Practical 7: Phytobacteriology

Activity 1: Morphology of Bacteria

OBJECTIVES

1. To observe the morphology of bacteria on slides provided under microscope.
2. To observe the shapes of bacteria colonies produce on NA plates. 

1. Different shapes of bacteria

2. Bacteria colonies on NA plate

Activity 2: Disease Symptoms 

OBJECTIVE

1. To observe the symptoms caused by bacteria on the speciimen provided

Disease Symptoms 

Disease	Symptoms
Citrus Canker Causal agent: Xanthomonas axonopodis pv. citri Host: citrus	Sunken spot surrounded by yellow halo. Leaves not wrinkled like those infected by scab.
Bacteria Wilt Causal agent: Ralstonia solanacearum Host: Chili, brinjal, groundnut, okra, tomato	Bacteria infecting vascular tissues and causing symptoms of overall wilt in host plant.
Soft Rot of vegetables  Causal agent: Erwina caratovora Host: Cabbage, brasicca and carrot	Infected tissues turned watery, rot and produce bad odor.
Stem rot of corn Causal agent: Erwina chrysanthemi pv. zeae Host: Corn	Bacteria infecting corn stem espeacialy at the nod showing symptoms of brown and watery rotting
Bacteria rot of crusifer Causal agent: Xanthomonas campestris pv. campestris Host: Brasicca, cabbage	Dry rot starting at the leaf edge.
Leaf  blight of paddy Causal agent: Xanthomonas oryzae pv. oryzae Host: Paddy	Dry spots starting from the edge of leaves due to infecting on hydathode openings.
Leaf streak of paddy Causal agent: Xanthomonas oryzae pv. oryzicola Host: Paddy	Necrotic narrow stripes parallel to leaf veins.

Activity 3: Bacteria Isolation from Plant Tissue

OBJECTIVE

1. To learn techniques on how to isolate bacteria from plant tissue. 

MATERIALS

Spirit lamp, petri dishes, inoculation needles, filter paper, pen knife, 70% alcohol, 10-20% chlorox, distilled water, citrus leaves and potato. 

METHODS

1. Direct Isolation

1. Pen knife is wipe with 70% alcohol and 10-20% chlorox and used to cut canker spots from disease tissues. 

2. The cut tissues are sterilized and placed in NYGA or NA media. The plates are incubated at 27-30 degrees celcius for 24-48 hours. 

b) Dilution Streak

1. Some of the cut out diseased tissues from earlier experiments in (a) is crushed in distilled water. 

2. Dilution streak is done on the provided petri dish plates containing appropriate medium.

RESULTS

Dilution streak result and direct isolation. 

The result shows that bacteria Xanthomonas axonopodis that were diluted from the plant tissue reproduce rapidly in the area that the streak is done. 

Activity 3: Diagnostic Test Using the Cut Plant Tissues

OBJECTIVE

1. To learn technique to isolate pathogen on plant tissue to confirm diagnosis.
2. To observe the ability of bacteria to soften the plant tissue and cause rot. 

MATERIALS

Spirit lamp, petri dishes, inoculation needles, pen knife, 70% alcohol, 10-20% chlorox, distilled water, forceps and potato. 

METHOD

1. Potato is washed, the skin is peeled off.
2. Diseased slices 7-9mm thick is cut out and arranged in sterilized Petri dish.
3. The “V” shaped is cut on the slice and a drop of sterile water is put on the slice. This slice is labelled as control.

4. The “V” shaped is cut on another slice and Erwinia caratovora subspecies caratovora is inoculate on the slice. 

5. All the plates in incubated for 24-48 hours AT 30°C. Symptoms is observed. 

RESULT

On the left side is the result for potato slice that act as control and on the right side is the result for the inoculation of Erwinia caratovora subspecies caratovora

DISCUSSION

The result shows that potato sliced that are infected by Erwina caratovora shows symptoms of soft rot. On the surface of potato seems like wet and mushy due to the infected tissues that become soft. The colour of the infected tissues turns dark brown or black. The bacteria first infect around the cut area and then spread around it. 

CONCLUSION

In conclusion, the symptoms of soft rot bacteria is the infected tissues turned watery, rot and produce bad odor. It will eventually turn from healthy normal colour into dark brown or black. It is proven by inoculating Erwina caratovora  on the potato sliced that shows the same symptoms as stated. Moreover, we also concluded that this diagnostic test using cut plant tissues is an easy method to be done and observe the outcomes accurately.