 to 6.1 μm (mean ± SD: 13.4 ± 1.0 × 5.4 ± 0.3 μm, n = 60). Morphological features were similar to N. parvum (Phillips et al. 2013). For further molecular identification, the internal transcribed spacer (ITS) region, partial translational elongation factor subunit 1-α (EF-1α), β-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F (Carbone and Kohn 1999)/EF-2 (O'Donnell et al. 1998), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences of the two isolates BJ-111.1 and BJ-111.4 were deposited in NCBI GenBank under the following accession numbers: OM348509-10, OM397537-40. The obtained ITS, EF1-α, and TUB2 sequences showed 99% (514/516, and 513/516 bp), 99% (275/276, and 274/275 bp), and 99% (429/431, and 429/430 bp) homology with several GenBank sequences of the ex-type strain N. parvum CMW 9081 (AY236943, AY236888, and AY236917, respectively) (Zhang et al. 2017). In addition, a phylogenetic analysis confirmed the isolates as N. parvum. Therefore, the isolates were identified as N. parvum based on morphological and molecular evidence. Furthermore, pathogenicity tests were carried out on 1.5-year-old B. striata plants. Healthy leaves on six plants (1 leaf per plant) were inoculated with a 10-μl droplet of conidial suspensions (106 conidia/mL). Three plants treated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C with a 12 h photoperiod. Six days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained healthy. The experiments repeated three times showed similar results. Finally, N. parvum was consistently re-isolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. No fungus was isolated from the controls. To our knowledge, this is the first report of N. parvum causing leaf spot of B. striata worldwide. This result will help develop disease management strategies against this pathogen.