Name: dry root rot disease assays in chickpea: a detailed methodology
Uploaded: 2021-01-17T13:00:00.000+00:00
Duration: 6 min 58 s
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 root with microsclerotia under the epidermis layer. While uprooting, a major part of the roots will remain inside the soil as the infected roots are brittle. Remove the coarse soil debris attached to the root. Cut the roots separately and collect them in paper envelopes (27.5 cm x 12 cm) with the proper label and place them in a sample collection box.</li>
		<li>After transporting samples to the laboratory, place the roots in a 200 mL beaker and cover with mesh (Nylon mesh with the pore size of 3 mm diameter), and wash the roots thoroughly with running tap water to remove adhering soil particles.</li>
		<li>Use reverse osmosis (RO) water to rinse once at the end.</li>
	</ol>
	</li>
	<li>Surface sterilization
	<ol>
		<li>Break the roots into four pieces with 2 cm length each with a scalpel blade and put them in a clean 200 mL beaker.</li>
		<li>Assemble autoclaved RO water, 2% NaOCl, discarding jar, and autoclaved blotting paper (5 cm2) inside the laminar flow chamber.</li>
		<li>Wash the roots with 50 mL of autoclaved RO water thrice and then with 50 mL of 2% NaOCl for 10 min. Wash the roots with 50 mL of RO water three times again to remove the NaOCl.</li>
		<li>Blot dry the roots by placing the roots on autoclaved blotting paper and leave until the roots are dried.</li>
	</ol>
	</li>
	<li>Media and incubation
	<ol>
		<li>Remove the edges of the roots with a sterilized scalpel blade. Split the roots using the blade and use the sterilized forceps to place them on a potato dextrose agar (PDA) media Petri plate containing streptomycin sulfate (50 mg L-1) and ampicillin (50 mg L-1).</li>
		<li>Close the plate, seal with parafilm, and incubate in an incubator at 28 &#176;C for two days in the dark.</li>
	</ol>
	</li>
	<li>Hyphal tip method
	<ol>
		<li>After two days of incubation, bring the plates into the laminar flow chamber. Cut the tip of the hyphal growth8 under a stereomicroscope (Leica EZ4 educational stereomicroscope) and transfer it into a fresh PDA plate with streptomycin sulfate and ampicillin.</li>
		<li>Incubate the plates in the incubator at 28 &#176;C for ten days in the dark.</li>
	</ol>
	</li>
	<li>Storage and maintenance of R. bataticola fungal inoculum
	<ol>
		<li>Make PDA slants in test tubes and transfer a fungal agar plug from a ten-days-old culture plate using the flame sterilized inoculation loop. Seal the test tube cap with parafilm.</li>
		<li>Incubate the slants at 28 &#176;C for ten days in the dark.</li>
		<li>Seal the cap with parafilm again and store it at 4 &#176;C in the refrigerator for the future use.</li>
		<li>Subculture every six months to maintain the fungus.</li>
	</ol>
	</li>
	<li>Maintenance of virulence
	<ol>
		<li>Infect the plants with pure fungal inoculum prepared as mentioned in the sick pot technique3. Isolate the same fungus from the infected plants (Koch&#8217;s postulates)9 and use for further experiments. 
		NOTE: In this study, a field isolated strain of the fungus was used (GenBank: MH509971.1 https://www.ncbi.nlm.nih.gov/nuccore/MH509971)</li>
	</ol>
	</li>
</ol>
2. Blotting paper technique
NOTE: The blotting paper technique entails the preparation of liquid fungal inoculum, seedling preparation, and disease assessment.
<ol>
	<li>Preparation of liquid R. bataticola inoculum 
	NOTE: Liquid R. bataticola fungal inoculum contains both mycelia and microsclerotia. Both these structures act as primary inoculum.
	<ol>
		<li>Prepare 500 mL of PDB media in a 1,000 mL flask and autoclave it at 121 lbs for 15 min.</li>
		<li>After the media is cooled down, inoculate the broth with a loopful of fungal agar plug from a fungal slant culture and incubate at 28 &#176;C for five days in a shaker at 180 rpm in the dark.</li>
		<li>Assemble an autoclaved glass or plastic funnel (10 cm), one-liter conical flask, and two layers of mesh (10 cm2 size) (Nylon mesh with the pore size of 3 mm diameter) on the table. Filter out the fungal mycelia and microsclerotia using the mesh. Blot dry the fungus in autoclaved blotting paper.</li>
		<li>Weigh 100 g mycelia for 50% inoculum and keep at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of chickpea plant
	<ol>
		<li>Select 50 healthy seeds (in this study, the DRR-susceptible genotype JG 62 was used), place them in a 100 mL beaker, and cover with a mesh.</li>
		<li>Wash the seeds with tap water first to remove soil debris.</li>
		<li>Take the beaker with seeds into the laminar flow chamber and wash them with sterilized 50 mL of RO water thrice for 1 min per wash.</li>
		<li>Sterilize the seeds with 50 mL of 2% aqueous NaOCl for 2 min with continuous shaking and wash the seeds with 50 mL of sterilized water five times for 1 min each.</li>
		<li>Fill a polythene bag (47.5 cm x 25 cm) with Soilrite (a mixture of horticulture grade expanded perlite, Irish Peat moss, and exfoliated vermiculite in equal ratio i.e., 1/3:1/3:1/3), sow the surface-sterilized 50 seeds two cm deep, and keep in a growth chamber/room with 28 &#176;C &#177; 2 &#176;C temperature, 16 h photoperiod with a light intensity of 150 &#956;mol m&#8722;2 s&#8722;1, and relative humidity of 70%. Water them with RO water and uproot the plants eight days after sowing.</li>
		<li>Wash the roots in tap water to remove the Soilrite particles. Rinse the roots with sterilized RO water, and keep them in the RO water in a 1000 mL beaker.</li>
	</ol>
	</li>
	<li>Preparation of blotting paper in trays
	<ol>
		<li>Take a blotting paper and cut them into pieces (30 cm &#215; 23 cm) in sufficient numbers to meet the replications.</li>
		<li>Pack the sheets in autoclavable polythene bag&#160;and autoclave them at 121 lbs for 15 minutes and keep it in a hot air oven for drying.</li>
		<li>Fold each blotting paper sheet in half.</li>
		<li>Place the paper on a clean, spirit-wiped plastic tray, as shown in the Figure 2i-iv.</li>
	</ol>
	</li>
	<li>Plant inoculation by dipping
	<ol>
		<li>Dissolve 100 g of fungal inoculum in 200 mL autoclaved RO water in a 200 mL beaker to obtain 50% inoculum.</li>
		<li>Dip the plant roots in the prepared inoculum for 1 min with intermittent up-and-down movement to ensure uniform attachment of fungal inoculum.</li>
	</ol>
	</li>
	<li>Placing the plants in the blotting paper
	<ol>
		<li>Wet the bottom side of the blotting paper placed on the tray with sterile RO water. Place the plants on the paper in a way where only the roots are covered by the paper, and shoots are left out.</li>
		<li>Close it by folding the top side of the blotting paper and wet the entire paper to provide enough water to sustain plant growth.</li>
		<li>Water the tray once a day and keep the trays at 28 &#176;C. Observe the symptoms such as necrosis, root rot, and leaf yellowing daily.</li>
	</ol>
	</li>
</ol>
3. Sick pot technique
NOTE: The sick pot technique entails the preparation of virulent inoculum and a sick pot, maintenance of moisture level, and assessment of disease symptoms.
<ol>
	<li>Preparation of substrate
	<ol>
		<li>Take one kg of any commercially available chickpea seeds. Remove infected seeds (seeds with fungal growth, infection spots, and insect damage). Place them in 5 L plastic beaker and wash with tap water thoroughly 3&#8211;4 times to remove major debris.</li>
		<li>Wash the seeds with 2 L of RO water thrice and soak the 1 kg seeds in a 5 L beaker with threefold more water for five h.</li>
		<li>Once the seeds imbibed the water, rewash them with RO water thrice to remove the seed exudates.</li>
		<li>Remove the water completely and pack the seeds in jam bottles (300 mL, 12 cm height, 6 cm diameter, and 155 g weight) to about 1/4th capacity (100 g per jam bottle) and close the bottles with caps. Pack ten jam bottles in an autoclavable plastic bag (48 cm x 30 cm) autoclave twice at 121 lbs for 15 min continuously.</li>
		<li>Dry the seeds at 40 &#176;C in an oven overnight to remove the water droplets inside the bottle.</li>
	</ol>
	</li>
	<li>Preparation of sick culture
	<ol>
		<li>Turn on the BSL-2 level laminar flow chamber. Wipe the floor thoroughly with 70% ethanol, and turn on UV for 15 min. Then, keep the seeds inside the laminar flow chamber.</li>
		<li>Take 10-day-old freshly isolated R. bataticola culture (part 1). Then, take three fungal agar plugs (4 mm diameter) using a sterile pipette tip or cork borer, and put them into jam bottles aseptically. Cover the bottles with caps and seal with parafilm.</li>
		<li>Shake the bottles to mix the fungal disc with the chickpea seeds uniformly.</li>
		<li>Incubate the bottles at 30 &#176;C for 15 days in the dark.</li>
		<li>Take jam bottles with black fungal growth (Figure 4iii) and transfer the sick culture (seeds with microsclerotia) from jam bottles to a glass Petri plate using sterile forceps. Dry them at room temperature for two days in a glass Petri plate.</li>
		<li>Powder the fungal mass using a mortar and pestle, and store the powder at 4 &#176;C.</li>
		<li>Autoclave Soilrite twice at 121 lbs for 15 min.</li>
		<li>Dry the autoclaved Soilrite mix under a sunshade.</li>
		<li>Mix the fungal powder with Soilrite at 50% w/w, fill the pots with the mixture, and keep them at room temperature for a day.</li>
		<li>Sow a surface-sterilized DRR-susceptible chickpea seed per pot (10 cm round pots) (Table of Materials) and maintain a moisture level of 80% field capacity (FC).</li>
		<li>Observe the symptoms such as necrosis and root rot by uprooting the plants after germination.</li>
	</ol>
	</li>
	<li>Assessment of sick pot efficiency 
	NOTE: Plants show yellow foliar symptoms when the roots are completely rotten.
	<ol>
		<li>Develop a disease score based on the lesion (necrotic spots) (Supplementary Figure 2C) numbers and severity of root rot. Sick pots showing 90% plant death can be used further.</li>
	</ol>
	</li>
	<li>Genotype screening
	<ol>
		<li>Prepare sick culture in large quantities and mix it either with sterilized Soilrite or field soil (5% w/w) in 30 cm-tall pots. Water the pots to wet the surface and leave them undisturbed for seven days.</li>
		<li>Sow one surface-sterilized seed per 10 cm round pot and three seeds per 30 cm round pots and water them adequately.</li>
		<li>Observe the yellow foliar and root rot symptoms.</li>
	</ol>
	</li>
	<li>Combined drought and R. bataticola infection
	<ol>
		<li>Impose drought stress by following the protocols mentioned in Sinha et al. (2019).</li>
	</ol>
	</li>
</ol>

<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>

We have nothing to disclose

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><tbody><tr><td>Fungus- &lt;em&gt;Rhizoctonia bataticola&lt;/em&gt;</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&amp;amp;view=article&amp;amp;&lt;br/&gt;id=1251&amp;amp;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&lt;br/&gt;&lt;br/&gt; https://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/p/leica-ez4/&lt;br/&gt;&lt;br/&gt; 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&amp;amp;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, ...

dry-root-rot-disease-assays-in-chickpea-a-detailed-methodology

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7.1K Views.  National Institute of Plant Genome Research. 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.

Video: Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Immunology and Infection

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The power of their influence is in extraradical mycelia that spread beyond nutrient depletion zones found near roots to ultimately interconnect individuals within a common mycorrhizal network (CMN). Most experiments, however, have investigated the role of AM fungi in plant interactions by growing plants with versus without mycorrhizal fungi, a method that fails to explicitly address the role of CMNs. Here, we propose a method that manipulates CMNs to investigate their role in plant interactions. Our method uses modified containers with conical bottoms with a nylon mesh and/or hydrophobic material covering slotted openings, 15N fertilizer, and a nutrient-poor interstitial sand. CMNs are left either intact between interacting individuals, severed by rotation of containers, or prevented from forming by a solid barrier. Our findings suggest that rotating containers is sufficient to disrupt CMNs and prevent their effects on plant interactions across CMNs. Our approach is advantageous because it mimics aspects of nature, such as seedlings tapping into already established CMNs and the use of a suite of AM fungi that may provide diverse benefits. Although our experiment is limited to investigating plants at the seedling stage, plant interactions across CMNs can be detected using our approach which therefore can be applied to investigate biological questions about the functioning of CMNs in ecosystems.

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The ...

Environment

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the southeastern USA, especially in Florida. Deployment of integrated pest management strategies, such as race-specific resistant cultivars, requires information on the diversity and population density of the pathogen in growers' fields. Despite some progress in developing molecular diagnostic tools to identify pathogen isolates, race determination often requires bioassay approaches.
Race typing was conducted by root-dip inoculation, infested kernel seeding method, and the modified tray-dip method with each of the four watermelon differentials (Black Diamond, Charleston Grey, Calhoun Grey, Plant Introduction 296341-FR). Isolates are assigned a race designation by calculation of disease incidence five weeks after inoculation. If less than 33% of the plants for a particular cultivar were symptomatic, they were categorized as resistant. Those cultivars with incidence greater than 33% were regarded as susceptible. This paper describes three different methods of inoculation to ascertain race, root-dip, infested kernel, and modified tray-dip inoculation, whose applications vary according to the experimental design.

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

Managing Fusarium wilt of watermelon requires knowledge of the pathogen races present. Here, we describe the root-dip, infested kernel seeding, and modified tray-dip inoculation methods to demonstrate their efficacy in race-typing of the pathogenic fungus Fusarium oxysporum f. sp niveum (Fon).

Fusarium wilt of watermelon (Citrullus lanatus), caused by Fusarium oxysporum f. sp. niveum (Fon), has reemerged as a major production constraint in the ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop fitness and productivity. Research on the cellular and subcellular mechanisms of PPNs infection and development can resort to field plants or seedlings under greenhouse conditions. Field studies are more representative of natural environments but are subjected to the unpredictability of environmental conditions that can heavily influence research outcomes. Greenhouse studies allow higher control over environmental variables and higher safety against contaminants or pathogens. However, in some hosts, genetic diversity becomes an important factor of variability and influences the host-parasite complex response. We have developed in vitro co-cultures of transgenic roots with PPNs as a reliable alternative that occupies less space, requires less time to obtain, and is free from contamination or from host genetic variability. Co-cultures are obtained by introducing aseptic PPNs to host in vitro transgenic roots. They can be maintained indefinitely, which makes them excellent support for keeping collections of reference PPNs. In the present work, a protocol is detailed for the controlled infection of in vivo potato roots with the root lesion nematode and for establishing in vitro co-cultures of potato transgenic roots with the root-knot nematode. The in vitro co-cultures provided a laboratory proxy for the natural potato infection condition and produced nematode life stages irrespective of season or climate conditions. Additionally, the methodology used for structural analysis is detailed using histochemistry and optical microscopy. The acid fuchsin dye is used to follow nematode attack sites on roots, while differential staining with Periodic acid-Schiff (PAS) and toluidine blue O highlights nematode structures in potato internal root tissue.

The protocol describes infection of Solanum tuberosum roots with plant parasitic nematodes under in vivo greenhouse conditions and potato in vitro transgenic roots for histochemical analysis of root structure through optical microscopy.

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Summary

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Chapters in this video

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

A Contrast of Three Inoculation Techniques used to Determine the Race of Unknown Fusarium oxysporum f.sp. niveum Isolates

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes