Introduction of an in vitro Pathogenicity Test of Ascochyta rabiei on Chickpea Leaves Detached from Resistant and Susceptible Cultivars

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.

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.

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.

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.