First Report of Bacterial Fruit Blotch Caused by Kosakonia cowanii on Trichosanthis fructus in Jiangsu Province, China

Abstract

Trichosanthis fructus is one of the most common medicinal plants in China. In September 2022, T. fructus fruit showed black necrotic spots and surface irregularities, with water-soaked lesions (Fig 1). The affected T. fructus fruit (five weeks after blossom) were located in a field in Huai’an Municipality, Jiangsu Province (33.85°N, 119.00°E). The incidence was approximately 50%, causing great losses in fruit production. To isolate the causal agent, two symptomatic fruit from different plants were surface-disinfested with 75% (v/v) ethanol for 1 min, washed three times with sterile distilled water, and cultured on Nutrient agar (NA) plates at 28°C for 24 h. The obtained colonies were light yellow and transferred to fresh NA plates using the conventional repetitive streaking technique to obtain pure cultures. The purified bacterial cells were rod shaped, 1.64 to 2.47 μm long (n = 45), and 0.58 to 0.74 μm wide (n = 45) (Figure S2). Three isolates were used for further characterization. Biochemical tests indicated that the three isolates were Gram negative. DNA was extracted from the three bacterial isolates and used to amplify the16S rRNA (27F/1492R primers) and partial gyrB (UP1/Up2r primers) genes (Marchesi et al. 1998; Yamamoto and Harayama 1995). PCR products were purified using the DNA Clean-up Kit (CW2301, CWBIO), ligated into the PMD-19 vector (6013, Takara), and sequenced by Beijing Tsingke Biotech. The obtained 16S rRNA (GenBank accessions: OQ923996-OQ923998) and gyrB sequences (OR140942-OR140944) showed the best match, over 99%and 98% identity with 100% coverage to the K. cowanii type strain JCM 10956 (CP019445.1). To fulfill Koch’s postulates, pathogenicity tests were conducted on healthy T. fructus fruit. T. fructus fruit showed no wounds or lesions, and were surface disinfected with 75% alcohol. The three isolates were grown in nutrient broth at 200 rpm in 28 oC for 24 h and re-suspended in sterilized ddH2O to OD600 = 0.6~1.0 (108~109cfu/mL). Five μL of bacterial suspension was inoculated into the healthy fruit surface with a sterile knife. For the control experiment, the same volume of sterilized ddH2O was used. In each treatment, four healthy T. fructus fruit were treated. All samples were incubated at 25°C and 75% humidity in a plant incubator (Bluepard, MGC-350BP-2). After 12 days, bacterial fruit blotch symptoms were observed in all the inoculated fruit. The pathogen was recovered from the infected fruit, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis. To further investigate the pathogenicity, four-week-old T. fructus plant leaves were inoculated with the above three isolated suspension (OD600=0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). Sterilized ddH2O was used as mock control. After 10 days, bacterial blight symptoms were observed in all inoculated leaves. To our knowledge, this is the first report of K. cowanii causing fruit blotch on T. fructus worldwide. This species has been previously associated with acute cholecystitis in humans (Berinson et al. 2020; Petrzik et al. 2021), but it was recently identified as the causal agent of bacterial wilt on patchouli, bacterial blight on soybean, and stalk rot in foxtail millet (Han et al. 2023; Krawczyk and Borodynko-Filas 2020; Zhang et al. 2022). China is the largest producer of T. fructus. This report reveals that K. cowanii has a greater host range than was known. This report will help to better understand the pathogens that affects T. fructus production in China.