First Report of Erwinia persicina Causing Stalk Rot of Celery in China.

Abstract

Species belonging to the genus Erwinia cause diseases in many economically important plants. In May 2021, celery (Apium graveolens var. dulce) plants (cv. Queen of France) showing soft rot symptoms were observed in greenhouses in Boye County, Baoding, Hebei Province (North China). Disease symptoms began with pinkish water-soaked lesions on the midrib of celery stalks, but at the same time the leaves and root were asymptomatic; and the infected celery plants rapidly developed brownish rotten stalks. The disease incidence in two greenhouses (0.15 ha in size) was more than 50%. Affected celery stalk pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were immersed in 200 µl 0.9% saline for 15 min. Aliquots (20 μl) of two tenfold dilutions of the tissue specimen soaking solution were plated onto Luria-Bertani (LB) medium and incubated at 28°C for 24 h. Single colonies were picked and restreaked onto LB agar three times for purity. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R and sequenced. Result of blastn analysis of the 16S rDNA amplicons (MZ614654, MZ614655) indicated that the bacterial isolates (BY21211 and BY21212) belonged to the genus Erwinia. Housekeeping genes including mdh, gapA, icdA, rpoS, acnA and proA were also amplified using a set of PCR primers (Ma et al. 2007) followed by sequencing (MZ643221-MZ643232). No sequence variation was observed at any MLSA locus between isolates BY21211 and BY21212. To determine the species of the Erwinia isolate BY21211 and BY21212, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). The result of phylogenetic analysis showed that the celery stalk rot isolate BY21211 and BY21212 was clustered with E. persicina type strain NBRC102418T (Hao et al. 1990). When celery plants (cv. Queen of France) have eight to nine true leaves, plants were inoculated with the isolate BY21211 by injecting 20 µl of bacterial suspensions (107 CFU·mL-1) into the celery stalks, and negative controls were injected with 20 µl of 0.9% saline. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacterial-inoculated seedlings showed disease symptoms resembled to those observed in greenhouses. Bacterial colonies were obtained from the infected stalks and were identified using the same PCR primers of housekeeping genes as described above. Therefore, E. persicina isolate BY21211 fulfill Koch's postulates for stalk rot of celery. Isolates BY21211 and BY21212 produce water-soluble pink pigment on sucrose-peptone agar. These isolates were gram negative and rod shaped, negative for oxidase, urease, indole production, gelatin liquefaction and acid production from xylose and glycerol. They were positive for catalase, citrate utilization, acid production from sorbitol, raffinose, glucose, arabinose, cellobiose, rhamnose, maltose, saccharose, inositol, lactose and esculin (hydrolysis). E. persicina has been reported to cause pink seed, crown and stem rot, soft rot or leaf spot on many plant hosts including pea (Pisum sativum), soybean (Glycine max), common bean (Phaseolus vulgaris), lucerne (Medicago sativa), barley (Hordeum vulgare), onion (Allium cepa), garlic bulbs (Allium sativum), tomato (Lycopersicon esculentum) and cucumber (Cucumis sativus) (Hao et al. 1990; González et al. 2005, 2007; Zhang and Nan 2014; Gálvez et al. 2015; Cho et al. 2019; Kawaguchi et al. 2021). To our knowledge, this is the first report of E. persicina causing stalk rot in celery. Stalk rot of celery has increased in prevalence over recent years in the Baoding region, it can cause significant yield loss and no cultivar has been found to be resistant to this disease so far. The stalk rot poses significant threat to local celery production, and further research on epidemiology and disease management options is needed.