The main goal of this study is to demonstrate a detailed methodology of dry root rot disease assays, such as blotting paper technique and sick pot technique. These techniques will be helpful to study the interaction between chickpea plant and Rhizoctonia bataticola. Understanding the chickweed plant interaction with the fungal pathogen Rhizoctonia bataticola is agriculturally important.
For this, versatile and highly reproducible assays are needed. Here, two assays that are widely used in our lab are demonstrated. One of them is blotting paper-based, and the other is sick pot-based assay.
To execute the blotting paper assay, prepare Rhizoctonia bataticola fungus inoculum by scaling up from slant culture to Petri plate and then to liquid culture. Prepare chickpea nursery by, sowing seeds in a big pot. Uproot them and put them in the liquid inoculum and place them on blotting paper.
Observe for disease symptoms, namely, necrosis on the root, root rot, and leaf yellowing eight days after inoculation. Shown here is a nutshell, the steps in blotting paper technique. To execute the sick pot assay, inoculate chickpea meal with fungus.
After incubation at 30 degrees Celsius for 15 days, sick culture is obtained. Then, powder them and mix with pot mix. Sow seeds on control pot and sick pot.
On the following days, observe for symptoms, namely necrosis on the root, root rot, and leaf yellowing on the infected plants, 10 days after sowing. Sick pot can be scaled up and can be used for large-scale screenings. Shown here is a nutshell of steps in sick pot technique.
Rhizoctonia bataticola is a developing pathogen. This fungus produces hyphae and microsclerotia, which act as primary inuclum during the plant infection. The pure culture applying through the isolative method is used for the assays.
The key point for these successful assays is optimum inoculum load. Demonstration of the blotting paper technique starts here. To prepare liquid Rhizoctonia bataticola inoculum, Inoculate the 500 ml broth with the fungal agar plug from three days old fresh plate culture and incubate the flask in an incubator at 28 degrees Celsius for five days.
Filter out the dark gray fungal mycelia with microsclerotia. Suspend in autoclaved water and remove the media. Again, filter out the mycelia and remove moisture by blot drying the mycelia for a few minutes.
Suspend 100 grams of fungal inoculum in 200 ml of autoclaved water in a jam bottle and mix it properly. Develop chickpea nursery by sowing surface sterilized seeds in solerant. Uproot the plants eight days after sowing, wash them and rinse them with autoclaved RO.Take a blotting paper and fold it once and place half of it on the tray.
Make it wet with autoclaved water. Take plants and dip into the inoculum for 30 seconds with intermittent up and down movement. Place the plants in such a way that roots are covered inside the sheet and folia stays out.
Fold the other half and bend the paper. For control, dip the plants in autoclaved water, keep the trays in a growth chamber for eight days.Results. Observe for the symptoms such as leaf yellowing, root rot, necrosis in root, and reduction in the lateral root number.
Demonstration of the sick pot technique starts here. Take one kilogram of commercial chickpea seeds. Wash the seeds with tap water and wash again with the RO water.
Soak the seeds for five hours. Again, wash the seeds with RO water add 300 gram in a jam bottle. Autoclave them at 121 LVS for 15 minutes twice continuously.
After cooling down, transfer the bottle through the laminar flow. Inoculate the bottles with three fungal agar plugs approximately four millimeter square sizes and shake it well to mix properly. Wrap the bottles and incubate them in an incubator at 30 degrees Celsius for 15 days.
Observe for the bottles with dark mycelial growth around the chickpea seeds and remove the seeds. Dry them at room temperature and powder them. Mix Rhizoctonia bataticola powder with solorite and keep them at room temperature for five days.
Surface sterilize the GT62 seeds and sow them at two centimeter depth.Results. Observe for the 90%mortality in sick pot. 10 days after sowing, observe for the symptoms such as necrosis in roots and reduction in lateral root number and leaf yellowing in the following days.
Sick pot can be prepared in field soil as well. Drought and Rhizoctonia bataticola infection. Impose drought stress in the sick pot by withholding the water.
Shown here are the plants subjected to control, drought, pathogen, and simultaneous drought and pathogen stress. Observe for the symptoms, namely, more necrosis, root rot, and reduction in the lateral root number, and root length in combined stress treatment as compared to the pathogen-only treatment.Conclusions. Blotting paper technique can be carried out In Vitro conditions.
Sick pot technique can be executed in In Vivo conditions. In sick port, abiotic stress, namely, drought stress, can be imposed along with the pathogen infection. Using these techniques, one can study the pathogen infection pattern.
Most of physiological changes occur during the chickpea-Rhizoctonia bataticola interaction, and also screen the chickpea genotypes for resistance. The blotting paper assay is very useful to generate quality samples for microscopy and molecular biology studies. The sick pot assay, which mimics the natural infection conditions, is applicable for screening chickpea germplasm.
