First Report of Causing Soft Rot on Broccoli in China.

In April 2023, soft rot symptoms were observed in broccoli (Brassica oleracea L. var. italica) commercial fields in Songming County, Yunnan province, China (103°12'E, 25°31'N). The disease incidence in these fields (6 ha in size) was high, exceeding 50%, and it caused significant yield loss. The affected plants displayed characteristic symptoms, with the roots and stems of broccoli becoming soft, yellowish-brown, rotten, and emitting a foul odor. To identify the causal agent, soft rot symptomatic stems were surface sterilized by dipping them in 75% ethanol for 30 seconds, followed by three successive rinses with sterile distilled water. Tissue specimens were then plated onto nutrient agar (NA) plates and incubated at 28°C for 24 hours. (Wang et al. 2022). Three representative bacterial isolates HYC22041801-HYC22041803 from broccoli were selected for further analysis. The colonies on NA plates appeared as white, small, round, and translucent with smooth edges. Physiological and biochemical tests were performed, along with 96 phenotypic screenings using the BIOLOG GENIII microplate system (Biolog, Hayward, CA, USA). Three isolates were negative for D-arabitol, maltose, and sorbitol, but were positive for cellobiose, α-D-glucose, sucrose, glycerol and gentiobiose tests, which are consistent with the reported type strain P. polaris NIBIO1006T (Chen et al. 2021). Total genomic DNA was extracted from three bacterial isolates using the QIAamp DNA Mini Kit (QIAGEN, USA). The 16S rRNA region and nine housekeeping genes (gapA, icdA, mdh, mtlD, pel, pgi, pmrA, proA and rpoS) were amplified with universal primers 27F/1492R (Monciardini et al., 2006) and designed specific primers (Xie et al., 2018), respectively. All amplicons were sequenced and deposited in GenBank with accession numbers ON723841-ON723843 and ON723846-ON723872. The BLASTn analysis of the 16S rRNA amplicons confirmed that the isolates HYC22041801-HYC22041803 belonged to the genus Pectobacterium. Phylogenetic trees based on 16S rRNA gene sequences and multilocus sequence analysis of other nine housekeeping genes of the three isolates were constructed and the results revealed that three isolates clustered with P. polaris type strain NIBIO1006T, which was previously isolated from potato (Dees et al., 2017). To confirm the pathogenicity, nine broccoli seedlings were stab inoculated with a bacterial suspension (108 CFU·ml-1), while sterile distilled liquid LB medium was used as a negative control. The seedlings were kept at 80% relative humidity and 28°C in a growth chamber. Three trials were conducted per isolate (HYC22041801-HYC22041803). After 3 days, the inoculated petioles showed soft rot symptoms similar to those observed initially in the field, while control plants remained asymptomatic. All three isolates were re-isolated successfully from symptomatic tissues to complete Koch's postulates. P. polaris has been previously reported as the causative agent of blackleg in potato in several countries, including Norway, Poland, Russia, and China (Handique et al. 2022; Wang et al. 2022). Additionally, it was reported to cause soft rot in Chinese cabbage in China (Chen et al. 2021). However, this is the first report of P. polaris causing soft rot disease in broccoli in China. This discovery is of great importance for vegetable growers because this bacterium is well established on Cruciferous vegetables in the local area, and effective measures are needed to manage this disease.