RESUMEN
Bok choy (Brassica rapa var. chinensis) is one of the most popular leafy green vegetables in Asia (Wang et al. 2019; Zhang et al. 2014). In May 2022, disease resembling bacterial soft rot was observed in a commercial greenhouse located in Xiluo, Yunlin County, Taiwan. Affected plants exhibited maceration, primarily close to the base of the plants (Fig. S1). Almost all bok choy plants (about 1,800 plants in total) on site were symptomatic. Macerated tissues were collected from six plants. The samples were homogenized in 10 mM MgCl2 and bacteria were isolated on nutrient agar (NA) by streak plating. After 1 day of culturing at 28°C, creamy-white, round colonies were consistently grown on all the plates, and six strains (Br1 to Br6) were obtained; each isolated from a different plant. The strains were able to ferment glucose and induced maceration on potato tuber slices (Schaad et al. 2001) but could not produce indigoidine on NGM medium (NA added with glycerol and MnCl2; Lee and Yu 2006). The DNA samples of these strains were tested with Pectobacterium-specific primers Y1 and Y2 (Darrasse et al. 1994) and all samples produced the expected amplicon. To identify the isolated pathogens, 1,592-bp sequences concatenated from fragments of the leuS (452 bp), dnaX (492 bp), and recA (648 bp) genes (GenBank accession nos. OP360013-OP360021) were obtained for each strain as previously described (Portier et al. 2019). Three genotypes were detected, the sequences of strains Br1, Br2, Br4, and Br5 were identical, while strains Br3 and Br6 each belong to a different genotype. The sequence identity between Br3 and Br6 was 98.2%. The concatenated sequences (dnaX-leuS-recA), along with those of type strains from known Pectobacterium species, were subjected to maximum likelihood analysis. The reconstructed trees showed that strains Br1, Br2, Br4, and Br5 grouped with P. carotovorum CFBP2046T (Fig. S2); the sequence identity between the isolated strains and the type strain was 98.7%. Strains Br3 and Br6 clustered with P. brasiliense CFBP6617T (Fig S2); the sequence identity between CFBP6617T and Br3 and Br6 were 97.5% and 98.4%, respectively. The six strains were inoculated onto 55-day-old bok choy plants using previously described prick inoculation methods (Wei et al. 2019). Autoclaved toothpicks, each carrying 9.3 x 106- 5.6 x 107 cfu of bacteria, were used to inoculate the base of plant leaves. All six strains were tested, and each strain had three replicates. Plants in the control group were stabbed with bacteria-free toothpicks. The plants were enclosed in clear plastic bags during the assay to maintain humidity and kept in a growth chamber (27/25°C day/night; 14-h photoperiod). After 1 d, all inoculated plants produced soft rot symptoms resembling those observed in the sampling site. No noticeable differences were observed among symptoms produced by different strains. The controls were symptomless. One strain was re-isolated from each treatment group and their identity were confirmed by sequencing the dnaX gene. All re-isolated strains shared the same sequences with those of the original strains tested. This is the first report of P. brasiliense and P. carotovorum causing bacterial soft rot of bok choy in Taiwan. Importantly, the findings showed that different Pectobacterium species and genotypes could induce symptoms on a crop in the same facility at the same time, highlighting the potential complexity of interactions among different soft rot bacteria in the environment.
RESUMEN
Pothos (Epipremnum aureum) is an Araceae foliage plant with great ornamental values, which has long been enjoyed by consumers (Chen et al. 2010). In September 2021, pothos showing soft rot symptoms were found in 2 nurseries in Taichung, Taiwan. The petioles of the infected plants were macerated; some lesions extended to the leaves (Figure S1). The disease incidence was 50% in one nursery and 37.5% in the other; two and three plants were respectively collected from the two sites. Macerated tissues were homogenized in 10 mM MgCl2 and the samples were observed microscopically without dyeing. Motile, rod-shaped bacteria were observed in the samples, and the bacteria were isolated onto nutrient agar (NA) and grown at 28°C for 2 days. Fast-growing, round, creamy colonies were isolated from all 5 plants. One strain was isolated from each plant and the strains were named Ea1 to Ea5. The bacteria could ferment glucose and induce maceration on potato tuber slices (Schaad et al. 2001), but did not produce indigoidine on NGM medium (Lee and Yu 2006) and were tested negative for phosphatase activity (Schaad et al. 2001). The bacteria's DNA samples were tested using primers specific to Pectobacterium (Y1/Y2; Darrasse et al. 1994). The expected 434-bp amplicon was amplified in all five strains. Multilocus sequence analysis was conducted as previously described (Portier et al. 2019). A concatenated sequence (1,592 bp) comprising partial dnaX (492 bp), leuS (452 bp) and recA (648 bp) sequences was obtained for each strain. Two genotypes were detected among the strains; Ea1 and Ea2 belonged to one genotype (i.e., they had identical sequences), while Ea3, Ea4 and Ea5 belonged to the other (GenBank accession nos. OK416015-OK416020). Phylogenetic analysis was conducted using these data and those of representative strains of known Pectobacterium species (Klair et al. 2022). A maximum-likelihood tree showed that Ea1 to Ea5 clustered with P. aroidearum CFBP8168T (Figure S2). Sequence comparison (Table S1) showed that the similarity between the two genotypes' concatenated sequences was 99.1% (Ea1 vs. Ea3; 1,578/1,592 bp); Ea1 and Ea3 shared 99.2% and 99.3% sequence similarity with P. aroidearum CFBP8168T, respectively. The sequences obtained in this work were searched against GenBank and all of their top hits were those of strains belonging to P. aroidearum (supplementary information). Koch's Postulates were fulfilled by stab inoculating cutting-propagated pothos (8-cm tall) using toothpicks carrying bacteria grown on NA. The pathogen loads used were estimated by suspending cells (attached to individual toothpicks) in 10 mM MgCl2 and spread-plating them onto NA (after dilution); the loads were 5.5 x 106 - 2.2 x 107 CFU. Three plants were inoculated for each strain (3 petioles per plant). Control plants were stabbed with sterile toothpicks. Each plant was then bagged and placed in a growth chamber (28°C; 14 h light). After 24 h, all inoculated plants produced symptoms resembling those found in the nurseries, and the controls did not. For every treatment group, a strain was re-isolated onto NA; each of them shared the same recA sequence with the original strain inoculated. This is first report of P. aroidearum causing pothos soft rot in Taiwan. Local nurseries often grow pothos and other Araceae plants together in humid areas. Since other Araceae species are also known to be susceptible to P. aroidearum (Xu et al. 2020), growers should be cautious of the pathogen's spread across hosts.