Reaction of advanced inbred lines of Habanero pepper to Ralstonia pseudosolanacearum and Phytophthora capsici

Habanero pepper is important in the international market and is becoming popular in the national market; however, few cultivars adapted and resistant to devastating diseases caused by the soilborne pathogens Ralstonia pseudosolanacearum (RP) and Phytophthora capsici (PC) are available in Brazil. The aim of this study was to evaluate the reaction of Habanero-type advanced inbred lines, developed by Embrapa Hortaliças breeding program, to RP and PC. Although not required for the process of protection, registration and release of new cultivars, this information is important. CNPH 15.737; CNPH 15.740; CNPH 15.744; CNPH 15.745; CNPH 15.749 and CNPH 15.750 were inoculated with three RP and one PC isolates. Plants showing wilted leaves (RP) and stem necrosis, leaf wilting and damping off (PC) were quantified. Total area under the disease progress curve (AUDPC) was obtained using incidence values and also severity index for RP. CNPH 15.740 and CNPH 15.737 were highly resistant to RP. CNPH 15.749 displayed considerable resistance levels to PC. CNPH 15.740 showed high resistance to RP isolates and intermediate resistance to PC isolates which also shows agronomic traits of interest to the sector, therefore with a high potential to be released as a new cultivar.

T h e b a c t e r i u m R a l s t o n i a pesudosolanacearum originates from the recent taxonomic reorganization of R. solanacearum, previously identified as race 1 biovar 3, phylotype I (Rossato et al., 2018). This species is more aggressive than R. solanacearum (races 1, 2, 3, biovars 1, 2, phylotype II) on Capsicum, and presents higher evolution rates and the largest geographical distribution worldwide (Lopes & Boiteux, 2004;Rossato et al., 2018). This fact was also noticed under natural conditions in the Amazon region (Coelho Netto et al., 2004). The bacterium penetrates the root system and proliferates in the xylem, causing irreversible wilting and, consequently, plant death (Lebeau et al., 2011). Hong et al. (2012 reported that bacterial wilt affects solanaceous yield in over 80 countries, with annual losses of more than one billion dollars. The oomycete Phytophthora capsici is found in practically all solanaceousproducing regions of the world and is considered one of the most destructive soilborne pathogens of Capsicum peppers (Sánchez-Chávez et al., 2017). This phytopathogen causes root rot and stem base rot, leading to sudden wilting, dark brown necrosis of stem base and plant death (Dunn et al., 2014). The variability of the species is represented by more than 45 physiological races (Barchenger et al., 2018). In Brazil, Ribeiro & Bosland (2012) identified eight physiological races of P. capsici in sweet pepper areas, and race 18 was the most common in the Central region. P. capsici reproduces both sexually and asexually, being characterized as a persistent problem, especially after consecutive cultivations of susceptible host plants (Naresh et al., 2019). Its establishment and propagation are favored by excessive humidity, poor drainage and high soil temperature (Granke et al., 2012;Petry et al., 2016).
The use of resistant cultivars is considered the best management strategy to control diseases caused by R. pseudosolanacearum and P. capsici (Naresh et al., 2019). Resistant cultivars are attractive since they are easy to be adopted by farmers and result in less environmental impacts than any other disease control strategy (Granke et al., 2012). Sources of resistance to these two diseases were identified in accessions of Capsicum spp. (Madeira et al., 2016;Petry et al., 2016, Rossato et al., 2018. Currently, the hybrid rootstock BRS Acará developed by Embrapa Hortaliças, has shown multiple resistance to several isolates of P. capsici and R. pseudosolanacearum, besides Meloidogyne incognita (Madeira et al., 2016).
Little emphasis has been given to developing disease resistant cultivars of Capsicum spp., such as C. baccatum, C. frutescens and C. chinense. Embrapa Hortaliças' Capsicum breeding program aims to develop Habanero pepper genotypes resistant to P. capsici and R. pseudosolanacearum, as well as to other pathogens which attack the crop, besides attributes such as high yield, nutritional quality, and adaptation to tropical growing conditions. In this breeding program, six new advanced inbred lines (S 5 ), from a base population of Habanero pepper with a wide genetic variability (Nass et al., 2015) were obtained after generations of selection using selfpollination methodology and individual plant selection with progeny test. These inbred lines are in the evaluation step for cultivar registration and protection of cultivars, mandatory by Brazilian legislation.
The aim of this study was to evaluate the reaction of six advanced inbred lines of Habanero pepper from Embrapa Hortaliças' breeding program to R. pseudosolanacearum and P. capsici.
Although not required, this information is important as additional data in the process of protection, registration and release of new cultivars.

Reaction to inoculation with R. pseudosolanacearum isolates
The reaction of the six Habanero inbred lines to R. pseudosolanacearum was compared with two C. annuum genotypes used as control, CNPH 143 (resistant) and Tico (susceptible). Inbred lines and controls were sown in expanded polystyrene trays filled with sterile commercial substrate Carolina Soil ® and kept in the greenhouse until inoculation and transplanting.
R. pseudosolanacearum isolates CNPH RS476, CNPH RS634 and CNPH RS639 were used in the inoculation. They were previously selected from the phytopathogenic bacteria collection of Embrapa Hortaliças for being highly virulent to pepper plants, as well as for the geographical diversity among isolates. These isolates were collected in different regions of Brazil, in Dom Pedro-MA, Guadalupe-PI and Altamira-PA, respectively. Inocula were prepared using the methodology described by Rossato et al. (2018).
At 45 days after sowing (DAS), seedlings of inbred lines and control genotypes were inoculated with the three R. pseudosolanacearum isolates, separately, by spraying 5 mL of a bacterial suspension, containing approximately 10 8 CFU mL -1 , directly on each plant root system exposed after removal from the tray (Lopes & Boiteux, 2004). After inoculation, the plants were transplanted to 1-liter plastic pots containing sterile soil and kept in a greenhouse during 27 days with night heating, preventing temperature drop below 20°C, which could increase the chance of escape. The temperature observed during the experimental period was 30±10°C.
Disease incidence was evaluated every three days. The evaluation started when the early symptoms appeared in the susceptible control [nine days after inoculation (DAI)]. Plants which presented wilting or the ones which died were counted, totalizing eight evaluations during the crop cycle (6,9,12,15,18,21,24 and 27 DAI), always in the afternoon in order to uniform the analysis. The last evaluation (at 27 DAI) occurred when the number of wilted plants stabilized.
Values of average disease incidence of each plot were used to calculate area under the disease progress curve (AUDPC) (Shaner & Finney, 1977). Disease severity was also evaluated at 26 DAI using a note scale from 1 to 5 (Winstead & Kelman, 1952), in which the lowest note corresponded to lack of wilting and the highest note to plant death. Notes 3 and above were attributed to plants which showed irreversible symptoms of wilting (susceptible) and notes between 1.5 to 2.5 were attributed to plants which showed light wilting symptoms, reversible after irrigation or at the coolest hours of the day.
A randomized complete block design, in factorial scheme 8 x 3 (six advanced inbred lines of Habanero and two controls CNPH 143 and Tico, and three R. pseudosolanacearum isolates), with four replicates, were used. Plots were composed of six plants, with two plants per pot.

Reactions of the six advanced
Habanero lines to P. capsici were compared to those of C. annuum controls CNPH 148 (resistant) and bell pepper cultivar Ikeda (susceptible). Seedlings of inbred lines and control genotypes were produced according to what was described for the R. pseudosolanacearum experiment. At 45 DAS, seedlings were transplanted to 3-L plastic pots containing sterile soil. The base of each plant received 3 mL of a solution containing a suspension of 2 x 10 4 zoospores/mL -1 of the Pcp 116 isolate, two days after transplant. The isolate was collected in the state of Goiás in 2007 and selected because of its high virulence. Inoculum was prepared based on the methodology described by Petry et al. (2016).
The experimental design was completely randomized, with eight treatments (inbred lines and controls) and five replications, three plants per experimental plot, totalizing 15 plants/ treatment.
Assessment of disease incidence began at six DAI when first wilted plants of susceptible control (cultivar Ikeda) were observed. The evaluation was repeated every two days, totalizing six readings during the crop cycle. The incidence was evaluated using the quantity of plants with wilting symptoms, damping off and stem base necrosis in each experimental plot. The values of the average incidence of each plot were used to calculate the AUDPC (Shaner & Finney, 1977).

Statistical analysis
Variance of severity caused by RP and AUDPC for RP and PC were estimated. Homogeneity and normality of residue of the mathematical model were tested, using Bartlett (Steel et al., 1997) and Jarque-Bera (1987) tests, respectively. ANOVA with F test was carried out and the means among treatments were compared by Tukey test. In all statistical analyses, p<0.05 was adopted. Statistical analyses were carried out using SISVAR v.5.6 statistical software (Ferreira, 2011), as well as Pearson correlation for AUDPC values and severity caused by RP.

Reaction of inbred lines to R. pseudosolanacearum
Significant effect among Habanero pepper lines and R. pseudosolanacearum isolates was detected for both AUDPC and a single reading of disease severity at 26 DAI. There was significance for interaction between genotypes and isolates for both evaluations. Significant correlation was also observed between AUDPC and severity, with R 2 values for CNPH RS476, CNPH RS634 and CNPH RS639 isolates of 0.80; 0.87 and 0.88, respectively. Thus, it is possible to state that AUDPC and severity were highly associated parameters.
Inoculation was successful for all RP isolates and environmental conditions were favorable for disease development, allowing the differentiation of incidence and resistance classes among genotypes. The disease progressed rapidly in the susceptible control, as expected, showing first wilting symptoms at five DAI. The standard resistance control, CNPH 143, was asymptomatic to all isolates, as previously observed by Different resistance levels of the evaluated inbred lines were observed in relation to R. pseudosolanacearum isolates. Inbred lines showed lower incidence of symptomatic plants for isolate CNPH RS476 in comparison to the highly virulent isolate CNPH RS634 (Figure 1). This fact reinforces the statement by Lopes et al. (2015) that the resistance to bacterial wilt is isolate specific rather than phylotype or biovar specific. This information should be taken into account in breeding programs aimed at resistance to bacterial wilt in different host plant species (Lopes & Boiteux, 2004;Wicker et al., 2007).
Among the inbred lines studied, CNPH 15.740 and CNPH 15.737 stood out for presenting high and moderate resistance levels, respectively, to the three R. pseudosolanacearum isolates tested. CNPH 15.744 also stood out for presenting moderate resistance to isolates CNPH RS476 and CNPH RS639.

Reaction of inbred lines to Phytophthora capsici
The viability of the Pcp 116 isolate and the experimental conditions were satisfactory, since high incidence and AUDPC values for wilting, damping off and necrosis were observed in the cultivar Ikeda, used as susceptible control (Figure 2, Table 3). Ribeiro & Bosland (2012) reported the high virulence of P. capsici race 18 based on pathogenicity reaction in pepper genotypes. The high virulence of this isolate was also verified in this study, considering that only the resistant control (CNPH 148) did not show symptomatic plants (Table 3).
All inbred lines evaluated, with exception of CNPH 15.744, showed higher levels of resistance to isolate Pcp116 than the susceptible control ( Figure 2, Infection by P. capsici may result in an expression of multiple disease symptoms, such as root rot, leaf wilt and stem necrosis, and each of these symptoms has a different resistance mechanism, requiring the presence of specific resistance genes to each one (Reeves et al., 2013;Barchenger et al., 2017). Therefore, the occurrence of stem base necrosis in the studied inbred lines may be related to the presence of genes that differ from those that determine partial resistance observed in most of the studied inbred lines, taking into consideration wilting and damping off symptoms ( Figure 2, Table 3). Steiner & Bosland (2008) report that breeding for resistance to P. capsici in peppers is difficult and complex mainly due to the quantitative nature of inheritance (Naresh et al., 2019). Naresh et al. (2019) highlight that the CM334 resistance (from the one which CNPH 148 was derived) to several P. capsici isolates is polygenic with additive and epistatic effect, so little success has been obtained in breeding programs using this genotype. The partial resistance of Habanero pepper lines in this study may be related to this complex effect, but further studies are necessary for its elucidation.
CNPH 15.749 stood out for presenting higher levels of resistance than other evaluated inbred lines and susceptible control, differing from resistance control CNPH 148 only in relation to the occurrence of stembase necrosis. CNPH 15.740 showed moderate resistance to the P. capsici isolate.
The use of resistant cultivars  is the most suitable way to control diseases due to its low cost, high efficiency and reduced environmental impact, especially when compared to other strategies that aim to control the pathogen after its establishment (Rossato et al., 2018).