In April 2021, typical bacterial symptoms of soft rot were observed in the graft cactus (cv. Yeonbit) in Goyang, Gyeonggi-do, Republic of Korea. Infected graft cactus developed a gray-green color and wilting, and these symptoms were similar to those of bacterial soft rot (Fig. 1A). An unusual outbreak of graft cactus with the same symptoms was observed in the surveyed field. These symptomatic features are similar to those of bacterial diseases, such as bacterial soft rot (Charkowski, 2018). To isolate pathogens, infected stem tissues were curled surface-sterilized with 70% ethanol for 30 sec and 1% sodium hypochlorite for 60 sec and then rinsed with sterile water. The stem tissue was placed on a glass slide, 1-2 drops of sterile water were added and left for 10 min, streaked on nutrient agar (NA; Difco, Detroit, MI, USA), and in-cubated at 28°C. After 3 days, the bacterial strain, designated KNUB-01-21, was isolated from NA and purified by streaking on King's medium B (KB; Difco) and NA.

For molecular analysis, total genomic DNA was extracted from strain KNUB-01-21 using the HiGene Genomic DNA Prep Kit (Biofact, Daejeon, Korea). The 16S rRNA regions were amplified using the primers 9F (5′-GAG TTT GAT CCT GGC TCA G-3′) and 1512R (5′-ACG GCT ACC TTG TTA CGA CTT-3′) (Weisburg et al., 1991). The polymerase chain reaction (PCR) cycling conditions were 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and synthesis at 72°C for 1 min 30 sec; post-synthesis was carried out at 72°C for 7 min. The amplified PCR products were purified using the ExoSAP-IT PCR Product Cleaning Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed using SolGent (Daejeon, Korea). A sequence of 1,352 bp was obtained from the 16S rRNA region (GenBank no. LC717492). A blast search of the National Center for Biotechnology Information (NCBI) database revealed similarities of 100% between the sequence of the 16S rRNA gene of KNUB-01-21 and several strains belonging to P. brasiliense (GenBank nos. MN393942, MN393919), and 99.93% with P. carotovorum subsp. carotovorum PDP201711 (GenBank no. MN394009), and P. versatile PJ17 (GenBank no. MN393903). These results indicated that comparative analysis based on the sequence of only the 16S rRNA gene did not allow precise identification of strain KNUB-01-21 at the species level.

The identification of Pectobacterium species and subspe-cies based on 16S rRNA was not accurate, and only the taxonomic relationship between other species at the genus level could be confirmed. Recently, phylogenetic analysis using three concatenated housekeeping genes, dnaX, leuS, and recA allowed for the assignment of 114 strains to a novel species of the P. carotovorum complex and, in particular, to describe P. brasiliense, P. versatile, P. actinidiae, and P. odoriferum (Portier et al., 2019). Following this approach, dnaX, leuS, and recA genes of strain KNUB-01-21 were amplified and se-quenced. The dnaX gene was amplified using dnaXF (5′-TAT CAG GTY CTT GCC CGT AAG TGG-3′) and dnaXR (5′-TCG ACA TCC ARC GCY TGA GAT G-3′) (Sławiak et al., 2009). PCR protocols and primers used were previously described by Portier et al. (2019). The PCR cycling conditions for the dnaX gene were 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and synthesis at 72°C for 2 min. The post-synthesis was performed at 72°C for 5 min. The leuS gene was amplified using leuSF (5′-TYT CCA TGC TGC CYT AYC CT-3′) and leuSR (5′-TCC AGT TRC GCT GCA TGG TT-3′) (Portier et al., 2019). The PCR cycling conditions for the leuS gene were 94°C for 10 min, followed by 31 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and synthesis at 72°C for 1 min. The post-synthesis was performed at 72°C for 5 min. The recA gene was amplified using recAF (5′-GGT AAA GGG TCT ATC ATG CG-3′) and recAR (5′-CCT TCA CCA TAC ATA ATT TGG-3′) (Waleron et al., 2002). The PCR cycling conditions for the recA gene were 94°C for 3 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 47°C for 1 min, and synthesis at 72°C for 2 min. The post-synthesis was performed at 72°C for 5 min. The amplified PCR products were purified using ExoSAP-IT PCR Product Cleaning Reagent.

Sequencing was performed using SolGent. A sequence of 528, 511, and 713 bp for dnaX (GenBank no. LC717494), leuS (GenBank no. LC717495), and recA (GenBank no. LC717493) were respectively obtained.

The dnaX sequence of strain KNUB-01-21 shared 99.41%, 98.24%, and 98.04% identity with closely related P. brasiliense HNP201719 (GenBank no. CP046380), P. carotovorum subsp. carotovorum PCC21 (GenBank no. CP003776) and P. atrosepticum IPO 998 (GenBank no. GQ904832), respectively. Based on leuS gene sequence similarity, the close relatives of KNUB-01-21 were identified as P. brasiliense BC1 (GenBank no. CP009769) (99.62%), P. carotovorum subsp. carotovorum PCC21 (GenBank no. CP003776) (99.62%), and P. quasiaquaticum A398-S21-F17 (GenBank no. CP065178) (98.86%). The recA sequence of the isolate showed 100% similarity with P. brasiliense CFBP7079 (GenBank no. MK517247), 98.60% with P. carotovorum subsp. carotovorum 333 (GenBank no. AY264787), 98.18% with P. aquaticum IFB5637 (GenBank no. MW660584), and 98.17% with P. quasiaquaticum A477-S1-J17 (GenBank no. CP065177).

All the results indicated that comparative analysis based on the sequence of any one gene did not allow precise identification of the bacterial strain at the species level; therefore, multilocus sequence analysis was performed using concatenated sequences of the three above-mentioned genes of strain KNUB-01-21. These combined three molecular mark-ers are highly effective in resolving species in the genus Pectobacterium (Portier et al., 2019). Sequences of the allied species were retrieved from the NCBI GenBank database (Table 1). Multiple sequence alignments were performed using MEGA7 (Kumar et al., 2016). A phylogenetic tree was con-structed using the maximum-likelihood method (Felsenstein, 1981). Maximum-likelihood analysis was performed using the nearest-neighbor interchange heuristic search method and Kimura's two-parameter model. Strain KNUB-01-21, P. brasiliense CFBP5392, P. brasiliense CFBP6607, P. brasiliense CFBP6615, and P. brasiliense CFBP6617 ^T clustered together in a monophyletic clade with high bootstrap value, strongly supporting their affiliation with the same species (Fig. 2).

The isolated P. brasiliense was characterized according to the methods described by Czajkowski et al. (2015) and Schaad et al. (2001). Strain KNUB-01-21 was gram-negative, motile, bacillar, and facultatively anaerobic. However, the isolate did not fluoresce in KB medium, did not grow at 37°C in NA, and did not utilize sucrose-reducing substances or in-dole. Growth was observed in the 5% NaCl treatment. KNUB-01-21 showed cavity formation on crystal violet pectate and produced acid from lactose, maltose, α-methyl glucoside, and trehalose. The strain was sensitive to antibiotics such as amikacin, ampicillin, cephaletin, cefinaxone, chlorampheni-col, enonaxin, gentamicin, notilmicin, and trimethoprim-sulfatomethoxasol, but it was resistant to penicillin, erythro-mycin, and dicloxacillin. The results of these morphological and biochemical tests for strain KNUB-01-21 were the same as those for P. brasiliense (Supplementary Table 1).

To determine the pathogenicity of P. brasiliense KNUB-01-21, pathogenicity tests were conducted on the grafted cactus. The surface of the grafted cactus was disinfected with 70% ethanol and washed with distilled water below inoculation. Graft cactus was inoculated with a 20 ml suspension (1×10⁸ cells/ml) of strain KNUB-01-21. Plants inoculated with 10 ml distilled water were used as mock-infected plants. The inoculated graft cactus was maintained under greenhouse conditions (25-30°C, relative humidity 80%). After inoculation, symptoms of soft rot were observed in the grafted cactus flowers after 2 days. The same symptoms, including gray-green color and wilting, occurred as the first discovered symptoms that appeared on the graft cactus 7 days after flowering (Fig. 1B). However, no symptoms were observed in the mock-infected graft cactus (Fig. 1C). The pathogen was re-isolated from each diseased graft cactus, and the isolated bacterial strain was re-identified as P. brasiliense (data not shown).

Various Pectobacterium species have been reported as crop pathogens. Among them, P. brasiliense is one of the most dangerous phytopathogens, and 19 different plant species belonging to 10 families have been reported as hosts of this agent (Oulghazi et al., 2021). The first isolation of P. brasiliense from Korea was reported in 2012 (Choi and Kim, 2012). Moreover, P. brasiliense has been identified in Korea as the causal pathogen of soft rot in amaranth, paprika, potato, and tomato (Choi and Kim, 2012; Jee et al., 2018; Lee et al., 2013).

In Korea, the bacterial pathogen causing soft rot on the cactus was reported to be P. carotovorum subsp. carotovorum (Kim et al., 2007). Our study is the first report of P. brasiliense as the causal agent of soft rot in graft cactus in Korea. Our results increase the awareness of P. brasiliense distribution in Korea, thereby improving our understanding of cactus soft rot associated with Pectobacterium species and can be used to develop control methods to prevent economic losses.