Materials and reagents

Biological materials

1. Virulent strain of Rhizoctonia solani OR230677.1 (origin: ARRI, Titabor, Assam, India)

2. Any susceptible rice (Oryza sativa) cultivar such as Swarna (MTU 7029), Samba Mahsuri (BPT 5204), or Pusa Basmati 1 (PB-1)

Reagents

1. Double-distilled water

2. Ethanol 99.5%, analytical grade (Changshu Hongsheng Fine Chemical Co., Ltd)

3. Ocimum gratissimum essential oil (extracted by hydrodistillation using Clevenger type apparatus [9])

4. Thymol (HiMedia, CAS number: GRM7581-100G)

5. Tween 80 (SRL, CAS number: 9005-65-6)

6. Propiconazole (Syngenta, Tilt, CAS number: 60207-90-1)

7. Potato dextrose agar medium (PDA) (HiMedia, CAS number: GM096-500G)

Solutions

1. Potato dextrose agar medium (PDA) (see Recipes)

2. Ethanol (70%) (see Recipes)

3. Tween 80 (0.25%) solution (see Recipes)

4. Ocimum gratissimum essential oil in 0.25% Tween 80 (1,000 ppm) (see Recipes)

5. Thymol in 0.25% Tween 80 (1,000 ppm) (see Recipes)

6. Propiconazole in 0.25% Tween 80 (1,000 ppm) (see Recipes)

Recipes

1. PDA medium

a. Weigh 39 g of commercially available PDA powder and transfer it to a sterile 1 L borosilicate flask.

b. Add double-distilled water to bring the total volume to 1,000 mL.

c. Stir thoroughly until the powder is completely dissolved. Heat gently if necessary.

d. Adjust the pH to 5.6 ± 0.2 using sterile 1 N NaOH or HCl.

e. Dispense the medium into autoclavable containers and sterilize at 121 °C, 15 psi for 15 min.

f. After sterilization, allow the medium to cool to approximately 50 °C by placing it on the laminar airflow bench with occasional gentle stirring; then, pour into sterile Petri dishes.

g. Store the plates inverted at 4 °C in the dark and use within 2–3 weeks.

2. Ethanol (70%)

a. To prepare 1,000 mL of 70% ethanol, measure 700 mL of absolute ethanol (≥99.5%) using a measuring cylinder and transfer it to a clean, sterile glass beaker.

b. Add 300 mL of double-distilled water to reach a final volume of 1,000 mL.

c. Mix thoroughly by swirling or gentle stirring.

d. Store the solution in a tightly sealed container at room temperature.

e. Label the container with preparation date and concentration.

f. Use freshly prepared solution or store for up to 6 months.

3. Tween 80 (0.25%) solution

a. To prepare 1,000 mL of 0.25% Tween 80 solution, measure 2.5 mL of Tween 80 using a sterile pipette and add it to approximately 900 mL of double-distilled water in a clean, sterile beaker.

b. Mix thoroughly using a magnetic stirrer or by gentle inversion until the solution is homogeneous.

c. Adjust the final volume to 1,000 mL with additional double-distilled water.

d. Sterilize the solution by autoclaving at 121 °C, 15 psi for 15 min.

e. Store at room temperature or 4 °C, protected from light.

4. Ocimum gratissimum essential oil in 0.25% Tween 80 (1,000 ppm)

a. To prepare 1,000 mL of Ocimum gratissimum essential oil solution at 1,000 ppm, first prepare 0.25% Tween 80 solution as described in Recipe 3.

b. Then, add 1.0 mL of Ocimum gratissimum essential oil to 999 mL of 0.25% Tween 80 solution.

c. Mix thoroughly using a magnetic stirrer or by vortexing until the oil is uniformly emulsified.

d. Use freshly prepared or store at 4 °C in a sealed amber bottle to protect from light.

5. Thymol in 0.25% Tween 80 (1,000 ppm)

a. To prepare 1,000 mL of thymol solution at 1,000 ppm, weigh 1.0 g of thymol and dissolve it in 999 mL of 0.25% Tween 80 solution, prepared as described in Recipe 3.

b. Mix thoroughly by stirring or vortexing until completely dissolved.

c. Store in a sealed container at 4 °C and protect from light.

6. Propiconazole in 0.25% Tween 80 (1,000 ppm)

a. To prepare 1,000 mL of propiconazole solution at 1,000 ppm, pipette 1.0 mL of commercial-grade propiconazole (active ingredient, a.i.) and mix into 999 mL of 0.25% Tween 80 solution prepared as described in Recipe 3.

b. Mix thoroughly using a magnetic stirrer or vortex to obtain a uniform solution.

c. Store at 4 °C in a tightly sealed, light-protected container and use within 2 weeks.

Laboratory supplies

1. Plastic pots (15 cm diameter)

2. Sterile Petri dishes (Tarson, catalog number: 100 mm PP Autoclavable Petri Dish 46203)

3. Sterile muslin cloth (Euqinu 1 × 1 Meter Cotton Muslin Cloth, Ultra Fine Fabric)

4. Sterile forceps (Vandersluys normal tip forceps)

5. Sterile scalpel or inoculation loop (Tarsons, catalog number: 920051)

6. Sterile needle (Thomas Scientific, needle with nichrome wire)

7. Hand sprayers (one per treatment) (1.5 L Garden Agriculture Spray Pump Atomizer Compression Pressure Sprayer)

8. pH indicator strips (Parshv, model number: P.H. [Universal] Indicator Paper)

9. Measuring cylinders (100 mL) (Borosil, catalog number: 3022016)

10. Transparent ruler or digital caliper (UniMarket, model number: UniMarketCH2-9MDPD26T)

11. Borosilicate glass flasks (1 L) (Borosil, catalog number: 5360029)

12. Sterile beakers (1,000 mL) (Borosil, model number: ZGB-08)

13. Micropipettes (10 µL, 20 µL, 200 µL, 1,000 µL) (Tarsons)

14. Corresponding sterile pipette tips (10 µL, 20 µL, 200 µL, 1,000 µL) (Tarsons)

15. Disposable gloves

16. Laboratory masks (AMPri, catalog number: 081307-L)

17. Lab coat

18. Waste disposal bags

Equipment

1. Greenhouse or plant growth chamber with environmental control (temperature, humidity, light cycle) (iGene, model number: IG-150GC)

2. Incubator (capable of maintaining 33 °C) (GENEXIS, model: GLA-109-2)

3. Autoclave (for sterilization at 121 °C, 15 psi) (EQUITRON, model: Portable PAD)

4. Magnetic stirrer with stir bars (REMI, model: 2-MLH)

5. pH meter (Mettler-Toledo GmbH, model: FP20)

6. Analytical balance (SCALETEC, model: SAB-203L)

7. Vortex mixer (Ambinova, model: AMBI-VM-01)

8. Laminar airflow cabinet (for aseptic work) (Ambinova, model: AMBI-LAF-3999HE)

9. Refrigerator (4 °C, for media and inoculum storage) (SAMSUNG, model: RT39B551ES8/HL)

10. Digital camera (for lesion imaging and documentation)

Software and datasets

1. RStudio (Posit, version 2025.05.1+513)

Procedure