Iranian Journal Pulses Research

Abstract Introduction
Chickpea (Cicer arietinum L.) is considered as one of the rich sources of plant protein in human diet and an important factor in soil fertility which has of special position in the crop rotation programs. In Iran, chickpea is grown in spring and autumn. Ascochyta blight is currently considered as the most important limiting factor of winter cultivation of chickpea in Iran. Identifying sources of resistance to Ascochyta blight by performing screening tests using artificial inoculation is one of the common methods in plant breeding. The cost and risks of spreading the diversity of pathogenic fungi due to the use of infected plots are among the limitations of alternative studies. Providing a reliable in vitro screening method besides speeding up the breeding programs can overcome many of these limitations.
 
Materials and Methods
Two chickpea seed samples were selected in the presented study which were including; MCC133 and ILC1929, respectively, as resistant and susceptible lines to Ascochyta blight. The seed samples were provided by the seed bank of the Research Center for Plant Sciences of Ferdowsi University of Mashhad, Iran. The fungal isolates called PI (Pathotype No. 1), related to Ascochyta rabiei with strain number of FUM 1001 was obtained from the Microorganisms Collection of Ferdowsi University of Mashhad (WDCM 1207), Iran. The germinated chickpea seeds were planted in pots containing equal proportions of leached sterilized coco peat and perlite. The pots were kept in a growth chamber at 24±2 °C and 16:8 h light/dark photoperiod and fertilized weekly by ZISTA nutritional plant solution up to 5 leaves. Three leaves were detached from each seedling and placed under sterile conditions in test tubes. The durability and growth potential of surface and endogenous infections on chickpea leaves detached from the seedlings grown in greenhouse condition were investigated after surface disinfection and culture under in vitro condition without nutrient medium. Moreover, the severity and rate of disease symptoms development as a result of applying two inoculation methods were compared on detached leaves separated from susceptible cultivar. In addition, the possibility of differentiation of resistant and susceptible genotypes was investigated in response to a virulent pathotype. In the final stage, the possibility of differentiation of different fungal pathotypes was evaluated based on pathogenicity on resistant cultivar.
 
Results and Discussion
Our daily visual observation showed that, no signs of fungal growth or symptoms of infection related to saprophytes were observed on detached leaves inside the test tube until the tenth day. The leaves were completely fresh until the tenth day and there were no symptoms of wilting on them. On the tenth day, a number of leaf samples had slightly reduced their greenness, but were perfectly healthy. The results showed that using this method the detached leaves can be stored safely in the test tube for at least 10 days. In addition, the method of preparing the leaves has prevented any surface infection. The results of pathogenicity test showed a clear difference between the disease symptoms in the two inoculation methods. Symptoms of the disease were visible on the leaves of susceptible cultivars in the immersion method from the third day in the form of very small spots resulting from the destruction of the cuticular layer on the leaf surface. These symptoms were strengthened on the fifth day. Disease development was increased rapidly in the whole leaf and the wounds were quite visible on the eighth day. In the drip method, no visible symptoms were visible until the eighth day, and on the tenth day, the wounds were observed at the site of inoculation. The results indicate that the immersion method induces quicker symptom development on leaflets, with a more rapid and widespread effect across the entire leaf. Additionally, owing to the limited survival period of detached leaves, this method facilitates faster symptom manifestation within a relatively shorter timeframe. Thus, inoculation by immersion method has a higher efficiency in the occurrence of Ascochyta blight disease symptoms. The disease symptoms observed on the leaves are due to the activity of a wide range of compounds produced by the fungus, including plant tissue degrading enzymes, toxins and fungal metabolites that are toxic to the plant. Investigation on gene expression pattern of A. rabiei in the early hours of plant infection has shown an increase in the expression of different gene families which can cause symptoms on the plant by destroying the cuticle of the leaf surface and cell wall in the early stages of infection.
 
Conclusions
Taken together, these results showed that the invented method of testing the pathogenicity inside the test tube is well able to distinguish resistant and sensitive cultivars from each other. Accordingly, this method can be used in selection studies and evaluation of resistance of chickpea samples against infection with A. rabiei isolates.
 

Abstract Introduction
Chickpea (Cicer arietinum L.) is an especial important crop among Pulse crops. One of the most important factors limiting the yield of chickpea is fungal diseases. Ascochyta blight is the most problematic disease of chickpea. The fungus Ascochyta rabiei (also called Didymella rabiei) causes ascochyta blight of chickpea. Pathogenesis related (PR) proteins are one of the ways to control plant pathogens. These proteins are encoded by the host plant, but their express done only in the presence of disease agent or the related conditions (Ghannadha et al., 2006). PR-proteins are a variety group of proteins including: glucanase, chitinases, endoproteinase, peroxidases, protease inhibitors, as well as small proteins such as osmotins, thionins, defensins, and lipid transfer proteins (Jain et al., 2012). So far seventeen PR proteins have been identified (Liu et al., 2006). PR-2 is the only PR-protein that identified in chickpea. The PR-10 has many functions. Direct evidence of antimicrobial activity induced by PR-10 is known in microbiological labs. The ability to connect to ligands is also observed. It also has an enzymatic activity in the secondary metabolism of plants and plays a role in abiotic stresses (Liu et al., 2006). This research was done to detect pr-10 gene in chickpea genome.
 
Materials & Methods
In this experiment, six genotypes of chickpea, including two sensitive genotypes (MCC506 and MCC507), two resistant genotypes (MCC142 and MCC528) and two tolerant genotypes (MCC20 and MCC150), against ascochyta blight were used. The seeds were surface sterilized using 2% sodium hypochlorite and germinated in sterile petri dishes. The germinated seeds were planted in pots (with 20 cm in diameter) containing combination of perlite, coco peat and vermicompost with a ratio of 1: 2: 1 at a depth of 2.5 cm. After one month, DNA extraction was down from leaves using CTAB method. RCR reaction was down using primers of PSH-91 and MN in 2 steps. These primers were designed using Primer software Ver.5. The first step was performed using PSH-91 primer on genomic DNA and second step using MN primer on first PCR product. The detected gene was extracted using a Gel extraction kit manufactured by Denazist Company. Then PR-10 gene constructs were sequenced. Sequencing results were analyzed using SeqMan software.
 
Results & Discussion
Germination of plants was almost simultaneously. The shoot production of resistant and tolerant plants was more than sensitive cultivars. Resistant and tolerant varieties reached to flowering stage earlier and produced more abundant seed rather than sensitive cultivars. The presence of a single-band 1350 bp in the first PCR and a single-band 1289 bp in the second PCR indicated to presence of the PR-10 gene in the chickpea genome. The results of this research indicated that there is no difference in sequence of coding gene of PR-10 protein in chickpea genome with different resistance levels.
 
 
 
Conclusion
Due to the lack of diversity in the nucleotide sequence of pr-10 gene among resistant and sensitive cultivars, it is likely that when the protein is expressed in the presence of the disease agent, the expression difference between the different varieties is shown. This seems reasonable considering research on the pr-10 rice genes (Babaiezadeh & Sayyari, 2012). The PR-10 gene in rice is naturally not expressed in leaves and does not respond to ulcers, but it has high expression potential in the presence of ethylene and jasmine acids (Heidarinejad et al., 2014). Also, in evaluating the resistant and susceptible cultivar of rice blight pods disease, PR-10 gene expression in resistant cultivars was significantly increased compared with susceptible cultivars (Babaiezadeh and Sayyari, 2012). It seems that additional experiments are necessary in disease condition on expression of pr-10 gene in chickpea.

Abstract Introduction Cold and freezing stress is one of the most important environmental stresses that affect the growth and yield of crops. One of the important processes in plant resistance to cold stress is known to acclimatize. In this process, the internal mechanisms of plants causes higher resistance to freezing stress after exposure to temperatures close to zero. Low temperature stress induce or inhibit the expression of certain genes. P5CS protein and transcription factor DREB (CBF) play an important role in adaptation and cold tolerance in plants. P5CS activates the enzymes in proline synthesis pathway and cause accumulation of this substance in plant that protects membranes and proteins against different effects of high concentrations of inorganic ions and high and low temperatures. Also DREB proteins with a conserved sequence bind to specific DNA sequences upstream of responsible genes in resistance to cold stress and leads to their expression. In this study the expression of P5CS and (DREB1)CBF genes that have an important role in the face of many plants to freezing and cold stress were examined in chickpea in both resistant and susceptible genotypes and used of Real time PCR method to evaluate the expression of these genes under stress and non-stress. Materials & Methods In the first experiment, the expression pattern of P5CS and CBF (DREB1) genes were evaluated at normal (23°C), acclimation (10°C) and freezing (-10°C)temperatures in two resistant (MCC426) and susceptible (MCC505) genotypes of chickpea, in two organs (leaf and stem). Briefly pots containing plants of both genotypes stored at 23°C for two weeks. After this period, plants are divided into two parts, half remained in the normal conditions and the other half were transferred to 10°C for cold acclimation. After 10 days, seedlings of normal and acclimation conditions transferred to a thermogradient freezer and after 15 minutes at -10°C, sampling was carried out from the leaves and stems. The simultaneous sampling of the control plants (normal conditions) were also done. After RNA extracting from samples and determine the concentration and quality by nanodrop and treatment with DNase I enzyme, cDNA synthesis was performed. After designing appropriate primers, the PCR process was optimized and Real time PCR was performed under optimized process conditions and gene expression levels were calculated. In the Second experiment free proline changes of seedlings in the acclimation phase were evaluated. Briefly after transferring of plants in the acclimation conditions, amount of proline at them were measured at appropriate intervals after exposure in acclimation conditions. Results & Discussion Results of analysis showed that the expression of these genes were significantly higher in resistant genotype than the susceptible one. Because of these genes are the important factors in plant resistance to stress especially cold and freezing stress, this can confirm that MCC426 genotype is resistant and MCC505 genotype is sensitive to cold. Also the interaction of genotype and organ sample in the control condition showed significant difference only for expression of P5CS gene. Since this gene is an important gene in freezing and cold stress, increased expression of this gene in MCC426 genotype that is resistant to cold not unexpected. But expression of CBF gene occurred only after cold stress induction and in the absence of stress is poor expression. In addition, it was revealed that the interaction of genotype and acclimation treatment on CBF genes in both genotypes had significant differences, but in P5CS gene only in MCC426, this trend was observed. However, changes of free proline as one of the mechanisms of cell protection in acclimation period in both genotypes showed that proline contents of MCC426 in response to acclimation treatment was gradually increased, but this increase in MCC505 genotype occurred during the early stages of acclimation, and proline has decreased over time. Conclusion According to the results, it generally seems, one of the main reason for tolerance to chilling is the high expression levels of the mentioned genes during chilling and freezing stress and therefore it can be used for inducing freezing tolerance into sensitive plants.