First Report of Flower Stalk Wilting Caused by Rhizopus oryzae on Chinese cabbage in China.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the main biennial vegetables in China and its flowers can be produced in the second year. In May 2021, approximately 50% of the flower stalks of Chinese cabbage wilted in a field in Laizhou, China. Water-soaked lesions were first observed on the lateral shoots of flower stalks, leading to wilting at a later stage. Small diseased tissues were excised from the margin of lesions, surface disinfected in 75% alcohol, rinsed in distilled water twice, and transferred onto potato dextrose agar (PDA) medium for incubation at 28 ℃. Five fungal isolates were obtained using single spore isolation method. The fungal colonies were initially white and became gray or black within 5 days. The columella was globose to subglobose and 82.86±5.25 μm (n=5) in diameter; sporangiophores were smooth-walled, simple or branched; the globose sporangia were 86.06±15.37 μm (n=5) in diameter and black; the sporangiospores were subglobose and abundant and 5.23±0.98 μm (n=5) in diameter; and the rhizoids were dark brown and 5.69±1.82 μm (n=5) wide. A cetyl tri-methyl ammonium bromide method was used to extract DNA from 3-day-old hyphae (Ausubel et al. 1987). PCR was performed for ITS (White et al. 1990), the RNA polymerase II large subunit (RPB1) gene (Voigt et al. 2000) and the actin (ACT) gene (Stiller et al. 1997). The DNA sequences of the five isolates were identical, therefore, the sequence of Isolate RO21 was submitted to GenBank. According to BLAST search, the ITS (MZ452687), RPB1 (OK431470), and ACT (OK431471) sequences showed 99.66% similarity to Rhizopus oryzae Strain CBS 112.07 (NR103595), 100% to Strain CBS 127.08 (KJ566325) and 100% to Strain CBS 102660 (KJ551423), respectively. A neighbor-joining phylogenetic tree was reconstructed based on the ITS of Isolate RO21 and 14 other Rhizopus species sequences obtained from GenBank. Isolate RO21 was found to be most closely related to R. oryzae and far from other species. Based on morphological and phylogenetic characteristics, Isolate RO21 was identified as R. oryzae (Dolatabadi et al. 2014, Kwon et al. 2015, Palemón-Alberto et al. 2020). Sporangiospores were harvested from 5-day-old PDA cultures, suspended in sterilized distilled water, adjusted to 106 spores/ml and amended with 0.1% Tween-80. Chinese cabbage inbred line "A54-1" was inoculated near the middle of the flower stalk by applying 20 μl of spore suspension (106 spores/ml) to each of three sites wounded using a sterilized knife or to the unwounded site. Sterilized distilled water was used as the control. Forty flower stalks (20 for the inoculation treatment and the rest for the control) selected from ten plants were used for pathogenicity test. All plants were incubated in a growth chamber at 28/22 °C (day/night), with 80 to 90% of relative humidity. Wilting symptoms similar to those in the field were observed in the wounded flower stalks after 5 days and in the non-wounded flower stalks after 15 days. All control flower stalks remained asymptomatic. The fungus was re-isolated from the artificially infected flower stalks and identified as R. oryzae by morphological characteristics and sequencing to fulfill the Koch's postulates. To our knowledge, this is the first report that R. oryzae causes flower stalk wilting on Chinese cabbage in China. The results can provide the basis for future studies on the occurrence, prevention and management of this disease.