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.     

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.     

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.)

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi‐type chickpea genome using next‐generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520‐Mb assembly covers 70% of the predicted 740‐Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27 571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA‐Seq reads identified several tissue‐specific and stress‐responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole‐genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.     

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.     

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).     

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.     

Enhanced Defence Responses of Chickpea Plants Against Rhizoctonia solani by Pre‐Inoculation with Rhizobia

The aim of this work was to study the antagonist effect of two Rhizobium strains Pch Azm and Pch S.Nsir2 to Rhizoctonia solani and for an evaluation of the relative impact of rhizobia on the expression of the plant's defence response against Rhizoctonia. First, these strains reduced fungal growth observed in vitro using the same or separately Petri dishes. Moreover, these isolates led to reduced chickpea infection by R. solani, resulting from the direct effect of rhizobia on pathogens and possible induced resistance in chickpea. Concomitantly, reduction in infection was accompanied by enhanced level of defence‐related enzymes, phenylalanine ammonia lyase (PAL) and peroxidase (POX). An increased level of phenol content was recorded in the roots of bacterized plants grown in the presence of pathogen. The results promise the use of rhizobia for protection of chickpea against R. solani.     

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.     

Interaction of acid exudates in chickpea with biological activity of Bacillus thuringiensis towards Helicoverpa armigera

The gram pod borer, Helicoverpa armigera, is one of the most important constraints to chickpea production. High acidity of chickpea exudates is associated with resistance to pod borer, H. armigera; however, acidic exudates in chickpea might influence the biological activity of the bacterium, Bacillus thuringiensis (Bt), applied as a foliar spray or deployed in transgenic plants for controlling H. armigera. Therefore, studies were undertaken to evaluate the biological activity of Bt towards H. armigera on chickpea genotypes with different amounts of organic acids. Significantly lower leaf feeding, larval survival and larval weights were observed on ICC 506EB, followed by C 235, and ICCV 10 across Bt concentrations. Leaf feeding by the larvae and larval survival and weights decreased with an increase in Bt concentration. However, rate of decrease in leaf feeding and larval survival and weights with an increase in Bt concentration was greater on L 550 and ICCV 10 than on the resistant check, ICC 506EB, suggesting that factors in the resistant genotypes, particularly the acid exudates, resulted in lower levels of biological activity of Bt possibly because of antifeedant effects of the acid exudates. Antifeedant effects of acid exudates reduced food consumption and hence might reduce the efficacy of Bt sprays on insect‐resistant chickpea genotypes or Bt‐transgenic chickpeas, although the combined effect of plant resistance based on organic acids, and Bt had a greater effect on survival and development of H. armigera than Bt alone.     

Approaches for enhancement of N2 fixation efficiency of chickpea (Cicer arietinum L.) under limiting nitrogen conditions

Chickpea (Cicer arietinum) is an important pulse crop in many countries in the world. The symbioses between chickpea and Mesorhizobia, which fix N₂ inside the root nodules, are of particular importance for chickpea's productivity. With the aim of enhancing symbiotic efficiency in chickpea, we compared the symbiotic efficiency of C‐15, Ch‐191 and CP‐36 strains of Mesorhizobium ciceri in association with the local elite chickpea cultivar ‘Bivanij’ as well as studied the mechanism underlying the improvement of N₂ fixation efficiency. Our data revealed that C‐15 strain manifested the most efficient N₂ fixation in comparison with Ch‐191 or CP‐36. This finding was supported by higher plant productivity and expression levels of the nifHDK genes in C‐15 nodules. Nodule specific activity was significantly higher in C‐15 combination, partially as a result of higher electron allocation to N₂ versus H⁺. Interestingly, a striking difference in nodule carbon and nitrogen composition was observed. Sucrose cleavage enzymes displayed comparatively lower activity in nodules established by either Ch‐191 or CP‐36. Organic acid formation, particularly that of malate, was remarkably higher in nodules induced by C‐15 strain. As a result, the best symbiotic efficiency observed with C‐15‐induced nodules was reflected in a higher concentration of the total and several major amino metabolites, namely asparagine, glutamine, glutamate and aspartate. Collectively, our findings demonstrated that the improved efficiency in chickpea symbiotic system, established with C‐15, was associated with the enhanced capacity of organic acid formation and the activities of the key enzymes connected to the nodule carbon and nitrogen metabolism.     

Embryo rescue and plant regeneration in vitro of selfed chickpea (Cicer arietinum L.) and its wild annual relatives

The main constraint to the transfer of desired traits into cultivated chickpea from wild Cicer relatives is the presence of post-zygotic barriers which result in abortion of the immature embryo following interspecific hybridisation. Rescue of hybrid embryos in vitro and regeneration of hybrid plantlets could allow chickpea breeders to transfer desirable traits from wild relatives of chickpea. The development of embryo rescue techniques using selfed chickpea and selfed wild relatives is being used as a first step to protocols for wide hybrids. Optical microscopy studies of embryogenesis, in both selfs and hybrids, identified deleterious changes in the fertilised hybrid seed as early as 2–4 days after pollination in some crosses. These observations suggest that the appropriate time to rescue chickpea × C. bijugum hybrids is at the early globular stage of embryogenesis (2–7 days old), which requires the development of a complex tissue culture medium. In contrast hybrids between chickpea × C. pinnatifidum abort later (up to 15–20 days old) at the heart-shaped or torpedo stages, and are easier to rescue in vitro. Genotype also plays a significant role in the ability of immature selfed ovules to germinate in vitro. In this paper we report on the optimisation of␣protocols for rescueing immature embryos using selfed chickpea and its wild relatives in ovule, and subsequently to regenerate plantlets.     

Assessment of genetic diversity of Iranian Ascochyta rabiei isolates using rep‐PCR markers

Genetic diversity and population structure among 29 isolates of Ascochyta rabiei (AR) obtained from diseased chickpea plants in six different geographical origins in Iran was characterized by MAT and rep‐PCR (BOX/ERIC/REP) markers. Both mating types were found in all six populations, and the frequencies of mating types were variable between populations. The majority of the isolates belonged to Mat1‐1 (58.12%) with the remainder (41.88%) being Mat1‐2. A dendrogram was calculated with Jaccard's similarity coefficients with unweighted pair group method clustering (UPGMA) for the combination of rep‐PCR results, AR strains were differentiated into four clusters (A–D) at 60% similarity level. ERIC, REP and BOX showed a total of 19, 37 and 24 alleles per locus, respectively. Gene diversity (He) and Shannon's information index (I) were the highest in the REP (He = 0.82; I = 2.11), while the lowest values were estimated for the ERIC (He = 0.42; I = 1.3). Our result showed that among the three techniques studied, REP‐PCR produced the most complex amplified banding patterns, which reflected a high degree of diversity among the Iranian AR strains. ERIC‐PCR was the least discriminating method, and BOX‐PCR was intermediate. To the best our knowledge, this is first study of assessment of genetic diversity of AR isolates by rep‐PCR markers.     

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.     

Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics

Molecular changes elicited by plants in response to fungal attack and how this affects plant–pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label‐free proteomics and NMR‐based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis‐related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea–Foc interactions.     

Improving chickpea yield by incorporating resistance to ascochyta blight

Ascochyta blight [Ascochyta rabiei (Pass.) Lab.] is the most destructive disease of chickpea (Cicer arietinum L.), but it can be managed effectively by the use of resistant cultivars. Therefore, a breeding programme was initiated during 1977–78 at ICARDA, Syria, to breed blight-resistant, high-yielding chickpeas with other desirable agronomic traits. Crosses were made in main season at Tel Hadya, Syria, and the F₁s were grown in the off season at Terbol, Lebanon. The F₂, F₄ and F₅ generations were grown in a blight nursery in the main season where blight epidemic was artificially created. The plants and progenies were scored for blight resistance and other traits. The F₃ and F₆ generations were grown in the off season under normal day length to eliminate late-maturing plants. The pedigree method of breeding was followed initially, but was later replaced by the F₄-derived family method. The yield assessment began with F₇ lines, first at ICARDA sites and later internationally. A total of 1584 ascochyta blight-resistant chickpea lines were developed with a range of maturity, plant height, and seed size not previously available to growers in the blight-endemic areas in the Mediterranean region. These included 92 lines resistant to six races of the ascochyta pathogen, and 15 large-seeded and 28 early maturity lines. New cultivars produced 33% more seed yield than the original resistant sources. The yield of chickpea declined by 340 kg ha^-1, with an increase in blight severity by one class on a 1–9 scale, reaching zero yield with the 8 and 9 classes. Development of blight-resistant lines made the introduction of winter sowing possible in the Mediterranean region with the prospect of doubling chickpea production. Twenty three cultivars have been released so far in 11 countries.Joint contribution from ICARDA and ICRISAT. ICRISAT Journal Article no. JA 1886.     

Low-Temperature Stress: Implications for Chickpea (Cicer arietinum L.) Improvement

Chickpea is the third major cool season grain legume crop in the world after dry bean and field pea. Chilling and freezing range temperatures in many of its production regions adversely affect chickpea production. This review provides a comprehensive account of the current information regarding the tolerance of chickpea to freezing and chilling range temperatures. The effect of freezing and chilling at the major phenological stages of chickpea growth are discussed, and its ability for acclimation and winter hardiness is reviewed. Response mechanisms to chilling and freezing are considered at the molecular, cellular, whole plant, and canopy levels. The genetics of tolerance to freezing in chickpea are outlined. Sources of resistance to both freezing and chilling from within the cultivated and wild Cicer genepools are compared and novel breeding technologies for the improvement of tolerance in chickpea are suggested. We also suggest future research be directed toward understanding the mechanisms involved in cold tolerance of chickpea at the physiological, biochemical, and molecular level. Further screening of both the cultivated and wild Cicer species is required in order to identify superior sources of tolerance, especially to chilling at the reproductive stages.     

Chickpea Ascochyta Blight: Disease Status and Pathogen Mating Type Distribution in Syria

Chickpea fields were surveyed in nine major chickpea‐growing provinces of Syria in 2008 and 2009 to determine the prevalence and severity of Ascochyta blight, and the distribution of Didymella rabiei mating types (MATs) in the country. A total of 133 Ascochyta rabiei isolates were assayed for mating type, including isolates from older collections that date back to 1982. Multiplex MAT‐specific PCR with three primers was used for MAT analysis. Out of the 133 tested isolates, 64% were MAT1‐1 and 36% were MAT1‐2. Both MATs were found in six provinces but MAT1‐1 alone was found in three provinces. Chi‐squared analysis was used to test for a 1 : 1 ratio of MAT frequencies in all samples. The MAT ratios in the six provinces were not significantly different from 1 : 1, suggesting that there is random mating of the pathogen population under natural conditions. The presence of the two MATs is expected to play a role in the evolution of novel virulence genes that could threaten currently resistant chickpea varieties. Overall analysis of the 133 isolates showed a significant deviation from the 1 : 1 ratio with almost twice as many MAT1‐1 isolates than MAT1‐2 isolates, which indicates a competitive advantage associated with MAT1‐1 in Syria. However, the overall picture of an unequal frequency in MATs indicates that there may be limited sexual recombination occurring in the Syrian population.     

First Report of Botrytis cinerea Pers. on Salicornia bigelovii Torr. in North‐West México

Grey mould has been detected on Salicornia bigelovii Torr plants in nursery and in the wild in north‐west Mexico. Sampling of the grey mould was performed in the state of Sonora, Mexico, of wild as well as cultivated S. bigelovii plants. The samples were isolated, and based on morphology, the species was identified as Botrytis cinerea Pers. Koch's postulates were fulfilled by pathogenicity tests carried out in plated petri dishes on branches from the 3‐month‐old potted S. bigelovii plants. To our knowledge, this is the first report of the isolation and identification of the fungal pathogen B. cinerea from S. bigelovii in the north‐west Mexico.