Lab 5

Objectives

 1. To isolate the plant pathogenic fungi and bacteria from symptomatic plant samples

Materials 

• Bunsen burner

• Wire loop

• 70% ethyl alcohol

• Potato Dextrose Agar(PDA)

• 10% sodium hyposhlorite

• Scalpel and forceps

• Sterile tissue paper

• Sterile distil water

• Plant disease sample (fungi/ bacteria)

Procedures

Fungal Isolation from Root, Stem, and Fruit

1. The work area was wiped with 70% ethyl alcohol.

2. Instruments (forceps and knife or scalpel) were dipped in 70% ethyl alcohol and flame-dried.

3. Leaf or stem tissue was rinsed in water to remove soil and other debris.

4. Leaf or stem tissue was surface sterilized by either wiping the surface with soft paper (paper tissue) dipped in 70% ethyl alcohol or briefly dipping thick leaves in 70% ethyl alcohol for 5 seconds, rinsing in sterile water, and damp-drying on sterile paper tissue.

5. Small pieces (approximately 2 × 2 mm) were aseptically cut from the margin of healthy and diseased tissue and transferred to PDA or a selective isolation medium, placing the pieces near the side of the plate.

6. The plates were incubated at approximately 30°C for 48 hours. If no growth appeared, the plates were incubated for 7 days.

7. Plates were checked daily, and when fungal colonies developed from the plant tissue pieces, material was transferred from the colony margins to a medium such as PDA or WA containing sterile pieces of plant tissue (e.g., green rice stem, carnation leaf, or bean pod). These sterile pieces of plant tissue encouraged sporulation, aiding in pathogen identification.

8. A final identification was made using pure cultures grown from a single germinated spore or a hyphal tip.

9. Fungal pathogens were identified based on morphological features, such as spores and spore-forming structures. For example, most fungal pathogens causing leaf diseases produced spore-forming structures such as perithecia, pycnidia, acervuli, sporangiophores, or conidiophores, which were examined microscopically. Characteristics of the fungal body (mycelium) were also considered.

10. The shape, size, color, and arrangement of spores on the sporophores or within the fruiting bodies, as well as the shape and color of the sporophores or fruiting bodies, were noted. These characteristics were sufficient for an experienced individual in fungal taxonomy to suggest the class, order, family, and genus to which the fungus belonged.

Bacteria isolation

1. The plant tissue was placed in a test tube containing 2-3 ml of a sterile liquid (such as sterile water, buffered saline, or quarter-strength Ringer’s solution) and allowed to diffuse at room temperature for 30-60 minutes.

2. The resulting leachate or macerate was streaked onto the appropriate selective agar media with a wire loop to obtain single colonies.

3. If bacterial wilt or soft rot was suspected, and large numbers of saprophytes could have been present, plating by dilution series was recommended.

4. A series of 1:10 dilutions of the leachate was made in sterile water, buffered saline, or Ringer's solution, and 0.1 ml of the diluted sample was plated by spreading on the surface of dried agar plates with a sterile L-shaped glass rod.

5. Separate, single colonies were more readily obtained in this way. The agar plates were incubated at about 30°C for at least 24 hours.

6. The colony morphology, color, margin, shape, and appearance were noted to identify specific phytopathogenic bacteria on the isolation media.

Results

Discussion

The process of isolating fungi and bacteria from plant tissues requires careful attention to detail to ensure accurate results. First, it’s essential to sterilize the work area and tools to avoid contamination. For fungal isolation, both healthy and diseased plant tissues are needed because the healthy tissue serves as a control, helping distinguish between the pathogen and other microorganisms. After sterilizing the plant material with 70% ethyl alcohol, small pieces of tissue are transferred onto selective media, such as PDA, and incubated. As the fungus grows, its spores and fruiting bodies are examined under a microscope to identify the pathogen based on characteristics like the size, shape, and arrangement of the spores. If more than one fungal species grows on the plate, subculturing is done to separate them. For bacterial isolation, plant tissues are soaked in a sterile solution to release bacteria, which are then plated onto selective media. If symptoms like bacterial wilt or soft rot are suspected, a dilution series is used to obtain pure bacterial colonies. The bacterial colonies are studied for their shape, size, color, and margin to identify the species. Fungal colonies tend to be fuzzy and filamentous, while bacterial colonies are smoother and more compact. The isolation methods for both fungi and bacteria stress the importance of maintaining sterile techniques and using the right media to ensure that the pathogens are properly identified. These steps ultimately help researchers and plant pathologists accurately diagnose and manage plant diseases.

Conclusion

In conclusion, the process of isolating and identifying plant pathogens, whether fungi or bacteria, is essential for understanding and controlling plant diseases. By following proper procedures, such as sterilizing work areas and tools, using selective media, and performing microscopic analysis, we are able to differentiate between the pathogens and other microorganisms present. The use of both healthy and diseased plant tissues ensures that we accurately isolate the target pathogens. The visual differences between fungal and bacterial colonies, where fungi often grow in a fuzzy, filamentous pattern, while bacteria form smoother, more compact colonies, are helpful in distinguishing them. These steps, combined with techniques like subculturing and dilution series, allow us to obtain pure cultures, which are crucial for precise pathogen identification. This approach not only enables accurate diagnosis but also provides valuable insight into the nature of the diseases affecting plants. Ultimately, this methodical process is essential for plant pathologists, as it helps develop effective strategies for managing and controlling plant diseases, ensuring healthier crops and more sustainable agricultural practices. By relying on these thorough isolation and identification methods, we can enhance our ability to prevent and address plant disease outbreaks.