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Inheritance of Resistance To Phytophthora Root Rot in Chili Pepper Depending On Inoculum Density and Parental Genotypes
Inheritance of Resistance To Phytophthora Root Rot in Chili Pepper Depending On Inoculum Density and Parental Genotypes
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Inheritance of Resistance to Phytophthora Root Rot in Chili Pepper Depending on Inoculum Density and Parental Genotypes
Jundae Lee1, Won Phil Lee1, Byoung-Cheorl Kang2, and Jae Bok Yoon1*
1
R&D Unit, Pepper & Breeding Institute, Business Incubator, College of Agriculture and Life Sciences, Seoul National University, Suwon 441-853, Korea 2 Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea
Abstract : Phytophthora capsici Leonian causes root rot and stem blight in pepper (Capsicum spp.) and is a serious threat to pepper production because of its ability to infect every root, stem, and leaf at any developmental stage. Recently, pepper F1 cultivars resistant to Phytophthora root rot have been commercially released in Korea. However, despite many studies, the inheritance of resistance remains controversial due to differences in experimental methods, including pepper materials, pathogen isolates, inoculation conditions, and evaluation methods. Our aim was to determine the inheritance of Phytophthora root rot resistance by using three different F2 populations derived from crosses between CM334 (a resistant male parent) and three Korean landraces, Subicho Daehwacho and Chilsungcho (susceptible female parents), and inoculating them with three 4 5 6 different pathogen densities (1 10 , 1 10 , and 1 10 zoospores/ml). The distribution patterns were varied, depending upon female parental susceptibility as well as inoculum densities. For example, as the inoculum density increased, pepper survival rates decreased. In all of the inheritance analyses, one common dominant resistant gene was participated in resistance to Phytophthora root rot. In addition, we found that a complementary gene, together with the major dominant gene, was necessary 6 for resistance at a high (10 ) inoculum density, based on a 9:7 (R:S) segregation ratio. This study will be helpful in developing molecular markers linked to genes that are resistant to Phytophthora root rot. Keywords : Capsicum annuum, Inheritance, Inoculum density, Phytophthora capsici, Resistance
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
*Corresponding author: (E-mail: [email protected], Tel: +8231-296-5797, Fax: +82-31-296-5794) (Received October 15, 2012. Revised November 29, 2012. Accepted December 4, 2012)
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
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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.
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 11cm 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
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 6 5 under inoculum densities of 1 10 , 1 10 , and 1 4 10 zoospores/ml, respectively.
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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 10 , 1 10 , and 1 10 zoospores/ml (Lee & Park 2002).
4 5 6
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 10 , 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).
4
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).
Table 1. Mean disease indices of Phytophthora root rot resistance, based on inoculum densities in parents and their F1 and F2 populations. Mean disease index Name CM334 Subicho Daehwacho Chilsungcho SF1 DF1 CF1 SF2 DF2 CF2
z y z
Generation Resistant parent Susceptible parent Susceptible parent Susceptible parent F1 of Subicho CM334 F1 of Daehwacho CM334 F1 of Chilsungcho CM334 F2 of SF1 F2 of DF1 F2 of CF1
1 105 zoospores/ml 1.3 0.3 5.0 0.0 5.0 0.0 5.0 0.0 2.5 0.0 2.0 0.1 2.1 0.1 2.2 1.2 1.8 0.6 1.7 0.7
1 106 zoospores/ml 1.3 0.2 5.0 0.0 5.0 0.0 5.0 0.0 2.8 0.1 2.0 0.1 3.0 0.1 2.9 0.3 2.1 0.7 3.4 1.0
4.9 0.1 4.8 0.2 5.0 0.0 1.6 0.0 1.1 0.1 2.0 0.1 1.8 0.6 1.1 0.3 1.3 0.5
1, no visible symptoms; 2, < quarter damaged; 3, half damaged; 4, three-quarters damaged with wilting; 5, fully damaged with wilting. Standard deviation resulted from three repeats.
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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 10 , 1 10 , and 1 10 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).
4 5 6
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:
Table 2. Segregation of Phytophthora root rot resistance in SF2, DF2, and CF2 populations.
SF2 population Inoculum density Expected Observed 2 Material ratio x -test P value R S (R:S) CM334 1 10 zoospores/ ml
4
DF2 population Material CM334 Daehwacho DF1 DF2 CM334 Daehwacho DF1 DF2 CM334 Daehwacho DF1 DF2 Expected Observed 2 ratio x -test P value R S (R:S) 1:0 0:1 1:0 63:1 1:0 0:1 1:0 3:1 13:3 1:0 0:1 1:0 3:1 13:3 6 0 6 96 6 0 6 0 6 0 1 0 6 0 0.178 1.520 0.045 0.593 5.830 0.673 0.218 0.833 0.441 0.016 Material CM334 Chilsungcho CF1 CF2 CM334 Chilsungcho CF1 CF2 CM334 Chilsungcho CF1 CF2
CF2 population Expected Observed 2 ratio x -test P value R S (R:S) 1:0 0:1 1:0 15:1 1:0 0:1 1:0 3:1 13:3 1:0 0:1 1:0 9:7 6 0 6 86 6 0 6 0 6 0 5 0 6 0 0.089 6.770 1.850 1.200 0.766 0.009 0.174 0.273
1:0 0:1 1:0 3:1 13:3 1:0 0:1 1:0 3:1 13:3 1:0 0:1 1:0 9:7
6 0 6
0 6 0
57 13 6 0 6 0 6 0
1 10 zoospores/ ml
50 19 6 0 5 0 6 1z
78 19 6 0 6 0 6 0
79 12 6 0 0 6
1 10 zoospores/ ml
5 1 z 46 45
36 34
68 27
507
6
1 10 , 1 10 , and 1 10 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.
SF2 (10 zoospores/ml) and DF2 (10 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 (10 ) 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 1104 in CF2 segregation ratios (Table 2), a recessive factor could be indicated by the 13:3 at 1 10 in SF2, 1 10 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
4 5 6
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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.
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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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