Evaluation of Bioassay Methods to Assess Bacterial Soft Rot Resistance in Radish Cultivars

무 품종의 세균성 무름병 저항성 생물검정법 평가

Abstract

Bacterial soft rot, caused by Pectobacterium carotovorum subsp. carotovorum (Pcc), is one of the destructive diseases of radish (Raphanus sativus) in Asian countries. The objective of this study was to establish 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 commercial radish cultivars was investigated. Six commercial radish cultivars were tested under various conditions: 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 strongest 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 breeding of soft rot resistance or could be used to determine varietal improvement for direct use by breeders, scientists, farmers, researchers, and end customers.

세균성 무름병 균[Pectobacterium carotovorum subsp. carotovorum (Pcc)]에 의해서 일어나는 무름병은 아시아 국가에서 재배되는 무에 있어서 심각한 질병 중의 하나로 알려져 있다. 이번 연구의 목적은 상업적으로 시판되는 무 품종의 세균성 무름병의 저항성에 대한 효율적인 생물검정법을 확립하고자 하였다. 첫째, 무 품종에 대해 세균성 무름병의 효율적인 생물검정 방법을 조사하였다. 6개의 무 품종을 다양한 조건[두 가지 온도(25℃와 30℃), 3가지 접종방법(관주, 분무, 침지), 두 발생단계(2와 4잎 단계)]으로 조사하였다. 연구 결과는 무름병균 1×10⁶ cfu/ml 농도를 분무한 4잎 단계와 30℃에서 배양한 생물검정 방법이 무 품종에 대해 가장 효율적인 방법임을 알 수 있었다. 둘째, 41개의 무 품종에 5가지 세균(KACC 10225, KACC 10343, KACC 10421, KACC 10458, KACC 13953)을 접종하여 저항성을 조사하였다. KACC 10421가 세균성 무름병의 감수성 및 저항성 질병 정도를 가장 잘 나타냈다. 41개의 무 품종 중 13개는 무름병 균에 대해 중도 저항성을 나타냈고 28개는 감수성을 나타냈다. 이 연구에서 중도저항성 무 품종은 세균성 무름병 저항성 육종을 위한 저항성 자원으로 활용 가능하고 육종가, 농민, 연구자, 최종 소비자에 의해서 다양한 목적을 위해 사용 가능할 것으로 사료된다.

Introduction

Radish (Raphanus sativus L.) is one of the most popular root vegetable crops in the Brassicaceae family, and it can be grown throughout the year in many parts of the world, including Asian countries such as China, Japan, and South Korea. According to the Korean Statistical Information Service (KOSIS), the area under radish cultivation was about 71,030 hectares and total production was 4.04 million tons, with an average productivity of 56.38 tons per hectare in South Korea [17]. Radishes are low in calories and sugar and high in fiber. In South Korea, radish is an important ingredient in foodstuffs such as kimchi (a traditional fermented food), dongchimi, kkakdugi, chonggak kimchi, nabak-kimchi, seokppakjji, and pickled radish. Some research indicates that radish can reduce the risk of chronic or life threatening illnesses including heart disease, diabetes, and colon cancer [8,28].

Bacterial soft rot is caused by Pectobacterium carotovorum subsp. carotovorum (Pcc). Pcc inflicts serious damage and economic losses in most vegetable crops, including radish, Chinese cabbage, cabbage, carrot, potato, and all Brassica spp. [9, 13, 14, 16, 24, 34]. It is one of the most destructive diseases affecting radish (Raphanus sativus) in China, Japan, and South Korea, where the crop is widely cultivated [5]. Pcc produces large amounts of extracellular plant cell wall-degrading enzymes (PCWDE, exoenzymes) including pectatelyases (Pel), polygalacturonases (Peh), proteases (Prt), and cellulases (Cel), which damage the cell membrane and thereby cause leakage of electrolytes, extensive tissue maceration, rotting, and subsequent plant death [3,29]. This results in losses in marketable yield in the field and also during transit, storage, and marketing [4].

Disease-resistant crops can reduce crop losses with minimum effort by growers in an environmentally safe and cost-effective manner. Additionally, disease-resistant crops can be combined with other control measures to optimize disease management. Identification of crops and genetic resources associated with disease resistance requires suitable screening techniques, and once these crops and genetic resources are discovered, it is necessary to develop effective inoculation methods [15, 30, 33]. Several screening techniques for identifying disease resistance in vegetables have been reported, including needle pricking, dipping, and detached leaves [25]; wooden toothpick pricking of stems of 3-4-week-old seedlings [32]; injection; and overhead spraying and drop-nozzle spraying of bacterial suspensions on plants in the field [1,2]. Several screening methods have been established for diverse germplasms; identification of the most effective screening method for a particular pathogen is crucial for resistance breeding [6]. We established an efficient bioassay method for bacterial soft rot of radish and identified sources of resistance to bacterial soft rot affecting 41 commercial radish cultivars.

Materials and Methods

Plant materials

We investigated resistance among 41 commercial radish cultivars purchased from seed companies in Korea. All experiments were conducted in a greenhouse and growth chamber at the National Agrobiodiversity Center (NAC), National Institute of Agricultural Sciences, Jeonju, Republic of Korea. Experiments used seedlings at the two-leaf (13 days old) and four-leaf (20 days old) stages.

Bacterial isolates and inoculum preparation

We used five Pcc isolates (KACC 10225, KACC 10343, KACC 10421, KACC 10458, and KACC 13953) from the Korean Agricultural Culture Collection (KACC) and confirmed their pathogenicity to radish plants. The bacterial isolates were stored at -70ºC. In preparation for experiments, bacteria were spread on nutrient agar (NA; Becton, Dickin- son, and Co., Sparks, MD) in a Petri dish and incubated for 1 day. Next, 5 ml of nutrient broth (NB; Becton, Dickinson, and Co.) were added and the cultures were mixed. Bacterial suspension (2 ml) was inoculated into 200 ml of fresh NB and cultured at 30℃ with shaking at 200 rpm for 36 hr. The bacterial culture was centrifuged at 4, 000 rpm at 4℃ for 10 min (Labogen, 1248R, South Korea), the supernatant was discarded, and sterile water was added to the pellet and shaken to generate a bacterial suspension. This suspension was diluted with water and the optical density at 600 nm (OD₆₀₀) was measured using a UV spectrophotometer (Optizen Pop, Daejeon, Korea). Bacterial density was adjusted to an OD₆₀₀ of 0.1 (1×10⁸ cfu/ml), and the bacterial suspension was diluted (1/10) with sterile water to yield a 1×10⁶ cfu/ml suspension.

Pathogen inoculation and disease monitoring

One or two seeds of each cultivar were planted in a 50 hole seedling tray (width 540 mm × length 280 mm) containing sterilized soil. After germination, the seedlings were thinned to one plant per pot. The pots were maintained in a greenhouse with photoperiod for 16 hr at 15-18℃. After the development of two leaves (13 days old) or four leaves (20 days old), seedlings were inoculated with Pcc by drenching, spraying, or root dipping. For drenching, a 10-ml sample of bacterial inoculum were drenched onto each plant. For spraying, 0.1% Tween 20 was added to the bacterial inoculum and shaken to mix well; a 10-ml sample of inoculum was then sprayed onto each plant using a fine atomizer. For root dipping, 10 plants were uprooted and dipped into 50 ml of suspension for 20 min; inoculated plants were replanted in the pot from which they were originally uprooted. After inoculation, all plants were incubated in a plant growth chamber at 25 or 30℃ and 80% relative humidity for 12 hr light and 12 hr dark periods. An equal number of plants serving as controls was inoculated with sterilized distilled water. After 5 days, the disease indices (DI; 0–4) of bacterial soft rot were recorded [18] (Fig. 1a–e). The scoring was as follows: 0 = healthy plant; 1 = chlorosis or rot 1-25%; 2 = chlorosis or rot 25-50%; 3 = chlorosis or rot 50-75%; 4 = chlorosis or rot 75-100% or plant dead. Disease response was also classified as resistant (R), DI ≤ 1.0; moderately resistant (MR), 1.1 < DI ≤ 2.0; or susceptible (S), DI > 2.0.

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.

Statistical analysis

The experiment involved a completely randomized design (CRD) with three replications: 20 plants per replication were used for each method. Statistical analysis was performed by Duncan’s new multiple range test at α = 0.05 using R software version 3.1.0 [21].

Results and Discussion

Effect of temperature, inoculation method, and growth stage on bacterial soft rot DI

To evaluate the bioassay methods, we used six commercial radish cultivars (Gangjamoojeok [SAM1], Gmchorong [SAK1], Seonbongaltari [S1], Worldminong [N₂], Jeonmuhoomu [K1], and Heongjaealtaimu [NS1]); two temperatures (25 and 30℃); three inoculation methods (drenching, spraying, and root dipping); and two growth stages (two- and four-leaf stages). Table 1 and Fig. 2 present the effects of temperature, inoculation method, and growth stage on the bacterial soft rot DI. Of the six cultivars, at the four-leaf stage Gangjamoojeok had the lowest DI of 3.8 and the others had a DI of 4.0 by spraying at 30℃; Jeonmuhoomu and Heongjaealtaimu also had a DI of 4.0 by spraying at 25℃. In contrast, at the two-leaf stage Gmchorong, and Jeonmuhoomu has a DI of 3.8 and 3.77, respectively, by spraying at 30℃. Environmental factors such as temperature and free moisture affect the development of soft rot disease [20]. High temperature plays an important role in soft rot infection and symptom development, and we found that 30℃ resulted in the highest DI (4) in each radish cultivar. Raju et al. reported that soft rot occurred at 20-35℃ and was most severe at 35℃ and 100% relative humidity [22]. Farrar et al. reported that 30-37℃ was optimum for soft rot development in various vegetable species [12]. However, Lee et al. found that a temperature of 25℃ promotes bacterial soft rot of radish [18], and Walker reported that bacterial soft rot caused the greatest damage at high humidity and 26℃ [31]. We found that the DI of soft rot on radish increased with increasing cultivation temperature (Table 1).

Table 1. Resistance of six commercial cultivars to Pectobacterium carotovorum subsp. carotovorum KACC 10421 by growth stage, inoculation method, and temperature

^xGrowth stage; two leaf stage (13-days-old) and four leaf stage (20-days-old).

^yMeans with the same letters are not significantly different based on Duncan’s multiple range test (DMRT) at p<0.05.

Fig. 2. Radish (Raphanus sativus) cultivar, Gmchorong (SAM1) showing soft rot symptoms at 25℃ (A) and 30°C (B) after inoculation by the drenching (a, d), spraying (b, e), and root-dipping (c, f) methods. Spraying and 30℃ resulted in the highest DI (DI 4). For drenching, a 10-ml sample of bacterial inoculum was drenched onto each plant; for spraying, a 10-ml sample of inoculum was sprayed onto each plant using a fine atomizer; for root dipping, each plant was dipped into a 50-ml sample of bacterial suspension for 20 min.

Spraying is the optimum inoculation method; it promotes rapid bacterial entry through natural openings such as lenticels or stomata, facilitating subsequent invasion, colonization, and expansion [19,27]. In contrast, root dipping and drenching methods involve drenching soil with the inoculum, thereby delaying colony growth and plant infection. Spraying inoculation accelerates disease development.

Tissue age, particularly leaf age, affects the susceptibility of radish to infection by Pcc. The DI was higher at the four-leaf stage (20 days old) compared with the two-leaf stage (13 days old). Lee at al. reported that radish seedlings at the four-leaf stage (20 days old) were more susceptible to bacterial soft rot [18]. Our results are consistent with previous reports that older leaves are more susceptible to disease [10, 11, 23].

Resistance of 41 radish cultivars to five Pcc isolates

We evaluated the resistance of 41 commercial radish cultivars to five Pcc isolates by spraying. The 41 radish cultivars were categorized into two groups based on their resistance level (Table 2). The bacterial soft rot DIs of Jeonmuhoomu (K1) and Heongjaealtaimu (NS1) were 1.3 and 3, 1.5 and 3.5, 2.3 and 4.0, 1.6 and 4.0, and 2.7 and 3.8 for KACC 10225, KACC 10343, KACC 10421, KACC 10458, and KACC 13953, respectively (Fig. 3). The mean DIs of bacterial soft rot were 2, 2.3, 2.7, 2.6, and 2.5 for KACC 10225, KACC 10343, KACC 10421, KACC 10458, and KACC 13953, respectively. Among the five Pcc isolates, KACC 10421 was the most pathogenic in the 41 radish cultivars. The commercial radish cultivars reacted differently to five Pcc isolates causing bacterial soft rot disease but were grouped into only two classes (moderate resistance, MR or susceptible, S) according to the classification system (Table 2). Of the 41 cultivars, 13 were classified as MR (mean DI 1.6 to 2.0) and 28 as S (mean DI 2.1 to 3.7) (Table 2); none were classified as R (no disease). The MR cultivars were Worldminong, Jeilbaegdong, Taecheong, Jeonmuhumu, Alpain goldae, Neulsaengbumu, Sanghwang, Gangjamoojeok, Cheongdumu, Sambogdabal, Gmchorong, Minongjosaengmu, and Khohaeyangsanchonmu. Lee et al. reported that the Jeonmuhumu, Cheongdumu, and Taecheong radish cultivars had moderate resistance to bacterial soft rot [18]. We used Jeonmuhum and Cheongdumu as controls. The variation in disease resistance could be the result of the complex interactions among the plant, pathogen, and environment that influence disease severity [7]. A few genotypes show resistance to pathogens by identifying and excluding them via a hypersensitivity response; others are susceptible because they cannot recognize the pathogen sufficiently early [26].

Table 2. Resistance of 41 commercial radish cultivars to five isolates of Pectobacterium carotovorum subsp. carotovorum^x

^x Four leaf stages (twenty-day-old seedlings) of radish cultivars were inoculated with a bacterial suspension (1.0×10⁶ cfu/ml) of five Pectobacterium carotovorum subsp carotovorum strains by spraying method. The inoculated plants were incubated in a growth chamber at 30°C and relative humidity 80% for 12 hr light and 12 hr dark periods. Five days after inoculation, disease index of each plant was score on a scale of 0-4.

^y DI, disease index; Each value represents the mean disease index (± standard deviation) of three runs with 10 replicates each.

^z RS, response; R, resistant, DI ≤ 1.0; MR, moderately resistant, 1.1 < DI ≤ 2.0; S, susceptible, DI > 2.0

Fig. 3. Radish (Raphanus sativus) Jeonmuhoomu (K1) (A) and Heongjaealtaimu (NS1) (B) exhibiting bacterial soft rot symptoms after inoculation with the Pcc isolates KACC 10225 (a, f), KACC 10343 (b, g), KACC 10421 (c, h), KACC 10458 (d, i), KACC 13953 (e, j). Among them, KACC 10421 exhibited the strongest resistance.

In conclusion, spraying at the four-leaf stage and incubation at 3ºC are optimal conditions for screening for radish germplasms resistant to bacterial soft rot; the MR accessions could serve as resistance donors for development radish varieties resistant to bacterial soft rot.