A New Identiﬁcation Method Reveals the Resistance of an Extensive-Source Radish Collection to Plasmodiophora brassicae Race 4

: Raphanus sativus , an important cruciferous vegetable, has been increasingly affected by clubroot disease. Establishing a stable and accurate resistance identiﬁcation method for screening resistant germplasms is urgently needed in radish. In this study, the inﬂuence of inoculum concentration, inoculation methods, and pH of the substrate on disease occurrence was studied. The result showed that the disease index (DI) was highest at 2 × 10 8 spores/mL, the efﬁciency of two-stage combined inoculation methods was higher than others, and pH 6.5 was favorable for the infection of P. brassicae . By using this new method, DIs of 349 radish germplasms varying from 0.00 to 97.04, presented signiﬁcantly different levels of resistance. Analysis showed that 85.06% germplasms from China were susceptible to P. brassicae , whilst 28 accessions were resistant and mainly distributed in east, southwest, northwest, and south-central China. Most of the exotic germplasms were resistant. Repeated experiments veriﬁed the stability and reliability of the method and the identity of germplasm resistance. In total, 13 immune, 5 highly resistant and 21 resistant radish accessions were identiﬁed. This study provides an original clubroot-tolerance evaluation technology and valu-able materials for the development of broad-spectrum resistant varieties for sustainable clubroot management in radish and other cruciferous crops. and H.Y.; formal analysis, H.Y. and X.L.; investigation, H.Y. and J.S.; resources, Y.Y., X.W. and H.W.; data curation, H.Y. and X.Z.; writing—original draft preparation, H.Y.; writing—review and editing, X.L.; supervision, X.L.; funding acquisition, X.L. All authors have read and agreed the version of the manuscript.


Introduction
Clubroot disease caused by the soil-borne obligate pathogen Plasmodiophora brassicae Woronin is one of the most common and serious diseases in cruciferous crops worldwide. It is widespread in Europe, North America, and Asia, especially in East Asia [1]. In China, approximately one-third of cultivated areas of Brassicaceae vegetables have been infected, leading to a loss of production of nearly 60% [2]. The production of radish (Raphanus sativus L.), an important vegetable crop, has also been seriously threatened by this disease in many growing areas of China, such as Henan, Sichuan, Hubei, and Yunnan provinces [3][4][5][6].
Historically, Plasmodiophora brassicae has been studied in many aspects [7,8] and is classified as an amoeboid protozoan in the infrakingdom Rhizaria [9][10][11][12]. The pathogen is a kind of eukaryote and belongs to the Plasmodiophorales [1]. The infection process of host plants comprises a primary phase and a secondary phase [13,14]. These two phases occur separately, mainly in root hairs and cortical tissue, while the first phase can also scale identification of clubroot-resistance in Brassicaceae crops so far. According to relative information, the artificial inoculation of P. brassicae in the seedling period is mainly affected by the inoculation method, inoculum concentration, pH of seedling substrate, etc. This has been reported in B. rapa and B. oleracea [83][84][85][86][87][88][89][90][91]. Clubroot-soil (mix substrate with inoculum), dipping root (dipping root in inoculum), injecting (injecting inoculum to surrounding seed, bud or seedling), and soaking (soaking seed or bud in inoculum) have comprised the main inoculation methods used in Brassicaceae crops. The Effect of the inoculation method varies in different species, as studies focusing on inoculation methods have shown that the clubroot-soil method is considered to be better than other methods in B. oleracea [84][85][86], while the dipping root method is better than the others in B. rapa [87][88][89][90]. In most cases, inoculation was given only once at the seedling stage, which may result in missed or underdosed inoculation. In addition, the optimal concentration was mainly setup at three magnitudes: 10 7 , 10 8 , 10 9 spores/mL varying in different species [85,89,92]. The optimal pH of seedling substrate was mainly between 5-6.5 [86,[91][92][93]. In radish, no research on the effect of different inoculation method, inoculation times, inoculation concentration and pH of seedling substrate has been conducted.
In the present study, the effects of different inoculum concentrations, inoculation methods, and pH of the seedling substrate were compared and a stable and reliable screening method was established. Based on the method optimized, 349 accessions of representative radish germplasms conserved in the National Mid-term Genebank of Vegetable Germplasm Resources (NMGVGR) were evaluated for their reactions to P. brassicae race 4. Understanding the resistance distribution and the acquisition of diverse resistant sources will be helpful for the discovery of new clubroot resistant genes and resistance genetic improvement in radish and other cruciferous crops.

Plant Materials
Radish cultivar 791 (known to be susceptible), provided by Henan Academy of Agricultural Sciences (HAAS), was used in the study of the identification method and in the large-scale evaluation as a susceptible control.
A total of 349 accessions of radish germplasms were studied, including 328 indigenous and 21 exotic germplasm accessions. The indigenous radish accessions were provided by the National Mid-term Genebank of Vegetable Germplasm Resources in Beijing (NMGVGR) and were originally collected from 230 counties or cities of 29 provinces or municipalities belonging to six geographic regions (https://en.wikipedia.org/wiki/List_of_regions_of_ China, accessed on 23 October 2020). They were classified into six groups corresponding to their original Chinese geographic regions. Exotic radish accessions were categorized into Group 7. Detailed information on all germplasms in this study is listed in Table S1.

Pathogen Preparation
P. brassicae race 4 [94] was used in this study for its high prevalence in China. The clubbed roots isolated from infected Chinese cabbage root tissues (collected from Nanyang City, Henan Province, China) were first washed with tap water and finally with sterile water, and then air-dried and stored at −20 • C. The preparation of the resting spore inoculum was based on the method of Luo et al. [95]. The swollen roots were homogenized with a two-fold volume of distilled water using a blender and were then filtered through four layers of cheesecloth. The filtrate was collected and centrifuged twice at 4000× g for 10 min. The precipitate/spores were washed twice with sterile water. Finally, the spores were resuspended and adjusted to the corresponding concentration using a hemocytometer. P. brassicae race 4 was identified using differential hosts [96].

Plant Cultivation and Treatment
Comparison of different inoculum concentration The seeds of "791" was pre-germinated in Petri dishes with 2 layers of moist filter paper at 25 • C until the length of bud measured 5 mm. Then, thirty germinated seeds of "791" for each spore concentration treatment were sown into small pots of 10 cm diameter with a drenched sterile peat-vermiculite mixture (1:1, v/v) as the seedling substrate. The inoculum concentration was adjusted to 2 × 10 9 , 2 × 10 8 , 2 × 10 7 , 2 × 10 6 spores/mL, respectively, and 5 mL of resting spore inoculum was injected onto the surface of germinating seeds in each pot. The control materials were injected with the same amount of sterile water.
Comparison of different artificial inoculation methods based on the concentration of 2 × 10 8 spores/mL Injecting bud: Thirty pre-germinating seeds of "791" according to the method mentioned above were sown into the seedling-raising pots. Meanwhile, 5 mL of resting spore inoculum was injected onto the surface of germinating seeds in each pot. Stirring of inoculum should be done during inoculation to avoid sedimentation. The control materials were injected with 5 mL sterile water.
Soaking bud: Thirty germinating seeds of "791" were soaked into resting spore inoculum for 45 min [91]. The control materials were soaked in sterile water for an equal amount of time.
Injuring roots: The seedling was grown according to the procedure used for the injecting method. The difference was that the inoculation was carried out at the two trueleaf stage. The lateral and fibrous roots of thirty seedlings were gashed down from the substrate surface at a distance of 2 cm away from the stem base of each seedling, followed by a separate injection of 5 mL inoculum. The control materials were injected in 5 mL sterile water.
Two-stage combined inoculation: When the length of the bud was 5 mm after pregermination, thirty germinating seeds of "791" were sown into small pots, and 5 mL of resting spore inoculum were injected onto the surface of germinating seeds in each pot. When the seedlings grew to the two true-leaf stage, the lateral and fibrous roots were gashed down from the substrate surface at a distance of 2 cm away from the stem base of each seedling, followed by a separate injection of 5 mL inoculum. The control materials were injected with 5 mL of sterile water at the first and second inoculation in the same way.
Clubroot-soil: The sterile peat-vermiculite mixture was drenched with resting spore inoculum. Then, 25-30 g of clubroot-soil was placed into a small hole made in the center of pots [85]. Subsequently, thirty germinating seeds without any subsequent inoculation were also planted.
Comparison of different pH of substrate based on the concentration of 2 × 10 8 spores/mL Thirty pre-germinated seeds of "791" per pH were sown into small pots with a drenched sterile peat-vermiculite mixture (1:1, v/v). The mixture pH was adjusted to 5.5, 6.0, 6.5, and 7.0, respectively. Meanwhile, 5 mL of resting spore inoculum was injected onto the surface of germinated seed in each pot. The pH of the substrate was adjusted by using Hoagland nutrient solution with hydrochloric acid or potassium hydroxide. Next, the seedlings were watered with nutrient solution of corresponding pH.
Resistance evaluation of a large-scale germplasm resources Based on the identification method established, 349 accessions of radish germplasms were inoculated with 2 × 10 8 spores/mL inoculum by using the two-stage inoculation of combining injecting bud and injury seedling root method, as above.
The germinated seeds or seedlings were kept moist and watered less in the first week after inoculation (to prevent the inoculum from being washing away), but watered more from the second week, ensuring that P. brassicae could infect the root hairs and cortical tissues adequately.
Experiment design A completely randomized block design was arranged in three biological replicates and 10 seedlings for each replicate (20 seedlings for each replicate of the control cultivar). All pots were placed in the isolation area in an identification chamber at the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences (Beijing, China), and protected by an insect-proof net. The average temperature was 20-25 • C and the plants were maintained under a 14 hL/10 hD photoperiod. In addition, Hoagland nutrient solution was watered according to the growth requirements of seedlings [97]. The seedlings of pH experiment were watered with nutrient solution of corresponding pH.
Verification of the new method and germplasm resistance All immune accessions and parts of highly susceptible accessions were arranged in a second assessment trial to verify the stability of their resistance and the reproducibility of the identification trials.

Data Collection
To ensure that the seedlings had been fully infected before investigation, two seedlings of the susceptible control cultivar 791 were pulled out and observed for disease status every week, starting from the third week after the second inoculation. As a result, disease severity was evaluated on the 65th day after sowing. The improved evaluation criteria of root symptoms in radish crops were established based on the study by Kamei et al. [82] and previous studies as follows: grade 0, all roots symptom-free; grade 1, taproots symptomfree, slightly swollen fibrous roots/lateral roots or 1-2 small clubs on fibrous roots/lateral roots; grade 3, slightly swollen taproots and/or multiple small galls on 1/3∼1/2 lateral roots; grade 5, medium swollen taproots or a few galls on taproots, or more irregular tumors on 1/2∼2/3 lateral roots; grade 7, lager and swollen taproots and/or many mallet-/clubroots on more than 2/3 lateral roots; grade 9, extremely swollen taproots with severe clubbed lateral roots, partial decay of roots. The disease index (DI) based on the disease severity was calculated according to Strelkov et al. [98] using the modified formula:

Data Analysis
Data analysis was conducted using statistical analysis software (SAS version 9.3). A paired t-test was used to test the stability of the DIs of immune/susceptible materials between the first inoculation and the re-inoculation (p < 0.05). Analysis of Variance (ANOVA) with Duncan's multiple range test (PROC GLM statistical procedure) was performed to determine whether there was a significant difference among different inoculation conditions (p < 0.05).

Influence of Different Inoculum Concentrations, Inoculation Methods, and pH of Substrate on Severity of Clubroot Disease in Radish
The influence of different inoculum concentrations on disease severity was studied by setting four levels: 2 × 10 9 , 2 × 10 8 , 2 × 10 7 , and 2 × 10 6 spores/mL. The disease incidence of four levels was 100%, while the disease severity varied significantly among different concentrations. The mean DIs of three biological replicates of 2 × 10 9 , 2 × 10 8 , 2 × 10 7 , and 2 × 10 6 spores/mL were 62.84, 70.00, 64.90, and 56.36, and the DI of 2 × 10 8 spores/mL was the highest (Figure 1). ANOVA indicated that the degree of disease severity of 2 × 10 8 spores/mL was significantly different from other concentrations, but there was no statistically significantly difference with 2 × 10 9 and 2 × 10 7 spores/mL (p < 0.05). Based on the concentration of 2 × 10 8 spores/mL, inoculation methods and pH of substrate were compared. All plants in these two experiments were infected. According to the DIs of different inoculation methods, the plants (DI = 87.30) of two-stage combined inoculation were most severely infected and significantly different from the other methods, as determined by ANOVA (p < 0.05). There were no statistically significant differences among the soaking bud (DI = 62.59), injuring root (DI = 59.79) and clubroot-soil methods (DI = 57.04); the injecting bud effect was the lowest (DI = 48.64). In the pH experiment, the disease severity of pH 6.5 (DI = 79.63) was the highest and significantly different from other pH values, as determined by ANOVA (p < 0.05). The optimal pH condition of plants infected by P. brassicae was weakly acidic (5.5-6.5) rather than neutral.
The influence of different inoculum concentrations on disease severity was studied by setting four levels: 2 × 10 9 , 2 × 10 8 , 2 × 10 7 , and 2 × 10 6 spores/mL. The disease incidence of four levels was 100%, while the disease severity varied significantly among different concentrations. The mean DIs of three biological replicates of 2 × 10 9 , 2 × 10 8 , 2 × 10 7 , and 2 × 10 6 spores/mL were 62.84, 70.00, 64.90, and 56.36, and the DI of 2 × 10 8 spores/mL was the highest (Figure 1). ANOVA indicated that the degree of disease severity of 2 × 10 8 spores/mL was significantly different from other concentrations, but there was no statistically significantly difference with 2 × 10 9 and 2 × 10 7 spores/mL (p < 0.05). Based on the concentration of 2 × 10 8 spores/mL, inoculation methods and pH of substrate were compared. All plants in these two experiments were infected. According to the DIs of different inoculation methods, the plants (DI = 87.30) of two-stage combined inoculation were most severely infected and significantly different from the other methods, as determined by ANOVA (p < 0.05). There were no statistically significant differences among the soaking bud (DI = 62.59), injuring root (DI = 59.79) and clubroot-soil methods (DI = 57.04); the injecting bud effect was the lowest (DI = 48.64). In the pH experiment, the disease severity of pH 6.5 (DI = 79.63) was the highest and significantly different from other pH values, as determined by ANOVA (p < 0.05). The optimal pH condition of plants infected by P. brassicae was weakly acidic (5.5-6.5) rather than neutral.

The Resistance of Radish Germplasms to P. brassicae Race 4
In this study, 349 radish germplasms were inoculated using the newly established two-stage combined inoculation method based on 2 × 10 8 spores/mL. The DIs from 0.00 to 97.04 (Table S1) varied significantly among all radish germplasms. According to the resistance classification standard mentioned above, most radish accessions (81.66%) were susceptible to P. brassicae, including 81 susceptible materials and 204 highly susceptible materials. Fortunately, 41 accessions (11.75%) exhibited resistance to clubroot. Among these materials, 21 accessions were resistant, 5 accessions showed high resistance, whilst 15 accessions revealed no disease symptoms and were identified as immune to P. brassicae (Table S1). A phenotype comparison of representative immune vs. highly susceptible radish materials is shown in Figure 2. materials. Fortunately, 41 accessions (11.75%) exhibited resistance to clubroot. Among these materials, 21 accessions were resistant, 5 accessions showed high resistance, whilst 15 accessions revealed no disease symptoms and were identified as immune to P. brassicae (Table S1). A phenotype comparison of representative immune vs. highly susceptible radish materials is shown in Figure 2.

Figure 2.
Comparison of the phenotypes of representative radish materials that are either immune or highly susceptible to P. brassicae race 4. CRR240, CRR297, and CRR330 illustrate the resistance of immune germplasms; CRR84, CRR71, and CRR108 show disease symptoms of highly susceptible germplasms.

Geographic Distribution of the Radish Resources with Different Resistance to P. brassicae Race 4
Resistance/susceptibility was also analyzed according to the geographical distribution of 328 accessions of indigenous radish germplasms, which were collected from 230 counties or cities belonging to 29 provinces or municipalities of six geographic regions. A total of 279 susceptible or highly susceptible accessions were distributed in all six geographic regions, especially concentrated in northeast and north China, where no resistant materials were found. The accessions with moderate resistance were mainly located in four geographic regions except northeast and northwest China. However, a total of 28 accessions of resistant materials (including I, HR, and R) were distributed in a dispersed fashion across several provinces of east, southwest, northwest, and south-central China ( Figure 3).

Figure 2.
Comparison of the phenotypes of representative radish materials that are either immune or highly susceptible to P. brassicae race 4. CRR240, CRR297, and CRR330 illustrate the resistance of immune germplasms; CRR84, CRR71, and CRR108 show disease symptoms of highly susceptible germplasms.

Geographic Distribution of the Radish Resources with Different Resistance to P. brassicae Race 4
Resistance/susceptibility was also analyzed according to the geographical distribution of 328 accessions of indigenous radish germplasms, which were collected from 230 counties or cities belonging to 29 provinces or municipalities of six geographic regions. A total of 279 susceptible or highly susceptible accessions were distributed in all six geographic regions, especially concentrated in northeast and north China, where no resistant materials were found. The accessions with moderate resistance were mainly located in four geographic regions except northeast and northwest China. However, a total of 28 accessions of resistant materials (including I, HR, and R) were distributed in a dispersed fashion across several provinces of east, southwest, northwest, and south-central China (Figure 3).
Compared with Chinese radish germplasms, most of the exotic radish germplasms were resistant to P. brassicae (Figure 4). A considerable portion of resistant accessions (61.90% of the total exotic materials) came from Japan, North Korea, South Korea, Russia, Germany, and Spain, especially the immune materials, which accounted for 61.54% of all immune materials. Compared with Chinese radish germplasms, most of the exotic radish germplasms were resistant to P. brassicae (Figure 4). A considerable portion of resistant accessions (61.90% of the total exotic materials) came from Japan, North Korea, South Korea, Russia, Germany, and Spain, especially the immune materials, which accounted for 61.54% of all immune materials.  Compared with Chinese radish germplasms, most of the exotic radish germplasms were resistant to P. brassicae (Figure 4). A considerable portion of resistant accessions (61.90% of the total exotic materials) came from Japan, North Korea, South Korea, Russia, Germany, and Spain, especially the immune materials, which accounted for 61.54% of all immune materials.

Verification of the New Method by Repeated Identification of the Resistance of Immune Materials and Representative Highly Susceptible Materials
In the second confirming trial-among all immune accessions and ten highly susceptible accessions with DIs varying from 70.85-87.65, identified in the first trial-most still appeared as being immune or highly susceptible in the second trial. There was no significant difference (p > 0.05) in disease severity between the two trials ( Figure 5) using a paired t-test. In more detail, thirteen materials remained immune, namely, CRR55, CRR221, CRR240, CRR274, CRR281, CRR297, CRR330, CRR340, CRR341, CRR342, CRR346, CRR348, and CRR349 still had no clubroot symptoms, indicating their resistance was stable over repeated trials. However, the resistance of two immune materials (CRR217 and CRR331) changed somewhat. CRR217 became susceptible as the DI increased to 39.51. CRR331 showed slight disease symptoms but remained resistant with a DI of 10.8. Figure 4. Percentage of indigenous and exotic radish germplasm resources with different resistance levels to P. brassicae. I, immune; HR, highly resistant; R, resistant; MR, moderately resistant; S, susceptible; HS, highly susceptible.

Verification of the New Method by Repeated Identification of the Resistance of Immune Materials and Representative Highly Susceptible Materials
In the second confirming trial-among all immune accessions and ten highly susceptible accessions with DIs varying from 70.85-87.65, identified in the first trial-most still appeared as being immune or highly susceptible in the second trial. There was no significant difference (p > 0.05) in disease severity between the two trials ( Figure 5) using a paired t-test. In more detail, thirteen materials remained immune, namely, CRR55, CRR221, CRR240, CRR274, CRR281, CRR297, CRR330, CRR340, CRR341, CRR342, CRR346, CRR348, and CRR349 still had no clubroot symptoms, indicating their resistance was stable over repeated trials. However, the resistance of two immune materials (CRR217 and CRR331) changed somewhat. CRR217 became susceptible as the DI increased to 39.51. CRR331 showed slight disease symptoms but remained resistant with a DI of 10.8.

Discussion
Previous studies have most commonly used single-inoculation methods to infect host plants with P. brassicae pathogens, such as the dipping root, clubroot-soil, or soaking inoculation methods [7,11,57,94,99]. In the process of inoculation, the clubroot-soil required the inoculum to be fully mixed with the substrate to ensure concentration uniformity of the mixture. Meanwhile, the dipping root method involved dipping the root into inoculum for some time (minutes or hours), and ensuring spore suspension during this time.

Discussion
Previous studies have most commonly used single-inoculation methods to infect host plants with P. brassicae pathogens, such as the dipping root, clubroot-soil, or soaking inoculation methods [7,11,57,94,99]. In the process of inoculation, the clubroot-soil required the inoculum to be fully mixed with the substrate to ensure concentration uniformity of the mixture. Meanwhile, the dipping root method involved dipping the root into inoculum for some time (minutes or hours), and ensuring spore suspension during this time. When a large number of seedlings were identified, these two methods became more complicated and time-consuming. Although the soaking bud method was suitable for large-scale identification, soaking also required preventing the inoculum from settling in a long time. In addition, during the single-inoculation process, no or only a slight infection often occurred in susceptible plants due to environmental change in different stages (such as temperature and humidity) or occasional missing or under-inoculation reasons. In our experiment, there were no statistically significant differences among several singleinoculation methods. In other words, the dipping root and clubroot-soil methods used for radish identification did not work as well as in B. rapa and B. oleracea. In contrast, the two-stage combined method was significantly more reliable than single-inoculation. This is probably because radish have a developed taproot with a few lateral roots and fibrous roots, which could result in radish seedlings being easily infected or take a longer time to exhibit symptoms. Among all methods, the two-stage combined inoculation method was the most reliable for the resistance identification of radish germplasm. This could be the reason why this approach prevents both the occasional missing and under-dosed inoculation effects, and also the influence of environmental or plant physiological factors at different developmental stages. The advantages of this method were proven by the results from the resistant evaluation of large-scale radish germplasms and repeated identification of representative resistant and susceptible germplasm resources, although there are a few exceptions that the resistance of CRR217 and CRR331 declined to different degrees in the second trial. These changes in resistance were presumed to be due to insufficient generations of purification or occasional disease escape.
Clubroot disease is hard to manage. Chinese cabbage (B. rapa L.) and cabbages (B. oleracea L.) are generally regarded as being highly susceptible to clubroot disease [7]. Researchers have identified resistant materials from different sources, such as turnip, and successfully bred a few resistant varieties by cross breeding, backcross transformation or pyramiding breeding [51,100]. It has been proven that utilization of resistant cultivars is the most effective strategy for clubroot disease control. In radish, early studies indicated that the majority of radish cultivars and inbred lines possess high levels of resistance [80,101]. However, the current study showed that most local Chinese radish cultivars were susceptible to P. brassicae. Indeed, clubroot disease outbreaks in radish production have been increasing in recent years in many regions of China [3,87], which reflects the lack of clubroot-resistant radish varieties in production. To screen and rationally use the clubroot resistant resources in radish is essential. After all, it is difficult to use Brassica resistant materials of clubroot because the genetic relationship between Raphanus and Brassica is distant.
In this study, we evaluated the resistance of large-scale of radish germplasms with the newly established method and screened a number of radish resistant germplasm resources from China, as well as some highly resistant exotic germplasms. These radish resistant materials could provide diverse resistant sources for managing clubroot by the following different approaches. One management strategy is to apply the resistant cultivars directly to the radish growing regions where race 4 is predominant. The other strategy is to indirectly apply resistant cultivars in radish crossbreeding programs for improving the resistance of varieties. It should be noted that these resistant materials to P. brassicae race 4 might be susceptible to other races due to their race specificity. Although at present, other races are less widespread and produce less damage compared with race 4, they might have the potential to become dominant races in the future. In addition, it has also been reported that the single-gene resistance of some clubroot-resistant cultivars has been overcome in B. rapa when genetic resistance was used as a management strategy [102][103][104][105]. Therefore, in order to improve the single-gene resistance of radish varieties, polymerizing multiple CR genes, especially with different race specificities, may be more efficient as a clubroot management strategy of genetic resistance utilization. This means that screening and utilization diverse resistant resources, and revealing their resistance genetic background is an urgent requirement for the effective utilization of resistant resources to manage clubroot. However, only a few CR loci have been preliminarily mapped by QTL analysis based on the linkage map in radish so far [82,106]. Thus, further studies on the resistant resources discovered in our study are desperately needed.
Through this research, we also discovered new characteristics of clubroot resistance distribution in radish, which will provide clues for us to effectively find and use new resistant resources, and adopt different prevention and control measures for different situations. Our results revealed that 71.58% (204/285) of susceptible materials showed high-susceptibility and displayed distinct swelling or fusiform clubroots, while only 3.72% (13/349) of materials had no symptoms. This indicates that there are more resistant resources in the indigenous materials from the east, southwest, and northwest of China, and exotic germplasms mainly from Korea, Japan and Russia. Specifically, most radish cultivars from northeast and northern China were identified as being highly susceptible, which was also found by Yoshikawa [81]. Some radish cultivars from southwest China have also been seriously infected, especially in Yunnan, Guizhou, and Chongqing provinces (Figure 3h Y, G1, C). Radish cultivars in the above areas have a high level of susceptibility, which is in accordance with clubroot severity in these areas [107]. In the most threatened areas, the soils were heavily polluted and in the short term there is great risk in growing cruciferous crops. In this situation, utilizing resistant cultivars might be the best measure for managing clubroot. In particular, it is noteworthy that native radish cultivars from Qinghai and Gansu provinces (Figure 3h Q, G2) have a high level of susceptibility, while these areas were reported to be clubroot-free in 2013-2014 [106]. One study showed that clubroot risks were low in Qinghai and Gansu since clubroot incidence requires higher resting spore concentrations in the soil [108]. The main clubroot management strategies in these areas should be to avoid disease-free soils from being polluted through various ways or media, considering that all the local cultivars in these areas are characterized by a potential high incidence.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11040792/s1, Table S1: Detailed information on the radish accessions that were tested in this study.