First Report of Enterobacter cloacae Causing Stem, Leaf and Fruit Rot on Tomato in China.

Tomato (Solanum lycopersicum L.), as one of the most economically important and highly nutritious vegetable crops across the world, is widely cultivated in China, one of the largest tomato-concuming countries in the world (Ye et al., 2020; Wang and Liu, 2021). At present, major bacterial diseases in tomato include bacterial speck disease, tomato bacterial wilt and bacterial canker, all of which affect the tomato production around the world (Rosli et al., 2021; Peritore-Galve et al., 2021; Wang et al., 2022). In April 2022, a new bacterial disease was discovered on leaves, stems and fruits of tomato in a farmer's greenhouse located in Longfeng District in DaQing (125°07`-125°15`E, 46°28`-46°32`N), Heilongjiang Province, China. This field had tomato disease incidences approximately 50%. Apparent brown discolorations were found on fruits, leaves and stems in tomato plants. Symptoms were similar to fungal brown spots caused by Phytophthora infestans of tomato (Zhi et al.,2021; Liu et al.,2021) (Supplementary Figure S1). To isolate and identify the pathogen, the tissues of infected fruits, leaves and stems with typical symptoms were excised from diseased plants separately, and were disinfected with 75% ethanol for 10 s followed by 2% NaClO for 3 min and then washed five to eight times with sterile water (Wang et al., 2017). Afterwards, the samples were plated on nutrient agar (NA) solid medium and incubated. After incubation at 30°C for 2-3 days, bacterial colonies were isolated, then purified on nutrient agar (NA) solid medium at least twice by a streak plate method (Dou et al., 2019; Li et al, 2021; Zhao et al., 2022). White colonies grew on the NA medium after incubating for 2 days, showing round, opaque and smooth, which was similar to characteristics described as Enterobacter cloacae (García-González et al., 2018; Li et al, 2021). To further confirm the speculation on the identity of the isolated bacterium, the fragments of 16S rRNA were amplified and sequenced. The sequence of 16S rRNA was uploaded into GeneBank with accession numbers (OP077195.1). BLAST analysis of the sequence showed 97.68% identity with one corresponding sequence of E. cloacae in GeneBank (namely MK937637.1). Furthermore, a phylogenetic tree based on the sequence of 16S rRNA gene revealed that the isolate was grouped in the same clade as E. cloacae (Supplementary Figure S2). Based on Koch postulates to test pathogenicity of isolated bacteria, bacteria were inoculated on 30 day-old healthy tomato plants with three leaves stages, and the re-isolation of bacteria were carried out after 2 days of inoculation. To confirm pathogenicity, the isolates were cultured on LB medium at 30℃ for 2 days to prepare suspensions and adjusted to an optical density (OD) of 0.2 at A600, with a final concentration of 1ⅹ108 CFU/ml. Eight potted tomato plants were sprayed with bacteria suspensions, and eight control potted plants were sprayed with sterile distilled water. These seedlings were incubated in a chamber at 30°C with a 12 h light/dark photoperiod, with 85% relative humidity. After 2 days, inoculated tomato seedlings showed irregular small spots in leaves and brown necrosis at blade tips, and 8 to 10 days later, the leaves of tomato plants browned and died. The symptoms were the same with those of the initial diseased leaves of tomato plants (Supplementary Figure S1). No symptoms were observed on the control leaves (Supplementary Figure S3). Pathogenicity tests were repeated three biological times with same results. Meanwhile, the bacteria strains were re-isolated from symptomatic inoculated seedlings and confirmed as E. cloacae by culture and sequence methods as above. In China, there are no detailed records about the causal agent of this disease on tomato in a published paper in Chinese and English. To our knowledge, this is the first report of Enterobacter leaf brown necrosis caused by E. cloacae on tomato in China. Those results are of great significance for the production and management of tomato in greenhouse and control of the disease.