Lab 3: Disease Diagnosis: Symptoms, Isolation and PathogenicityTest

Introduction

In the studies of plant pathology, it is define as a science that studies the plant disease and the causes. This is included in the elements of causal agents, mechanisms of infection and also the methods to control those diseases. One of the steps that is need to be done studying plant pathology is through the identification of plant disease on the host plant. It involves in the determination of the environment conditions and also the causal agents of the diseases. The identification is done through the observation on the disease symptoms or signs of causal agents on plant parts. Next is to know the techniques to isolate the plant pathogens from diseased materials. This usually depends on the growth, reproduction and ecology of the microorganism. 

Other than that, it is important to know the steps in Koch Postulates. The association of a microorganism with a plant disease does not necessarily proven  that the microorganism is the causal agent. The identification of causal agent should be confirmed by a related manually and if not, the pathogenicity of the microorganism must be confirmed using the Koch Postulates.

Objective

1. To identify the disease of the plant through symptom observation.
2. To differentiate the isolation techniques depending on the type of pathogen from diseased materials.
3. To identify the disease of the plant through Koch Postulates.

Materials
Disease samples, Petri dish, slides, and culture media.

Methods
Activity 1: Identification of plant diseases through sign and symptoms

1. Diseased samples were identified and observed 

Activity 2: Isolation techniques of pathogens from diseased materials

1. The causal agents from diseased specimens were isolated
2. The isolated causal agents were cultured in PDA plated and Na for further observation

Activity 3: Koch Postulates

1. Pure culture of 2 fungal pathogens, Colletrichum truncatum and C.capsici , diseased chilli fruit, healthy chilli fruit, inoculation needle, plastics container, filter paper and PDA plates were given.
2. Slides from both pure culture were prepared and observation was made under light microscope.
3. The causal agent was isolated from the disease tissue into PDA plates by using aseptic technique. The plates at room temperature for observation in the next practical are labelled and incubated
4.  Aseptically, 1 agar block containing hyphae from each pure culture provided were cut and each species of pathogen on a sterilized chilli fruit were inoculate. The third chilli fruit are uninoculates as a control. The fruit in a moist tray were keep and covered with a pastics sheet. The fruit was incubate for 3 – 5 days and the symptom were observed.
5.  The isoaltion processes in (3) were repeat using fruit that demonstrated similar symptom as in (3) above (step 4). 

Result and Discussion

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

General necrosis:

Soft root	Most symptoms are along the lines of watery and soft decay of the tissue.Often there is a change in colour and in the case, the whole taproot can be decayed leaving just the epidermis.
Vascular wilt	Avascular wilt disease appears first as premature yellowing or other discoloration of the leaves, while the stems and leaf petioles remain green. Plants may show bunching of upper leaves and shortening of stem internodes, creating a rosetting symptom.
Damping-off	Affects seeds and new seedlings, damping off usually refers to the rotting of stem and root tissues at and below the soil surface. In most cases, infected plants will germinate and come up fine, but within a few days they become water-soaked and mushy, fall over at the base and die.
Blight	Symptoms include sudden and severe yellowing, browning, spotting, withering, or dying of leaves, flowers, fruit, stems, or the entire plant.
Blast	Initial symptoms appear as white to gray-green lesions or spots, with dark green borders.Older lesions on the leaves are elliptical or spindle-shaped and whitish to gray centers with red to brownish or necrotic border.
Dieback	Common symptom or name of disease, especially of woody plants, characterized by progressive death of twigs, branches, shoots, or roots, starting at the tips. Staghead is a slow dieback of the upper branches of a tree; the dead, leafless limbs superficially resemble a stag’s head.

Local necrosis:

Scab	Leaves of affected plants may wither and drop early. Plant diseases characterized by crustaceous lesions on fruits, tubers, leaves, or stems. The term is also used for the symptom of the disease.
Leave spot	Leaf spot initially resembles drought or insect damage, and it can be difficult to tell the difference.Leaf spot grows in random patterns on lawns, not in any particular, recognizable shapes.
Anthracnose	Anthracnose causes the wilting, withering, and dying of tissues. It commonly infects the developing shoots and leaves.Symptoms include sunken spots or lesions (blight) of various colours in leaves, stems, fruits, or flowers, and some infections form cankers on twigs and branches.
Rust	Early on, look for white, slightly raised spots on the undersides of leaves and on the stems. After a short period of time, these spots become covered with reddish-orange spore masses. Later, leaf postules may turn yellow-green and eventually black. Severe infestations will deform and yellow leaves and cause leaf drop.
Downy mildew	Older lesions turn brown and appeared shrivelled. Mycelium of fungus forms mats and appears as white, grayish white or tan colored patches on leaves, buds, stems or young fruit. Fruiting bodies (cleistothecia) appear as small black or brown specks on the mycelial mats. Infected leaves often appear chlorotic due to decreased photosynthesis. Infected fruit and flowers are often aborted or malformed. Early signs include small chlorotic spots or blistering on leaves or flowers
Powdery mildew	Infected plants display white powdery spots on the leaves and stems. The lower leaves are the most affected, but the mildew can appear on any above-ground part of the plant.
Canker	Symptoms include round-to-irregular sunken, swollen, flattened, cracked, discoloured, or dead areas on the stems (canes), twigs, limbs, or trunk. Cankers may enlarge and girdle a twig or branch, killing the foliage beyond it.

Hypertrophy and hyperplasia:

Gall	Symptoms include roundish rough-surfaced galls (woody tumourlike growths), several centimetres or more in diameter, usually at or near the soil line, on a graft site or bud union, or on roots and lower stems. The galls are at first cream-coloured or greenish and later turn brown or black.
Smut	Smut is characterized by fungal spores that accumulate in sootlike masses called sori, which are formed within blisters in seeds, leaves, stems, flower parts, and bulbs. The sori usually break up into a black powder that is readily dispersed by the wind. Many smut fungi enter embryos or seedling plants, develop systemically, and appear externally only when the plants are near maturity. Other smuts are localized, infecting actively growing tissues.
Witches broom	Can be easily identified by the dense clusters of twigs or branches, which grow from a central source resembling a broom. It is best seen on deciduous trees or shrubs when they are not in leaf.

Hypoplasia:

Mosaic

Symptoms are variable but commonly include irregular leaf mottling (light and dark green or yellow patches or streaks). Leaves are commonly stunted, curled, or puckered; veins may be lighter than normal or banded with dark green or yellow. Plants are often dwarfed, with fruit and flowers fewer than usual, deformed, and stunted.

Activity 2: Isolation techniques from diseased materials

Figure 3.0

After 4 days, the isolated causal agents from diseased specimen (Chilli) have grown in the Petri Dishes respectively (Figure 3.0). However, the fungus that we cultured in the PDA is neither alike as ColletotrichumcapsicinorColletotrichumtruncatumbased on the physical appearance of the pure culture (Figure 3.1) and (Figure 3.2)

Figure 3.1: Pure culture of Colletotrichumtruncatum

Figure 3.2: Pure culture of Colletotrichumcapsici

The slides (Figure 3.3) that we had made from our cultured media show the mixture types of fungus instead of ColletotrichumcapsiciorColletotrichumtruncatum. Therefore, this shows that the cultured media is contaminated with other type of fungi.

Figure 3.3

Based on the slide of our cultured media, the morphology of the fungus is not the same as the targeted fungus which are ColletotrichumcapsiciorColletotrichumtruncatumif compare to the slide from the pure culture (Figure 3.4 &3.5). For example, the fungus does not have the sickle-like shape conidia as in Colletotrichumcapsici.

Figure 3.4: Slide from pure culture of Colletotrichumcapsici

Figure 3.5: Slide from pure culture of Colletotrichumtruncatum

Activity 3: Koch Postulates

Pure culture

Colletotrichumtruncatum	Colletotrichumcapsici
Slide	Slide

Figure 3.6

In Figure 3.6, this is the result of the Koch Postulate process after 4 days. The healthy chillies that are wounded and inoculated with the fungi disease now has shown the sign and symptoms of infection of Colletotrichum capsica and Colletotrichumtruncatumfungi.

Figure 3.7

Figures 3.8

The closer look of the chili that is inoculated with Colletotrichumcapsici after 4 days( figure 3.7 and 3.8). The infected chili started to show the infected symptom which is sunken at infected part and growth of whitish mycelium within the wound.

Figure 3.9

Figure 3.9 shows the closer look of chili that is inoculated withColletotrichumtruncatum for 4 days. The wounded part of the chili show little or no infected symptoms of Colletotrichumtruncatum. This is because the fungus pathogen, Colletotrichumtruncatumused for inoculation is too little in the amount. Therefore, the infection process of the pathogen require a longer period of time. Hence in Figure 3.9, the wounded part of the chili did not appear the symptoms of infection.

Conclusion

In conclusion, disease in plant can identify though observation on disease symptoms or signs of the presence of causal agents on plant parts. In most diseases, the pathogens live or produce various kind of structures on the surfaces of host. These structures include mycelia, sclerotia, sporophores, fruiting bodies and spores which are called signs and they are different symptoms which show visible responses on the infected part of the host plant.