Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 under field conditions^1,2,3. 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 use^1,13. 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 technique^1,4. 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 stress^6,7. 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—inherent to DRR disease—can be addressed using the sick pot technique^1,3,7.

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 Table of Materials.

Protokół

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 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.
   2. 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.
   3. Use reverse osmosis (RO) water to rinse once at the end.
3. Surface sterilization
   1. Break the roots into four pieces with 2 cm length each with a scalpel blade and put them in a clean 200 mL beaker.
   2. Assemble autoclaved RO water, 2% NaOCl, discarding jar, and autoclaved blotting paper (5 cm²) inside the laminar flow chamber.
   3. 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.
   4. Blot dry the roots by placing the roots on autoclaved blotting paper and leave until the roots are dried.
4. Media and incubation
   1. 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).
   2. Close the plate, seal with parafilm, and incubate in an incubator at 28 °C for two days in the dark.
5. Hyphal tip method
   1. 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.
   2. Incubate the plates in the incubator at 28 °C for ten days in the dark.
6. Storage and maintenance of R. bataticola fungal inoculum
   1. 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.
   2. Incubate the slants at 28 °C for ten days in the dark.
   3. Seal the cap with parafilm again and store it at 4 °C in the refrigerator for the future use.
   4. Subculture every six months to maintain the fungus.
7. Maintenance of virulence
   1. Infect the plants with pure fungal inoculum prepared as mentioned in the sick pot technique³. Isolate the same fungus from the infected plants (Koch’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)

2. Blotting paper technique

NOTE: The blotting paper technique entails the preparation of liquid fungal inoculum, seedling preparation, and disease assessment.

1. Preparation of liquid R. bataticola inoculum
   NOTE: Liquid R. bataticola fungal inoculum contains both mycelia and microsclerotia. Both these structures act as primary inoculum.
   1. Prepare 500 mL of PDB media in a 1,000 mL flask and autoclave it at 121 lbs for 15 min.
   2. 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 °C for five days in a shaker at 180 rpm in the dark.
   3. 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.
   4. Weigh 100 g mycelia for 50% inoculum and keep at room temperature.
2. Preparation of chickpea plant
   1. 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.
   2. Wash the seeds with tap water first to remove soil debris.
   3. 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.
   4. 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.
   5. 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 °C ± 2 °C temperature, 16 h photoperiod with a light intensity of 150 μmol m⁻² s⁻¹, and relative humidity of 70%. Water them with RO water and uproot the plants eight days after sowing.
   6. 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.
3. Preparation of blotting paper in trays
   1. Take a blotting paper and cut them into pieces (30 cm × 23 cm) in sufficient numbers to meet the replications.
   2. Pack the sheets in autoclavable polythene bag and autoclave them at 121 lbs for 15 minutes and keep it in a hot air oven for drying.
   3. Fold each blotting paper sheet in half.
   4. Place the paper on a clean, spirit-wiped plastic tray, as shown in the Figure 2i-iv.
4. Plant inoculation by dipping
   1. Dissolve 100 g of fungal inoculum in 200 mL autoclaved RO water in a 200 mL beaker to obtain 50% inoculum.
   2. Dip the plant roots in the prepared inoculum for 1 min with intermittent up-and-down movement to ensure uniform attachment of fungal inoculum.
5. Placing the plants in the blotting paper
   1. 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.
   2. Close it by folding the top side of the blotting paper and wet the entire paper to provide enough water to sustain plant growth.
   3. Water the tray once a day and keep the trays at 28 °C. Observe the symptoms such as necrosis, root rot, and leaf yellowing daily.

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.

1. Preparation of substrate
   1. 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 3–4 times to remove major debris.
   2. 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.
   3. Once the seeds imbibed the water, rewash them with RO water thrice to remove the seed exudates.
   4. 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 1/4^th 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.
   5. Dry the seeds at 40 °C in an oven overnight to remove the water droplets inside the bottle.
2. Preparation of sick culture
   1. 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.
   2. 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.
   3. Shake the bottles to mix the fungal disc with the chickpea seeds uniformly.
   4. Incubate the bottles at 30 °C for 15 days in the dark.
   5. 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.
   6. Powder the fungal mass using a mortar and pestle, and store the powder at 4 °C.
   7. Autoclave Soilrite twice at 121 lbs for 15 min.
   8. Dry the autoclaved Soilrite mix under a sunshade.
   9. 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.
   10. 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).
   11. Observe the symptoms such as necrosis and root rot by uprooting the plants after germination.
3. Assessment of sick pot efficiency
   NOTE: Plants show yellow foliar symptoms when the roots are completely rotten.
   1. 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.
4. Genotype screening
   1. 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.
   2. Sow one surface-sterilized seed per 10 cm round pot and three seeds per 30 cm round pots and water them adequately.
   3. Observe the yellow foliar and root rot symptoms.
5. Combined drought and R. bataticola infection
   1. Impose drought stress by following the protocols mentioned in Sinha et al. (2019).

Wyniki

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...

Dyskusje

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...

Materiały

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