First Report of Pectobacterium brasiliense Causing Soft Rot on Graft Cactus in Korea

Article information

Res. Plant Dis. 2022;28(3):172-178
Publication date (electronic) : 2022 September 30
doi : https://doi.org/10.5423/RPD.2022.28.3.172
1 School of Applied Biosciences, Kyungpook National University, Daegu 41566, Korea
2 Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Korea
3 Gumi Floriculture Research Institute, Gyeongsangbuk-do Agricultural Research and Extension Services, Gumi 39102, Korea
4 Institute of Plant Medicine, Kyungpook National University, Daegu 41566, Korea
5 Department of Horticultural Science, Kyungpook National University, Daegu 41566, Korea
Corresponding author Tel: +82-53-950-5760 Fax: +82-53-950-6758 E-mail: heeyoung@knu.ac.kr
Received 2022 July 21; 2022 September 3; Accepted 2022 September 6.

Trans Abstract

The graft cactus (Gymnocalycium mihanovichii) continues to be exported to more than 20 countries worldwide. In April 2021, typical bacterial symptoms of soft rot were observed in the graft cactus (cv. Yeonbit) in Goyang, Gyeonggi-do, Korea, resulting in economic losses in cactus production. The stems turned dark brown and the flowers were covered with black rot. The bacterial strain designated as KNUB-01-21 was isolated from infected stems and flowers. The results of the morphological and biochemical tests of the isolate were similar to those of Pectobacterium brasiliense. For molecular analysis, the 16S rRNA region and three housekeeping genes (dnaX, leuS, and recA) of the strain KNUB-01-21 were amplified. Based on t he results of the molecular analysis and morphological and biochemical tests, KNUB-01-21 was identified as P. brasiliense. The pathogenicity of KNUB-01-21 on graft cactus was confirmed by an inoculation test. Artificial inoculation using P. brasiliense KNUB-01-21 produced soft rot symptoms on the grafted cactus, and the same bacterium was re-isolated and re-identified. This is the first report of P. brasiliense causing soft rot in graft cactus in Korea.

In 2019, 43.2 ha of cactus was cultivated and the graft cactus (Gymnocalycium mihanovichii) continues to be exported to more than 20 countries worldwide (Ministry of Agriculture, Food and Rural Affairs, 2020). In addition, cactus accounted for 27.2% of Korea's total flower exports by 2020 (Korea Agro-Fisheries and Food Trade Corporation, 2020). Various diseases of the cactus have been reported worldwide. Bipolaris cactivora, Glomerella cingulata, and Fusarium oxysporum are the three major fungal stem decay diseases known in Korea (Chang et al., 1998; Hyun et al., 1998; Kim et al., 2000, 2004). Pectobacterium species are also known as causing agents of cactus diseases. P. brasiliense was reported in Mexico as the causal pathogen of soft rot in cactus (Mejía-Sánchez et al., 2019). P. cacticida was identified as the causal pathogen of soft rot of cactus in the United States (Alcorn et al., 1991), and P. carotovorum subsp. carotovorum was identified as the causal agent of soft rot in cactus in Korea (Kim et al., 2007). It is known that bacteria belonging to Pectobacterium species that cause soft rot in various plant hosts produce various plant cell wall-degrading enzymes, such as cellulase, polygalacturonase, and pectinase (Lee et al., 2013).

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.

Fig. 1.

Bacterial soft rot symptoms of graft cactus caused by Pectobacterium brasiliense KNUB-01-21. (A) Soft and black rot symptoms observed in the greenhouse. (B) Symptom induced by artificial inoculation using Pectobacterium brasiliense KNUB-01-21. (C) Sterilized water was used as a control.

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).

Pectobacterium species used in this study for phylogenetic analysis and GenBank accession numbers

Fig. 2.

Maximum-likelihood phylogenetic tree, based on concatenated partial sequences of dnaX, leuS, and recA genes, showing the phylogenetic position of strain KNUB-01-21 among related species of the genus Pectobacterium. Bootstrap values (based on 1,000 replications) greater than 70% are shown at branch points. Pectobacterium cacticida CFBP3628 T was used as the outgroup. Scale bar, 0.020 substitutions per nucleotide position.

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.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and For-estry (IPET) through the Agriculture, Food and Rural Affairs Convergence Technologies Program for Educating Creative Global Leader Program funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (no. 321001-03).

Electronic Supplementary Material

Supplementary materials are available at Research in Plant Disease website (http://www.online-rpd.org/).

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Article information Continued

Fig. 1.

Bacterial soft rot symptoms of graft cactus caused by Pectobacterium brasiliense KNUB-01-21. (A) Soft and black rot symptoms observed in the greenhouse. (B) Symptom induced by artificial inoculation using Pectobacterium brasiliense KNUB-01-21. (C) Sterilized water was used as a control.

Table 1.

Pectobacterium species used in this study for phylogenetic analysis and GenBank accession numbers

Species Strain no. GenBank accession no.
dnaX leuS recA
Pectobacterium aroidearum CFBP1457 MT683925 MT684072 MT684219
Pectobacterium aroidearum CFBP2573 MT683941 MT684088 MT684235
Pectobacterium aroidearum CFBP6725 MT684029 MT684176 MT684323
Pectobacterium aroidearum CFBP8737 MT684054 MT684201 MT684348
Pectobacterium atrosepticum CFBP1526T MK516904 MK517048 MK517192
Pectobacterium betavasculorum CFBP1539T MK516905 MK517049 MK517193
Pectobacterium brasiliense KNUB-01-21 LC717494 LC717495 LC717493
Pectobacterium brasiliense CFBP5392 MK516927 MK517071 MK517215
Pectobacterium brasiliense CFBP6607 MK516954 MK517098 MK517242
Pectobacterium brasiliense CFBP6615 MK516955 MK517099 MK517243
Pectobacterium brasiliense CFBP6617T MK516956 MK517100 MK517244
Pectobacterium cacticida CFBP3628T MK516923 MK517067 MK517211
Pectobacterium carotovorum subsp. carotovorum CFBP1364 MK516896 MK517040 MK517184
Pectobacterium carotovorum subsp. carotovorum CFBP2046T MK516909 MK517053 MK517197
Pectobacterium carotovorum subsp. carotovorum CFBP6071 MK516950 MK517094 MK517238
Pectobacterium carotovorum subsp. carotovorum CFBP7351 MK516962 MK517106 MK517250
Pectobacterium odoriferum CFBP1878T MK516907 MK517051 MK517195
Pectobacterium odoriferum CFBP3259 MK516920 MK517064 MK517208
Pectobacterium odoriferum CFBP3297 MK516921 MK517065 MK517209
Pectobacterium odoriferum CFBP5539 MK516929 MK517073 MK517217
Pectobacterium fontis CFBP8629T MK516878 MK517022 MK517166
Pectobacterium parmentieri CFBP8475T MK516972 MK517116 MK517260
Pectobacterium peruviense CFBP5834 MK516935 MK517079 MK517223
Pectobacterium polaris CFBP1403 MK516898 MK517042 MK517186
Pectobacterium polaris CFBP6058 MK516945 MK517089 MK517233
Pectobacterium polaris CFBP7360 MT684038 MT684185 MT684332
Pectobacterium polaris CFBP8603T MT684046 MT684193 MT684340
Pectobacterium punjabense CFBP8604T MK516877 MK517021 MK517165
Pectobacterium versatile CFBP1118 MK516888 MK517032 MK517176
Pectobacterium versatile CFBP2138 MK516912 MK517056 MK517200
Pectobacterium versatile CFBP6051T MK516938 MK517082 MK517226
Pectobacterium versatile CFBP8656 MK516973 MK517117 MK517261
Pectobacterium wasabiae CFBP3304T MK516922 MK517066 MK517210

The isolated strain is shown in bold.

Fig. 2.

Maximum-likelihood phylogenetic tree, based on concatenated partial sequences of dnaX, leuS, and recA genes, showing the phylogenetic position of strain KNUB-01-21 among related species of the genus Pectobacterium. Bootstrap values (based on 1,000 replications) greater than 70% are shown at branch points. Pectobacterium cacticida CFBP3628 T was used as the outgroup. Scale bar, 0.020 substitutions per nucleotide position.