First Report of Pectobacterium brasiliense causing Soft Rot on Conophytum luiseae in China.

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Conophytum luiseae is native to the Namaqualand region of Cape, South Africa. It is a lovely plant with many short succulent spines on ingot-shaped fleshy leaf surfaces, and a high-value ornamental plant in China. In August to October 2021, a serious soft rot disease on Conophytum luiseae plants was observed in four greenhouses at a horticultural farm in Songjiang District, Shanghai, China. 70% of Conophytum luiseae plants on this farm had severe rot symptoms. Initially, wilting and soft rot symptoms appeared on fleshy leaves, then progressed into browning and withering symptoms of all fleshy leaves. To isolate and identify the causal agent, small pieces of lesion tissues were sterilized by 75% ethanol for 30 s, and rinsed three times with sterile water. Later, the tissues were crushed in sterile 2.0 mL centrifuge tube with 100 μl of sterile water. The suspension was serially diluted and spread on Luria-Bertani agar (LB) medium. After incubation at 28°C for 48 h, the bacterial colonies were tiny and streaked on LB plate for purification. After purification, five independent representative colonies were used for further confirmation. Genomic DNA from the bacterial isolate was extracted and used as the template to amplify 16s rDNA with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes, dnaX with primers dnaXF/ dnaXR (Slawiak et al. 2009), and leuS with primers leuSF/ leuSR (Portier et al. 2019), respectively, by a polymerase chain reaction (PCR). The 16S rRNA sequences of one bacterial isolate was deposited in GenBank (GenBank accession OM333246) and showed a 99% similarity to that of Pectobacterium brasiliense (syn. Pectobacterium carotovorum subsp. brasiliense, Pcb) strains HG1501090309 (KU997683), BC1(CP009769), KC08 (KY021029). The dnaX (OM320998) and leuS (OM321306) sequences showed high similarity (> 99%) to P. brasiliense sequences. To further validate this identification, Pcb-specific primers BR1f/L1r was used for PCR, and it produced a predicted amplicon of 322 bp expected for P. brasiliense (Duarte et al. 2004). All five isolates could be detected by BR1f/L1r primer. To fulfill Koch's postulates, five healthy Conophytum luiseae were inoculated by spraying bacterial inoculum (108 CFU/ml), meanwhile five additional healthy Conophytum luiseae were implemented with sterilized distilled water as a negative control. The plants were then kept at 70% humidity and 25ºC. Seven days after inoculation, the inoculated plants showed serious soft rot, while the control samples remained healthy. Bacteria were re-isolated from rot of inoculated tissues, and the isolates were identified as the original pathogen by the 16S rRNA gene sequences. P. Brasiliense has been reported to cause soft rot on diverse plant hosts, like sweet potato, radish, tobacco (Liu et al. 2019; Voronina et al., 2019; Wang et al., 2017). Best to our knowledge, this is the first report that P. Brasiliense causes soft rot on Conophytum luiseae in China.