First report of spotting disease caused by Fusarium proliferatum infection of Alisma orientale in China.

Alisma orientale (Alismatidae) is highly valued for both its pharmaceutical and nutritional properties. The tubers are in Chinese herbal medicine and the leaves and stems for several Chinese delicacies. Intercropping A. orientale and Nelumbo nucifera may increase quality, yield, and other economic benefits. In July 2021, a novel spotting disease was observed in these plants in the White Lotus Science and Technology Expo Park in Guangchang County, Fuzhou City, Jiangxi Province (26.79°N, 116.31°E). The symptom was round to regular black spots on the stems during the early stage of infection. Over time, the larger spots merged, resulting in stem breakage and eventually death. A. orientale spot disease arose in July of 2021, causing approximately 50% of leaves to die, and leading to 10 to 25% yield loss. To identify the pathogenic organism, 5×5 mm samples were taken from affected tissue adjoining healthy tissue, sterilized in 75% ethanol for 30 s, and immersed in 0.1% mercury chloride for a further 30 s, before washing in sterile water and transfer to potato glucose agar (PDA) plates. After culturing at 28℃±1℃ for seven days, aerial mycelia were identified. At the start of culture, the mycelia were white but later turned purple-red. Three to five straight or partially bent septa were visible on the macroconidia, which were 28.8(19.1~38.6) ×2.9(1.9~4.0) µm in size (n=50). In contrast, the microconidia appeared glassy and elliptical, with sizes of 9.8(4.9~14.8) ×2.7(1.2~4.1) µm (n=50). These features suggested F. proliferatum (Zhao et al., 2019). To verify this, various primers, including universal ITS1/ITS4, Fusarium-specific EF1T/EF2T, PRO1/PRO2 (Mulè et al., 2004), and Bt2a/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1998) primers were used for amplification of the 5.8S rRNA/ITS, α-elongation factor, calmodulin, and β-tubulin genes. The resulting sequences were between 99% and 100% identical to those of F. proliferatum in GenBank (accession numbers MW721116.1, KR071735.1, KU604008.1, and MH398186.1, respectively). The present sequences were uploaded with accession numbers of OK047496, OL448294, OL448295, and OM280358, with sequence lengths of 549 bp, 725 bp, 594 bp, and 325bp, respectively. A maximum likelihood-phylogenetic tree was created in MEGA5 based on ITS+TEF+PRO sequences. Pathogenicity was tested by hyphal inoculation. Needles, cotton, and water were sterilized under high temperature and pressure. Five-millimeter punches were taken from infected and uninfected PDA plates and three uninfected stems of A. orientale were inoculated with the pathogen with a fourth used as the control. Plants were maintained in experimental field of the Bailian Science and Technology Expo Park. Infected wounds were gently wetted with sterile water and sealed with sellotape. After 10 days, the infected stems displayed symptoms while the controls did not. The same pathogen was recovered from the infected stems, fulfilling Koch's requirements. This appears to be the only report describing F. proliferatum infection of A. orientale stems. These results are useful for the recognition and avoidance of F. proliferatum infections in A. orientale and other plants.