Ascochyta blight infected chickpea fields were chosen in Kermanshah and Illam province (west of Iran). Thirty-two separate fields (10 km distance) were chosen and infected leaves stem and pods were collected during 2017 and 2018 growing season. To isolate the A. rabiei fungus, infected samples were surface-sterilized with hypochlorite sodium (0.5%) for 2 min, and washed twice with sterilized distilled water. Samples were plated on CSMDA (chickpea seed meal 40 g, dextrose 20 g and agar 18 g in 1 l sterilized distilled water). Plates were incubated for 7-10 days at 20-22°C. Single pycnidia isolates were purified from plates and stored on CSMDA for pathogenicity tests.

The pathogenicity test was conducted on susceptible cultivar “Bivanij” under controlled greenhouse. Pre-cultures of 32 A. rabiei isolates were prepared on CSMDA medium. 2-4 mm of these pre-cultures was then used to inoculate 100 ml Erlenmeyer flask containing 40 ml of potato dextrose broth (PDB) and incubated on shaker for five days at 20-22°C. Ten days oil plants of susceptible check cultivar “Bivanij” were inoculated using A. rabiei medium adjusted to 5 × 10⁵ spores/ml supplemented with 0.15% of Tween 20 (MERCK^®, Nottingham, UK). The inoculated plants were covered by dark plastic which was removed after 24 h. The inoculated plants were kept in greenhouse for 14 days. During the experiment, environmental conditions were kept at 22/18°C (day/night rhythm) with 14-h photoperiod and 90% humidity. 14-days after inoculation, disease reaction of susceptible check against A. rabiei isolates was assessed using 0-9 rating scale as described previously (Chongo et al., 2004; Nene et al., 1981; Pande et al., 2011) as follow: 0 = no symptom or small lesions (highly resistant); 1= few and small lesions on the apical stems (highly resistant); 2 = small girdling stem lesions low down on some branches (resistant); 3 = lesions up to 5-6 mm in size and 1-2 branches broken (resistant); 4 = lesions (2-5 mm in size) obviously on most part of plant and several branches broken (moderately resistant); 5 = many large lesions and defoliation initiated and half of branches broken (moderately susceptible); 6 = lesions as in 5, with more defoliation, broken stems and dry branches (susceptible); 7 = many large lesions as in 5 and 6, with obviously defoliation and broken branches (up to 70%) and up to 25% of plants killed (susceptible); 8 = symptoms as in 7 and up to 50% of plants killed (highly susceptible); 9 = symptoms as in 8 and all plants killed (highly susceptible).

Seven chickpea genotypes (Table 1) were obtained from ICRISAT and ICARD as identified previously (Chen et al., 2004; Kanouni et al., 2011; Shahriari et al., 2016) were utilized as differentials representing resistant (ILC202 and ILC72), moderately resistance (ILC5928 and ICC3996), moderately susceptible (ILC194) and susceptible (ILC1929 and PCH215) genotypes. The pathogenicity test of 32 A. rabiei isolates were assessed on seven chickpea differentials based on their virulence/avirulence responses with different A. rabiei isolates. Five seeds form each genotype was sown in pods containing soil, peat and sand (1:1:2, v/v/v). Pathogenicity test procedures (inoculation and disease reaction) were same as described for pathogenicity test. 14-days after inoculation differentials were assessed for their reaction against 32 A. rabiei isolates based on 0-9 rating scale as described previously (Chongo et al., 2004; Nene et al., 1981; Pande et al., 2011). Physiologic races of 32 isolates of A. rabiei collected were determined using seven chickpea standard differential cultivars as described by Shahriari et al. (2016). Experiment was carried out in randomized complete factorial design with three replications.

One hundred and sixty five chickpea genotypes (20 Iranian landrace Desi accessions and 145 Kabuli accessions) were assessed for resistance/susceptibility pattern against different A. rabiei races. Genotypes name, origin and pedigree of chickpea genotypes summarized in Supplementary Table 1.

Six isolates that representing different races of A. rabiei were chosen from first experiment (AR9, AR13, AR18, AR23, AR28 and AR32). To evaluate 165 chickpea genotypes against A. rabiei races, five seeds form each genotype was sown in pods containing soil, peat and sand (1:1:2, v/v/v) in randomized complete factorial design with three replications under greenhouse condition. Fourteen days old seedlings were spray-inoculated with 5 × 10⁵ spores/ml suspension. During the experiment, environmental conditions were kept at 22/18°C (day/night rhythm) with 14-h photoperiod and 90% humidity. Two weeks after inoculation, disease ratings of Ascochyta blight were made by using 0-9 rating scale (Chongo et al., 2004; Nene et al., 1981; Pande et al., 2011).

The experimental design of the present study was a randomized complete factorial design with three replications. Analysis of variance (ANOVA) of the data for differential and chickpea germplasm was performed using SAS program (SAS Institute Inc, 2001). Principal component analysis (PCA) and cluster analysis of differential and chickpea genotypes according to disease severity rate data by each isolate as well as for tested isolates were performed using the un-weighted pair-group method (UPGMA) and the dissimilarity matrix was measured using ward’s method implemented in SAS JMP software (SAS 2012).

ANOVA analysis showed high significant effects (P ≤ 0.01) of isolates, differentials and isolate × differentials interactions based on Ascochyta blight scores on 0-9 scale (Table 1). The resistant and susceptible pattern of seven differential genotypes and susceptible cultivar “Bivanij” are given in Table 2. Results from susceptible cultivar showed high aggressiveness (grades from 7 to 9) for all tested A. rabiei races. For all isolates, symptoms initially appeared as irregular white spots on leaves and light green lesions on stems between 4 to 7 days after inoculation. Diseases severity increased with time and after 14 days dried broken stems and killed plants were observed in susceptible check (Bivanij cv.). Increasing the disease severity for all isolates was same, although symptoms for isolates belonged to race VI appeared 2-3 days earlier. The blight severity was recorded on a 1-9 scale as suggested by Chongo et al. (2004) and Pande et al. (2011). In order to score differentials and to identify the pathogentic groups of isolates, plants with 0 to 4 score were categorized as resistant and 5-9 susceptible making simple the count of resistant and susceptible plant. Based on diseases severity rate of differentials cultivars, isolated grouped into six pathogenic groups as described by Shahriari et al. (2016).

The differential genotypes, ILC1929 was highly susceptible to all isolates, while ILC202 was the most resistant genotype that showed resistance responses to all isolates except against three isolates belonging to race VI. Differential genotypes, ILC5928 showed resistance reaction to most of isolates, except for 8 isolates belonging to pathotype III (Table 2). The most and least frequent races were race I and race V, respectively. Isolated collected from “Kermanshah” province characterized for six races, while isolated from “Illam” province characterized only for race I, II and IV. Overall, results from differential genotypes grouped A. rabiei isolates into six pathotypes including 9 isolates for race I, 5 isolates for race II, 8 isolates for race III, 5 isolates for race IV, 2 isolates for race V and 3 isolates for race VI (Table 2). Principal component analysis (PCA) and cluster analysis of A. rabiei tested on differentials grouped isolates into three clusters (Fig. 1). Isolated belonged to race II and III grouped in first cluster, race I in second cluster and race IV, V and VI in third cluster.

ANOVA analysis showed high significant (P ≤ 0.01) differences between chickpea genotypes for their interactions with A. rabiei races (Table 3). The chickpea-isolates interaction effect analysis was highly significant (P ≤ 0.01). Significant (P < 0.01) differences among the chickpea genotypes indicated that they varied in their responses to infection by the different A. rabiei races.

The resistance spectra of 165 chickpea genotypes against six A. rabiei races presented in Supplementary Table 2. In order to characterize the susceptible and resistance genotypes based on score 0-9, plants with 0 to 4 score were categorized as resistant and 5-9 susceptible, as described previously by Labdi et al. (2013) and Shahriari et al. (2016). Among all interactions (n = 990), 259 resistance responses (DS ≤ 4) were identified, which 129 responses were considered as highly resistances (DS ≤ 2) (Supplementary Table 2). Resistance spectra of chickpea genotypes showed more resistance rate to race I (49.70%) and race III (35.15%), while there were no resistance genotypes to race VI. Out of 165 chickpea genotypes, 38 genotypes (23%) showed no race specific responses and were susceptible to all races. In contrast, two genotypes (FLIP02-04C and FLIP06-65C) were resistant (disease severity ≤ 4) to all races, except for race VI, while FlIP02-04C was also partially resistant to race VI. Nine genotypes showed specific resistance to four races (Supplementary Table 2). Cluster analysis and principal component analysis (PCA) based on mean disease severity data of each genotype against six A. rabiei races grouped 165 chickpea genotypes in four distinct clusters (Fig. 2 and 3). First cluster contained 46 genotypes including three Iranian cultivars (Arman, Hashem and Kaka). Mean diseases severity for genotypes belonging to this cluster showed resistance spectra to race I and highly susceptible to other races (Table 4). Cluster II comprised 47 genotypes that mean diseases severity of genotypes showed resistance interaction to race III, moderate resistance to race I and IV and highly susceptible to race II, V and VI. Cluster III comprised 35 genotypes including three Iranian cultivars (ILC482, Adel and Azad). Genotypes belonged to this cluster showed resistance interaction to race I, race II and race III, moderately susceptibility to race IV and V and high susceptibility to race VI. Mean diseases severity of genotypes belonged to this cluster ranged from 2.24 (race II) to 7.59 (race VI). Cluster IV comprised 34 genotypes that all genotypes showed highly susceptibility to all A. rabiei races (Table 4). All ‘desi’ chickpea accessions grouped in cluster IV and showed highly susceptibility to all A. rabiei races.