First report of a new bacterial stem rot disease of strawberry (Fragaria×ananassa) caused by Pantoea ananatis in Jiangsu, China

Strawberry (Fragaria×ananassa Duch.) is an important economic fruit crop in the world. With the continuous expansion of strawberry planting area, strawberry disease is one of the most important limiting factors, which seriously affects the agronomic performance and leads to significant economic losses. In November 2020, an infected stem rot disease of strawberries was detected in the strawberry growing area of Donghai County, Jiangsu Province, China. The disease incidence ranged from 30 % to 45 %. Initially, infected plants included stunted growth of new leaves, leaflet asymmetry, and holes in the vertical section of the stem, resulting in leaf blight and death in severe cases. To isolate the pathogen, two symptomatic plants were randomly collected. And then infected plants were surface sterilized with 75 % ethanol for 1 min, followed by 2 % sodium hypochlorite for 6 minutes. After that, the infected plants were washed 4-5 times with double sterilized distilled water, cut into 3-5 mm small pieces, and soaked in 2 ml of sterile water for 15 min, after which 100 µl of liquid suspension were spread onto Luria-Bertani medium (LB) and incubated at 28 °C for 12-16 h. All isolates showed yellow, viscous, round, and smooth (Figure S1, C) and the isolates were designated as JX1 and JX2. To identify the pathogen, the genomic DNA were extracted from isolates using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China) and the fragments of gyrB, rpoB and leuS gene were amplified using the primer pairs UP-1S/UP-2Sr (Yamamoto and Harayama 1995), rpoB-F/rpoB-R and leuS-F/leuS-R (Yu et al. 2022), respectively. Sequence analyses showed that the nucleotide sequences of gyrB, rpoB, and leuS fragments of the isolates shared 99.72 %, 99.67 % and 98.37 % identity with the Pantoea ananatis type strain LMG 2665 (KF482590.1, EF988972.1 and KF482626.1, respectively ), which suggests that the isolate could be Pantoea ananatis. To further verify that P. ananatis was identity of these isolates, the whole genome was sequenced using PacBio sequel II technology. The Average Nucleotide Identity (ANI) calculation showed that the whole-genome sequence was 99.0% similar to that of the Pantoea ananatis type strain LMG 2665 (Jain et al. 2018). The isolates were therefore recognized as P. ananatis. To confirm pathogenicity, roots of strawberry plants were inoculated by wounding as described (Wang et al. 2017) with bacterial suspensions (108 CFU/ml) for 30 min, and transpl