Leaf-to-Whole Plant Spread Bioassay for Pepper and Ralstonia solanacearum Interaction Determines Inheritance of Resistance to Bacterial Wilt for Further Breeding

Bacterial wilt (BW) disease from Ralstonia solanacearum is a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance to R. solanacearum in peppers. Capsicum annuum ‘MC4′ displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible ‘Subicho’. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F1 and 90 F2 progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.


Introduction
Chili pepper (Capsicum spp.) is an important economic crop that belongs to the Solanaceae family alongside potatoes, tomatoes, and eggplants. Pepper is widely consumed as fresh, dried, or processed products and provides many essential vitamins, and capsaicin is used as a major spicy source in most global cuisines [1]. The consumption of pepper has increased in the last 40 years, with production ranging from 9 to approximately 41 million tons and the cultivation area increasing from 2.4 to approximately 3.8 million ha [2]. The world trade value of hot peppers has consistently increased during the last decade, with the second-largest quantity after the tomato in Solanaceae crops [3]. Pepper production is continuously challenged by biotic stresses such as fungi, viruses, and bacteria [4]. Ralstonia solanacearum is the causal agent of bacterial wilt (BW), one of the most destructive soilborne bacterial pathogens in tropical and subtropical areas, with a wide host range of more than 400 plant species, especially the Solanaceae family including peppers [5]. BW by R. solanacearum is widely prevalent in peppers across much of Asia [6][7][8]. China accounted for approximately half of the world's production of peppers in 2017 (FAOSTAT), and the yield loss of BW from peppers is estimated to be approximately 20%-50% in its cultivation area [9].
R. solanacearum species is divided into five races according to host range and five biovar according to the utilization of disaccharides and hexose alcohols [10]. R. solanacearum is also classified based on geographical origin: phylotype I from Asia, phylotype II from America, phylotype III from Africa, and phylotype IV from Indonesia [11]. Recently, a few studies proposed to classify R. solanacearum into three species based on phylotype: R. psedosolanacearum (phylotype I and III), R. solanacearum (phylotype II), and R. syzygii (phylotype IV) [12,13]. Thus, the R. solanacearum species complex includes phenotypically diverse and heterogeneous strains causing BW in a variable host range. This is one of the constraint factors of resistance studies on R. solanacearum. The pathogen can invade the plant through root wounds and subsequently resides in the xylem vessels to block water transport and ultimately kills the plant host [8,14].
Most studies on resistance to R. solanacearum in plants used two screening methods of R. solanacearum, i.e., root cut (soil)-drench and root-dipping inoculation [15][16][17][18]. However, both methods are difficult to determine the resistance degree according to the size of artificial root wounds, which leads to a large standard deviation due to low uniformity after inoculation [17]. The stem-puncture inoculation method also has limitations as it is difficult to apply this approach depending on the crop [19]. The leaf-inoculation method by syringe is a commonly used method for bacteria inoculation. In tobacco, the leaf inoculation method to R. solanacearum was already optimized and has been reported in several studies [20,21]. But this has not yet been reported to optimize a reliable bioassay in the resistance screening to R. solanacearum studies in peppers. This assay can infiltrate a relatively equal quantity of R. solanacearum into infected leaves and evaluate the quantification of pathogen growth in a plant. Additionally, leaf infiltration can recognize the inoculated leaves and non-inoculated systemic organs and establish disease scoring according to disease transmission in the whole plant.
To date, developed management programs of R. solanacearum are not sufficiently effective because chemical and biological controls are limited and ineffective in preventing the spread of R. solanacearum to the host plant [22,23]. One of the most effective BW control methods is the development of a resistance cultivar in the crops. Presently, several resistance sources of BW resistance have been evaluated to develop resistant cultivars in Capsicum spp. Several pepper accessions were reported among them, C. annuum 'MC4 , C. annuum 'MC5 , C. annuum 'LS2341 , C. annuum 'PBC473 , C. annuum 'PBC 1347 , and C. annuum 'PBC631 are well known as the most strong BW resistant cultivars in various pathogens [24][25][26]. BW resistance is generally quantitatively inherited and is controlled by at least two genes in the pepper cultivar C. annuum 'Mie-Midori' [27]. Additionally, a pepper line C. annuum 'PM687 reported additive effects with two to five genes to control the BW resistance [28]. The pepper line C. annuum 'LS2341 is reportedly polygenic and linked to a major quantitative trait loci (QTL) named Bw1 on chromosome 1 [29]. Recently, a major QTL named qRRs-10.1 in chromosome 10 was revealed as a resistance pepper line C. annuum 'BVRC1 [30].
Among them, C. annuum 'MC4 is a well-known accession with a strong level of resistance to various R. solanacearum strains [15,24,31,32]. However, despite reports of C. annuum 'MC4 resistance to BW, genetic inheritance analysis of BW resistance in C. annuum 'MC4 has not been determined yet because of pathogen strain complexity and a lack of an efficient bioassay of R. solanacearum in peppers. Here, we developed a fast and reliable bioassay for phenotype evaluation against R. solanacearum in pepper germplasms. Using this method, BW resistance and susceptible symptoms were distinctly confirmed, and we successfully detected disease symptoms through whole plant wilting and validation for pepper cultivars. Through this, a genetic inheritance analysis of BW resistance was investigated in the parents, F 1 and F 2 progeny populations. The BW resistance trait in 'MC4 confirmed to be affected with at least two major complementary genes.

Identification of Leaf Wilt Symptoms between Resistant and Susceptible Pepper
To identify the response of pepper plants on leaf wilting by R. solanacearum, we performed an infiltration of R. solanacearum SL1931 (hereafter SL1931) with 10 6 CFU/mL in resistant 'MC4 and susceptible 'Subicho' to BW. We observed phenotypes of the infiltrated area for both cultivars from day 1 to day 4 after inoculation. Disease symptoms, leaf wilting, and yellowing with necrosis were observed in 'Subicho' at 3 days after inoculation (dai), whereas 'MC4 displayed less disease symptoms within 4 dai ( Figure 1A). To confirm the resistant response between 'MC4 and 'Subicho', we quantified the level of bacterial cell growth in both cultivars. The differences in bacterial growth were observed at 2 dai but were significant from 3 to 5 dai, displaying 10 to 100 times more bacterial growth in 'Subicho' than in 'MC4 ( Figure 1B). Although no differences were observed during infection until 3 dai, the resistant response of R. solanacearum-inoculated leaves changed dramatically within a day between the two pepper cultivars ( Figure 1C). We measured the transcript expression of defenserelated genes, CaHIN1, CaCDM, and CaHsr203J, which were expressed during the resistant response related by various pathogens [33][34][35]. The expression level of the CaHIN1 gene was significantly increased in 'MC4 than in 'Subicho' at 12 h after inoculation (hai), and the CaCDM gene was also significant at 6 and 24 hai. We confirmed the transcript expression levels of the CaHsr203J gene was significantly increased in 'MC4 than in 'Subicho' at all three-time points ( Figure 1C). Additionally, we conducted quantitative RT-PCR with other defense-related genes including PR4, PR10, and CaAccOX [36]. The expression levels of these genes in 'MC4 were significantly higher than those in 'Subicho' (Supplementary Figure S1), which are similar to the result as shown in Figure 1C. Collectively, these data strongly indicated that 'MC4 also has a suitable resistance to leaf wilting disease by R. solaneacerum alongside BW disease through root infection [15].

BW Symptoms by R. solanacearum through Leaf-to-Whole Plant Spread Bioassay (LWB)
To further understand the spectrum of defense responses to BW disease, the difference in phenotype of whole plants after leaf infection in the two cultivars was observed during 15 dai (Figure 2). 'Subicho' started to display wilt disease symptoms with the injected leaf abscising at 5 dai, whereas no differences in 'MC4 were observed until 10 dai. On the 15 dai, 'MC4 had a symptom of shedding and/or yellowing only with the inoculated leaves, while 'Subicho' had wilted and the whole plant died, which is a common BW disease symptom (Figure 2A,B). We confirmed the same wilt symptoms as the soil (root)-drenching inoculation method, although the leaf infection was conducted. We also represent the wilting rate (%) data that analyzed two replicate experiments using 30 plants for each cultivar ( Figure 2C). With consistency, 'Subicho' started to wither 6 dai, and rapid wilting progressed until 10 dai, and almost all the plants died on the 15 dai. Conversely, the 'MC4 was healthy with no wilting symptoms until two weeks after inoculation. Collectively, through the LWB, we could demonstrate quantified resistance and susceptible phenotypes to BW disease ( Figure 2C).

Development of an Efficient Evaluation System for Resistance to R. solanacearum in Pepper
A clear score criterion for resistant evaluation was established on the disease severity index (DSI) from 0 to 4 using LWB, which demonstrated identical BW symptoms with other methods ( Figure 3A-E) [30,37]. Additionally, we measured closely examine the abscission of leaves in the stem after wilting ( Figure 3F-H). A score of one of the DSI represents the 3rd and 4th leaf abscission that is injected leaves simultaneously, the wilt of 2 leaves stands for 25% wilt symptoms (1 score of DSI) in total 8-leaf stage ( Figure 3B,F). The DSI of 2 scores designated when three or/and four leaves wither or abscission, which is a symptom of 50% wilt in 8-leaf-stage ( Figure 3C,G). The degree of more than half of the leaves wilted and a few alive is determined as DSI of 3 ( Figure 3D,H). A plant with a DSI of <2 was considered resistant (R), 2 ≤, a DSI of < 3 was moderate resistance (MR), and susceptible (S) was defined as a DSI of ≥3 in 15 dai based on Figure 2A,C results. Next, to ensure the optimal evaluation for BW resistance in peppers, we determined the optimal conditions of LWB. Among the environmental conditions, temperature most affects the vitality of R. solanacearum that inhabits tropical and subtropical areas. Appropriate temperature conditions (28-32 • C) of screening for bacterial wilt were identified in several studies on various crops and R. solanacearum strains [17,38,39]. We followed the above temperature and plant growth conditions and experimented to confirm the suitable inoculum concentration. Here, we compared four inoculum concentration levels from 10 3 CFU/mL to 10 6 CFU/mL at 10-fold intervals ( Figure 4). Differences in BW symptoms between the two cultivars can be verified at all concentrations of 10 3 CFU/mL to 10 6 CFU/mL according to statistical analysis. The DSI of 10 3 CFU/mL concentration scored an average 2.6 in 20 dai, which does not represent a completely susceptible phenotype, and we considered it unsuitable. In the case of 10 4 CFU/mL, the disease progression was similar with 10 3 CFU/mL until 11 dai, and after that disease progression was similar with 10 5 CFU/mL from the 15 to 20 dai. The 10 6 CFU/mL concentration was represented as the most suitable result. Resistance in 'MC4 maintained a DSI score of less than 1, whereas 'Subicho' displayed a fast-wilting symptom that scored a mean value of 3.8 until 20 dai (Figure 4). The 10 6 CFU/mL concentration displayed relatively quick and clear phenotypic differences between resistant and susceptible cultivars than others at 10 dai, and the condition was maintained until 20 dai.
To further confirm and validate the LWB method, 12 commercial cultivars were reevaluated for resistance to R. solanacearum. The DSI of BW symptoms was checked daily according to LWB ( Figure 5 and Table 1), which displayed R, MR, and S groups. We observed that 'PR-Daedeulbo' and 'Supermanidda' wilt in most individuals scored 3.3 and 3.9, respectively, of which 'Supermanidda' is as susceptible as 'Subicho' ( Figure 5). 'Suppermanidda' started to wilt early at 4 dai, also its disease progression is similar to 'Subicho', an S-control cultivar. 'PR-Daedeulbo' was a MR phenotype until 14 dai, but then exceeded a score of 3 with over 70% of individuals dead and was thus identified as an S cultivar. By contrast, 'PR-Jangwongeunje' and 'PR-Chengyang' belonged to the resistance category with the same DSI score of 1.8 in 20 dai but did not display the resistance of 'MC4 (0.6 score). The other 8 pepper accessions were denoted MR with scores between 2.0 to 2.5, and a wilt rate (%) at approximately half of the total tested plants for each (data not presented). Additionally, we conducted an experiment with another R. solancearum strain, HWA to 'MC4 and 'Subicho' using LWB. The HWA strain that is known as a highly strong pathogenic strain [16] showed similar disease symptoms and wilt rate (%) with the SL1931 strain in 'MC4 and 'Subicho' (Supplementary Figure S2). These results indicate that LWB is a stable and reliable screening method for R. solanacearum in pepper.    Furthermore, on the LWB method, we compared the BW phenotype with the previous root and soil inoculation methods (Table 1). We also calculated the area under the disease progress curve (AUDPC) and relative (r) AUDPC based on DSI scores at 7, 10, and 15 dai. Not only the DSI for wilting evaluation, but also the rAUDPC (%) value was able to distinguish between 0%-30% R, 30%-40% MR, and 40%-100% as S in 15 dai [15]. The AUDPC and rAUDPC (%) were distributed as 3.5% and 8.9% in 'MC4 , and 'Subicho' was 38.5% and 100%, displaying significant results as controls. Of the 12 commercial pepper cultivars, the rAUDPC (%) of 'Supermanidda' (100%) and 'Muhanjilju' (20.9%) had greater results for BW susceptibility and resistance, respectively. We compared the traits with the other inoculation methods and analyzed the DSI score of the BW phenotype 15 dai when 'Subicho' was in a saturating state. The 'Gangryeokjosenggeon' (R), 'Meotjinsanai' (MR), 'PR-Daedeulbo' (S), and 'Supermanidda' (S) have the same traits in either inoculation method (Table 1). However, the traits of the root-drenching method in 'PR-Cheongyang', 'Ilsongjung', 'Muhanjilju', and PR-'Jangwongeubje' were MR or S phenotypes [15], but in this study represented all R phenotypes. 'Muhanjilju' and 'Meotjinsanai' also displayed previously different traits with S, MR, or R on infection methods and/or R. solanacearum strains [16], whereas we observed R and MR uniformly in each cultivar, respectively (Table 1 and Figure 5). Even though it could be difficult to determine the exact traits to BW, our results suggested that the LWB could be a simple and reliable evaluation method for BW resistant screening in peppers.

Inheritance Analysis of Resistance to R. solanacearum in Pepper
To analyze the inheritance of resistance to R. solanacearum in 'MC4 , the parents, F 1 and F 2 , progenies were evaluated until the disease progressed at 30 dai (Table 2 and Table  S2, Figure 6). The parents, 'MC4 and 'Subicho', maintained resistance and susceptibility, respectively. The wilting progression of F 1 plants was conspicuously slower than in the susceptible parent, and the wilt rates of F 1 until 20 dai were closer to the resistant parent. In generation F 2 , the individuals were distributed on most DSI scores, but resistant plants were most common both at 15 and 20 dai. However, these BW symptoms in parents, F 1 and F 2 , developed continuously until the end of the experiment at 30 dai ( Figure 6 and Table 2). These results suggested that BW resistance acts as a QTL with a few genes in 'MC4 .  We measured the segregation ratio of BW resistance with the chi-square analysis in the F 2 population with disease progression. At 15 dai, segregation in F 2 yielded 63 resistant and 27 susceptible plants that fitted closely to a 11:5 (p > 0.5) and 3:1 ratio (p > 0.1). It appeared more closely at an 11:5 ratio than 3:1, which demonstrated that BW resistance was predominantly controlled by at least one major factor and/or two major alleles around two weeks after inoculation. At 20 dai, resistant plants in the F 2 prevailed with 61 resistant plants versus 29 susceptible, which nearly matched a 9:7 ratio (p > 0.5) and 11:5 ratio (p > 0.1). Lastly, the segregation was represented as a 9:7 ratio (p > 0.05) with 42 resistant plants versus 48 susceptible at 30 dai ( Figure 6 and Table 3). According to these chisquare tests, there were significant differences in the segregation ration during pepper-R. solanacearum interaction. The BW resistance in 'MC4 may be affected by a major dominant factor until 15 dai alongside at least two factors controlling the resistance after the 20 dai. Additionally, the separation ratios of 11:5 and 9:7 were consistently represented with a high p-value closest at 20 and 30 dai, which indicated that two complementary dominant genes could mainly control the resistance to BW in 'MC4 . p value indicate according to * p > 0.05, ** p > 0.1, *** p > 0.5.

Discussion
As global warming continues, the damage of BW is spreading beyond tropical and subtropical regions worldwide. The interaction between R. solanacearum and its plant hosts has been studied as plant resistance to bacterial phytopathogens for more than two decades [22,40,41]. To study various interactions with plants, it is important to establish accurate screening. Accordingly, the inoculation method that makes good use of the infection characteristic of the bacteria was dominated since R. solanacearum is a soil-dwelling bacterium. Soil-drench or/and root-dipping inoculation is mostly used to investigate bacterial wilt disease progress on peppers, tomatoes, eggplants, potatoes, and the model plants Medicago and Arabidopsis [15,38,40,[42][43][44]. Using this root-infection method requires a wound of the root; however, there is uncertainty regarding the infections before the symptoms alongside difficulty in knowing the exact resistance phenotype depending on the degree of artificial root wound. Consequentially, variation and deviation of the BW symptom appear large in plants [15][16][17][18]. To overcome these problems, we developed an LWB assay for BW on peppers.
In this study, we confirmed the different symptoms in leaves after inoculation to discover if the method is suitable for resistant 'MC4 and susceptible 'Subicho'. Additionally, the transcript levels of defense-related genes and bacterial cell growth were significantly different in the resistant or susceptible cultivars following R. solanacearum infection. Although the strains and cultivars were different from our study, the result was consistent with the real-time visualization of the bioluminescent R. solanacearum strain BL-Rs7 colonization of grafted peppers in Du et al. (2019) that demonstrated more aggregation of the pathogen in susceptible cultivar (BVRC 1) then resistance (BVRC 25) [30]. Likewise, in our study, 'MC4 inhibited the proliferation of R. solanacearum and displayed a higher expression level of cell-death related genes compared with 'Subicho'. The cell-death markers used in this study were related to the resistant response and defense-related pathway [34,45]. As a result, it can be assumed that the resistance-related factor acts for the defense as 'MC4 has a higher expression value than that of 'Subicho'. Through these results, we confirmed that 'MC4 was a clear BW resistance cultivar compared with 'Subicho'. According to the study of Akinori et al. (2007), the same BW phenotype was also represented in tobacco when leaf-infiltration and root-inoculation were performed, similar to our studies [20]. The leafinfiltration method is more useful to elucidate molecular events than root (soil)-drenching to better understand the interaction between plants and pathogens since it is possible to inoculate equally [20,21,46]. In conclusion, the wilting symptoms appeared on the whole plant even when inoculated to the leaves, which confirmed the same symptoms as the root infection.
The temperature was the main environmental factor in which R. solanacearum affects crops [47,48]. An experiment was conducted to confirm the most suitable temperature conditions for LWB before the inoculum concentration experiment. As a result of our experiments at 25, 28, and 32 • C, two suitable temperatures were revealed except for 25 • C (data not shown). Additionally, the studies derived that the temperature of 25 • C was not suitable for peppers and tomatoes, respectively, in the screening research for optimization condition [15,38]. Therefore, the temperature was fixed at 28-30 • C in the experimental conditions, and the inoculum concentrations were tested to identify the most suitable for the LWB. The most appropriate concentration was 10 6 CFU/mL indicating that it was sufficiently able to confirm the phenotypic difference between two control cultivars with a lower concentration and less volume than the drenching method.
We executed the LWB in eleven commercial pepper cultivars with BW phenotype information and one commercial pepper cultivar with no information. As a result, five and two cultivars represented R-phenotype and S-phenotype, respectively, and the others were the MR-phenotype. Among them, the cultivars of 'Muhanjilju', 'PR-Jangwongeubje', and 'PR-Gukgadeapyo' demonstrated susceptibility in Hwang et al. (2017), but our results demonstrated the resistance of BW phenotypes, which is an opposite result. These results could affect the metabolic activity of the host due to artificial wounds in the root, making it difficult to identify the accurate BW phenotype. In case of 'Muhanjilju', it represented R, MR, or S-phenotypes according to inoculation with various R. solanacearum strains in Lee et al. (2018) [16]. Additionally, the 'Subicho' was inoculated by soil-drenching without root wounds and represented 0.7 DSI scores (0 to 4 scale scores) with very low disease incidence 15 dai [15], in which the BW phenotype is dependent on the root wound in pepper. For this reason, the study of interactions with pepper-R. solanacearum is exceptionally difficult. An accurate and reliable bioassay (LWB) can identify the exact BW phenotype in pepper through the equal inoculate without any wound of the root.
One of the most effective control managements is developing a resistance cultivar in the crops by integrating a resistance gene. Until now, a few sources of BW resistance have been reported in Capsicum spp. including C. annuum 'MC4 , 'MC5 , 'LS2341 , and 'PBC631 [24][25][26]. In previous studies on the resource of resistance to BW, different QTL studies for only a few were determined that a major QTL (qRRs-10.1) in 'BVRC1 accession and one major (Bw1) in 'LS2341 accession were identified at different chromosome 10 and 1 for each resource, respectively [29,30]. Despite the above reports of resistance to bacterial wilt, there are no useful cultivars comprised of high resistance with good yield and desirable agronomic traits. In this regard, understanding the genetic control for resistance to BW disease in plant breeding programs is essential and required to increase their efficiency, especially for planning a proper breeding method [49,50].
'MC4 is well-known to have high-level resistance to the species of the R. solanacearum complex [24,26,31], but the genetic inheritance of 'MC4 for BW resistance has not been identified yet. In this study, we constructed the F 2 population with 'Subicho' (susceptible) and performed an analysis of the inheritance of BW resistance through the LWB. We identified BW resistance as dominant and over susceptible, and at least two pairs of genes appeared to control the trait in a complementary manner. Matsunaga et al. (1998) studied the mode of inheritance of BW resistance by crossing the resistant sweet pepper cultivar 'Mie-Midori' with the susceptible 'AC2258 and found that bacterial wilt resistance demonstrated incomplete dominance, and at least two genes were involved in resistance [27]. This result is similar to our segregation ratio date representing two major genes affected in the BW resistance of 'MC4 in this study. Additionally, Denis et al. (2005) concluded that two to five genes with additive effects were estimated to control the resistance. Tran et al. (2010) reported various dominance genetic effects as polygenic or oligogenic for R. solanacearum using six resistant pepper lines and five susceptible pepper lines [51]. Recently, Heshan et al. (2019) represented the disease index and wilt rate (%) using the F 2 plants (n = 440), in which the wilting pattern of segregation was similar to our result [30]. Especially, the disease symptoms kept progressing over time alongside no represented complete dominance resistance like 'MC4 (R-parent). In the F 1 and F 2 generation, and which indicated to appear epistasis dominant like our result. These studies indicated that the inheritance of BW resistance is complicated, and a minimum of two genes interact to express resistance traits in the pepper germplasm. Our data suggest that the LWB method may determine a more exact BW resistance phenotype of pepper germplasms and reveal the interaction of plant-pathogens at the molecular level. Further investigations of inheritance factors could provide insights into QTL analysis and the development of BW resistance-related molecular markers.

Plant Materials and Growth Conditions
Two varieties of peppers, Capsicum annuum 'MC4 with resistance to R. solanacearum and C. annuum 'Subicho' with susceptibility to R. solanacearum, were provided by Dr. Seon-Woo Lee (Dong-A University, Korea). The 12 commercial pepper cultivars (5 resistant, 5 moderately resistant, and 2 susceptible cultivars; Table 1) were used. The 'MC4 was crossed with 'Subicho' to get F 1 plants. The F 2 population was obtained by self-pollination of F 1 plants. The pepper plants were kept in a growth chamber at 29 ± 1 • C under a 16 h light/8 h dark cycle with 50% humidity for 3-4 weeks. We inoculated R. solanacearum onto the 3rd and 4th leaves of fully expanded four-leaf-stage on pepper plants.

Bacteria Inoculation and Quantification
The strain R. solanacearum SL1931 (race1, phylotype I) was obtained from Dr. Seon-Woo Lee (Dong-A University, Korea). Bacterial cells were streaked and grown on Kelman's tetrazolium chloride gar medium and maintained at 28 • C for 48 h. A single fluidal colony of R. solanacearum was grown on CPG broth and shaken at 250 rpm at 28 • C for 24 h. A bacterial culture suspension was diluted with distilled water to adjust the concentration to 10 8 CFU/mL (OD 600 = 0.3) [15]. Ten-fold serial dilutions of bacteria from 10 3 CFU/mL to 10 6 CFU/mL per leaf were used for inoculation. Seedlings of fully expanded four-leaf-stage were inoculated with 0.1 mL bacteria/leaf using a needleless syringe. Disease symptoms were observed under controlled conditions of 29 ± 1 • C under 16 h of light a day with 50% humidity for 20 days. The leaf-inoculation assay was performed with three independent tests, and each consisted of at least 8 plants per cultivar. Inoculum concentration was performed with 10 6 CFU/mL per leaf for the inheritance analysis of the F 2 population.
Bacterial quantification was performed like below with modification described by Yi et al. (2009) [52]. To determine in plant bacterial growth, pepper plants (C. annuum 'MC4 and 'Subicho') were leaf-inoculated with bacterial suspensions (1 × 10 4 CFU/mL). Inoculated leaves were harvested at various time points for further analysis. Two independent assays were performed, which consisted of 6-8 samples for each time point in an experiment. Bacterial growth was measured by grinding inoculated samples in distilled water, plating serially diluted tissue samples with two replicates on CPG agar with 0.1% gentamicin (v/v), and counting colony-forming units.

Disease Evaluation and Data Analysis
Disease evaluations were assessed daily after inoculation with R. solanacearum as described below. The disease severity index (DSI) of individual inoculated plants was rated on a scale of 0 to 4 as five phases in which 0 is no wilt disease symptoms observed; 1 is minor symptoms with less than 25% wilted leaves; 2 is moderate symptoms with 25%-50% wilted leaves; 3 is severe symptoms with 50%-75% wilted leaves; 4 is 75%-100% wilted leaves or dead plant. The area under the disease progress curve (AUDPC) was calculated during the disease observation (0 to 15 dai) with a DSI value. [53]. Wilting rate (%) was calculated [The number of wilt plant/the number of total plants] x 100. The differences between the mean values of disease scores of the pepper cultivars were analyzed using Duncan's multiple range tests, and p < 0.05 was considered a significant difference. Statistical analysis used SAS (SAS 9.1, SAS Institute Inc., Cary, NC, USA).

Quantitative RT-PCR of Defense-Related Genes
Total RNA was extracted from pepper leaves inoculated with the pathogen using the Trizol reagent (Invitrogen, Carlsbad, USA), and 2 ug of total RNA were reverse transcribed using Superscript IV (Invitrogen, Carlsbad, USA). To confirm the plant response against R. solanacearum infection, quantitative RT-PCR was performed using the defense-related genes (Supplementary Table S1) [34,36,54]. The following cycling conditions were used: 1 cycle of 94 • C for 3 min; 28 cycles or 30 cycles of 95 • C for 30 s, 58 • C for 30 s, and 72 • C for 30 s; 72 • C for 5 min. The actin gene (designated CaACT) was used as an endogenous control to normalize the expression levels. Expression levels were reported as three replicates as mean values with standard errors.

Conclusions
Breeding a resistant cultivar is most effective in controlling bacterial wilt that causes serious yield losses in peppers worldwide. An accurate and reliable evaluation method is necessary to evaluate disease severity and reveal the genetic inheritance for BW resistance. We established a simple LWB to evaluate BW disease and then, using this, analyzed the inheritance of BW resistance through a 'Subicho' × 'MC4 F 2 population. The BW resistance response of 'MC4 represents lower disease symptoms in leaves than susceptible 'Subicho', and we observed the spreading of wilt symptoms from leaves to a whole susceptible plant, similar to the drenching method. As a result, we optimized the evaluation method of resistance to BW with 12 commercial pepper cultivars. Using LWB, we confirmed the two major complementary genes related to the BW resistance trait through the analyzed genetic inheritance in 90 F 2 progenies. This bioassay could promote an accurate evaluation of BW disease phenotype, and the two inheritance factors of 'MC4 could provide useful information for further QTL analysis in pepper breeding.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding authors upon reasonable request.