Lab 7

Objectives

 1. To demonstrate Koch's postulate using red chili fruit infected by anthracnose, Colletotrichum spp.

Materials 

• infected and healthy red chili fruit

• sterilized scalpel and blade

• Potato dextrose agar (PDA)

• Petri dish

• Inoculation needle

• Streile pin-pricking needle

• 70% ethanol

• 2% sodium hypochlorite (NaOCl)

• Sterile distilled water

• Sterile filter paper

• Lactophenol cotton blue (LCB)

• Glass slide and glass slip

• Stereo microscope

• Compound light microscope

Procedures

A. Isolation of Pathogen from the Infected Tissue (According to Sanders and Korsten, 2003, with modifications)

1. The red chili fruit infected tissue was cut into 1 cm x 1 cm sections.

2. The tissue was soaked in a 70% ethanol solution for 3 minutes, followed by soaking in 2% sodium hypochlorite (NaOCl) for another 3 minutes. It was then rinsed in two changes of sterile distilled water.

3. The sterilized infected tissue was dropped onto sterile filter paper and left to dry before plating it onto Potato Dextrose Agar (PDA). The plates were incubated for 3-4 days at room temperature. The hyphae were subcultured onto fresh PDA to obtain a pure culture and incubated for 7-10 days.

4. The hyphae were scraped from the colony and placed on a glass slide with Lactophenol Cotton Blue (LCB). The pure culture was identified under both stereo and compound light microscopes. The classification was based on conidia and colony morphology, as referenced by P.P. Than et al. (2008) in Plant Pathology (57, 562–572).

B. Inoculation of Healthy Red Chili Fruit

1. Six healthy red chili fruits were prepared, swabbed with 70% ethanol, and left to dry for 3-4 minutes. Three of the healthy chili fruits were wounded with a sterile needle, while the other three were kept as controls.

2. A 7-day-old Colletotrichum spp. colony, grown in PDA, was used as the inoculum source. A few plugs (5 mm diameter) were cut from the actively sporulating areas near the colony periphery using a sterilized cork borer and placed over the wounded part of the chili fruits.

3. The fruits were incubated in the dark for 7-10 days in a moist chamber or sterilized container.

C. Re-isolate the Pathogen and Examine

1. The anthracnose symptoms on the red chili fruit (from step B) were observed using a stereo microscope.

2. Step A was repeated to re-isolate the pathogen.

Results

Discussion
In this experiment, we applied Koch's Postulates to confirm that Colletotrichum spp. is the causal agent of anthracnose in red chili fruit. First, we isolated the pathogen from infected fruit by cutting tissue into small pieces, sterilizing them, and placing them on a Potato Dextrose Agar (PDA) plate to promote fungal growth. After incubating, we obtained pure cultures of the fungus and identified the pathogen by observing its colony characteristics and conidia under both stereo and compound microscopes. The next step involved inoculating healthy chili fruit with the pathogen. We carefully wounded the fruit and applied fungal plugs from the active colony to these wounds. The inoculated fruit showed clear symptoms of anthracnose after several days of incubation, replicating the disease symptoms initially observed in the infected fruit. Finally, we re-isolated the pathogen from the inoculated fruit and confirmed its identity through microscopy. This step verified that the same organism was present in both the original infection and the inoculated fruit, fulfilling all four of Koch's Postulates. By following this procedure, we demonstrated that Colletotrichum spp. is indeed the cause of anthracnose in red chili fruit. The successful application of Koch's Postulates underscores the critical role of pathogen isolation, identification, and re-inoculation in understanding plant diseases. This experiment also emphasizes the importance of using accurate and consistent techniques in diagnosing and confirming the cause of plant diseases. Through these steps, we were able to establish a clear connection between the pathogen and the disease, reinforcing the value of Koch’s Postulates as a foundational tool in plant pathology.

Conclusion
In conclusion, this experiment successfully demonstrated Koch's Postulates in confirming Colletotrichum spp. as the causative agent of anthracnose in red chili fruit. By isolating the pathogen from infected tissue, inoculating healthy fruit, and re-isolating the pathogen from the symptomatic fruit, we were able to fulfill all four criteria set by Koch. The reappearance of identical disease symptoms in the inoculated fruits and the identification of the same pathogen upon re-isolation reinforced the role of Colletotrichum spp. in causing anthracnose. This process not only validated the application of Koch’s Postulates but also highlighted the importance of rigorous isolation and identification techniques in plant disease diagnostics. Ultimately, this experiment emphasizes the significance of Koch's Postulates as an essential tool for identifying plant pathogens and understanding their role in crop diseases, offering a reliable foundation for disease management strategies in agriculture.