Jul 09, 2025
Isolation of Pyrenophora teres f. teres from Infected Barley Leaves and Re-inoculation of Barley Leaves in a Glasshouse
- Lilian Sanglard1,
- Sabrina M Cuellar1,
- Robabeh H Ghavamabad1,
- Julie Lawrence1,
- Nola D'Souza1,
- Jordi Muria Gonzalez1,
- Fatima Naim1,
- Ciara Gifford1
- 1Curtin University
Protocol Citation: Lilian Sanglard, Sabrina M Cuellar, Robabeh H Ghavamabad, Julie Lawrence, Nola D'Souza, Jordi Muria Gonzalez, Fatima Naim, Ciara Gifford 2025. Isolation of Pyrenophora teres f. teres from Infected Barley Leaves and Re-inoculation of Barley Leaves in a Glasshouse. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl462qrgo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 07, 2025
Last Modified: July 09, 2025
Protocol Integer ID: 221917
Keywords: Pyrenophora teres f. teres, barley, fungal isolation, sporulation, conidia, single spore isolation, plant inoculation, leaf infection, cereal pathology, fungal stock preparation, infected barley leaf, infected leaf sample, applicable to pyrenophora tritici, conidia production, leaf sterilisation, inoculation of barley plant, pyrenophora tritici, conidia harvest, pathogen isolation, barley leaf, grown barley plant, barley plant, wheat inoculation, pyrenophora, single spore isolation, conidiophore growth, pathogen, isolation of pyrenophora
Funders Acknowledgements:
Grains Research and Development Corporation
Grant ID: CUR1403-002BLX
Abstract
This protocol provides a detailed, step-by-step guide for the isolation of Pyrenophora teres f. teres (Ptt) from infected barley leaves, followed by culturing for vegetative growth, sporulation (conidiophore and conidia production), and inoculation of barley plants under glasshouse conditions. The protocol is also applicable to Pyrenophora tritici-repentis (Ptr), with optimisations for sporulation and wheat inoculation. Infected leaf samples may be sourced from either field-grown or glasshouse-grown barley plants.
Protocol timeline overview:
Week 1: Seed sowing, leaf sterilisation, pathogen isolation, and initial sporulation
Weeks 2–3: Single spore isolation and culture stock preparation
Week 4: Conidiophore growth, maturation, and experimental plate setup
Week 5: Conidia harvest and plant inoculation
Guidelines
You must have read and understood the SDS of all chemicals listed below in the 'Safety Warnings' section. Safety warnings: This protocol requires the following hazardous substances: Ethanol, Sodium hypochlorite. Greatest care needs to be given when using scissors, scalpel blades, needles, glass slides, and cover slips. You must always use appropriate personal protective equipment, including lab coats, gloves, safety glasses, closed-toe shoes, and hair tied back, when handling chemicals and biological agents.
Materials
Ethanol, Sodium hypochlorite, PDA (Potato Dextrose Agar), V8PDA (V8-Potato Dextrose Agar), Sterile Milli-Q water, Tween-20, Potting mix (Richgro: 50% fine composted pine bark, 20% coco peat, 30% river sand, with added nutrients: Osmoform NXT 2 kg/m³, dolomite lime 2 kg/m³, fine lime 1 kg/m³, gypsum 1 kg/m³, iron sulfate 0.8 kg/m³, and iron chelate 0.2 kg/m³).
Troubleshooting
Week 1: Seed Sowing, Leaf Sterilisation, Pathogen Isolation, and Initial Sporulation
Sow barley or wheat seeds in pots or trays filled with potting mix. Water and fertilise as required to promote healthy plant growth under glasshouse conditions.
Note: in our laboratory we use Richgro: 50% fine composted pine bark, 20% coco peat, 30% river sand, with added nutrients: Osmoform NXT 2 kg/m³, dolomite lime 2 kg/m³, fine lime 1 kg/m³, gypsum 1 kg/m³, iron sulfate 0.8 kg/m³, and iron chelate 0.2 kg/m³.
Prepare culture media:
PDA (Potato Dextrose Agar): 40 g/L PDA powder.
V8PDA (V8-Potato Dextrose Agar): 150 mL/L V8 Juice, 3 g/L CaCO₃, 10 g/L PDA, 15 g/L agar.
Autoclave both media at 121 °C for 30 min. In sterile conditions, pour into Petri dishes and store plates at 4 °C until use.
Set up all subsequent sterile procedures (Steps 4–7) in a Class II Biological Safety Cabinet (BSCII).
Using sterile scissors or a scalpel, cut 3–4 pieces (1 cm²) of infected leaf tissue through symptomatic lesions to expose infected margins.
Transfer leaf pieces to a 15 mL Falcon tube containing 10 mL of sterilisation solution (1% sodium hypochlorite, 5% ethanol, 0.05% Tween-20). Shake manually for 2–3 minutes. Transfer leaf pieces to a new tube containing 15 mL sterile Milli-Q water.
Rinse by shaking gently for 30 seconds, then decant the water. Repeat the rinse step with fresh sterile Milli-Q water a total of four times.
Using sterile forceps, transfer the sterilised leaf pieces onto PDA plates, ensuring that the infected edge is flat and in contact with the agar surface.
Incubate plates at 20 °C for 2–4 days, or until a visible mycelial clump forms around the leaf margin. Use a dissecting microscope (e.g., Nikon SMZ 745T) to monitor growth.
Under the microscope, use a sterile needle to transfer a portion of the mycelium to a fresh V8PDA plate. Prepare three replicate plates and incubate at 20 °C for four days.
Note: Insufficient mycelium transfer may result in failed culture establishment. Ensure visible transfer of actively growing mycelia.
Once the plate is fully colonised, pipette 1 mL of 1 M glucose (for Ptt) or 1 mL sterile Milli-Q water (for Ptr) onto the plate. Use a sterile Petri spreader to flatten the mycelial surface evenly.
Note: You could use water in this step for both Ptt and Ptr.
Incubate the plates with lids off under UV light (Philips TL-D 15W ACTINIC BL) for ~20 hours to induce conidiophore and conidia formation.
After UV exposure, incubate the plates with lids on at 15 °C in the dark overnight to allow conidia to mature.
Weeks 2–3: Single Spore Isolation and Culture Stock Preparation
Inspect sporulated plates and select the one with the best visible conidia production. Use a microscope to identify mature conidia around the edge of the mycelial growth.
Under a dissecting microscope, use a sterile needle to transfer a single mature spore to a fresh V8PDA plate. Repeat this process to obtain three replicate plates to increase the likelihood of single-spore isolation. Incubate plates at 20 °C for 5–6 days.
Note: Transferring a single spore requires practice. If needed, pipette sterile water onto the plate to gently wash spores toward the edge and then transfer an individual spore to a fresh plate.
Monitor mycelial growth.
Select one plate with healthy growth for sporulation.
In a BSCII, use a sterile scalpel to cut ~1 cm² agar sections from the outer white mycelial edge and the dark inner region of the culture (Figure 1a). Transfer the agar plugs to the Petri dish lid and allow them to dry overnight (Figure 1b).
Transfer dried agar plugs into individual 2 mL Eppendorf tubes (one plug per tube). Store tubes at -70°C. This constitutes the first isolated stock.
Figure 1. Reference images for stock preparation (a) Mycelial colony derived from single spore isolation. The outer edge of the colony was selected for plug preparation. (b) Agar plugs drying overnight under a BSCII.
To generate additional isolate stocks, remove one plug from the first stock and transfer it to a fresh V8PDA plate, ensuring the mycelial surface contacts the agar. Incubate at 20°C for 5-6 days.
Repeat Steps 16-17 to produce the second isolate stock. Store the plugs at -70°C or -20°C, depending on storage availability.
Week 4: Conidiophore Growth, Maturation and Experimental Plate Setup
In a BSCII, retrieve one frozen plug of your isolate (first or second isolated stock) and transfer it to a fresh V8PDA plate. Ensure the mycelial surface is pressed firmly against the agar. Prepare additional plates as required. Incubate at 20 °C for 5 days.
Note: If planning to use the spore spray inoculation method, ensure you prepare enough sporulated plates to achieve the required spore concentration to suit your experimental design.
After incubation, identify the perimeter of vegetative growth. Use the wide, sterile end of a 200 µL pipette tip to punch circular plugs from just inside this perimeter (Figure 2a).
Pipette 1 mL of 1 M glucose (for Ptt) or 1 mL sterile Milli-Q water (for Ptr) onto the surface. Use a sterile Petri spreader to flatten the mycelial surface and distribute the liquid evenly.
Transfer the vegetative plugs to a new V8PDA plate, arranging them in a spaced ring near the edge of the plate (Figure 2b). Incubate plates with lids off under UV light (Philips TL-D 15W ACTINIC BL) for 20 hours to induce conidiophore and conidia development.
After UV exposure, incubate the plates with lids on at 15 °C in complete darkness to allow for conidia maturation (Figure 2b). Times may vary depending on isolate vigour. Approximate maturation times are as follows:
Pyrenophora teres f. teres, ~32 hours
Pyrenophora tritici-repentis, ~24 hours
Once mature, store sporulated plates at –20 °C until ready for inoculation.
Figure 2. Reference images for sporulation of Ptt (a) The edge of the colony punched for isolation of plugs and transfer to a clean V8 PDA plate. (b) Sporulated plugs prepared for use for leaf inoculation.
Week 5: Conidia Harvest and Plant Inoculation
On the day before or morning of inoculation, prepare plants in the glasshouse. Secure a single, mature, fully expanded leaf from each plant to a polystyrene board (Figure 4a). Schedule the inoculation for late afternoon to optimise humidity and infection success.
In the laboratory, retrieve frozen sporulated plugs. Place the plugs on one side of the Petri dish lid and pipette 2 mL of sterile Milli-Q water onto the opposite side of the lid (Figure 3).
Using sterile tweezers and a soft paintbrush, submerge each plug into the water and gently brush its surface to release conidia into suspension (Figure 3). Collect the resulting spore suspension into a 50 mL Falcon tube and keep on ice. Repeat for all isolates required for the experiment.
Figure 3. Reference image for spore harvesting. Conidia released from sporulated plugs into sterile water using a sterile paintbrush.
Choose an inoculation method.
Option A: Spot inoculation.
Prepare spore suspension: Homogenise the spore suspension by gently swirling the tube and then pipette 10 µL onto a microscope slide. Add a coverslip and count mature spores under a compound microscope (e.g., Nikon Eclipse E200). Adjust the concentration using sterile Milli-Q water to achieve:
Ptt: ~2,000 spores/mL and
Ptr: ~3,000 spores/mL.
Note: These are starting concentrations and can be adjusted for your isolate and experiment as appropriate.
Transport the suspension to the glasshouse on ice. Switch off the air conditioning to reduce evaporation. Homogenise the spore suspension by gently swirling. Pipette a 10 µL droplet on either side of the leaf midrib, avoiding placement on the midrib (Figure 4b).
Activate the misting system ((e.g., Idrobase‱ Fog Extra). Adjust the misting schedule and duration according to environmental conditions and the duration of the experiment. Use Table 1 as a general guideline for temperature and relative humidity.
Note: Optimal Ptt infection requires intermittent misting to maintain humidity.
Table 1. Glasshouse guideline for relative humidity (%) and temperature (°C) for Ptt inoculation and disease phenotype development over 7-10 days.
Note: Ensure misting settings are optimal to maintain high humidity without washing off the spore solution from the leaf surface within the first 12 hours.
Before sample collection, turn off the mister and remove leaves from the polystyrene board, allowing them to air dry.
Option B: Spray inoculation (optimised for Ptt only).
In the glasshouse, set up appropriate spray guards to prevent cross-contamination between isolates if using multiple isolates for spray inoculation.
Prepare spore suspension: Count spores using a microscope as described in Step 30.1 and adjust the concentration to ~1,000 spores/mL. Add Tween-20 to a final concentration of 0.02%. Transfer the solution to a 1 L Nylex trigger spray bottle directly before proceeding to the glasshouse for spray inoculation.
Note: The solution must be aerated if not used straight away.
Spray inoculation: Turn off the air conditioning and gently shake/swirl the spore suspension to homogenise it. Spray ~4 mL over a 30 cm² area of the secured leaves (Figure 4c). Repeat as needed according to your experimental design.
Activate the misting system as described in Step 30.3.
Before sample collection, turn off the mister and remove leaves from the Polystyrene board, allowing them to air dry and assess disease phenotype (Figure 4d).
Figure 4. Representative images of leaf inoculation in a glasshouse. (a) Leaves secured on a polystyrene block with double-sided tape. (b) A representative leaf was imaged for six days to capture droplet inoculation and disease development. (c) Image of leaves sprayed for the whole-leaf spray inoculation method. (d) Disease phenotype eight days post inoculation using the whole-leaf spray inoculation method.
Protocol references
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Hassett, K., Ellwood, S. R., Zulak, K. G., & Muria-Gonzalez, M. J. (2020). Analysis of apoplastic proteins expressed during net form net blotch of barley. Journal of Plant Diseases and Protection, 127(5), 683-694.
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Muria-Gonzalez, M. J., Lawrence, J. A., Palmiero, E., D'Souza, N. K., Gupta, S., & Ellwood, S. R. (2023). Major susceptibility gene epistasis over minor gene resistance to spot form net blotch in a commercial barley cultivar. Phytopathology, 113(6), 1058-1065.
Acknowledgements
This work was supported by a co-investment between the Grains Research and Development Corporation (GRDC) and Curtin University with grant [CUR1403-002BLX]. Research activities were undertaken by the Centre for Crop and Disease Management.