First Report of Melon Soft Rot Disease Caused by Pectobacterium brasiliense in Korea

Article information

Res. Plant Dis. 2023;29(3):310-315

Publication date (electronic) : 2023 September 30

doi : https://doi.org/10.5423/RPD.2023.29.3.310

Kyoung-Taek Park¹, Leonid N. Ten², Chang-Gi Back^3,4, Soo-Min Hong¹, Seung-Yeol Lee^1,2, Jeung-Sul Han⁵, Hee-Young Jung^1,2,

¹School of Applied Biosciences, Kyungpook National University, Daegu 41566, Korea

²Institute of Plant Medicine, Kyungpook National University, Daegu 41566, Korea

³National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Korea

⁴Department of Environmental Horticulture and Landscape Architecture, Environmental Horticulture, Dankook University, Cheonan 31116, Korea

⁵Department 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 2023 July 11; Revised 2023 August 14; Accepted 2023 August 18.

Abstract

Melon (Cucumis melo) belongs to the family Cucurbita-ceae, subfamily Cucurbitoideae, tribe Melothrieae, subtribe Cucumerinae, and the genus Cucumis. The fruit exhibits many morphological colors, shapes, flavors, and textures, along with diverse biochemical properties that vary de-pending on region and climate (Kim et al., 2021). In Korea, melons are grown on 3,595 hectares of land, with an annual production of 144,834 tons in 2021 (Food and Agriculture Organization Corporate Statistical Database, 2021). However, globally, bacterial diseases can affect melon production and are highly prevalent. In Serbia, Pectobacterium brasiliense has been identified as the causal agent of melon soft rot, while P. carotovorum subsp. carotovorum was identified as the causal agent of melon soft rot in Korea (Yi and Kim, 1996; Zlatković et al., 2019). As a highly aggressive phytopathogen, P. brasiliense has been reported in several global regions, including Asia, Africa, Europe, Oceania, and North and South America (Charkowski, 2018; Gottsberger and Huss, 2016; Hua et al., 2020; Jaramillo et al., 2017; Naas et al., 2018; Panda et al., 2012; Park et al., 2022, 2023).

In May 2021, typical melon soft rot symptoms, including soft, watery, slimy, black rot, wilting, and collapse were observed in Gokseong, Jeollanam-do, Korea. Affected plants showed soft rot symptoms on the infected tissue surfaces and water-soaked lesions on the infected stems (Fig. 1A, B). Disease incidence was approximately 15% of the melons in the investigated region, as observed in the comprehensive survey.

Fig. 1.

Soft rot symptoms caused by Pectobacterium brasiliense KNUB-06-21 on melon stems. (A) Soft rot symptoms on infected tissue surfaces and water-soaked infected stems. (B) Stem surface turned dark brown.

To isolate pathogens from infected plants, the affected stem surfaces sterilized in 1% hypochlorite solution for 90 sec and rinsed in sterile distilled water. Bacterial isolation was car-ried out on selective crystal violet pectate medium (Hyman et al., 2001) at 26°C for 48 hr. Strains positive for pectolytic cavity formation were selected and purified on nutrient agar (NA; Difco, Franklin Lakes, NJ, USA). A total of 2 isolates with identical colony morphology were obtained from the affected stem. Among these isolates, a single colony, designated as KNUB-06-21, was selected in a random manner for further investigation.

Genomic DNA of strain KNUB-06-21 was extracted using a commercial extraction kit (HiGene Genomic DNA Prep Kit, Biofact, Daejeon, Korea) following manufacturer's instructions. The 16S rRNA gene underwent polymerase chain reaction (PCR) using 9F/1512R primers (Weisburg et al., 1991) and yielded a 1,352 base pair (bp) fragment (GenBank no. LC773684), which after sequencing, was BLAST searched in the National Center for Biotechnology Information (NCBI) database. BLAST analyzes revealed 100% 16S rRNA gene similarity with several P. brasiliense strains (GenBank nos. CP009769, CP020350, MK910250, MN393919, and MN393942).

For accurate identification of the strain KNUB-06-21, three housekeeping genes (dnaX, leuS, and recA) were amplified using PCR protocols and primers as described by Portier et al. (2020). A 521 bp fragment was generated for dnaX gene, 511 bp for leuS gene, and 714 bp for recA gene (GenBank nos. LC773681, LC773682, and LC773683). The highest dnaX gene homology was 100% with P. brasiliense (GenBank no. LC717494). Similarly, the strain KNUB-06-21 exhibited a high level of sequence homology 100% with P. brasiliense (GenBank nos. CP059959, LC717493, and MK517247) for the recA gene and with P. brasiliense (GenBank no. LC717495) for the leuS gene, confirming their close genetic relationship. Con-catenated dnaX, leuS, and recA gene sequences were used in phylogenetic analyses and sequences from closely related neighbors were retrieved from NCBI (Table 1). Using MEGA7, a phylogenetic tree was constructed using the maximum-likelihood method with a bootstrap of 1,000 replicates (Kumar et al., 2016). As depicted in Fig. 2, strain KNUB-06-21 grouped with P. brasiliense clade in the phylogenetic tree, ex-hibiting a bootstrap value of 100%. This result indicates that it is a strain belonging to P. brasiliense.

Table 1.

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

Fig. 2.

Maximum-likelihood phylogenetic tree, based on concatenated sequences (dnaX+ leuS+ recA), shows the phylogenetic position of strain KNUB-06-21 among the closest members of the Pectobacterium. The isoated strain is shown in bold. KNUB-06-21 is in bold. Bootstrap values (based on 1,000 replications) >70% are shown at branch points. Dickeya solani CFBP 7704 was used as an outgroup. Scale bar=0.020 substitutions/nucleotide position.

The API ID 32 GN system (Biomérieux, Marcy l'Etoile, France) was used to investigate compound utilization by strain KNUB-06-21 according to manufacturer's instructions. KNUB-06-21 responded positively to N-acetyl-glucosamine, L-alanine, D-glucose, inositol, D-maltose, D-mannitol, D-mel-ibiose, L-rhamnose, salicin, L-serine, and sucrose utilization. However, it responded negatively to L-fucose, L-histidine, 3-hydroxybutyric acid, propionic acid, and D-sorbitol utilization. These results were consistent with the characteristics of the type strain of P. brasiliense (Supplementary Table 1) reported by Portier et al. (2019).

Strain KNUB-06-21 pathogenicity was tested by inoculating the strain onto melon plant stems. Bacterial colonies, grown for 48 hr on NA at 26°C, were picked using sterile toothpicks, inserted into stems at 5 cm above the stem base, and inoculation sites covered with vaseline. Control plants were treated with toothpicks dipped in sterile water. Inoculated plants were maintained in greenhouse conditions (25–30°C and relative humidity=80%). Typical soft rot symptoms appeared at 3–4 days after inoculation and were represented by wilting leaves on stems (Fig. 3A), water-soaked lesions on leaves (Fig. 3B), and detailed examination of a cross-section of an infected melon stem revealed the presence of a hollow stalk (Fig. 3C). In contrast, control plants showed no infectious symptoms (Fig. 3D–F). From infected plants, pathogen was successfully re-isolated and, based on its morphological characteristics and the 16S rRNA, dnaX, leuS, and recA gene sequences, was identified as P. brasiliense (data are not shown).

Fig. 3.

Pectobacterium brasiliense KNUB-06-21 pathogenicity results. (A) Inoculated melon plants show leaf wilting symptoms. (B) Water-soaked lesions on melon plants. (C) Cross-section of an infected melon stem showing a hollow stalk. (D–F) Control melon stems showing no infectious symptoms.

P. brasiliense has been reported as a soft rot pathogen on different crops (Meng et al., 2017; Onkendi and Moleleki, 2014). The isolated strain KNUB-06-21 was identified using 16S rRNA and housekeeping gene sequencing, biochemical analyses, and pathogenicity studies on melon stems. To the best of our knowledge, this is the first report of P. brasiliense causing melon soft rot in Korea. Our findings contrib-ute to an increased understanding of P. brasiliense diversity associated with melon, and highlight the importance of early detection and management strategies preventing pathogen spread. Further research is required to investigate P. brasiliense epidemiology and ecology in melon production areas, and develop effective control measures to minimize economic losses caused by this pathogen.

Electronic Supplementary Material

Article information Continued