Practical 8: Plant Virology

Introduction

Viruses are sub-microscopic, infectious particle that multiply only inside living host cells. Viruses are for the most part beyond the resolution capabilities of a light microscope. Consequently, virus structures have been determined primarily by electron microscopy. The basic shapes of plants viruses are elongate (rigid rods or flexuous threads), cylindrical rods (Bacillus-like) and spherical (isometric and polyhedral). Plant viruses  are made up of two components- a protein coat and the nucleic acid center. the nucleic acid is the infectious component of a virus. Viruses are obligate parasites, meaning that they must be within living tissue before they can reproduce themselves. They require a wound to gain entrance to plant cell. In nature, they depend primarily on biological agents such as nematodes, insects, man  for their dissemination. Once duplication starts, the virus is translocated from cell to cell through the plasmodesmata and to distant plant parts by the phloem. 

In general, viruses severely affect the host both in quantity, quality and longevity. To diagnose plant disease caused by viruses, several characterization aspects have to be done. Symptoms may often be very characteristic for a specific virus on a specific host. Symptoms along with other criteria are used to identify virus diseases, which depends on several factors:

1. Symptoms are important indicators of a plant virus disease
2. Symptoms on a diagnostic indicator plants assists in characterizing the virus.
3. The ability to cause symptoms confirms the biological activity of the viruses

A. Virus Morphology

B. Disease Symptoms

C. Mechanical Inoculation

Virus like other plant disease causing agents can be transmitted from plant to plant by sap (mechanical) inoculation. Every virus has a characteristics range of plants species which it is able to infect and in which it induces characteristics symptoms. Thus, the host range and symptomatology of a virus can provide valuable information about the identity of the virus. 

a. Material/equipment required:

• Diseased leaf
• Test host plants
• Mortar and pestle
• Carborundum
• Buffer/Distilled water

Mortar and pestel, Carborundum

Distilled water

Diseased chilli leaf: Mosaic leaf

Test Host Plant

b. The virus inoculum was prepared by crushing the diseased leaf using mortar and pestle until a uniform extract obtained. One or two volume was added (0.5 to 1 ml) of distilled water/buffer to the extract.

Diseased leaf was crushed and distilled water was added.

The diseased leaf was crushed until it become paste.

c. The host plant leaf surface was dusted (to injure the leaf surface) on which virus mix will be inoculated with carborundum abrasive.

Spread carborundum evenly on leaf surface.

Carborundum causes injury on the leaf result in minute openings on the leaf surface.


 

d. The virus inoculum that has been prepared was applied on the test leaf surfaces of the host plant using soft piece of cloth or forefinger. The leaves was inoculated by gently wipe the inoculum acrross the epidermis several time.

The crushed diseased leaf was spread evenly on healthy host leaf.

Distilled water used to uniformly apply the virus into host plant.

e. The leaves was washed that were inoculated with virus using water. This is to remove the excessive leaf pieces and sap from the leaf surface.

The leaf was washed. 

Result

The host plant is easily infected with virus after one day infected with disease.

Practical 7: Phyto bacteriology

Practical 7: Phyto bacteriology

Introduction

Bacteria are a group of microorganisms classified as prokaryote. They have cell walls which are consisted of peptidoglycan layers. Bacteria are smaller than fungal spores and can only be seen clearly using the highest magnification of a light microscope. The classification of bacteria depends on the shape, composition of cell wall and mobility. According to shape, bacteria can be divided into 3 main types it is coccus, Bacillus, and spiral. Gram satin divides bacteria into 2 groups according to the composition of cell wall it is gram positive (with peptidoglycan layer) and gram-negative (with peptidoglycan and an additional outer membrane)

Objective

1.         i.            To study the taxonomy and pathological important of bacteriology.
2.       ii.            To learn the characteristics are shown by each of bacterial and microorganisms.
3.     iii.            To observer the disease cycles by bacterial microorganisms.

Method and material

1.      Morphology

a)      Observe the slide of bacteria.

b)      Draw the shape of bacteria on the Petri plates provided.

c)      Draw the shape of bacteria colonies produced on NA using dilution streak plate.

d)      Observe the color of Gram bacteria positive and negative (purple and red).

2.      Disease symptom

a)      Take it the picture

b)      Observe the symptom by bacteria

3.      Disease cycle

a)      Take the picture.

b)      Observe the cycle of various bacteria.

4.      Bacterial isolation

a)      Wipe a knife, alcohol and Clorox

b)      Cut out canker spot from disease tissue. (direct isolation)

c)      Cut out disease from (b) above is distilled water 10 min (dilution streak)

d)      Sterilize the cut tissue and place on the provided NYGA and NA.

e)      Incubate the petri plate at temperature 27- 30 ˚C for 24- 48 hours.

5.      Diagnostic test using cut plant tissue.

a)      Wash the potato and peel off the skin.

b)      Cut out two potato slices of 7-9 mm thick aseptically.

c)      Cut ‘V’ shape of two potatoes on slices.

d)      Put a drop of sterile water on it. (control)

e)      Inoculate it with Erwinia caratovora subspecies on the ‘V’.

f)       Incubate both plates for 24-48 hours at 30˚C.

g)      Observe for symptom developed.

Result and discussion

Morphology

1	NYGA
2	NA

Disease symptom

Types of disease symptom	Description	Disease
Local necrosis scab	Scab is a serious disease of all lemon varieties the disease also affects Rangpur lime and rough lemon rootstocks. Scab is caused by the fungus Elsinoe fawcettii. Symptoms. Citrus scab attacks the fruit, leaves, and twigs, producing slightly raised, irregular scabby.
Local necrosis Leave spot	Leaf spot is a common descriptive term applied to a number of diseases affecting the foliage of ornamentals and shade trees. The majority of leaf spots caused by bacteria. Some insects also cause damage that appears like a leaf spot disease
Local necrosis Downy mildew	It is caused by the fungus-like water mold Pseudoperonospora cubensis. Once established in a region, the disease can spread rapidly, causing significant loss of fruit quality and yield. Downy mildew symptoms on cucumber leaves.
Local necrosis Canker	Citrus canker is a disease affecting Citrus species caused by the bacterium Xanthomonas. The infection causes lesions on the leaves, stems, and fruit of citrus trees
Local necrosis Powdery mildew	Powdery mildew, mainly caused by the Erysiphe cichoracearum, infects all cucurbits, including muskmelons, squash, cucumbers, gourds, watermelons, and pumpkins. In severe cases, powdery mildew can cause the premature death of leaves, and reduce yield and fruit quality.
Hypertrophy Hyperplasia-gall	Crown gall is a common plant disease caused by the soil-borne bacterium Agrobacterium tumefaciens. It is found throughout the world and occurs on woody shrubs and herbaceous plants including grapes, raspberries, blackberries and roses.
General necrosis Soft rot	Bacterial soft rots are caused by several types of bacteria, but most commonly by species of gram-negative bacteria, Erwinia, Pectobacterium, and Pseudomonas. It is a destructive disease of fruit, vegetables and others.
Local necrosis Rust leaf	Rust on the leaf are very easy to identify on the plant. The disease can be characterized by a rust color on plant leaves and stems. The rust will start out as flecks and will eventually grow into bumps. The plant rust will most likely appear on the underside of the leaves of the plant.

Disease cycle

a)      Erwinia sp.

b)      Xanthomonas oryzae (campestris) oryzae 

c)   Ralstonia (Psuedomonas) solanacearum

d)    Psuedomonos syringae pv. Phaseolicola

e)  Xanthomonas axonopodis (campestris) pv. citri

bacterial isolation

1	NYGA
2	NA

Diagnostic test using cut plant tissue

1	Erwinia
2	Control

Practical 6: Higher Fungi - Taxanomy and Pathological Importance (Phylum Basidiomycota)

INTRODUCTION

The phylum basidiomycota is considered as the most developed group among fungi consisting among others of the mushrooms, toad tools, puffballs and stink horns. The rust and smut fungi are included although considered to be lower within the group. The rust and smut fungi are considered as plants pathogens that caused millions of losses to crops annually. Also included are the root disease pathogens that caused serious damages to many shade and other tree crops. On the positive side, many species of mushrooms are edible and contribute to the food industry.

The basidiomycota differs from the other groups of fungi in that they generally produce sexual spores called basidiospores (usually 4 in number) on a structure called the basidium. This may be formed within a fruiting body called the basidiocarp altough this may be absent in many species.

   A.      PLANT DISEASE SPECIMEN

1)      Coffee leaf rust

Pathogen: Hemileia vastatrix underside of coffee leaves (yellowish spores)

2)      Maize/Corn rust

Pathogen: Puccinia polysora on maize leaf (rust coloured pustule)

DISEASE SYMPTOM

1)      Rust

a.       Groundnut rust

Pathogen: Puccinia arachidis

Symptom: Yellowish coloured pustules on leaves.

b.    Long bean rust

       Pathogen: Uromyces vignae

       Symptom: Yellowish coloured pustules on leaves.

                     

2)      Smut

a. Corn smut

Pathogen: Ustilago maydis

Symptom: Hypertrophy of seeds on the cob.

  

b. Sugarcane smut

Pathogen: Ustilago scitaminae

Symptom: Abnormal shoots, whip like with balck spores.

3. Blister blight of tea

Pathogen: Exobasidium vexans

Symptom: Spots forming blisters on the underside of tea leaves

    

4.  Pink disease

Pathogen: Corticium salmonicolor

Infects stems of rubber, cocoa, tea, mango, coffee

Symptoms: White mycelia turning to pink in color gave disease its name. Fungus does not produce basidiocarp.

5. Horse hair blight

Pathogen: Marasmius equi-crinus

Minor problem on stems rubber and cocoa

Symptoms: Observe black, hair-like rhizomorphs.

5. White thread blight

Pathogen: Marasmiellus scandens

Infects stems of plantation crops such as cocoa and fruit tree crops

Symptoms: White rhizomorphs on stems and leaves.

6. White root disease

Pathogen: Rigidoporus lignosus

Symptoms: White rhizomorphs on roots infected by disease. Serious on rubber and cocoa. Basidiocarps (wooden brackets) formed

7. Red root disease

Pathogen: Ganoderma philippii

Symptoms: Reddish rhizomorphs on roots mainly on rubber. Basidiocarps formed on above.

                                           

8. Brown root disease

Pathogen: Phellinus noxius

Symptoms: Infects roots of rubber and cocoa. May infect damaged stems/trunk and causes upper stem rot in oil palm. Basidiocarps formed.

9. Vascular-streak dieback (VSD) of cocoa

Pathogen: Oncobasidium theobromae

Symptoms: Basidiospores formed on leaf scars during wet weather. Infects young cocoa leaves with symptoms appearance usually only 2-3 months later. Caused stunting and death of seedlings, Leaf clorosis with green islands, rough bark appearance and streaking within vascular tissues. No basidiocarps formed.

10. Bunch rot of oil palm

Pathogen: Marasmiellus palmivorus

Symptoms: Infect oil palm fruits causing rotting of the mesocarps. White rhizhomorph on leaf fronds and fruits bunches. White basidiocarps (soft and mushroom like) on rotten bunches.

11. Basal stem rot (BSR) of oil palm

Pathogen: Ganoderma boninensis

Symptoms: Rots base of palms causing tress to fall. Basidiocarps formed (wooden brackets).