Genotyping with RAPD and microsatellite markers resolves pathotype diversity in the ascochyta blight pathogen of chickpea

 The poor definition of variation in the ascochyta blight fungus (Ascochyta rabiei) has historically hindered breeding for resistance to the chickpea (Cicer arietinum L.) blight disease in West Asia and North Africa. We have employed 14 RAPD markers and an oligonucleotide probe complementary to the microsatellite sequence (GATA)₄ to construct a genotype-specific DNA fragment profile from periodically sampled Syrian field isolates of this fungus. By using conventional pathogenicity tests and genome analysis with RAPD and microsatellite markers, we demonstrated that the DNA markers distinguish variability within and among the major pathotypes of A. rabiei and resolved each pathotypes into several genotypes. The genetic diversity estimate based on DNA marker analysis within pathotypes was highest for the least-aggressive pathotype (pathotype I), followed by the aggressive (pathotype II) and the most-aggressive pathotype (pathotype III). The pair-wise genetic distance estimated for all the isolates varied from 0.00 to 0.39, indicating a range from a clonal to a diverse relationship. On the basis of genome analysis, and information on the spatial and temporal distribution of the pathogen, a general picture of A. rabiei evolution in Syria is proposed. Received: 10 January 1998 / Accepted: 23 January 1998     

Use of a Mini-Dome Bioassay and Grafting to Study Resistance of Chickpea to Ascochyta Blight

A mini‐dome bioassay was developed to study pathogenicity of Ascochyta rabiei and relative resistance of chickpea (Cicer arietanium). It was determined that the best condition for assaying pathogenicity of A. rabiei was to use 2 × 10⁵ spores/ml as inoculum and to maintain a leaf wetness period of 24 h under mini‐domes at a temperature between 16 and 22°C. This mini‐dome pathogenicity assay was used to determine relative resistance of six chickpea cultivars (cvs) to isolates of two pathotypes of A. rabiei. Grafting was employed to detect any translocated factors produced in the chickpea plant that mediate disease response, which could help elucidate possible resistance mechanisms to Ascochyta blight. The six chickpea cv. were grafted in all possible scion–rootstock combinations, and then inoculated with isolates of two pathotypes of A. rabiei using the mini‐dome technique. Results showed that self‐grafted‐resistant plants remained resistant and self‐grafted‐susceptible plants stayed susceptible, indicating the grafting procedure did not alter host response to infection by A. rabiei. Susceptible scions always exhibited high and similar levels of disease severity regardless of rootstock genotypes, and resistant scions always showed low and similar levels of disease severity when they were grafted onto any of the six rootstock genotypes. Orthogonal contrasts showed that scion genotypes determined disease phenotype, and that rootstock genotypes had no contribution to disease phenotype of the scions. The pathogenicity assay did not detect any translocated disease‐mediating agents responsible for susceptibility or resistance in chickpea. Disease phenotypes of Ascochyta blight of chickpea were conditioned locally by scion genotypes.     

Virulence diversity of Ascochyta rabiei the causal agent of Ascochyta blight of chickpea in the western provinces of Iran

Chickpea is the third most important food legume in the world. The most important limiting factor for the chickpea production in the world, including Iran, has been the Ascochyta blight. The pathogenic variation of 40 Ascochyta rabiei isolates from the western provinces of Iran was assessed on eight chickpea differential lines. The results revealed that A. rabiei population is diverse in the western provinces of Iran and the virulence rating of isolates across differential lines showed a large but continuous pathogenic variability. Based on the statistical analysis and the continuous response in differential lines, it was not possible to categorise A. rabiei isolates in the present study into pathotypes or races. Information obtained from the current study can be valuable in developing quarantine methods aimed to prevent dissemination of highly virulent isolates and in the development of durable resistant cultivars against the Ascochyta blight of chickpea.     

Peroxidase: a marker for Ascochyta blight resistance in chickpea

On the basis of incidence of appearance of Ascochyta blight symptoms after artificial inoculation of 25-day-old chickpea seedlings with 10 different pathotypes of Ascochyta rabiei, GL94011, PBG5 and C214 have been classified as resistant, moderately resistant and susceptible, respectively, to Ascochyta blight. In none of the pathotypes, peroxidase (PO) activity could be detected in culture medium and mycelium. Healthy tissues of GL94011 have almost three times the PO activity in comparison with that of C214. Resistant and moderately resistant genotypes showed 30–60% upregulation of PO activity against infection by A. rabiei whereas it was only 3–6% in susceptible genotype C214. These results indicate the possibility of using PO as a marker of Ascochyta blight resistance.     

Detection of two quantitative trait loci for resistance to ascochyta blight in an intra-specific cross of chickpea (Cicer arietinum L.): development of SCAR markers associated with resistance

Two quantitative trait loci (QTLs), (QTL_AR1 and QTL_AR2) associated with resistance to ascochyta blight, caused by Ascochyta rabiei, have been identified in a recombinant inbred line population derived from a cross of kabuli×desi chickpea. The population was evaluated in two cropping seasons under field conditions and the QTLs were found to be located in two different linkage groups (LG4a and LG4b). LG4b was saturated with RAPD markers and four of them associated with resistance were sequenced to give sequence characterized amplified regions (SCARs) that segregated with QTL_AR2. This QTL explained 21% of the total phenotypic variation. However, QTL_AR1, located in LG4a, explained around 34% of the total phenotypic variation in reaction to ascochyta blight when scored in the second cropping season. This LG4a region only includes a few markers, the flower colour locus (B/b), STMS GAA47, a RAPD marker and an inter-simple-sequence-repeat and corresponds with a previously reported QTL. From the four SCARs tagging QTL_AR2, SCAR (SCY17₅₉₀) was co-dominant, and the other three were dominant. All SCARs segregated in a 1:1 (presence:absence) ratio and the scoring co-segregated with their respective RAPD markers. QTL_AR2 on LG4b was mapped in a highly saturated genomic region covering a genetic distance of 0.8 cM with a cluster of nine markers (three SCARs, two sequence-tagged microsatellite sites (STMS) and four RAPDs). Two of the four SCARs showed significant alignment with genes or proteins related to disease resistance in other species and one of them (SCK13₆₀₃) was sited in the highly saturated region linked to QTL_AR2. STMS TA72 and TA146 located in LG4b were described in previous maps where QTL for blight resistance were also localized in both inter and intraspecific crosses. These findings may improve the precision of molecular breeding for QTL_AR2 as they will allow the choice of as much polymorphism as possible in any population and could be the starting point for finding a candidate resistant gene for ascochyta blight resistance in chickpea.     

Genetic dissection of pathotype-specific resistance to ascochyta blight disease in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) using microsatellite markers

Ascochyta blight is an economically important disease of chickpea caused by the fungus Ascochyta rabiei. The fungus shows considerable variation for pathogenicity in nature. However, studies on the genetics of pathotype-specific resistance are not available for this plant-pathosystem. The chickpea landrace ILC 3279 has resistance to pathotype I and II of the pathogen. In order to understand the inheritance of pathotype-specific resistance in this crop, both Mendelian and quantitative trait loci analyses were performed using a set of intraspecific, recombinant inbred lines derived from a cross between the susceptible accession ILC 1272 and the resistant ILC 3279, and microsatellite markers. We identified and mapped a major locus (ar1, mapped on linkage group 2), which confers resistance to pathotype I, and two independent recessive major loci (ar2a, mapped on linkage group 2 and ar2b, mapped on linkage group 4), with complementary gene action conferring resistance to pathotype II. Out of two pathotype II-specific resistance loci, one (ar2a) linked very closely with the pathotype I-specific resistance locus, indicating a clustering of resistance genes in that region of the chickpea genome.     

Genetic analyses and conservation of QTL for ascochyta blight resistance in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Ascochyta blight (AB) caused by Ascochyta rabiei (teleomorph, Didymella rabiei) Pass. Lab. is an important fungal disease of chickpea worldwide. Only moderate sources of resistance are available within the cultivated species and we hypothesized that the available sources may carry different genes for resistance, which could be pyramided to improve field resistance to AB. Four divergent moderately resistant cultivars CDC Frontier, CDC Luna, CDC Corinne, and Amit were each crossed to a highly susceptible germplasm ICCV 96029. Parents, F₁ and F₂ generations were evaluated under controlled conditions for their reactions to AB. A total of 144 simple sequence repeat (SSR) markers were first mapped to eight linkage groups (LG) for the CDC Frontier × ICCV 96029 population. Then based on the evidence from this population, 76, 61, and 42 SSR markers were systematically chosen and mapped in CDC Luna, CDC Corinne, and Amit populations, respectively. Frequency distributions of the AB rating in the F₂ generation varied among the four populations. Composite interval mapping revealed five QTLs (QTL1–5), one on each of LG 2, 3, 4, 6, and 8, respectively, distributed across different sources, controlling resistance to AB. CDC Frontier contained QTL2, 3, and 4 that simultaneously accounted for 56% of phenotypic variations. CDC Luna contained QTL 1 and 3. CDC Corinne contained QTL 3 and 5, while only QTL 2 was identified in Amit. Altogether these QTL explained 48, 38, and 14% of the estimated phenotypic variations in CDC Luna, CDC Corinne, and Amit populations, respectively. The results suggested that these QTLs could be combined into a single genotype to enhance field resistance to AB. Y. Anbessa and B. Taran contributed equally to this work.     

Ascochyta blight of chickpea reduced 38% by application of Aureobasidium pullulans (anamorphic Dothioraceae,Dothideales) to post-harvest debris

In 2004–2005, application of non-amended suspensions of Aureobasidium pullulans conidia to post-harvest chickpea debris resulted in 37.9% fewer Ascochyta blight lesions on chickpea test plants relative to controls. Analogous tests in 2006–2007 resulted in 38.4% fewer lesions. Ascospores released from debris were predominantly Davidiella sp. (anamorph, Cladosporium sp.), followed by Didymella rabiei (anamorph, Ascochyta rabiei, agent of Ascochyta blight).     

Genetic marker discovery,intraspecific linkage map construction and quantitative trait locus analysis of ascochyta blight resistance in chickpea (Cicer arietinum L.)

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Labr., is a highly destructive disease of chickpea (Cicer arietinum L.) on a global basis, and exhibits considerable natural variation for pathogenicity. Different sources of ascochyta blight resistance are available within the cultivated species, suitable for pyramiding to improve field performance. Robust and closely linked genetic markers are desirable to facilitate this approach. A total of 4,654 simple sequence repeat (SSR) and 1,430 single nucleotide polymorphism (SNP) markers were identified from a chickpea expressed sequence tag (EST) database. Subsets of 143 EST–SSRs and 768 SNPs were further used for validation and subsequent high-density genetic mapping of two intraspecific mapping populations (Lasseter × ICC3996 and S95362 × Howzat). Comparison of the linkage maps to the genome of Medicago truncatula revealed a high degree of conserved macrosynteny. Based on field evaluation of ascochyta blight incidence performed over 2 years, two genomic regions containing resistance determinants were identified in the Lasseter × ICC3996 family. In the S95362 × Howzat population, only one quantitative trait locus (QTL) region was identified for both phenotypic evaluation trials, which on the basis of bridging markers was deduced to coincide with one of the Lasseter × ICC3996 QTLs. Of the two QTL-containing regions identified in this study, one (ab_QTL1) was predicted to be in common with QTLs identified in prior studies, while the other (ab_QTL2) may be novel. Markers in close linkage to ascochyta blight resistance genes that have been identified in this study can be further validated and effectively implemented in chickpea breeding programs.     

Characterisation of Ascochyta rabiei isolates and evaluation of genotypic stability in chickpea

Ascochyta rabiei isolates were characterised for their variability using a set of host differentials following cloth chamber screening technique. Sixty chickpea genotypes were evaluated against the characterised 10 individual pathotypes separately to identify genotypes with stable resistance during 2007–2008. Twenty four genotypes showed resistance to all the pathotypes; whereas 18 genotypes were resistant to moderately resistant to these pathotypes. The above genotypes can be considered good sources of stable resistance and recommended as donors or for direct cultivation in north western plain zone of India.     

Genetic mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.) using a simple sequence repeat linkage map.

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Lab., is one of the most devastating diseases of chickpea (Cicer arietinum L.) worldwide. Research was conducted to map genetic factors for resistance to ascochyta blight using a linkage map constructed with 144 simple sequence repeat markers and 1 morphological marker (fc, flower colour). Stem cutting was used to vegetatively propagate 186 F2 plants derived from a cross between Cicer arietinum L. 'ICCV96029' and 'CDC Frontier'. A total of 556 cutting-derived plants were evaluated for their reaction to ascochyta blight under controlled conditions. Disease reaction of the F1 and F2 plants demonstrated that the resistance was dominantly inherited. A Fain's test based on the means and variances of the ascochyta blight reaction of the F3 families showed that a few genes were segregating in the population. Composite interval mapping identified 3 genomic regions that were associated with the reaction to ascochyta blight. One quantitative trait locus (QTL) on each of LG3, LG4, and LG6 accounted for 13%, 29%, and 12%, respectively, of the total estimated phenotypic variation for the reaction to ascochyta blight. Together, these loci controlled 56% of the total estimated phenotypic variation. The QTL on LG4 and LG6 were in common with the previously reported QTL for ascochyta blight resistance, whereas the QTL on LG3 was unique to the current population.     

Mapping quantitative trait loci in chickpea associated with time to flowering and resistance to Didymella rabiei the causal agent of Ascochyta blight

Drought is the major constraint to chickpea (Cicer arietinum L.) productivity worldwide. Utilizing early-flowering genotypes and advancing sowing from spring to autumn have been suggested as strategies for drought avoidance. However, Ascochyta blight (causal agent: Didymella rabiei (Kov.) v. Arx.) is a major limitation for chickpea winter cultivation. Most efforts to introgress resistance to the pathogen into Kabuli germplasm resulted in relatively late flowering germplasm. With the aim to explore the feasibility of combining earliness and resistance, RILs derived from a cross between a Kabuli cultivar and a Desi accession were evaluated under field conditions and genotyped with SSR markers. Three quantitative trait loci (QTLs) with significant effects on resistance were identified: two linked loci located on LG4 in epistatic interaction and a third locus on LG8. Two QTLs were detected for time to flowering: one in LG1 and another on LG2. When resistance and time to flowering were analyzed together, the significance of the resistance estimates obtained for the LG8 locus increased and the locus effect on days to flowering, previously undetected, was significantly different from zero. The identification of a locus linked both to resistance and time to flowering may account for the correlation observed between these traits in this and other breeding attempts.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

Host plant resistance of different chickpea genotypes against Ascochyta rabiei (Pass.) Lab. under tunnel conditions

Host plant resistance is the most efficient and easy way to manage chickpea blight caused by Ascochyta rabiei (Pass.) Lab. For this purpose, 374 chickpea lines/varieties from various research organisations were evaluated in plastic tunnels. None of the line showed immune response against the blight; however, one line (K-01005) was found to be highly resistant. Moreover, 15 entries were resistant, 136 exhibited moderate resistant reaction, 150 were susceptible and 72 showed highly susceptible response. The genotypes found that resistance against blight can serve as a source of resistance for breeding programmes, and they could be released for commercial production directly.     

RAPD Analysis of Pathogenic Variability in Ascochyta rabiei

Thirty Italian isolates of the phytopathogenic fungus Ascochyta rabiei (Pass.) Labr., the causal organism of Ascochyta blight on chickpea (Cicer arietinum L.), were analysed by a random oligonucleotide primer dependent polymerase chain, reaction (PCR) technique called random amplified polymorphic DNA analysis (RAPD) using three decamer primers. In previous investigations these isolates had been differentiated in six pathogenic groups. RAPD results were summarized in an analysis using the program PAUP. With each of the primers several amplification products were observed which were common to all isolates. The results of the RAPD analyses also showed that all isolates could be identified by a unique RAPD pattern. No correlation between RAPD patterns and the division of the isolates in pathogenic groups could be established. The application of the RAPD technique for cataloguing isolates and to obtain specific genetic markers for all isolates of the species Ascochyta rabiei is discussed.     

A linkage map of chickpea (Cicer arietinum L.) based on populations from Kabuli × Desi crosses: location of genes for resistance to fusarium wilt race 0

Two recombinant inbred line (RIL) populations derived from intraspecific crosses with a common parental line (JG62) were employed to develop a chickpea genetic map. Molecular markers, flower colour, double podding, seed coat thickness and resistance to fusarium wilt race 0 (FOC-0) were included in the study. Joint segregation analysis involved a total of 160 markers and 159 RILs. Ten linkage groups (LGs) were obtained that included morphological markers and 134 molecular markers (3 ISSRs, 13 STMSs and 118 RAPDs). Flower colour (B/b) and seed coat thickness (Tt/tt) appeared to be linked to STMS (GAA47). The single-/double-podding locus was located on LG9 jointly with two RAPD markers and STMS TA80. LG3 included a gene for resistance to FOC-0 (Foc0₁/foc0₁) flanked by RAPD marker OPJ20₆₀₀ and STMS marker TR59. The association of this LG with FOC-0 resistance was confirmed by QTL analysis in the CA2139 × JG62 RIL population where two genes were involved in the resistance reaction. The STMS markers enabled comparison of LGs with preceding maps.     

Pathotype-specific QTL for stem rust resistance in Lolium perenne

A genetic map populated with RAD and SSR markers was created from F1 progeny of a stem rust-susceptible and stem rust-resistant parent of perennial ryegrass (Lolium perenne). The map supplements a previous map of this population by having markers in common with several other Lolium spp. maps including EST-SSR anchor markers from a consensus map published by other researchers. A QTL analysis was conducted with disease severity and infection type data obtained by controlled inoculation of the population with each of two previously characterized pathotypes of Puccinia graminis subsp. graminicola that differ in virulence to different host plant genotypes in the F1 population. Each pathotype activated a specific QTL on one linkage group (LG): qLpPg1 on LG7 for pathotype 101, or qLpPg2 on LG1 for pathotype 106. Both pathotypes also activated a third QTL in common, qLpPg3 on LG6. Anchor markers, present on a consensus map, were located in proximity to each of the three QTL. These QTL had been detected also in previous experiments in which a genetically heterogeneous inoculum of the stem rust pathogen activated all three QTL together. The results of this and a previous study are consistent with the involvement of the pathotype-specific QTL in pathogen recognition and the pathotype-nonspecific QTL in a generalized resistance response. By aligning the markers common to other published reports, it appears that two and possibly all three of the stem rust QTL reported here are in the same general genomic regions containing some of the L. perenne QTL reported to be activated in response to the crown rust pathogen (P. coronata).     

QTL analysis for ascochyta blight resistance in an intraspecific population of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

In both controlled environment and the field, six QTLs for ascochyta blight resistance were identified in three regions of the genome of an intraspecific population of chickpea using the IDS and AUDPC disease scoring systems. One QTL-region was detected from both environments, whereas the other two regions were detected from each environment. All the QTL-regions were significantly associated with ascochyta blight resistance using either of the disease scoring systems. The QTLs were verified by multiple interval mapping, and a two-QTL genetic model with considerable epistasis was established for both environments. The major QTLs generally showed additive gene action, as well as dominance inter-locus interaction in the multiple genetic model. All the QTLs were mapped near a RGA marker. The major QTLs were located on LG III, which was mapped with five different types of RGA markers. A CLRR-RGA marker and a STMS marker flanked QTL 6 for controlled environment resistance at 0.06 and 0.04 cM, respectively. Other STMS markers flanked QTL 1 for field resistance at a 5.6 cM interval. After validation, these flanking markers may be used in marker-assisted selection to breed for elite chickpea cultivars with durable resistance to ascochyta blight. The tight linkage of RGA markers to the major QTL on LG III will allow map-based cloning of the underlying resistance genes.Communicated by P. Langridge     

Biocontrol of Ascochyta blight by Azospirillum sp. depending on the degree of resistance of chickpea genotypes

Throughout arable land that was devoted to chickpea (Cicer arietinum L. (Family: Leguminosae) production, Ascochyta blight (Ascochyta rabiei (Pass.) L. (Order: Sphaeriales; Family: Mycosphaerellaceae) is a widespread disease that would lead to significant loss of chickpea yield. This study's purpose was to explain the responses of a resistant chickpea cultivar (ICC 12004) and a susceptible cultivar (Bivanij) in terms of disease resistance, disease symptoms appearance and expression pattern of two defence‐related genes (DEF0442 and Snakin2) after the Azospirillum brasilense seeds inoculation. In this research, the Snakin2 gene expression was affected by Azospirillum inoculation. The gene expression has been enhanced in plants inoculated with Azospirillum in both cultivars in comparison with non‐inoculated plants, but this change in ICC 12004 and Bivanij were significant and non‐significant, respectively. Although, Azospirillum would up regulate the DEF0422 gene expression in ICC 12004, but it would down regulate the expression of this gene in Bivanij. A. brasilense inoculation decreased the A. rabiei disease severity, regardless of the chickpea cultivar. Bivanij still could be classified as susceptible, even if treated with A. brasilense.     

Characterization and genetic analysis of an EIN4-like sequence (CaETR-1) located in QTL(AR1) implicated in ascochyta blight resistance in chickpea

Two alleles of a chickpea (Cicer arietinum L.) ethylene receptor-like sequence (CaETR-1) were sequence-characterized using synteny analysis with genome sequences of Medicago truncatula L. The full length of the sequence obtained in the accession FLIP84-92C resistant to ascochyta blight (CaETR-1a) span 4,428?bp, including the polyadenylation signal in the 3'-untranslated region (UTR), whereas it has a 730?bp deletion in the 3'-UTR region in the susceptible accession PI359075 (CaETR-1b). The deduced protein belongs to subfamily II of the ethylene receptors and contains all the domains that define EIN4 homologs in Arabidopsis. The EIN4-like sequence (CaETR-1) has been mapped using a recombinant inbred line (RIL) population derived from an intraspecific cross between ILC3279 and WR315, resistant and susceptible to blight, respectively. The locus was located in LGIVa of the genetic map, flanked by markers NCPGR91 and GAA47 (at distances of 11.3 and 17.9?cM, respectively). This is the first potentially functional sequence identified under a QTL peak for ascochyta blight resistance in chickpea (QTL(AR1)). This EIN4-like (CaETR-1) sequence explained up to 33.8% of the total phenotypic variation. This sequence could be directly related to blight resistance, together with other QTLs that have been found to be involved in resistance to this major chickpea disease.