First report of Fusarium solani causing Fusarium wilt of Bletilla striata (hyacinth orchid) in Zhejiang province of China.

Abstract

Bletilla striata, a member of the family Orchidaceae, is a perennial herbaceous plant used in Chinese medicine. It is a commonly cultivated economic crop in the Yangtze River Basin provinces of China, as its roots are used to treat bleeding and inflammation. In Zhejiang province, Bletilla striata has a planting area of 1400 hectares with a total production of approximately 2.6×106 kg. In October 2021, over 40% of B. striata plants showed severe wilt in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. In July, leaf curling, crinkling, and leaf-edge browning of the diseased plants were first noticed in the field. Then, necrotic streaks gradually spread to the roots. Stems displayed chlorosis and withering and when they were cut vertically, symptoms such as vascular bundle discoloration, appeared. After October, the individual plants slowly wilted and died, their aboveground parts became filamentous, and the epidermis detached from the corm's fibrous roots. Diseased plants were easily removed as the corm root had fractured. White mycelia were clearly seen in the stem. Three symptomatic leaves and three stems were cut, their surfaces disinfected, and plated on potato dextrose agar (PDA). Six strains were subsequently isolated from all samples. Fungal colonies with white to cream-colored mycelia from all tissues appeared after 3 d of incubation at 26 °C. Pure cultures obtained after monospore isolation were examined for their morphological characteristics. The colonies grew rapidly, were fluffy and appressed, and had cottony white to pale cream coloration. Microconidia were hyaline, oval to reniform, with zero or one-septate (4.0-12.0 × 1.0-5.5 µm), and usually formed on elongated monophialidic conidiogenous cells. Macroconidia were wide, fusiform, or slightly curved with one or three septa (23.0-36.0 × 4.5-7.0 μm). Chlamydospores were spherical and were abundant on carrot agar (CA) medium within 2 wk. Fresh mycelia and conidia that grew at 26 ℃ for 7 d were collected from PDA plates. Next, DNA was extracted using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). We amplified a portion of RNA polymerase II second largest subunit gene (RPB2) using primers 5f2/7cr (O'Donnell et al. 2010), the internal transcribed spacer (ITS) region using primers ITS1F/ITS4 (White et al. 1990), and the partial translation elongation factor-1α gene using primers EF1/ EF2 (O'Donnell et al. 1998) from the genomic DNA and sent the PCR amplicons for sequencing at Tsingke Biotechnology Co., Ltd., Wuhan, China. A BLAST search of the obtained sequences (GenBank accessions OP743920, OP913183, and OP913180) showed 99-100% homology with the respective sequences of the Fusarium solani reference isolate NRRL46702 (O'Donnell et al. 2008). Based on the morphological and molecular characteristics and BLAST search, the fungus was identified as F. solani (Leslie and Summerell 2006). Pathogenicity of the purified F. solani isolate was assessed by inoculateing a F. solani spore suspension of 1×106 conidia/mL (20 mL per seedling) on corm wounds made with a toothpick. Four inoculated and three non-inoculated seedlings (sterilized water as a negative control) were grown in a greenhouse at 26 °C under natural sunlight and covered with plastic bags to maintain humidity for 72 h. After 15 d, leaf browning on leaf edges, new leaf bases, and corm epidermis was observed. Symptoms, similar to those detected in the original sample, developed on the inoculated leaves, whereas the controls remained asymptomatic. Fusarium solani was successfully re-isolated from all four inoculated seedlings, and their identity confirmed by generating partial Tef1 and RPB2 sequences, thereby fulfilling the Koch's postulate. To our knowledge, F. solani has not been previously reported as a pathogen of B. striata.