ISSN (Online) 2287-3406 Journal of Life Science 2021 Vol. 31. No. 7. 609~616 DOI : https://doi.org/10.5352/JLS.2021.31.7.609 Evaluation of Bioassay Methods to Assess Bacterial Soft Rot Resistance in Radish Cultivars Tania Afroz1, Onsook Hur1, Nayoung Ro1, Jae-eun Lee1, Aejin Hwang1, Bichsaem Kim1, Awraris Derbie Assefa1, Ju Hee Rhee1, Jung Sook Sung2, Ho-sun Lee3* and Bum-Soo Hahn1* 1National Agrobiodiversity Center, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54874, Korea 2National Institute of Crop Science, Rural Development Administration, Miryang 50424, Korea 3International Technology Cooperation Center, Rural Development Administration, Jeonju 54875, Korea Received April 22, 2021 /Revised July 25, 2021 /Accepted July 27, 2021 Bacterial soft rot, caused by Pectobacterium carotovorum subsp. carotovorum (Pcc), is one of the destruc- tive diseases of radish (Raphanus sativus) in Asian countries. The objective of this study was to estab- lish an efficient bioassay method for the evaluation of bacterial soft rot resistance in commercial radish cultivars. First, an efficient bioassay method for examining resistance to bacterial soft rot in commer- cial radish cultivars was investigated. Six commercial radish cultivars were tested under various con- ditions: two temperatures (25℃ and 30℃), three inoculations methods (drenching, spraying, and root dipping), and two growth stages (two- and four-leaf stages). The results suggested that spraying with 1×106 cfu/ml of bacterial inoculums during the four-leaf stage and incubating at 30℃ could be the most efficient screening method for bacterial soft rot resistance in commercial radish cultivars. Second, we investigated the degree of resistance of 41 commercial radish cultivars to five Pcc isolates, namely KACC 10225, KACC 10343, KACC 10421, KACC 10458, and KACC 13953. KACC 10421 had the stron- gest susceptibility in terms of moderately resistant disease response to bacterial soft rot. Out of the 41 radish cultivars, 13 were moderately resistant to this pathogen, whereas 28 were susceptible. The moderately resistant radish cultivars in this investigation could serve as resistance donors in the breed- ing of soft rot resistance or could be used to determine varietal improvement for direct use by breed- ers, scientists, farmers, researchers, and end customers. Key words : Bacterial soft rot, disease, evaluation, Raphanus sativus, resistance Introduction ingredient in foodstuffs such as kimchi (a traditional fer- mented food), dongchimi, kkakdugi, chonggak kimchi, na- Radish (Raphanus sativus L.) is one of the most popular bak-kimchi, seokppakjji, and pickled radish. Some research root vegetable crops in the Brassicaceae family, and it can indicates that radish can reduce the risk of chronic or life- be grown throughout the year in many parts of the world, threatening illnesses including heart disease, diabetes, and including Asian countries such as China, Japan, and South colon cancer [8, 28]. Korea. According to the Korean Statistical Information Ser- Bacterial soft rot is caused by Pectobacterium carotovorum vice (KOSIS), the area under radish cultivation was about subsp. carotovorum (Pcc). Pcc inflicts serious damage and eco- 71,030 hectares and total production was 4.04 million tons, nomic losses in most vegetable crops, including radish, Chi- with an average productivity of 56.38 tons per hectare in nese cabbage, cabbage, carrot, potato, and all Brassica spp. South Korea [17]. Radishes are low in calories and sugar [9, 13, 14, 16, 24, 34]. It is one of the most destructive diseases and high in fiber. In South Korea, radish is an important affecting radish (Raphanus sativus) in China, Japan, and South Korea, where the crop is widely cultivated [5]. Pcc *Corresponding authors produces large amounts of extracellular plant cell wall-de- *Tel : +82-63-238-1118, Fax : +82-63-238-4859 grading enzymes (PCWDE, exoenzymes) including pectate- *E-mail : [email protected] (Ho-sun Lee) *Tel : +82-63-238-4930, Fax : +82-63-238-4859 lyases (Pel), polygalacturonases (Peh), proteases (Prt), and *E-mail : [email protected] (Bum-Soo Hahn) cellulases (Cel), which damage the cell membrane and there- This is an Open-Access article distributed under the terms of by cause leakage of electrolytes, extensive tissue maceration, the Creative Commons Attribution Non-Commercial License rotting, and subsequent plant death [3, 29]. This results in (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction losses in marketable yield in the field and also during transit, in any medium, provided the original work is properly cited. storage, and marketing [4]. 610 생명과학회지 2021, Vol. 31. No. 7 Disease-resistant crops can reduce crop losses with mini- bacterial culture was centrifuged at 4,000 rpm at 4℃ for 10 mum effort by growers in an environmentally safe and cost- min (Labogen, 1248R, South Korea), the supernatant was dis- effective manner. Additionally, disease-resistant crops can carded, and sterile water was added to the pellet and shaken be combined with other control measures to optimize dis- to generate a bacterial suspension. This suspension was di- ease management. Identification of crops and genetic re- luted with water and the optical density at 600 nm (OD600) sources associated with disease resistance requires suitable was measured using a UV spectrophotometer (Optizen Pop, screening techniques, and once these crops and genetic re- Daejeon, Korea). Bacterial density was adjusted to an OD600 sources are discovered, it is necessary to develop effective of 0.1 (1×108 cfu/ml), and the bacterial suspension was di- inoculation methods [15, 30, 33]. Several screening techni- luted (1/10) with sterile water to yield a 1×106 cfu/ml ques for identifying disease resistance in vegetables have suspension. been reported, including needle pricking, dipping, and de- tached leaves [25]; wooden toothpick pricking of stems of Pathogen inoculation and disease monitoring 3-4-week-old seedlings [32]; injection; and overhead spray- One or two seeds of each cultivar were planted in a 50- ing and drop-nozzle spraying of bacterial suspensions on hole seedling tray (width 540 mm × length 280 mm) contain- plants in the field [1, 2]. Several screening methods have ing sterilized soil. After germination, the seedlings were been established for diverse germplasms; identification of thinned to one plant per pot. The pots were maintained in the most effective screening method for a particular patho- a greenhouse with photoperiod for 16 hr at 15-18℃. After gen is crucial for resistance breeding [6]. We established an the development of two leaves (13 days old) or four leaves efficient bioassay method for bacterial soft rot of radish and (20 days old), seedlings were inoculated with Pcc by drench- identified sources of resistance to bacterial soft rot affecting ing, spraying, or root dipping. For drenching, a 10-ml sam- 41 commercial radish cultivars. ple of bacterial inoculum were drenched onto each plant. For spraying, 0.1% Tween 20 was added to the bacterial in- Materials and Methods oculum and shaken to mix well; a 10-ml sample of inoculum was then sprayed onto each plant using a fine atomizer. For Plant materials root dipping, 10 plants were uprooted and dipped into 50 We investigated resistance among 41 commercial radish ml of suspension for 20 min; inoculated plants were replanted cultivars purchased from seed companies in Korea. All ex- in the pot from which they were originally uprooted. After periments were conducted in a greenhouse and growth inoculation, all plants were incubated in a plant growth chamber at the National Agrobiodiversity Center (NAC), chamber at 25 or 30℃ and 80% relative humidity for 12 hr National Institute of Agricultural Sciences, Jeonju, Republic light and 12 hr dark periods. An equal number of plants of Korea. Experiments used seedlings at the two-leaf (13 serving as controls was inoculated with sterilized distilled days old) and four-leaf (20 days old) stages. water. After 5 days, the disease indices (DI; 0–4) of bacterial soft rot were recorded [18] (Fig. 1a–e). The scoring was as Bacterial isolates and inoculum preparation follows: 0 = healthy plant; 1 = chlorosis or rot 1-25%; 2 = We used five Pcc isolates (KACC 10225, KACC 10343, chlorosis or rot 25-50%; 3 = chlorosis or rot 50-75%; 4 = chlo- KACC 10421, KACC 10458, and KACC 13953) from the rosis or rot 75-100% or plant dead. Disease response was Korean Agricultural Culture Collection (KACC) and con- also classified as resistant (R), DI ≤ 1.0; moderately resistant firmed their pathogenicity to radish plants. The bacterial iso- (MR), 1.1 < DI ≤ 2.0; or susceptible (S), DI > 2.0. lates were stored at -70℃. In preparation for experiments, bacteria were spread on nutrient agar (NA; Becton, Dickin- Statistical analysis son, and Co., Sparks, MD) in a Petri dish and incubated for The experiment involved a completely randomized de- 1 day. Next, 5 ml of nutrient broth (NB; Becton, Dickinson, sign (CRD) with three replications: 20 plants per replication and Co.) were added and the cultures were mixed. Bacterial were used for each method. Statistical analysis was per- suspension (2 ml) was inoculated into 200 ml of fresh NB formed by Duncan’s new multiple range test at α = 0.05 us- and cultured at 30℃ with shaking at 200 rpm for 36 hr. The ing R software version 3.1.0 [21]. Journal of Life Science 2021, Vol. 31. No. 7 611 Fig. 1. Disease index (0–4) of radish soft rot. a, DI = 0; b, DI = 1; c, DI = 2; d, DI = 3 and e, DI = 4. Scale bar, 4.8 cm. Results and Discussion jaealtaimu also had a DI of 4.0 by spraying at 25℃. In con- trast, at the two-leaf stage Gmchorong, and Jeonmuhoomu Effect of temperature, inoculation method, and growth has a DI of 3.8 and 3.77, respectively, by spraying at 30℃.