Breeding host resistance to Ascochyta rabiei, a destructive pathogen of chickpea worldwide is considered the most practical, effective and environmentally friendly method [11]. However, resistance breeding to this disease can be time-consuming and challenging because of the complex nature of host resistance, the evolving of continuously new pathotypes within the pathogen populations and the breakdown of resistance in commercially available cultivars [11, 26]. Comprehensive breeding studies by different research institutions in many countries have been continuously conducted for identification of new resistance sources and screening breeding materials [18, 26, 27, 28]. The reliable and repeatable screening techniques are essential for the identification and screening of chickpea genotypes resistant to Ascochyta blight. Several assays developed for screening chickpea genotypes are now widely used [11]. However, these techniques have serious restrictions, especially the visual assessment of disease symptoms may show significant variations both between researchers and between different assessments of the same researcher [11, 18, 29]. In a previous study, we observed a strong relationship between disease assessment and quantification of pathogen infection in resistant and susceptible cultivars based on a quantitative real-time PCR assay [23]. Our results indicated that this approach may be a useful tool for the evaluation of breeding material for disease resistance during initial phases of infection as an optional approach to visual scoring and disease management. In this study, some chickpea genotypes were evaluated for the level of resistance to Ascochyta blight by detached leaf inoculation and quantitative real-time PCR assays.
Eighty-four chickpea genotypes selected from field yield trials were inoculated with detached leaflet method, and eight days after inoculation, leaflets were scored for disease severity. Significant variations were statistically found among disease severity of chickpea genotypes (LSD: 13.2, P: 0.05), ranging from 0–95.24% (Table 1, Fig. 1). The highest disease severity was observed in Tüb-108 genotype, following by Tüb-72 and Tüb-67 chickpea genotypes with disease severity ratings of %72.81 and %72.38, respectively. The lowest disease severity was determined in the genotypes of Tüb-47, Tüb-69, Tüb-26, Tüb-44, Tüb-22, Tüb-51 and Tüb-82 with a disease reaction of < 2%, respectively, while seven genotypes had disease severities of 2%. Twenty-four genotypes were resistant and forty-four were highly resistant, while sixteen genotypes were found susceptible or highly susceptible. Similar work was carried out by Chaudhry and Muhammad [30], who screened 867 samples to Ascochyta blight. 119 chickpea samples were susceptible, while 60 samples were found to be resistant. Toker and Cancı [31], screening 41 genotypes found 7 genotypes to be resistant. Akalın et al. [32], screening 50 chickpea genotypes to Ascochyta blight in Turkey, found that 5 genotypes were immune, 5 genotypes highly resistant, and 19 genotypes resistant or moderately resistant. The other 21 genotypes were regarded as tolerant or susceptible. Sahi et al. [33] evaluated 52 lines and indicated that three lines were resistant, 30 moderately resistant. Also, 12 and 3 lines exhibited susceptible and moderately susceptible reaction, respectively. Similar studies on the screening of chickpea materials for resistance to Ascochyta rabiei were performed by many researchers [28, 34, 35].
Table 1
Disease reaction of chickpea genotypes inoculated with Ascochyta rabiei
Chickpea genotypes | Disease severity %* | Disease reaction | | Chickpea genotypes | Disease severity % | Disease reaction |
Tüb-01 | 23.33 | R | | Tüb-48 | 14.67 | R |
Tüb-02 | 56.19 | HS | | Tüb-49 | 10.67 | R |
Tüb-03 | 16.67 | R | | Tüb-50 | 15.33 | R |
Tüb-04 | 24.67 | R | | Tüb-51 | 1.33 | HR |
Tüb-05 | 8 | HR | | Tüb-52 | 21.9 | R |
Tüb-06 | 8.67 | HR | | Tüb-53 | 5.33 | HR |
Tüb-07 | 8.67 | HR | | Tüb-54 | 45.24 | MS |
Tüb-08 | 10 | HR | | Tüb-55 | 2.67 | HR |
Tüb-09 | 11.33 | R | | Tüb-56 | 2.67 | HR |
Tüb-10 | 8.67 | HR | | Tüb-57 | 6.67 | HR |
Tüb-11 | 2 | HR | | Tüb-58 | 28 | R |
Tüb-12 | 2 | HR | | Tüb-59 | 22 | R |
Tüb-13 | 6 | HR | | Tüb-60 | 16.67 | R |
Tüb-14 | 8 | HR | | Tüb-61 | 9.33 | HR |
Tüb-15 | 33.33 | MS | | Tüb-62 | 59.05 | HS |
Tüb-16 | 4 | HR | | Tüb-63 | 26.67 | R |
Tüb-19 | 4.67 | HR | | Tüb-64 | 22.86 | R |
Tüb-20 | 10.67 | R | | Tüb-65 | 2 | HR |
Tüb-21 | 2 | HR | | Tüb-67 | 72.38 | HS |
Tüb-22 | 1.33 | HR | | Tüb-68 | 37.14 | MS |
Tüb-23 | 2.67 | HR | | Tüb-69 | 0 | HR |
Tüb-24 | 4 | HR | | Tüb-70 | 2.67 | HR |
Tüb-25 | 2 | HR | | Tüb-71 | 14 | R |
Tüb-26 | 0.67 | HR | | Tüb-72 | 72.81 | HS |
Tüb-27 | 16.67 | R | | Tüb-76 | 21.9 | R |
Tüb-28 | 9.33 | HR | | Tüb-78 | 34.29 | MS |
Tüb-29 | 5.33 | HR | | Tüb-79 | 10 | HR |
Tüb-30 | 6 | HR | | Tüb-82 | 1.43 | HR |
Tüb-31 | 12 | R | | Tüb-84 | 44.05 | MS |
Tüb-33 | 14 | R | | Tüb-86 | 39.17 | MS |
Tüb-35 | 2 | HR | | Tüb-87 | 58.57 | HS |
Tüb-36 | 5.33 | HR | | Tüb-93 | 37.14 | MS |
Tüb-37 | 3.33 | HR | | Tüb-96 | 4 | HR |
Tüb-38 | 6.67 | HR | | Tüb-97 | 24 | R |
Tüb-39 | 4.67 | HR | | Tüb-100 | 11.33 | R |
Tüb-40 | 51.7 | HS | | Tüb-105 | 65.71 | HS |
Tüb-41 | 14.67 | R | | Tüb-108 | 95.24 | HS |
Tüb-42 | 8.67 | HR | | Tüb-114 | 4 | HR |
Tüb-43 | 2 | HR | | Tüb-119 | 29.52 | R |
Tüb-44 | 0.67 | HR | | Tüb-121 | 23.33 | R |
Tüb-45 | 4 | HR | | Tüb-124 | 46 | MS |
Tüb-46 | 4.67 | HR | | ILC482 | 9.33 | HR |
Tüb-47 | 0 | HR | | SARI | 62.67 | HS |
LSD (P = 0.05): | 13.02 | | | | | |
*Disease severity was assessed with 0–5 scale (0: no lesion. 1: 10%; 2: 25%; 3: 50%. 4: 75% and 5: 100% of leaflet areas affected). Highly resistant (HR): 0–10%. Resistant (R): 11–29%. Moderately susceptible (MS): 30–49% and Highly susceptible (HS): >50%. Data are means of three replicates. Means compared with the least significant difference (LSD) (P = 0.05) |
Disease reaction of the genotypes evaluated to Ascochyta blight was also quantified by using a standard curves constructed by amplifying known amounts of target DNA by real-time PCR method developed in a previous study [23]. The primer pairs HEF1/2 amplified a PCR fragment of 82 bp in size from plant materials infected with A. rabiei. The slope and intercept value of the regression line for standards was 3.36 and 23.53, respectively, and the r2 value was 1.00. The presence of a single melt peak confirmed the amplification of a specific product with a dissociation temperature of 83°C. The assay showed significant variation among chickpea genotypes, and the amount of the target DNA in highly resistant genotypes was predominantly less than in more susceptible genotypes (Fig. 1). The quantity of pathogen DNA in the inoculated leaflet samples changed from 0.004–83.37 ng. The amount of target DNA quantified in cultivar ILC 482, highly resistant to Ascochyta blight was 1.87 ng, while DNA quantity in highly susceptible cultivar Sarı-98 was found as 43.07 ng. The results showed a highly correlation (r = 0.82) between the quantities of pathogen DNA and the levels of disease severity in chickpea genotypes. Among the chickpea genotypes tested, Tüb-35, Tüb-47, Tüb-26, Tüb-82, Tüb-65, Tüb-14, Tüb-16 and Tüb-69 genotypes with DNA quantities of ≤ 0.1 ng were classified as the most resistant to Ascochyta blight, while Tüb-105, Tüb-72, Tüb-67 and Tüb-108 genotypes were highly susceptible based on the quantification of the target DNA in leaflet tissues, respectively. Also, the average DNA quantities in highly susceptible, moderately susceptible, resistant and highly resistant genotypes exhibited significant differences (P ≤ 0.05). The average quantity of pathogen DNA in chickpea genotypes, exhibiting highly resistant response on visual disease scoring was 1.0 ng, while the average DNA quantity in chickpea genotypes regarded as resistant was determined as 5.37 ng. The average DNA quantity in moderately susceptible and highly susceptible genotypes was found to be 11.08 and 46.85 ng, respectively. Similarly, many studies on the assessments of the resistance levels of different host cultivars or genotypes by using quantitative real time PCR assay have been performed [21; 22]. Brouwer et al. [36] reported that the quantitative-real time PCR assay can be used for quick and reliable assessment of the susceptibility of Arabidopsis genotypes to different fungal pathogens. Similar work was conducted by Jimenaz et al. [37], who assessed the potential uses of qPCR for differentiating susceptible from resistant chickpea reactions to Fusarium wilt. A strong correlation (r = 0.87) between disease assessment and DNA quantity, suggested that qPCR assay may be used for screening resistance chickpea germplasm to Fusarium wilt. Daniëls et al. [38] which developed Venturia inaequalis-specific real-time PCR assay to analyze resistance of apple cultivars, found a significant correlation between the amount of DNA of Venturia inaequalis quantified in apple leaves and host resistance. They indicated this assay was a more objective and sensitive method than visual disease assessments to evaluate host resistance of apple cultivars. Leiminger et al. [39] evaluated relationship between visual disease assessments and DNA levels of Alternaria solani and A. alternata in potato leaves by quantitative PCR. They found a closely correlation (r = 0.71) between DNA quantities and the ratio of necrotic area caused by A. solani in agreement with the results of this study.
This study demonstrated the utility of real-time PCR assay for quick and reliable assessment of disease severity on chickpea genotypes to Ascochyta blight. A strong correlation was observed between DNA quantities and visual disease scoring in different chickpea genotypes. Tüb-35, Tüb-47, Tüb-26, Tüb-82, Tüb-65 and Tüb-69 genotypes were classified as resistant to Ascochyta blight based on both the levels of disease severity and the quantification of target DNA in infected plant tissues. These genotypes should be considered in detail in breeding studies as sources of resistance to Ascochyta blight. This assay could be used for selecting breeding material during initial phases of disease infection as an optional approach to visual score by quantifying the pathogen presence in host plant.