Name: dry root rot disease assays in chickpea: a detailed methodology
Uploaded: 2021-01-17T13:00:00
Description: Watch this Scientific Journal Video about Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology at JoVE.com

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.

1. Isolation of R. bataticola and storage
<ol>
	<li>Details of the chickpea genotype and DRR symptoms
	<ol>
		<li>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 pith1,3.</li>
	</ol>
	</li>
	<li>Collection and washing
	<ol>
		<li>Uproot plants showing symptoms such as dry straw-colored foliar and brittle primary roo...

<ol>
	<li>Sharma, M., Ghosh, R., Pande, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+root+rot+(Rhizoctonia+bataticola+(Taub.)+Butler):+an+emerging+disease+of+chickpea+-+where+do+we+stand.">Dry root rot (Rhizoctonia bataticola (Taub.) Butler): an emerging disease of chickpea - where do we stand.</a> Archives of Phytopathology and Plant Protection. 48 (13-16), 797-812 (2015).</li><li>Sinha, R., Irulappan, V., Mohan-Raju, B., Suganthi, A., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+drought+stress+on+simultaneously+occurring+pathogen+infection+in+field-grown+chickpea.">Impact of drought stress on simultaneously occurring pathogen infection in field-grown chickpea.</a> Scientific Reports. 9 (1), (2019).</li><li>Nene, Y., Haware, M., Reddy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chickpea+diseases:+resistance+screening+techniques,+information+bulletins+No.+10.+Patancheru.">Chickpea diseases: resistance screening techniques, information bulletins No. 10. Patancheru.</a> Information Bulletin No. 10. Patancheru, A.P., India: International CroDS Research Institute for the Semi-Arid Tropics. , 1-10 (1981).</li><li>Pande, S., Krishna Kishore, G., Upadhyaya, H. D., Narayana Rao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+sources+of+multiple+disease+resistance+in+mini-core+collection+of+chickpea.">Identification of sources of multiple disease resistance in mini-core collection of chickpea.</a> Plant Disease. , (2006).</li><li>Pandey, P., Irulappan, V., Bagavathiannan, M. V., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+combined+abiotic+and+biotic+stresses+on+plant+growth+and+avenues+for+crop+improvement+by+exploiting+physio-morphological+traits.">Impact of combined abiotic and biotic stresses on plant growth and avenues for crop improvement by exploiting physio-morphological traits.</a> Frontiers in Plant Science. 8, (2017).</li><li>Irulappan, V., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morpho-physiological+traits+and+molecular+intricacies+associated+with+tolerance+to+combined+drought+and+pathogen+stress+in+plants.">Morpho-physiological traits and molecular intricacies associated with tolerance to combined drought and pathogen stress in plants.</a> Biotechnologies of Crop Improvement, Volume 3: Genomic Approaches. , (2018).</li><li>Jensen, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+methods+for+fungal+brood+disease+research.">Standard methods for fungal brood disease research.</a> Journal of Apicultural Research. 52 (1), 1-39 (2013).</li><li>Agrios, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology: Fifth Edition. , 9780080473 (2004).</li><li>Coley-Smith, J. R., Cooke, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+germination+of+fungal+sclerotia.">Survival and germination of fungal sclerotia.</a> Annual Review of Phytopathology. , (1971).</li><li>Nagamma, G., Saifulla, M., Sab, J., Pavitra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Screening of chickpea genotypes against dry root rot caused by Macrophomina phaseolina (tassi) goid. 10 (4), 1795-1800 (2015).</li><li>Infantino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+techniques+and+sources+of+resistance+to+root+diseases+in+cool+season+food+legumes.">Screening techniques and sources of resistance to root diseases in cool season food legumes.</a> Euphytica. 147 (1-2), 201-221 (2006).</li><li>Khaliq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+control+of+dry+root+rot+of+chickpea+caused+by+Rhizoctonia+bataticola+under+the+natural+field+condition.">Integrated control of dry root rot of chickpea caused by Rhizoctonia bataticola under the natural field condition.</a> Biotechnology Reports. 25, 00423 (2020).</li></ol>

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...

Dry root rot (DRR) is one of the economically significant diseases in chickpea1,2. It is a root-specific disease caused by Rhizoctonia bataticola (teleomorph, Macrophomina phaseolina). Infected plants lack lateral roots and possess brittle taproots and yellow foliage1,3. DRR under drought stress has been reported to be an emerging threat to chickpea cultivation1,2,3. Moreover, DRR incidence is reported to be aggravated under drought stress under field conditions1,2,3. DRR is more prevalent in rainfed areas than in irrigated fields4. The utilization of resistant varieties is the way to overcome the disease and circumvent fungicide use1,13. Because chickpea germplasm available across the globe harbors genetic variation for the trait5, the screening and identification of resistant/susceptible genotypes are critical for molecular breeding for crop improvement.
Robust, easy, and cost-effective disease assays are essential to investigate R. bataticola infection patterns in chickpea. The primary disease assay used to observe the response of chickpea genotypes to R. bataticola infection is the blotting paper technique1,4. It is a simple technique and can be executed using liquid fungal inoculum, seedlings with roots, and sterile blotting paper. However, this technique has not been utilized to its maximum because no step-by-step-protocol is available in the literature.
Meanwhile, the sick pot technique involves the preparation of a potential sick culture and the imposition of drought stress. Given that drought stress aggravates DRR disease incidence3, it is essential to study the plant-pathogen interaction under drought stress6,7. The sick pot technique provides the platform for such a simultaneous study, promoting better possibilities for germplasm screening and understanding the mechanistic basis of the interaction. Pathomorphological changes such as an increase in root length and reduction in lateral root number&#8212;inherent to DRR disease&#8212;can be addressed using the sick pot technique1,3,7.
Herein, a detailed protocol for blotting paper and sick pot techniques, which can be used to study the interaction between chickpea and R. bataticola and screen chickpea germplasm, is presented. The details of the materials used in the study are given in the&#160;Table of Materials.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fungus- Rhizoctonia bataticola</td>
			<td>Pathogen inoculum</td>
			<td>Indian Type Culture Collection No. 8365</td>
			<td>GenBank: MH509971.1, ITCC 8635 (https://www.iari.res.in/index.php?option=com_content&#38;view=article&#38; id=1251&#38;Itemid=1370)</td>
		</tr>
		<tr>
			<td>Soilrite mix</td>
			<td>Soil medium in the lab</td>
			<td>Keltech Energies Limited, Bangalore, India</td>
			<td>http://www.keltechenergies.com/</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Blotting paper to support the plant growth</td>
			<td>Himedia</td>
			<td>http://himedialabs.com/catalogue/chemical2017/index.html#374</td>
		</tr>
		<tr>
			<td>Pot</td>
			<td>Growing plants</td>
			<td>10 and 30 cm size pots</td>
			<td>Routinely used nursery pots, for example, https://dir.indiamart.com/impcat/nursery-pots.html</td>
		</tr>
		<tr>
			<td>Potato dextrose agar/broth</td>
			<td>Culture and maintain the fungus</td>
			<td>Cat# 213400, DifcoTM, MD, USA</td>
			<td>https://www.fishersci.com/shop/products/bd-difco-dehydrated-culture-media-potato-dextrose-agar-3/p-4901946</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Culture the fungus</td>
			<td>LOM-150-2, S/N AI13082601-38, MRC, incubator, and shaker</td>
			<td>http://www.mrclab.com/productDetails.aspx?pid=91131</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Growing plants in controlled condition</td>
			<td>Model No. A1000, Conviron, Canada</td>
			<td>https://www.conviron.com/products/gen1000-reach-in-plant-growth-chamber</td>
		</tr>
		<tr>
			<td>Laminar airflow</td>
			<td>Carrying out aseptic exercises</td>
			<td>Telstar, Bio II advance, Class II cabinet, EN-12469-2000</td>
			<td>https://www.telstar.com/lab-hospitals-equipment/biological-safety-cabinets/bio-ii-advance-plus/, http://www.atlantisindia.co.in/laminar-air-flow.html</td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>Filtering the fungal mycelia</td>
			<td>Nylon mosquito net</td>
			<td>Mesh with 0.6-1 mm diameter pore size</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Autoclaving media and chickpea seeds</td>
			<td>Autoclave</td>
			<td>http://www.scientificsystems.in/autoclave</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td>Visualizing the infection ang fungal mycelia</td>
			<td>SMZ25 / SMZ18, Research Stereomicroscopes, Leica EZ4 educational stereomicroscope</td>
			<td>https://www.microscope.healthcare.nikon.com/products/stereomicroscopes-macroscopes/smz25-smz18 
			https://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/p/leica-ez4/ 
			https://www.microscopyu.com/museum/eclipse-80i</td>
		</tr>
		<tr>
			<td>Weighing balance</td>
			<td>Weighing fungus and chemicals</td>
			<td>Sartorius Electronic Weighing Balance, BSA 4202S-CW</td>
			<td>https://www.sartorius.com/en/products/weighing/laboratory-balances</td>
		</tr>
		<tr>
			<td>WGA-FITC</td>
			<td>Fungus staining</td>
			<td>Sigma</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/l4895?lang=en&#38;region=IN</td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>Fungus staining</td>
			<td>Himedia</td>
			<td>http://www.himedialabs.com/intl/en/products/Chemicals/Dyes-Indicators-and-Stains/Aniline-blue-Water-soluble-Practical-grade-GRM901</td>
		</tr>
	</tbody>
</table>

dry root rot disease assays in chickpea: a detailed methodology

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&#8212;plant, pathogen, and environment&#8212;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.

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 symptoms1,3,4 were collected from a chickpea field, and the fungus was isolated using the hyphal tip method8. R. bataticola fungal culture appears dark gr...

Watch this Scientific Journal Video about Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology at JoVE.com

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;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, ...

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