Practical 3: Symptom, Isolation and Pathogenecity Test

Activity 1: Identification of plant disease through signs and symptoms observation

OBJECTIVES

1.      To identify plant disease through signs and symptoms

2.      To learn how to describe signs and symptoms of plant disease.

METHODS

1.      The sample of symptoms of plant disease is observed.

2.      The symptoms are described and host plant and causal agent are identified.

3.      Slides to observed causal agent under light microscope are prepared.

RESULTS

1)      Plant disease symptoms

Hypoplasia

Disease	Healthy Sample	Infected Sample
Chlorosis (chili plant)  Causal agent: virus trasmit by aphid vectors		colour on leaves change (discolouration)
Mosaic (chili plant) Causal agent: virus transmit by aphid vectors		leaves curl downward along with midrib

Hyperplasia

Disease	Symptoms	Causal Agent
a.       Witches broom Host: Cocoa	growth of vertical and numerous axillary shoots and branches.	Crinipellis perniciosa

Hypertrophy

Disease	Symptoms	Causal Agent
Smut Host: Corn	Mushroom-like tumors or galls began to grow on the ears. The expanding of cells began with creating small, whitish grey then become bigger and turn black.	Ustilago maydis
b.      Gall Host: Roses	The disease first appears as a small overgrowth or galls on root, crowns, trunk. In early stages of development, the gall appears as tumor-like swellings white or flesh-coloured, rough, spongy (soft) and wart-like. They turn dark brown to black as they aged.	Agrobacterium tumefaciens

General Necrosis

Disease	Symptoms	Causal Agent
a.       Vascular wilt Host: tomato, tobacco, legumes, cucurbits, sweet potato, banana	Wilting and death of leaves due to water blockage cause by pathogenic fungi or bacteria that enter xylem vessels of the plant.	Fusarium oxysporum
Dieback Host: Cocoa	In early stage, the yellowing and browning of young leaves at the shoots occur. The leaves will then fall.	Oncobasidium theobrome
Blast Host: Paddy Plant	Lesions that can be found on all parts of the plant, including leaves, leaf collars, necks, panicles, pedicels, and seeds.	Magnaporthe oryzae
Damping-off Host: wide range of host, example tomato	Seedling stems become water soaked and thin, almost thread like, where infected. Young leaves wilt and turn green-gray to brown. Roots are absent, stunted or have grayish-brown sunken spots. Fluffy white cobweb-like growth on infected plant parts under high humidity.	Pythium spp. Rhizoctonia spp.
Soft Rot Host: Cabbage	Infected plant tissue first develops a water-soaked lesions that enlarges rapidly. The affected area become soft and mushy and generally turns dark. An offensive odor usually present.	Erwina carotovora

Local Necrosis

Disease	Symptoms	Causal Agent
Canker Host: citrus	Sunken spots surrounded by yellow halo on leaves, branches and fruits.	Xanthomonas citri
Anthracnose Host: Mango	lesions start as small, angular, brown to black spots that can enlarge to form extensive dead areas. The lesions may drop out of leaves during dry weather.	Colletotrichum gloeosporoides
Powdery Mildew Host: Cucurbit	Pale yellow spots in early stage on leaves. White powdery spots appear on upper and lower leaves and enlarge into big blotches. Plants weakens and fruit ripen prematurely.	Erysiphe cichoracearum
d.      Downy Mildew Host: Cucurbit	Only occur on cucurbit leaves. Appear as angular, yellow spots on upper leaf surface. Spots are often bounded by leaf veins. During humid weather, a grayish growth of fungus appears on the underside of this spots.	Pseudoperonospora cubensis

DISCUSSION

There are many types of disease symptoms that occur in plants. The symptoms include general necrosis, local necrosis, hypoplasia, hypertrophy and hyperplasia. Necrosis often describes as death of cells or tissues and can be divided into general necrosis and local necrosis. General necrosis is more general and affect all part of the host. Examples of general necrosis are damping-off, blight, blast, dieback, vascular wilt and soft rot. Besides, local necrosis is more limited to only cells surrounding the invading pathogen. The examples are anthracnose, powdery mildew, downy mildew, canker, and rust.

Furthermore, disease symptom hypoplasia involves the reducing size of cells and plant growth retarded. The plant experience under development which include stunting of leaves, shortened internodes and inadequate chlorophyll production. It is caused by many types of pathogen and the examples include chlorosis and mosaic. Moreover, hypertrophy and hyperplasia are symptoms for overgrowth. Hypertrophy involves cell enlargement that leads to symptoms such as smut and gall. Hyperplasia involves fast cell division that results in symptom called witches broom.

Activity 2: Koch’s Postulates

OBJECTIVES

1. To observe disease symptoms of the casual agents on chilli parts.
2. To determine pathogenicity of the causal agent of the plant disease on chilli, whether Collectotrichum truncatum or Collectotrichum capsici by Koch’s postulate.
3. To observe the presence of disease on chilli

MATERIALS

Pure culture of 2 fungal pathogen specimen (Collectotrichum truncatum and Collectotrichum capsica), diseased and healthy chilli fruit, petri dish, filter paper, PDA plates, forceps, inoculation needle, plastic container, slides, spirit lamp, scalpel, coverslips, microscope and Lactophenol Cotton Blue (LCB)

METHODS

1.  Slides from both cultures are prepared and observed under light microscope.
2. The causal agent is isolated from the disease tissues onto PDA (Potato Dextrose Agar) plates. The plates are labelled and incubated at room temperature for observation.

 

3. Aseptically, 1 agar containing hyphae is cut from each pure culture provided and being inoculate each species of pathogens on sterilized chilli fruit. Third chilli is uninoculated and used as control. The fruit is kept in moist tray and is covered with plastic sheet. The fruit is incubated for 3-5 days and the symptoms exhibit is observed.

The agar containing hyphae is cut using inoculation needle The healthy chili is injured by cutting knife

The chili is sterilized with sodium hypochloride the cut hyphae is put on the injured part and close with cotton and tape.

Incubate for 3-5 days in moist tray covered with plastic sheet

4. The isolation process as in step 2 above is repeated using fruit that demonstrate similar symptoms as in step 2 above. 

RESULTS

1. Inoculation from disease infected chili into PDA plates

Symptoms on specimen provided shows black swirling, dieback ripe fruit rot and brown necrotic lesions.

The result shown characteristic of Collectotrichum capsici

2. Koch’s Postulates

Chili infected by Collectotrichum truncatum

Chili infected by Collectrotrichum capsici

 Controlled chili

DISCUSSION

In the experiment, we use two cultured fungi which is Collectotrichum truncatum and Collectrotichum capsica to carry out Koch’s Postulates. These two fungi are in the same genus Collectotrichum sp. Koch’s Postulates that we carry out involve inoculation and isolation. Isolation method is method to isolate suspected causal agent from disease host plant and grown in pure culture. Inoculation method involve pure culture of the suspected causal agent is inoculated into healthy susceptible host plant. The host must able to shows the symptoms of the suspected disease.

In the result, it shows that after 5 days, healthy chili that infected by Collectrotichum capsica shows the same symptoms as the infected chili specimen provided which is black swirling spots, dieback ripe fruit rot and the development of brown necrotic lesions around the black spot began to take place. The infection shows invasive and severe. While healthy chili that is infected by Collectotrichum truncatum shows only mild infections on the injured area. Therefore, chili fruit is more susceptible host to causal agent Collectrotichum capsica than Collectotrichum truncatum.

CONCLUSION

In conclusion, the disease symptoms of the causal agent shown on the chili are black swirling spot, dieback ripe fruit rot and the development of brown necrotic lesions around the black spot on the fruit. Moreover, from the Koch’s Postulates procedure, we could determine the pathogenicity of fungus Collectrotrichum capsici that infect the healthy chili. The disease that cause the symptoms shown on the infected chili is anthracnose disease.