Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea–Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components—plant, pathogen, and environment—involved in the disease triangle.

Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

Dry root rot (DRR) is one of the economically significant diseases in chickpea^1,2. It is a root-specific disease caused by Rhizoctonia bataticola (teleomorph, Macrophomina phaseolina). Infected plants lack lateral roots and possess brittle taproots and yellow foliage^1,3. DRR under drought stress has been reported to be an emerging threat to chickpea cultivation^1,2,3. Moreover, DRR incidence is reported to be aggravated under drought stress un....

1. Isolation of R. bataticola and storage

1. Details of the chickpea genotype and DRR symptoms
   1. Use chickpea plants (genotype, JG 62) that generally show typical DRR symptoms, such as dry, brittle primary root with no lateral roots and microsclerotia beneath the bark and inside the pith^1,3.
2. Collection and washing
   1. Uproot plants showing symptoms such as dry straw-colored foliar and brittle primary roo.......

This study aimed to demonstrate techniques such as blotting paper and sick pot techniques to facilitate pathomorphological and molecular understanding of plant-pathogen interaction under drought stress. To accomplish this, plants exhibiting DRR symptoms^1,3,4 were collected from a chickpea field, and the fungus was isolated using the hyphal tip method⁸. R. bataticola fungal culture appears dark gr.......

The blotting paper technique provides a straightforward approach to screen chickpea genotypes under laboratory conditions. Dip inoculation enables the investigation of interaction on a temporal basis with easy control over inoculum load (Supplementary Figure 1) and facilitates in vitro screening. Furthermore, even young seedlings can be used. Five-day-old fungal culture (Figure 1B) can yield enough inoculum to infect the plants. Liquid inoculum contains both mycelia and micr.......

Projects at the M.S.K lab are supported by the National Institute of Plant Genome Research core funding. VI acknowledges DBT- JRF (DBT/2015/NIPGR/430). We thank trainee students, Miss. Rishika, Mr. Jayachendrayan, and Miss. Durgadevi for technical help during video shooting and Mr. Sandeep Dixit, Miss. Anjali and Dr. Avanish Rai for critically assessing raw data and the manuscript files. We thank Mr. Rahim H Tarafdar and Mr. Sunder Solanki for their help in the laboratory. We acknowledge DBT-eLibrary Consortium (DeLCON) and NIPGR Library for providing access to e-resources and NIPGR Plant Growth Facility for plant growth support/space.