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 transplanted into 10 cm ×8.5 cm pots filled with substrate (peat: perlite: vermiculite =3:1:1). The negative control plants were inoculated with sterile distilled water (20 individual plants per group). All infected plants were placed in a greenhouse under the following environmental conditions: 30 ℃/25 ℃ day/night, >70 % relative humidity, 16-h/8-h photoperiod. The experiment was carried out three times. After 3 to 4 weeks of inoculation, the new leaves of the plants were smaller and asymmetrical, while the negative plants remained healthy. After 8 weeks, a significant stem rot pocket developed on all inoculated plants, similar to the symptoms observed in the field. In contrast, no symptoms were observed in negative plants (Figure S2). To fulfill Koch's postulates bacteria were further isolated, purified and identified from the greenhouse inoculated plants. The results proved that the causative agent of strawberry stem rot was P. ananatis. In recent decades, P. ananatis has been found to cause bacterial leaf blight in strawberries (Bajpai et al. 2020). It has also caused other crop diseases, such as maize white spot, peach soft rot and others (Cui et al.2022; Liao et al. 2015). Although other crop diseases caused by P. ananatis, a bacterial pathogen, there has been no report of P. ananatis causing the symptoms of stem rot disease in strawberry. To our knowledge, this is the first report of P. ananatis causing stem rot in strawberry. This study provides solid evidence that strawberry stem rot disease in China can also be caused by the novel pathogen Pantoea ananatis. In conclusion, this report will provide a theoretical reference for the prevention and control measures of P. ananatis causing strawberry stem rot disease in the future.