*Corresponding author. Phone) +82-42-860-7434, FAX) +82-42-861-4913, E-mail) Introduction
The soilborne biotrophic pathogen Plasmodiophora brassicae (Woronin) causes clubroot disease in crucifers worldwide, which has led to significant economic losses in crucifer production (Strelkov and Hwang, 2014). The roots of susceptible plants infected by this pathogen develop large clubs that interfere with the transportation of water and nutrients, which result in the stunting, discoloration, and wilting of plants (Hwang et al., 2012). Numerous strategies have been proposed to control clubroot disease, such as pH adjustments of the soil, crop rotation, chemical treatments, and biological controls using antagonistic microorganisms (Dixon, 2009; Voorrips, 1995). Currently, the development of resistant cultivars has been recognized as the most desirable method for controlling clubroot disease (Rahman et al., 2014). However, despite considerable efforts to develop clubroot-resistant (CR) cultivars, breakdown in the resistance to P. brassicae has been reported in numerous Brassica spp., which probably resulted from the selection pressure imposed on the pathogen (Hatakeyama et al., 2004; Jo et al., 2011; Kuginuki et al., 1999; Piao et al., 2004; Zhang et al., 2015). Therefore, an understanding of the virulence phenotypes of the P. brassicae population and detailed information of the host responses to the pathogens have been required for the development of effective breeding strategies.
Pathological specialization has been known to occur in P. brassicae through which the pathogens differ in their ability to infect a host (Honig, 1931; Hwang et al., 2012). Various sets of differential hosts, such as the Williams’ differential set and the European Clubroot Differential set, have been proposed in order to identify the pathotypes of P. brassicae in several countries (Buczacki et al., 1975; Kuginuki et al., 1999; Some et al., 1996; Williams, 1966). In Korea, it has been reported that 14 of the 16 races exist based on the Williams’ differential set, which consists of cabbage (Brassica oleracea L. var. capitata L. ‘Jersey Queen’ and ‘Badger Shipper’) and rutabaga (B. napo-Brassica Mill. ‘Laurentian’ and ‘Wilhelmsburger’) (Cho et al., 2003; Williams, 1966). Although the Williams’ differential set has been primarily used as a differential host set, Hatakeyama et al. (2004) suggested that two CR F1 cultivars of Chinese cabbage, Super CR Hiroki and Ryutoky, can be used as differential hosts to determine the races of P. brassicae isolates in Japan rather than those of the Williams’ differential set. Thus, more efficient systems are required for the identification of pathotypes of P. brassicae isolates, which is useful in breeding.
Since clubroot disease of Chinese cabbage (B. rapa) was first reported at 1928 in Korea, it has emerged as a significant concern in Chinese cabbage cultivation with spreading rapidly in cultivated areas (Cho et al., 2003). In order to overcome this problem, many Korean seed companies have bred CR cultivars of Chinese cabbage using CR genes identified from European fodder turnip (B. rapa subsp. rapifera), and their clubroot resistances have been evaluated using P. brassicae isolates that were classified by the Williams’ host responses (Jo et al., 2011; Piao et al., 2009; Zhang et al., 2015). Despite considerable efforts, clubroot disease by P. brassicae has been observed on Chinese cabbage in Korea. To obtain a better understanding of the pathogenic diversity of the P. brassicae isolates, this study investigated the pathotypes of Korean P. brassicae field isolates with various cultivars of Chinese cabbage, including 22 cultivars from Korea, China, and Japan, and 15 inbred lines. We found that four differential hosts can distinguish four pathotype groups of P. brassicae field isolates in Korea, which provides a new foothold for breeding strategies for CR Chinese cabbage.
Materials and Methods
Plant materials and P. brassicae field isolates
A total of 37 plants, including 22 Chinese cabbage F1 cultivars and 15 inbred lines, were used to evaluate clubroot resistance to P. brassicae. Of the 22 F1 cultivars, twenty-one have been developed as CR cultivars, and are currently cultivated in Korea, China, and Japan. The non-CR cultivar ‘Noranggimjang’ was used to verify the pathogenicity of the P. brassicae isolates and to maintain the P. brassicae field isolates. The differential hosts proposed by Williams, such as cabbage cultivars (‘Jersey Queen’ and ‘Badger Shipper’) and rutabaga cultivars (‘Laurentian’ and ‘Wilhelmsburger’), were used for race identification of the 12 P. brassicae field isolates (Williams, 1966). The P. brassicae isolates were obtained from naturally occurring clubroot galls and were used in the pathogenicity assays (Table 1).
Preparation of the inoculum for the pathogenicity assays
To prepare the inoculum, the resting spores of P. brassicae were obtained as previously described (Jo et al., 2011). Briefly, infected roots (approximately 1 g) from each field were collected and blended into a slurry with 500 ml of water in a blender. Next, the slurry was passed through four layers of cheesecloth and the concentration of the spore suspension was adjusted to 1 × 106 resting spores/ml. The spore suspension (5 ml) of each field isolate was inoculated onto the non-CR cultivar ‘Noranggimjang’ grown in 6 × 6 × 6 cm plastic pots (one seedling per pot) for 30 days. The potting mixture (Punong, Gyungju, Korea) was kept saturated with water for the first week after inoculation and fertilized as required. After seven days of incubation in a growth chamber (20°C), the inoculated single plants were transplanted into a plastic cup (18 cm of diameter) and grown in a greenhouse (20-25°C). The club roots were collected 60 days after incubation and stored at −80°C until required for the pathogenicity assay.
Plant inoculation and disease assessment
Five milliliters of spore suspensions (1.0 × 107 spores/ml) of each field isolate were prepared as described above, and then they were inoculated via drenching onto the Chinese cabbage cultivars and inbred lines that were grown in 6 × 6 × 6 cm plastic pots (one seedling per pot) for 10 days. The inoculated plants were kept in a growth chamber for seven days with a 12 h photoperiod, and then transferred to the greenhouse. After four weeks, the plants were dug out and the roots were washed with tap water. The severity of the club development on each plant was rated using a scale of 0-4, where 0 = no clubbing, 1 = one or a few small clubs on the lateral roots, 2 = a few medium and separate globular clubs on the lateral roots, 3 = medium sized clubs on the main roots, and 4 = severe clubbing on the lateral roots and main roots (Kuginuki et al., 1999; Suwabe et al., 2003). The experimental units consisted of 20 individual plants and the experiments were repeated three times independently. Host responses developing disease severity (DS) ≤ 1 were regarded as ‘resistant’, while those developing a DS ≥ 2 were regarded as ‘susceptible’.
Results
Race determination of P. brassicae isolates using the Williams’ host set
A differential host set of Williams was proposed in order to identify the pathotypes of P. brassicae, which is one of the most commonly used hosts among the differential hosts proposed (Williams, 1966). When each field isolate was inoculated onto a Williams’ host set, it was observed that the host responses are clearly either susceptible or resistant. All Williams’ hosts were highly susceptible to HN1 and HN2 isolates, and resistant to DJ, KS, and SS isolates (Table 1). The GS, GN1, JS, and PC isolates caused typical symptoms on the rutabaga cultivar ‘Laurentian’ and ‘Wilhelmsburger’; however, they did not cause these symptoms on the cabbage cultivar ‘Jersey Queen’ and ‘Badger Shipper’. The HS and YC isolates led to resistant responses on the ‘Wilhelmsburger’ cultivar only, and the GN2 isolate exhibited resistant responses in interactions with the ‘Badger Shipper’ cultivar. According to the reactions on the Williams’ differentials, it was found that the 12 field isolates could be classified into five races: race 1 (GN2), race 2 (HS and YC), race 4 (HN1 and HN2), race 5 (DJ, KS, and SS), and race 9 (GS, GN1, JS, and PC) (Table 1).
Disease responses of Chinese cabbage cultivars using P. brassicae isolates
To investigate the disease responses of Chinese cabbage to the P. brassicae isolates, the spore suspensions of 12 field isolates were inoculated against various CR cultivars of Chinese cabbage. The non-CR cultivar ‘Noranggimjang’ exhibited the typical clubroot disease with all P. brassicae field isolates, which included severe clubbing on the lateral roots and main roots; this indicates that the field isolates are highly pathogenic (Table 2). All Korean CR cultivars exhibited resistant responses to the GN, GN2, GS, HS, JS, DJ, and KS isolates, whereas they exhibited susceptible responses to the PC, YC, HN1, HN2, and SS isolates (Table 2). In addition, all Korean CR cultivars exhibited similar disease responses to the 12 field isolates, suggesting that the CR Korean cultivars may carry similar sources of resistance. In contrast to the Korean CR cultivars, the Chinese CR cultivars exhibited resistant responses to the GN, GN2, GS, HS, JS, HN1, PC, and YC isolates, but exhibited susceptible responses to the DJ, KS, HN2, and SS isolates (Table 2). The two Japanese CR cultivars also exhibited resistance to all isolates, except the HN2 and SS isolates (Table 2). Therefore, the 22 Chinese cabbage cultivars used in this study were classified into four different groups related to their disease resistance. Moreover, based on the ability to cause clubroot disease, the 12 field isolates were reclassified into four different groups: pathotype 1 (GN1, GN2, GS, JS, and HS), pathotype 2 (DJ and KS), pathotype 3 (HN1, PC, and YC), and pathotype 4 (HN2 and SS) (Table 2). These results also presented that several isolates cause different disease responses in the CR cultivars of Chinese cabbage even though the isolates belong to the same race according to Williams’ host responses.
Disease responses of inbred lines of Chinese cabbage using P. brassicae isolates
To clarify the disease responses on the CR cultivars, we examined the plant responses of 15 inbred lines of Chinese cabbage to the P. brassicae isolates. Since there are four different pathotype groups that cause similar disease responses in CR cultivars, a representative isolate was selected from each pathotype group: the GN1, DJ, YC, and HN2 isolates. When these isolates were inoculated onto the 15 inbred lines, either highly resistant or highly susceptible plant responses were observed in the inbred lines, which were similar to the disease responses on Chinese cabbage cultivars (Table 3). The inbred lines of WR02, WR09, and WR11 exhibited severe clubroot disease by all isolates, which indicates that these inbred lines were similar to the disease response of the non-CR cultivar. When each representative isolate was inoculated on the inbred lines (SS002, SS004, WR03, WR05, and WR06), the inbred lines exhibited resistant responses to the GN1 and DJ isolates, whereas they exhibited susceptible responses to the YC and HN2 isolates. These results were similar to the disease responses of the Korean CR cultivars. The inbred lines of SS001, WR01, WR04, and WR10 exhibited resistant responses to the GN1 and YC isolates, and susceptible responses to the DJ and HN2 isolates, which were similar to the disease responses of the Chinese CR cultivars. The inbred lines of SS003, WR07, and WR08 exhibited exclusively susceptible responses to the HN2 isolate from the four representative isolates, which was comparable with the Japanese CR cultivars. Combining these results, it was demonstrated that the 15 inbred lines consisted of four different groups based on the disease response to the four representative isolates (Table 3). In addition, it was observed that other isolates in the same pathotype group as the representative isolates exhibited similar results compared with the disease responses from the representative (data not shown).
Chinese cabbage differential hosts with P. brassicae isolates
On the basis of the plant disease responses of the Chinese cabbage cultivars and inbred lines to P. brassicae isolates, it was determined that there are four different host groups. Thus, a representative cultivar was selected from each group: the non-CR cultivar ‘Noranggimjang’ and the CR cultivars ‘CR-Cheongrok’, ‘DegaoCR1016’, and ‘Akimeki’ (Table 4). With four pathotypes, a new model was proposed for disease reactions between Chinese cabbage and P. brassicae. Pathotype 1 led the resistant reactions for all CR cultivars, whereas pathotype 4 was highly virulent on all CR cultivars. Pathotype 2 and 3 only exhibited pathogenicity on the ‘DegaoCR1016’ and ‘CR-Cheongrok’ cultivars, respectively (Table 4, Fig. 1).
Discussion
The ability of P. brassicae strains to infect the host has been distinguished depending on host genotypes (Honig, 1931). In the clubroot pathosystem, strain of the pathogen has been referred to as ‘race’, but when populations rather than single-spore isolates of P. brassicae are examined, the term ‘pathotype’ has been proposed instead of ‘race’ (Parlevliet, 1985; Voorrips, 1996). In this study, P. brassicae field isolates differing in virulence were referred to as ‘pathotype’, although ‘races’ of Williams (1966) are often referred to as ‘pathotypes’ in several literatures (Xue et al., 2008; Zhang et al., 2015).
Disease resistance to P. brassicae has become an important issue in the breeding of cruciferous crops worldwide; however, there is limited information related to host resistance resources (Hwang et al., 2012). In B. rapa, resistance is known to be controlled through a qualitative major resistance gene, which results in pathotype-specific reactions by P. brassicae (Hirai et al., 2004; Hwang et al., 2012; Suwabe et al., 2003; Wit and van de Weg, 1964). However, resistance based on single genes is not durable in general. In this study, the disease responses of Chinese cabbage to the P. brassicae isolates were investigated, and it was found that there are differences in pathogenicity among the P. brassicae isolates. In particular, all CR cultivars and inbred lines used in this study exhibited considerable DS to pathotype 4 isolates HN2 and SS, suggesting that a major gene related with resistance seems no longer effective to pathotype 4 isolates.
Considering that the virulence of P. brassicae depends on the host cultivars and inbred lines, the differential hosts are essential to distinguish the pathogen populations even in the same location (Hatakeyama et al., 2004; Kuginuki et al., 1999). Since the genetic heterogeneity of P. brassicae populations were reported in the field, the results obtained regarding the resistance or susceptibility of a set of differentials through inoculation with field isolates of P. brassicae might not provide precise information on the occurrence of pathotypes (Jones et al., 1982; Manzanares-Dauleux et al., 2001). Despite the coexistence of multiple isolates in the field, a system for determining the pathotypes of the P. brassicae field populations could provide practical information to breeders (Hatakeyama et al., 2004). Our results showed complete reactions of either resistance or susceptibility on all Chinese cabbage and inbred lines by P. brassicae field isolates; intermediate resistances were not observed. These results suggested that the investigated field isolates are genetically uniform and stable, although we cannot exclude a possibility that one pathotype might be dominant and others might be present at a low frequency and easily overlooked. Strehlow et al. (2014) presented that the 59 field isolates exhibited a unique genotype pattern depending on the regions or fields. The authors proposed that the geographic differentiation resulted from low levels of gene flow due to the limited dispersal of the soilborne pathogen P. brassicae and from the localized selection pressure as a unifying force on the genotypes of the host (Strehlow et al., 2014). Thus, the collection and characterization of field isolates showing different pathogenicities would be useful for breeding of CR Chinese cabbage.
In this study, pathotypes of P. brassicae isolates that differed from the results based on the Williams’ host reactions were determined. For example, the HN1, PC, and YC isolates in pathotype 3 were identified as belonging to races 4, 9, and 2, respectively, using the Williams’ host set. In contrast, several isolates identified as the same race by Williams’ host responses led to different disease severities in Chinese cabbage, which resulted in reclassification of these isolates into different pathotype groups (e.g., DJ and SS isolates were reclassified into pathotypes 2 and 4, respectively). Moreover, the disease responses of the CR cultivars to the P. brassicae isolates were more explicit than those of Williams’ differential hosts, suggesting that the fluctuating results of the Williams’ hosts might result from their genetic heterogeneity (Kuginuki et al., 1999). Similarly, Hatakeyama et al. (2004) proposed that a differential host set of Chinese cabbage, which consisted of CR cultivar ‘Super CR Hiroki’, ‘Ryutoky’, and non-CR cultivars, would provide more precise plant disease responses compared to the Williams’ differential set. Therefore, these results support our demonstration that the Williams’ differential hosts could not appropriately classify the field isolates of Korea.
Consequently, we suggest that three CR cultivars (‘CR-Cheongrok’, ‘DegaoCR1016’, and ‘Akimeki’) and the non-CR cultivar ‘Noranggimjang’ can be used as differential hosts to determine the pathotype of P. brassicae field isolates in Korea. Furthermore, the P. brassicae field isolates of the four pathotype groups can be also used to screen resistant Chinese cabbage. Taken together, it is anticipated that with the pathotypes of P. brassicae, the differential system suggested in this study will be valuable in breeding for resistance and management of clubroot disease in Chinese cabbage.
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