Practical 3: Disease Diagnosis: Symptom, Isolation and Pathogenicity Test

DISEASE DIAGNOSIS: SYMPTOM, ISOLATION AND PATHOGENICITY TEST

INTRODUCTION

In general, A symptom of plant disease is a visible effect of disease on the plant. Symptoms may include a detectable change in color, shape or function of the plant as it responds to the pathogen. Pathogens cause 3 types of symptoms which is necrosis (host cell), overgrowth for example hypertrophy (abnormal cell enlargement) and hyperplasia (fast cell division) and hypoplasia (stunting of host organs or overall). In most disease,   the pathogens live or produce various kind of structures on the surface of the host plant. These structures include mycelia, sclerotia, sporophores, fruiting bodies and spores which called signs and they are different from symptoms which show visible responses on the infected part of the host plant. Other than that, for isolation techniques of a pathogen from diseased materials, it is used depends on the growth, reproduction, and ecology of the microorganism. There is four Isolation technique of pathogen which are isolation technique for fungi, isolation technique for bacteria, a technique to produce pure culture for fungi and technique to produce pure culture for bacteria. Last but not least, Pathogenicity tests of fungi or bacteria are done by artificial inoculation of the pathogen inoculum applied on the host surface. Depending on the penetration mechanism of the pathogen one has to make wounds on the host surface but others that penetrate through natural openings or directly through the epidermis, the inoculum can be applied directly without a wound. After inoculation, the inoculated plants have to be incubated under favorable conditions for infection (moist chamber, controlled temperature and some times in the dark).

OBJECTIVE

1. To identify plant disease through symptom observation.
2. To study the isolation technique of pathogen from diseased materials.
3. To learn the Koch postulate method for identifying the pathogenicity of the microorganisms.

MATERIAL AND APPARATUS

    1.   Pure culture of 2 fungal pathogen specimen

    2.   Chilli fruit

    3.   Petri dish

    4.   Forceps

    5.   Inoculation needle

    6.   Plastic container

    7.   Filter paper

    8.   PDA plates

    9.   Slides

   10.  Spirit lamp

   11.  Scalpel

   12.  Coverslips

   13.  Lactophenol Cotton Blue (LCB)

   14.  Microscope

METHODS

1)     Identification of plant disease through signs and symptoms observation

    i. The various sign and symptoms shown in the provided specimens have been observed and captured.

       ii.   We briefly described the symptoms and wrote the names of the host plant and causal agent.

2)     Isolation techniques of pathogens from diseased materials

        i.    The causal agents from diseased specimens provided have been isolated. Used the PDA plates for isolation from leaf or fruit lesions and NA for wilt symptom.

3)     Koch Postulates

      i.    We are given pure culture of 2 fungal pathogens, Colletotrichum truncatum and Colletotrichum capsici, diseased chili fruit, healthy chili fruit, inoculation needle, plastic container, filter paper, and PDA plates.

     ii.       Slides from both pure cultures have been prepared. The slide has been observed under the light microscope. We captured the image shown inside the microscope and label the structure produced by pathogens including microscopic characteristics of the colonies.

    iii.    The sign and symptoms of diseased chili fruit have been captured and observed. We used the aseptic technique, the causal agent from the diseased tissues has been isolated onto PDA plates. The plates were labeled and incubated at room temperature for observation in the next practical.

     iv.    Aseptically cut 1 agar block containing hyphae from each of the pure cultures third chili fruit which is un-inoculated is used as a control. The fruits kept in a moist tray and covered with a plastic sheet. After that, the fruit incubated for 3-5 days and observed the symptom exhibited if any suspected causal agent is inoculated into a healthy susceptible host plant, the host must reproduce the specific disease.

       v.    All results and observations that we have done has been recorded in a table and us discussed the conclusion of the experiment.

RESULT AND DISCUSSION

ACTIVITY 1: IDENTIFICATION OF PLANT DISEASE THROUGH SIGNS AND SYMPTOMS OBSERVATION

General necrosis:

Soft rot	Soft rot diseases are caused by pathogens that secrete enzymes capable of decomposing cell wall structures, thereby destroying the texture of plant tissue which the tissues become soft with a water-soaked appearance that often spreads rapidly, followed by the development of fuzzy gray mycelium and black spores. Soft rots commonly occur on fleshy vegetables such as potato, carrot, eggplant, and tomato.
Vascular wilt	Vascular wilt results from the bacterial invasion of the plant’s vascular system. The subsequent multiplication and blockage prevent movement (translocation) of water and nutrients through the xylem of the host plant. Drooping, wilting, or death of the aerial plant structure may occur. For example bacterial wilt of sweet corn, alfalfa, tobacco, tomato, and cucurbits
Damping-off	Species of Pythium and Rhizoctonia are most important among the fungi which cause damping-off disease of seedlings. Either the stem is attacked near the soil level or the crown of roots. The attacked region becomes weak and thus is not able to bear the load of the upper portion of the seedling. Consequently, the seedling collapses. It topples down and dies. The seedling of many plants such as chili, tobacco, tomato, and mustard are prone to damping-off disease.
Blight	In many cases the leaves, stems or twigs, in response to the attack of the pathogen undergo rapid discoloration followed by death. The dead parts become dark or brown in color. This condition of the affected organ is called the blight.

Blast

The sudden death of unopened buds or inflorescence is referred to a blast. Magnaporthe grisea, is a plant-pathogenic fungus that causes a serious disease affecting rice. Initial symptoms are white to gray-green lesions or spots with darker borders produced on all parts of the shoot, while older lesions are elliptical or spindle-shaped and whitish to gray with necrotic borders. Lesions may enlarge and coalesce to kill the entire leaf.

Dieback

Necrosis of woody tissue often brings about various types of die-back symptoms. Dieback is the extensive necrosis of a shoot from its tip downwards.

Local necrosis:

Scab	Citrus scab is causes by pathogen named Elsinoë fawcettii. The early symptom is Lesions on young leaves begin as minute water-soaked spots which subsequently evolve into amphigenous, creamy-yellowish or variously bright-colored pustules. These grow as irregular, globose or conical excrescences which coalesce and extend mostly along the main veins to cover a large part of the leaf blade, particularly on the lower surface. The central area of these wart-like outgrowths is depressed and becomes drab, greyish and velvety when the fungus is fruiting.
Leave spot	Leaf spots are classically thought of as tan to brown, local lesions with distinct borders or delimited borders on leaves or flower parts. When the spots become numerous and begin to touch each other the disease is called a blight or blotch or less commonly, scorch. This category can be confused with anthracnose diseases, many of which are also leaf spots.
Anthracnose	Mango anthracnose is caused by Glomerella cingulate. The anthracnose pathogen invades inflorescences, fruit, leaves, and stems of mango. Leaf anthracnose appears as irregular-shaped black necrotic spots on both surfaces of the mango leaf. Lesions often coalesce to form large necrotic areas, frequently along the leaf margins. Severely affected leaves usually curl.
Rust	Rust, plant disease caused by more than 4,000 species of fungi and funguslike organisms of the phylum Oomycota. Rust affects many economically important plant species and usually appears as yellow, orange, red, rust, brown, or black powdery pustules on leaves, young shoots, and fruits. Plant growth and productivity are commonly reduced, and some plants wither and die back.

Downy mildew	Downy mildew is a disease of the foliage, caused by a fungus-like (Oomycete) organism. It is spread from plant to plant by airborne spores. It is a disease of wet weather as the infection is favored by prolonged leaf wetness. The downy mildew is all internal obligate parasites. They are characterized by superficial downy growth consisting of conidiophores and conidia on the host lesions in damp and warm weather.
Powdery mildew	Powdery mildew is a fungal disease that affects a wide range of plants. Powdery mildew diseases are caused by many different species of fungi in the order Erysiphales, with Sphaerotheca fuliginea) being the most commonly reported cause. The powdery mildew is external parasites in which the mycelium forms whitish patches on the surface of the leaves of the host plant. The patches appear dusty or powdery with the formation of numerous white conidia which form a coating on the host surface.
Canker	Canker is a dead lesion. Sometimes it is surrounded by a raised margin. Cankers may be due to slow rots of the outer parts of herbaceous and woody stems. They may also be restricted in extent due to the formation of cork around and edges of the wound in the woody plants. When the canker partly encircles the branch its growth is subnormal. The branch is underdeveloped. If the canker completely encircles the branch the part of the branch beyond the canker dies.

Hypertrophy and hyperplasia:

Gall

Galls are abnormal growths that occur on leaves, twigs, roots, or flowers of many plants. Most galls are caused by irritation and/or stimulation of plant cells due to infections by bacteria, fungi, or nematodes. Galls may appear as balls, knobs, lumps, or warts, each being characteristic of the causal organism. Oaks are one of the most susceptible plants. Plant gall damage is usually an aesthetic problem and is not considered serious. Affected trees ordinarily show little injury, although foliage of young trees is sometimes completely deformed. On ornamental trees, this condition can be unsightly.

Smut

Smut, plant the disease primarily affecting grasses, including corn (maize), wheat, sugarcane, and sorghum, caused by several species of fungi. 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

Witches’-broom, a symptom of plant disease that occurs as an abnormal brush like a cluster of dwarfed weak shoots arising at or near the same point; twigs and branches of woody plants may die back. Susceptible plants include alder, alfalfa, Amelanchier, birch, and California buckeye,

Hypoplasia:

Mosaic

Mosaic, plant disease caused by various strains of several hundred viruses. 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 the 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 Colletotrichum capsici nor Colletotrichum truncatum based on the physical appearance of the pure culture (Figure 3.1) and (Figure 3.2)

Figure 3.2: Pure culture of  Colletotrichum capsici

The slides (Figure 3.3) that we had made from our cultured media show the mixture types of fungus instead of Colletotrichum capsici or Colletotrichum truncatum. Therefore, this shows that the cultured media is contaminated with another 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 Colletotrichum capsici or Colletotrichum truncatum if 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 Colletotrichum capsici.

Figure 3.4: Slide from a pure culture of Colletotrichum capsici

Figure 3.5: Slide from a pure culture of Colletotrichum truncatum

ACTIVITY 3: KOCH POSTULATES

Figure 3.6

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

Figure 3.7

Figure 3.8

The closer look of the chili that is inoculated with Colletotrichum capsici 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 with Colletotrichum truncatum for 4 days. The wounded part of the chili shows little or no infected symptoms of Colletotrichum truncatum. This is because the fungus pathogen, Colletotrichum truncatum used for inoculation is too little in the amount. Therefore, the infection process of pathogen requires 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, from this experiment, we get to know how to identify plant disease through symptom observation from our naked eyes. Other than that, we have studied the technique to isolate the pathogen from diseased material through some different techniques taught in the class. Next, from this experiment, we also get to learn how to prepare the slides which are using for identifying and knowing the plant disease symptom. We also learn how to use the microscope properly. Finally, Koch Postulate method is an important technique to confirm the pathogenicity of the microorganisms and plant disease symptoms. 

REFERENCE

Online

1.   https://www.britannica.com/science/plant-disease/Symptoms-and-signs

2.   https://homeguides.sfgate.com/necrosis-plant-diseases-39504.html

3.   https://www.planetnatural.com/pest-problem-solver/plant-disease/mosaic-virus/

Literature cited

1.   Susheel Kumar Pandey, A.C. Mathur, and Manisha Srivastava, 2010. Management of Leaf Curl Disease of Chilli (Capsicum annuum L.). International Journal of Virology, 6: 246-250. Retrieved from, https://scialert.net/abstract/?doi=ijv.2010.246.250