Pathogen Isolation Methods PDF

Isolation of Fungal and Bacterial Plant Pathogens Objective: To isolate the fungal and bacterial plant pathogens from affected plant parts Preparing for isolation 1. Procure already sterilized glassware, such as petri dishes, test tubes, and pipettes 2. Prepare solutions for treating the surface of...

Isolation of Fungal and Bacterial Plant Pathogens Objective: To isolate the fungal and bacterial plant pathogens from affected plant parts Preparing for isolation 1. Procure already sterilized glassware, such as petri dishes, test tubes, and pipettes 2. Prepare solutions for treating the surface of the infected or infested tissue to eliminate or markedly reduce surface contaminants that could interfere with the isolation of the pathogen. The most commonly used surface sterilants is 70 per cent ethyl alcohol, which is used for leaf dips for three seconds or more. The tissues must be blotted dry with a sterile paper towel. 3. Prepare culture media on which the isolated fungal or bacterial pathogens will be grown. The most commonly used medium is potato dextrose agar (PDA), which is good for most, but not all fungi; water agar or glucose agar (1–3% glucose in water agar) for separating some oomycetes (Pythium) and fungi (Fusarium) from bacteria; V-8 and other less rich media, which encourage fungal sporulation; and nutrient agar, which contains beef extract and peptone and is good for isolating bacterial plant pathogens. Fungi can also be separated in culture from bacteria by adding 1 or 2 drops of a 25 per cent solution of lactic acid to 10 milliliters of the medium before pouring it in the plate, which inhibits the growth of bacteria. Pouring the plates Properly sterilized culture media are melted (if stored as in the previous exercise), allowed to cool somewhat, and are subsequently poured from the flasks into sterilized petri dishes, test tubes, or other appropriate containers. If agar was added, the medium will soon solidify and is then ready to be used for growth of the fungus or bacterium. Pouring of the culture medium into petri dishes, tubes, and so on is carried out as aseptically as possible either in a separate culture room or in a clean room free from drift and dust. In either case, the work table should be wiped with ethyl alcohol, hands should be clean, and tools such as scalpels, forceps, and needles should be dipped in alcohol and flamed to prevent introduction of contaminating microorganisms. Working in a laminar flow hood greatly helps to grow the desired fungus free of airborne contaminants. Methods of isolation Methods to be adopted for isolation of plant pathogens depend on the nature of the latter particularly in relation to its presence in the host cells and the form in which it is present. Isolating the Pathogen From Leaves i) Fungi 1. Cut across several small sections of 5 to 10 mm square size from the margin of the infected lesion so that they contain both diseased and healthy looking tissue. 2. Place them in one of the surface sterilant solutions, making sure that the whole surface gets wet. After about 15 to 30 seconds, the sections are taken out aseptically one by one and at regular intervals (e.g. every 10–15 seconds) so that each of them has been surface sterilized for different times. 3. The sections are then washed in three changes of sterile water; blotted dry on clean sterile paper towels, and are finally placed on the nutrient medium, usually three to five bits per petri dish aseptically. 4. Incubate the inoculated petri dishes at 25°C for 3-5 days. 5. If fruiting structures (pycnidia, perithecia) are present on the leaf, it is sometimes possible to pick them out, drop them in the surface sterilant for a few seconds, and then plate them on the nutrient medium. ii) Bacteria The serial dilution method is often used to isolate pathogenic bacteria from diseased tissues contaminated with other bacteria. After surface sterilization of sections of diseased tissues from the margin of the infection, the sections are ground aseptically but thoroughly in a small volume of sterile water and then part of the homogenate is diluted serially in equal volumes or 10 times the volume of the initial water. Finally, plates containing nutrient agar are streaked with a needle or loop dipped in each of the different serial dilutions, and single colonies of the pathogenic bacterium are obtained from the higher dilutions that still contain bacteria.