RESUMO
Philodendrons are important foliage ornamentals planted worldwide (Chen et al. 2010). In November 2021, soft rot symptoms were observed on Philodendron selloum (now known as Thaumatophyllum bipinnatifidum; Sakuragui et al. 2018) grown in a nursery in Taichung, Taiwan. On symptomatic plants, the petioles were macerated; leaf lesions were also found on some plants (Figure S1). About 60% of the plants on site were symptomatic; these plants tended to cluster together. Four plants were sampled. Infected tissues were soaked and cut into pieces in 10 mM MgCl2 (using scalpels); undiluted samples were streak-plated onto nutrient agar (NA) and grown for 24 h at 28°C. Translucent, creamy-white colonies were isolated from all of the tissues examined, and 4 isolates, PHIL1 to PHIL4, were obtained (each from a different plant). All isolates exhibited typical phenotypes of bacteria belonging to Dickeya; they could cause maceration symptoms on potato slices, ferment glucose and produce phosphatase (Schaad et al. 2001); they could also produce indigoidine on NGM medium (NA added with glycerol and MnCl2; Lee and Yu. 2006). Polymerase chain reactions using Dickeya-specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon in all 4 isolates. The 16S rDNA of PHIL1 to PHIL4 were amplified using primer pair 27f/1492r (Lane 1991) and the amplicons were sequenced; all 4 isolates shared the same 1,395-bp sequence (accession nos. ON203122, ON479664-ON479666). Among the strains belonging to known species (in GenBank), PHIL1 to PHIL4 shared the highest sequence identity (99.93%) with D. dadantii 3937; they also shared 98.78% sequence identity with D. dadantii CFBP 1269T. Multilocus sequence analysis (MLSA) targeting fragments of PHIL1 to PHIL4's dnaA (720 bp), dnaJ (672 bp), dnaX (450 bp), gyrB (822 bp), and recN (762 bp) genes (Marrero et al. 2013) were conducted. The five-gene concatenated sequences (3,426 bp) of the 4 isolates (accession nos. ON227444-ON227448, ON494509-ON494523) were identical. A maximum-likelihood phylogenetic analysis including these sequences and those of type strains of other known Dickeya species revealed that PHIL1 to PHIL4 clustered with strains belonging to D. dadantii (Figure S2). Koch's postulates were fulfilled with an inoculation test conducted on T. bipinnatifidum (17 cm in aboveground height; 7-months-old). Stab inoculation using sterile toothpicks was conducted on petioles. Three plants were tested for each isolate and 2 petioles were inoculated for each plant; all 4 isolates were included in the assay. The pathogen loads inoculated were quantified by the spread plate method and were 3.22 - 4.81 x 107 colony forming units. Three plants were stabbed with bacteria-free toothpicks, serving as controls. All plants were bagged post inoculation and kept in a growth chamber (28°C; 14 h light). After 72 h, all of the inoculated petioles exhibited symptoms resembling those observed in the nursery. Bacteria were re-isolated from the symptomatic tissues (one isolate from each treatment), and all of their five-gene concatenated sequences were the same as those of PHIL1 to PHIL4. This is the first formal report of the occurrence of D. dadantii infecting T. bipinnatifidum in Taiwan. Studies have shown that D. dadantii could affect other Araceae plants in Taiwan (Lee and Chen 2021). Since different Araceae ornamentals are often planted together in gardens and nurseries, growers should be aware of potential transmission of D. dadantii among them.
RESUMO
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.