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ISSN : 0250-3360(Print)
ISSN : 2287-5174(Online)
Korean Journal of Breeding Science Vol.44 No.4 pp.503-509
DOI : https://doi.org/10.9787/KJBS.2012.44.4.503

Inheritance of Resistance to Phytophthora Root Rot in Chili Pepper Depending on Inoculum Density and Parental Genotypes

Jae Bok Yoon1*, Jundae Lee1, Won Phil Lee1, Byoung-Cheorl Kang2
1R&D Unit, Pepper & Breeding Institute, Business Incubator, College of Agriculture and Life Sciences, Seoul National University
2Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University
Received October 15, 2012, Revised November 29, 2012, Accepted December 4, 2012

Abstract

Phytophthora capsici Leonian causes root rot and stem blight in pepper (Capsicum spp.) and is a serious threat topepper production because of its ability to infect every root, stem, and leaf at any developmental stage. Recently, pepper F₁cultivars resistant to Phytophthora root rot have been commercially released in Korea. However, despite many studies, theinheritance of resistance remains controversial due to differences in experimental methods, including pepper materials, pathogenisolates, inoculation conditions, and evaluation methods. Our aim was to determine the inheritance of Phytophthora root rotresistance by using three different F₂ populations derived from crosses between ‘CM334’ (a resistant male parent) and threeKorean landraces, ‘Subicho’ ‘Daehwacho’ and ‘Chilsungcho’ (susceptible female parents), and inoculating them with threedifferent pathogen densities (1×104, 1×105, and 1×106 zoospores/ml). The distribution patterns were varied, dependingupon female parental susceptibility as well as inoculum densities. For example, as the inoculum density increased, pepper survivalrates decreased. In all of the inheritance analyses, one common dominant resistant gene was participated in resistance toPhytophthora root rot. In addition, we found that a complementary gene, together with the major dominant gene, was necessaryfor resistance at a high (106) inoculum density, based on a 9:7 (R:S) segregation ratio. This study will be helpful in developingmolecular markers linked to genes that are resistant to Phytophthora root rot.

44(4)503(2012.12).pdf303.1KB

INTRODUCTION

 Chili pepper (Capsicum annuum L.), which originated in the South American tropics, is one of the most economically important vegetable crops in Korea (Surendra et al. 2010). However, pepper production is affected by numerous factors, such as insects, various pathogens, and climate change. Phytophthora capsici Leon, which causes root rot, stem and foliar blight, has been one of the most damaging pathogens to pepper production in Korea for decades (Leonian 1922).

 Numerous studies have reported about resistance to Phytophthora root rot among C. annuum accessions, including ‘AC2258’ ‘AC311’ ‘CM334’ ‘Fyuco’ ‘Line29’ ‘P51’ ‘PI 123469’ ‘PI 201232’ ‘PI 201234’ and ‘PI 201238’ (Kimble & Grogan, 1960; Kim et al., 2010). In some cases, it was reported that single or two genes qualitatively control resistance to Phytophthora root rot (Bnejdi et al. 2009, Monroy-Barbosa & Bosland 2008, Sy & Bosland 2005, Walker & Bosland 1999). In other cases, however, the resistance was reported to be controlled by polygenes (Bonnet et al. 2007, Lefebvre & Palloix 1996, Minamiyama et al. 2007, Ogundiwin et al. 2005, Sugita et al. 2006, Thabuis et al. 2003). The difference in reporting resistance as qualitative or quantitative traits might result from the use of different parents, inoculation methods, inoculum densities, evaluation times, or other conditions. For example, the relationship between infections and inoculum concentration was studied to be low in resistant peppers but high in susceptible peppers (Palloix et al. 1988). Moreover, Lee and Park (2002) reported that mean disease index and survival rate to Phytophthora root rot were varied with seedling age and inoculum density. Nevertheless, these analyses commonly concluded that the major quantitative trait locus (QTL) for resistance to Phytophthora blight is on chromosome 5 of the pepper linkage map (Kim et al. 2008, Quirin et al. 2005, Truong et al. 2012).

In this study, we evaluated the effects of inoculum density and susceptible parental line on inheritance of resistance to Phytophthora root rot and determined a suitable inheritance model for resistance in chili pepper.

MATERIALS AND METHODS

Plant materials

Three Korean landraces, C. annuum ‘Subicho’ ‘Daehwacho’ and ‘Chilsungcho’, were used as female susceptible parents. The male resistant parent was C. annuum ‘Criollo de Morelos 334’ (‘CM334’), which was developed by the French National Institute for Agricultural Research. Three F2  populations were generated by self-pollination of F1  plants derived from cross combinations between the resistant and susceptible parents; ‘Subicho’ × ‘CM334’ (SF), ‘Daehwacho’ × ‘CM334’ (DF), and ‘Chilsungcho’ × ‘CM334’ (CF).

 

Inoculum preparation

An isolate of P. capsici was provided by Nongwoo Bio Co., Ltd. (Suwon, Korea). Potato dextrose agar (PDA) (Sigma #70139, St.Louis, KS, USA) medium was used to preserve and subculture the isolate in a clean bench. A mat of mycelia completely covered the PDA plates in one week, and we cut 1×1cm block samples (plugs) using a surgical blade. The mycelial plugs were used as subculture materials. V8 juice agar medium, used as a sporulation medium, was composed of V8-juice (Campbell Soup Co., Cambden, NJ, USA), CaCO3 , agar, and distilled water. At 7 to 10-day, a spreader to scratch the mycelial mats growing on the V8-juice agar medium to remove aerial mycelia and then the mycelia mat was incubated in a clean bench or growth chamber under continuous light to induce the formation of zoosporangia. The zoosporangia were harvested by spraying distilled water on the plates and filtering with two layers of cheesecloth. The final concentration of zoospores was calculated under a hemacytometer and adjusted to 1 × 104 , 1 × 105 , and 1 × 106  zoospores/ml (Lee & Park 2002).

 

Inoculation method

All parents, F1 , and F2  plants were grown in a greenhouse in 2007. A slightly modified soil drenching method was used to evaluate resistance against P. capsici (Bosland & Lindsey 1991, Lee & Park 2002). At the 6 to 8-leaf stage of the plants, 5 ml of pathogenic suspension were dispensed using a solution dispenser (Labmax #D5370-5; Coherent, Santa Clara, CA, USA).

Evaluation of resistance

Disease symptoms were scored in 2 weeks after inoculation and classified into five degrees based on the following root symptoms: 1, no visible symptoms; 2, less than a quarter damaged; 3, half damaged; 4, three-quarters damaged with wilting; 5, fully damaged with wilting (Fig. 1; Table 1).

Fig. 1. Frequency distribution of Phytophthora root rot resistance of different susceptible female parents and inoculum densities in three F2 populations: SF2 (A), DF2 (B), and CF2 (C). Black, gray, and light-gray bars indicate percentages of resistant individuals in each population under inoculum densities of 1 × 106, 1 × 105, and 1 ×104 zoospores/ml, respectively.

Table.1. Mean disease indices of Phytophthora root rot resistance, based on inoculum densities in parents and their F1 and F2 populations.

 

RESULTS

Effects of inoculum density and parent genotype on resistance to Phytophthora root rot

The mean disease index of the resistant male parent ‘CM334’ was 1.3 and those of the three susceptible female parents, ‘Subicho’ ‘Daehwacho’ and ‘Chilsungcho’, were 4.8-5.0 at all inoculum densities (Table 1). The disease index of DF1  (1.1, 2.0 and 2.0) was lower than that of SF1  (1.6, 2.5 and 2.8) and CF1  (2.0, 2.1 and 3.0) at 1 × 104  , 1 × 105   and 1 × 106   inoculum densities, respectively (Table 1). Some plants of ‘CM334’ and their F1  progenies showed susceptible symptoms (between disease indices 2 and 3) at a high (1 × 106 ) density of inoculum (Table 1). The mean disease indices of F1  progenies increased as inoculum density increased (Table 1). For example, CF1  became more susceptible at a high inoculation density (1 × 106  zoospores/ml). In F2  populations, the mean disease indices for Phytophthora resistance ranged from 1.1 to 1.8 at low inoculum density, 1.7 to 2.2 at intermediate density, and 2.1 to 3.4 at high density (Table 1).

 

Frequency distribution of resistance to Phytophthora root rot in three F₂ populations

The proportion of resistant plants in each F2  population ranged from 50.5-69.4% at high inoculum density, 72.5- 86.8% at intermediate density, and 81.4-99.0% at low density (Fig. 1). The correlation between inoculum density and mortality in each population was positive (r=0.967 in SF2 , r=0.989 in DF2 , and r=0.936 in CF2 ). Symptoms in CF2  was visible 3 days after inoculation, in the meanwhile the symtoms in DF2  was visible 5 days after inoculation, implying rapid symptom development in CF2  population compared to DF2  population. In all concentrations and populations, the frequency distribution of resistance was bimodal, which distribution has two peaks with one resistant peak and one susceptible peak (Fig. 1).

 

Inheritance of resistance to Phytophthora root rot

Disease indices 1 and 2 were considered as resistant and 3, 4, and 5 as susceptible (Lee & Park 2002, Table 2). In the SF2  population, the segregation ratios of resistant to susceptible plants (R:S) were 13:3 at low, 3:1 at intermediate, and 9:7 at high density (Table 2). At low density, the ratio was more appropriately 13:3 than 3:1 (Table 2). However, in the DF2  population, the R:S ratios were 63:1, 13:3, and 3:1 at 1 × 104 , 1 × 105 , and 1 × 106  zoospores/ml, respectively (Table 2). In the CF2  population, the ratios were 15:1, 13:3, and 9:7 at low, intermediate, and high inoculum densities, respectively (Table 2). The inheritance modes of resistance to Phytophthora root rot varied depending on inoculum density and parent genotype (Table 2).

Table.2. Segregation of Phytophthora root rot resistance in SF2, DF2, and CF2 populations.

DISCUSSION

Factors affecting resistance to Phytophthora root rot

In this study, the effects of inoculum density and susceptible parental genotypes were determined because of their controllability (Table 2). To elucidate their effects on resistance, we generated three F2  populations by self-pollinating three different F1  peppers (SF1 , DF1, and CF1) derived from crossing a resistant male parent, ‘CM334,’ with three susceptible female parents, ‘Subicho,’ ‘Daehwacho,’ and ‘Chilsungcho’. We then inoculated them at three different inoculum densities: 1 × 104 , 1 × 105 , and 1 × 106  zoospores/ml (Fig. 1; Tables 1 and 2). The inheritance mode of resistance in each population varied according to inoculum density and female parental genotypes (Table 2). These results are consistent with previous reports that various segregation ratios appeared in populations derived from a common resistant parent and different susceptible parents (Kim & Hur 1990, Kim & Park 1997, Lee & Park 2002, Reifschneider et al. 1992, Sy & Boland 2005, Walker & Bosland 1999) and that QTLs for Phytophthora root rot resistance originated from the susceptible parent as well as the resistant parent (Nahm & Kim 2001). Pochard & Daubeze (1980) reported that the genetic background of the susceptible parent was an important factor in Phytophthora root rot resistance.

Inheritance mode of resistance to Phytophthora root rot

To analyze the inheritance mode of the resistance to Phytophthora root rot in chili pepper, a total of nine combinations of three inoculum densities (1 × 104 , 1 × 105 , and 1 × 106  zoospores/ml) and three susceptible parents (‘Subicho’ ‘Daehwacho’ and ‘Chilsungcho’) were designed, and a chi-square test was performed to identify an appropriate inheritance model (Tables 2).

In all concentrations, the frequency distribution of resistance was bimodal (Fig. 1), thus implying that Phytophthora root rot resistance might be controlled by one or two major genes in a resistant parent. This assumption is consistent with previous reports (Kim & Park 1997, Walker & Bosland 1999). In this study, resistance was also controlled by one or two genes in all cases except 1 × 104  inoculum density (three genes) in the DF2  population (Table 2). Continuous variation in resistance was also observed in the resistant plants (Fig. 1; Table 2), suggesting that minor factors are also present, as previously reported (Barksdale et al. 1984, Kim & Shon 1992, Sugita et al. 2006).

 

In all of the chi-square test results, one common dominant resistant gene participated in resistance to Phytophthora root rot (Table 2), which we believe to be the major QTL gene on chromosome 5 of the pepper linkage map (Kim et al. 2008, Quirin et al. 2005, Truong et al. 2012). Hence, SF2  (105  zoospores/ml) and DF2  (106  zoospores/ml) populations with a 3:1 segregation ratio can be used to develop molecular markers linked to the major QTL for resistance (Table 2).

 All of the results showed the following tendency: as the inoculum density increased, fewer plants survived (Fig. 1; Table 2). This implies that pepper requires more resistant genes to survive at a high (106 ) inoculum density. In the inheritance analysis of SF2  and CF2  populations at high inoculum density, the segregation ratio was 9:7 (Table 2), thus indicating that a complementary gene together with a major dominant resistant gene is necessary for resistance at a high density (106 ) of Phytophthora pathogen (Gil Ortega et al. 1992, Walker & Bosland 1999). Thus, SF2  and CF2  populations with a 9:7 segregation ratio can be used to develop molecular markers linked to the complementary resistant gene (Table 2). In the DF2 population, the susceptible parent, ‘Daehwacho,’ appeared to have a complementary resistant gene; thus, the segregation ratio was 3:1 (Table 2). Moreover, DF1  was more resistant than SF1 or CF1 (Table 1), and the survival rate of DF2 plants was the highest among the three F2 populations (Fig. 1; Table 2).

The chi-square test of low and intermediate inoculum density indicated that at least two additional minor factors, one dominant and one recessive, might be present (Table 2). Whereas a dominant factor could be inferred from the 63:1 at 1 × 104  in DF2  and 15:1 at 1×104 in CF2  segregation ratios (Table 2), a recessive factor could be indicated by the 13:3 at 1 × 104  in SF2 , 1 × 105  in DF2 , and 1 × 105  in CF2  segregation ratios (Table 2). It is difficult to distinguish between 13:3 and 3:1 ratios, due to the small difference between them. We chose the 13:3 segregation ratio as more suitable on the basis of the chi-square values (at 1 × 104  in SF2  and 1 × 105  in DF2 ; Table 2).

 In conclusion, the inheritance mode of Phytophthora root rot resistance varied, based on inoculum density and parent genotype, and it is speculated that the resistance was controlled by at least four resistant genes. One is a common major dominant gene that might be identified on chromosome 5 of the pepper linkage map of a previous QTL analysis (Kim et al. 2008, Quirin et al. 2005, Truong et al. 2012). Another is a complementary gene that, together with the major dominant resistant gene, confers pepper resistance at high inoculum densities. The others are one dominant and one recessive gene, which are related to resistance at low and intermediate inoculum densities.

 To further confirm the existence of the resistant genes, a study of a set of near-isogenic lines or recombinant inbred lines carrying none, one, or both of the genes associated with resistance is needed. Such a study will be helpful to develop molecular markers linked to the resistant genes for Phytophthora-resistant pepper breeding.

ACKNOWLEDGMENTS

This research was partially supported by a grant from the Next Generation BioGreen 21 Program (No. PJ008056012012), Rural Development Administration, Republic of Korea and by a grant (#609001-05) of Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

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