First Report of Blight on Fritillaria thunbergii Caused by Fusarium oxysporum in Zhejiang Province, China.

Abstract

Fritillaria thunbergii Miq. (Zhe beimu), is an oldest known homeopathic traditional folk medicinal plant in Zhejiang Province, China. The bulbs are medicinally important curing cough, inflammation, gastric ulcers, hypertension, diarrhea, and bronchitis (Nile et al. 2021). In April of 2018 and 2019, symptoms of blight on F. thunbergii were observed with an incidence of 20-25% in Cixi city, Zhejiang Province. At the early stage of this disease, the stalk turned brown, then the whole bulbs rotted within a few days. Symptomatic bulbs were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30 seconds and 1% NaClO for 30 seconds under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA). After incubation at 28 ℃ for 7 days in the dark, the hyphae were observed white fluffy, spreading from the middle to the whole plate. Macroconidia were falciform with zero to four septa and (11.0-39.0) × (3.0-5.0) μm in size. Microconidia were fusiform with zero to two septa (4.0-7.0) × (2.6-3.0) μm in size. Based on morphological characteristics of macroconidia, and microconidia, isolates were identified as F. oxysporum (Lombard, L. et al., 2019). The internal transcribed spacer (ITS) region, translation elongation factor (EF-1α), and RNA polymerase second largest subunit (RPB2) gene were amplified and sequenced respectively using ITS1/ITS4, EF1/EF2 and 5f2/7cr primers (O'Donnell et al., 2010). BLASTN analysis of FUSARIUM-ID using ITS (Accession NO.MZ268594), EF-1α (Accession NO.MZ292517) and RPB2(Accession NO.MZ292516) showed 95.2%, 100%, and 99.11% identity to F. oxysporum species complex isolates NRRL43730, NRRL38599 and NRRL38302, respectively. Based on the morphological and molecular characters, the pathogen was identified as F. oxysporum. To verify pathogenicity, ten healthy F. thunbergii plants were used for inoculation tests. One milliliter of a conidial suspension (106 conidia ml-1) was pipetted onto the soil around the base of F. thunbergii plants per pot. Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70-80% relative humidity and a photoperiod of 16:8 [L: D] h). Seven days later, all inoculated plants showed typical symptoms of blight identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as F. oxysporum by morphology observation and sequence analysis. To our knowledge, this is the first report of blight caused by F. oxysporum on F. thunbergii in Zhejiang Province, China. Acknowledgments: The author(s) declare no conflict of interest. Funding: This work was supported by Zhejiang Provincial Program for Science and Technology Development (2017C32006, 2018C02030) and the Student Science and Technology Innovation Project of China Jiliang University (2021YW95).