First Report of Bacterial Leaf Spot of Cucurbita pepo Caused by Erwinia persicina in China

In February 2020, the common symptoms of water-soaked spots on Cucurbita pepo L. cotyledon were observed in Guangrao county in Shandong province, China. Field investigation showed that 40% of the Cucurbita pepo cotyledons in an area of approximately 0.8 ha were infected. The disease resulted in a severe loss in seedling production. Samples of C. pepo with water-soaked leaf spots were collected and prepared for pathogen analysis. Symptomatic cotyledon tissue was surface disinfested in 75% ethanol for 30 sec, then rinsed three times in sterilized water. Bacteria were released in sterile water in Petri dish for 2 min by cutting symptomatic tissue into small sections and stirring the plant tissue mixture fully. The diffusate was streaked onto plates containing nutrient agar (NA) and plates were incubated at 28℃ for 2 days. Three representative isolates were purified eventually from each of the plates. Colonies on NA were small, round and with smooth margins. All bacterial isolates characterized as gram-negative, white to cream color, and pink pigment was formed on the plates over long-term culture. The isolates were positive for catalase, Voges­Proskauer, potato rot, methyl red, acetoin production, nitrate utilization and citrate utilization, and acid production from maltose, glucose, melezitose, sucrose, D-arabinose, D-trehalose, cellobiose, lactose, raffinose, mannitol, D-sorbitol, melibiose and xylitol. KOH production was demonstrated according to strand formation within the potassium hydroxide test (Suslow et al. 1982). Isolates were negative for oxidase, arginine dihydrolase, phenylalanine deaminase, gelatinase, esculine, indole production and H2S production. Total genomic DNA was extracted from isolate XHL2002230201 with TIANamp Bacteria DNA Kit (TIANGEN). Universal primers 27F and 1492R (Monciardini et al. 2002) were used in PCR to amplify a 1,307-bp DNA fragment of the 16S rRNA region for molecular identification. Furthermore, four additional housekeeping genes (gyrB, atpD, rho, and rpoS) were selected and amplified using specially designed primers. The amplification products of 16S rRNA were sequenced and submitted to GenBank under accession number (MT568607.1). Sequence analysis showed 99% similarity to Erwinia persicina strains B57 (LM651373.1) and B64 (CI789_17875) by BLAST search in GenBank database (Gálvez et al. 2015; Cho et al. 2019). A phylogenetic tree was constructed, and the taxonomic position of strain XHL2002230201 was determined from the multilocus sequence analysis (MLSA) on 16S rRNA and other four housekeeping genes with E. persicina and not with other closely related Erwinia species. Pathogenicity tests and re-isolation and re-identification of the bacteria were performed to confirm the isolate and fulfill the Koch' postulates. The strain XHL2002230201 suspensions (108 CFU ml−1) were spray inoculated onto fifteen Cucurbita pepo seedlings with two true leaves, and the same number of control plants were inoculated with water. Experiments were repeated three times. All inoculated plants were kept in a moist chamber placed in a greenhouse at 28℃. Initial symptoms were observed on leaves of inoculated plants at 5 days post-inoculation, whereas no symptoms appeared on the plants inoculated with sterile distilled water. Based on morphological and biochemical characteristics, phylogenetic analysis, and Koch's postulates, the bacterial isolates were identified as E. persicina. To our knowledge, this is the first report of E. persicina causing leaf spot disease on Cucurbita pepo in China.

Characterization and Control of Erwinia spp. and Pluralibacter sp. in Tuna Salad Preparations

ABSTRACT Histamine-producing Erwinia and Pluralibacter spp. capable of producing toxic histamine levels were isolated from ingredients commonly used in tuna salad preparations. The characterization and control of these histamine-producing bacteria are necessary to prevent illness from tuna salad consumption. We confirmed the identity of two Erwinia spp. and one Pluralibacter sp. previously isolated from tuna salad ingredients through whole genome sequencing and phylogenic analysis and characterized them for growth and histamine production at different temperatures, pH values, and salt concentrations. In addition, we examined the effects of dried vinegar (DV) powder on growth and histamine production of these strains in inoculated tuna salad preparations. Optimum growth temperatures in tryptic soy broth (TSB) for the two Erwinia spp. and one Pluralibacter sp. were 30.1, 31.1, and 33.9°C, respectively, and growth in TSB was observed at 5°C for both genera. Optimum histamine production of Erwinia persicina, Erwinia spp., and Pluralibacter spp. in TSB with histidine occurred from 25 to 30°C, pH 4 to 6, and 0 to 4% NaCl. No significant growth or histamine production was observed in tuna salad preparations stored at 4°C. Growth and histamine production by Erwinia or Pluralibacter spp. was inhibited in tuna salad containing celery and onion and 2% DV, whereas significant growth and histamine production occurred in tuna salad without DV. Understanding optimum growth conditions and histamine production can provide guidance to tuna salad manufacturers in formulating products and adjusting processing conditions that minimize hazards from these histamine-producing bacteria. Addition of 2% DV to tuna salad preparations may prevent histamine production in the event of temperature abuse. HIGHLIGHTS

Whole-Genome Sequence of Erwinia persicina B64, Which Causes Pink Soft Rot in Onions

Erwinia persicina B64 was isolated from rotten onions in cold-storage facilities. Here, we report the complete genome sequence of E. persicina B64, which contains 5,070,450 bp with 55.17% GC content. The genome of this isolate is composed of one chromosome and two plasmids.