Onion (Allium cepa L.) is one of the most cultivated vegetables throughout the world. With an average annual production quantity of 18 million kg in recent 21 years, China is the world’s biggest onion producer (Hanci F., 2018). Among them, onion is mainly cultivated in the provinces of Gansu, Shandong, Yunnan, Jiangsu, Zhejiang and Henan. A survey in Gansu province in last several years showed that the incidence of onion bulb rot was 30%-80%. In April 2020, bulbs displayed water-soaked, and then rot symptoms observed during storage in Lanzhou City, Gansu Province, China. The initial symptoms of bulb rot disease were yellowish brown, and produced an abundant exudate in the inner bulb scales when cut. Gradually, symptomatic bulbs became soft, watery and decayed. In severe infections, the onions showed total rot of the bulb. Therefore, we sampled some diseased onions and isolated pathogenic bacteria from the junction of lesion along with healthy parts on Luria-Bertani (LB) medium. Three representative single colonies were obtained on LB medium, and the culture characteristics were raised elevation, mucoid texture, round, and smooth with entire margin, the brown at the beginning and turned yellow later, and scanning electron microscopy (SEM) observations showed that these isolates were short rod-shaped (Fig. 1A). The physiological and biochemical determination revealed that the isolates were positive for yellow pigment, v-p test, growth at 37 ℃, nitrate reduced, catalase, glucose, sucrose, D (-)-salicin, starch hydrolysis, motility, pellicle. On the contrary, they were negative for indole production, methyl red, lactose, gelatin liquefaction, glycerol, gram staining (Gavini et al., 1989; Nabrdalik et al., 2018). Based on these morphological, physiological and biochemical characteristics, three isolates were initially identified as Pantoea agglomerans (Guo et al., 2020). A representative isolate L1 was selected to extract DNA, and the conserved sequences of the pathogen gene were sequenced according to 23S ribosomal RNA (23S rRNA), DNA gyrase subunit B (gyrB), elongation factor G (fusA) (She et al., 2021) housekeeping gene. The sequence alignment of the 23S rRNA gene (P. agglomerans, MZ314289, 930bp) showed that the homology between the strain L1 and P. agglomerans (CP016889) with similarity of 99.54%, and based on the sequence alignment of gyrB (P. agglomerans, MZ337547, 1189bp) and fusA (P. agglomerans, MZ350961, 1037bp) genes, the similarity with P. agglomerans (FJ617386 and MG845872) was 100%. A phylogenetic analysis based on the 23S, gyrB, fusA housekeeping gene sequences was performed by using the neighbor-joining methods in MEGA 7.0 under the p-distance (Kumar et al, 2016), which included P. agglomerans strains AR1a, TH81, L15, ASB05, P. eucalypti strain LMG 24197, P. dispersa strains BJQ0007 and DSM 32899, P. ananatis strains LMG 20103 and AJ13355, P. vagans strains C9-1, LMG24199 and PV989. The phylogenetic distribution generated five primary phylogroups, and strain L1 formed a clade with the other four P. agglomerans strains (Fig. 2). Thus, the strain L1 was identified as P. agglomerans. To satisfy Koch's postulates, ten onions were divided into two groups, five in each group, and needle punctured wound on the surface of each onion. In the experimental group, 400 μL bacterial suspensions were injected with sterile syringe, and the other five onions were injected with the same amount of sterile distilled water as the negative conrol. Inoculated onions were incubated in the greenhouse incubator (28 ℃, humidity > 80%). After 4 days of incubation, all onions inoculated with strain L1 appeared water-soaked, rot symptoms, and no symptoms were observed in the negative control (Fig. 1B). Subsequently, pathogens were re-isolated from inoculated bulbs and identified as P. agglomerans according to molecular identification described above. To the best of our knowledge, this is the first report that the bulb rot disease of stored onion caused by P. agglomerans in China.
