Fungal and Bacterial Pathogen Isolation Techniques

What is the primary purpose of placing tissue sections in a surface sterilant solution?

How long should the tissue sections typically be incubated at 25°C?

What method is recommended for isolating pathogenic bacteria from contaminated tissues?

What should be done with sections of diseased tissue after surface sterilization?

Why might fruiting structures be isolated directly in the sterilization process?

What type of sterilant is most commonly used for surface sterilization of infected plant tissue?

Which culture medium is best known for isolating bacterial plant pathogens?

What method can be employed to inhibit bacterial growth in a fungal culture medium?

Which process must be performed aseptically to prevent contamination when pouring culture media?

What type of agar is typically used for separating certain fungi from bacteria?

Why is it important to dry the tissues with a sterile paper towel after surface sterilization?

What is the purpose of using a laminar flow hood during isolation procedures?

What must be done to glassware and tools used in the isolation process?

Fungal and Bacterial Plant Pathogen Isolation

Objective: Isolate fungal and bacterial plant pathogens from affected plant parts.

Preparing for Isolation

Sterilize glassware: Use petri dishes, test tubes, and pipettes.
Surface sterilization: Use 70% ethyl alcohol for leaf dips (3 seconds or more) to eliminate surface contaminants. Blot dry with sterile paper towels.
Culture media preparation:
- Potato dextrose agar (PDA): Suitable for most fungi.
- Water agar or glucose agar: For separating oomycetes (Pythium) and fungi (Fusarium) from bacteria.
- V-8 and other less rich media: Encourage fungal sporulation.
- Nutrient agar: Contains beef extract and peptone, good for isolating bacterial plant pathogens.
- Lactic acid addition: Use 1-2 drops of a 25% lactic acid solution to 10mL of medium before pouring to inhibit bacterial growth.

Pouring Plates

Sterile media: Melt and cool before pouring into sterilized containers.
Aseptic techniques: Work in a clean room or laminar flow hood. Wipe surfaces, wash hands, sterilize tools to prevent contamination.

Isolation Methods

Method selection: Depends on the pathogen's presence within host cells and its form.

Isolating from Leaves

Fungi

Cut sections: 5 to 10 mm square sections from the margin of the infected lesion, including diseased and healthy tissue.
Surface sterilization: Dip sections in sterilant for varied durations (15 to 30 seconds).
Washing and plating: Wash sections in sterile water, blot dry, and place 3-5 sections per petri dish.
Incubation: Incubate at 25°C for 3-5 days.
Fruiting structures: If present, pick them out, sterilize briefly, and plate them.

Bacteria

Serial dilution: Used to isolate pathogenic bacteria from contaminated diseased tissues.
Grinding: Aseptically grind surface-sterilized sections in sterile water.
Serial dilution: Dilute homogenate in equal volumes or 10 times the initial volume of water.
Streaking: Streak plates with nutrient agar using a needle or loop dipped in various dilutions.
Single colonies: Obtain single colonies of pathogenic bacteria from the higher dilutions still containing bacteria.

This quiz focuses on the isolation techniques for fungal and bacterial plant pathogens from affected plant parts. It covers topics such as sterilization, culture media preparation, and the process of pouring plates. Test your knowledge on best practices and methodologies in plant pathology.