BSc 2nd Year Viruses as Plant Pathogens Notes Study Material

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BSc Viruses as Plant Pathogens Notes Study Material

Viruses are too small to be seen with light microscope. They multiply only in live cells, and have the ability to cause disease. They parasitise unicellular plants or animals to large trees and mammals. Viruses attack man and/or animal (causing diseases like influenza, rabies, polio, smallpox, AIDS) as well as bacteria and mycoplasmas. More than half of all known viruses (total number more than a thousand) attack and cause diseases of plants. A single virus can infect different species of plants, and one plant may be attacked by many viruses.

Viruses are remarkable in the sense that they behave as chemical molecules. Each virion consists of nucleic acid and protein, the latter wrapped around the nucleic acid. There is only RNA or DNA, never both in a virus, and in most plant viruses, only one kind of protein. Viruses do not divide and do not produce any reproductive structures as spores. But they multiply by inducing the host cells to form more virus. They cause disease not by consuming cells or killing them with toxins, but by upsetting the cell metabolism, which in turn leads to production of abnormal substances by the cell. These are injurious to life of the plant.

General Characteristics of Plant Viruses

Plant viruses differ greatly from other plant pathogens (fungi, bacteria, etc.) not only in size and shape but also in the simplicity of their chemical and physical structure, methods of infection, multiplication, translocation within the host, dissemination and the symptoms they produce on their hosts.

1. Detection. Mere presence of a virus in the infected cell does not mean that it causes a disease. Some viruses may be seen in sections of cells or crude sap from infected part under electron microscope. For most plant viruses, it has to be proved that characteristic symptoms are caused by that particular virus (proof of pathogenicity). This job requires (i) the elimination of every other possible cause of disease, and (ii) transmission of the virus from diseased to healthy plants in a way that would exclude transmission of any other causal agents.

The current methods for detection of plant viruses involve primarily transmission of the virus from a diseased to a healthy plant by budding, gratis or by rubbing with plant sap. Certain other methods of transmission, as dodder insect vectors, are also used to demonstrate the presence of virus. Out of these, only transmission through plant sap is considered as proof of the viral nature of the pathogen. The most authentic proof of the presence of a virus in a plant is provided microscopy and or serology. We shall describe some later under “Techniques in plant virology”.

2. Morphology. Plant viruses differ in shapes and size. Usually they are elongate (rigid rods or flexous threads), as rhabdoviruses (bacillus-like), or spherical (isometric or polyhedral). The elongated ones are as tobacco mosaic virus, maize dwarf mosaic virus, barley stripe mosaic virus etc., whereas the spherical forms include cowpea chlorotic mottle virus, potato yellow dwarf virus, wheat mosaic virus, lettuce necrotic yellow virus etc. Some elongated viruses as TMV and barley stripe mosaic virus measure about 15 X 300 nm and 20 X 130 nm respectively, whereas most elongated viruses are usually 10 to 13 nm wide, and from 480 nm to 2000 nm in length.

Among rhabdoviruses potato yellow rhabdoviruses potato yellow dwarf virus measures 75 X 380 nm wheat mosaic virus, 65 x 270 nm and the lettuce necrotic yellow virus 52 x 300 nm.(Viruses as Plant Pathogens Notes Study Material)

Most spherical viruses are polyhedral, ranging in diameter from 17 nm to 60 nm. Most plant viruses consist of more than one component. Tobacco rattle virus has two rods, a long (195 X 25 nm) and a shorter (43-110 X 25 nm), alfalfa mosaic virus consists of five components measuring 58 X 18,54 X 18,42 x 18,30 X 18 and 18 x 18 mm. Most isometric viruses have two or three different components.

The surface of plant viruses consist of a definite number of protein subunits, which are spirally arranged in elongated viruses, and packed on the sides of polyhedral particles of the spherical viruses. In cross-sections, elongated viruses appear as hollow tubes with the protein subunits forming the outer coat and the nucleic acid, also spirally arranged, embedded between the inner ends of two successive spirals of the protein subunits. The spherical viruses may or may not be hollow, the visible shell consisting of the protein subunits, with the nucleic acid inside the shell and arranged in yet unknown manner. The rhabdoviruses, potato yellow dwarf, lettuce necrotic yellow virus etc. are provided with an outer envelope or membrane bearing surface projections. Inside the menibrane is the nucleocapsid, consisting of helically arranged nucleic acid and associated protein subunits.(BSc Viruses as Plant Pathogens Notes Study Material)

3. Composition and structure. A plant virus consists of at least a nucleic acid (NA) and a protein (P). Some viruses consist of more than one size of nucleic acid and proteins, and some of them contain additional chemical compounds as lipids, polyamines or specific enzymes.

The proportions of NA and P vary with each virus, NA making up 5 to 40% making up the rest 60 to 95%. Normally, elongated viruses contain higher protein percentages and lower nucleic acid, whereas the reverse is true for spherical viruses. The total weight of nucleoprotein of different viral particles varies from 4.6 million mol. wt. units (bromegrass virus) to 39 million mol. wt. unit (TMV) to 73 million mol. wt. units (tobacco rattle virus). The weight of nucleic acid alone ranges only between 1 and 3 million (1-3 X 10⁶) mol. wt. units per virus particle.

The protein components are composed of repeating subunits. Amino acid content and sequence varies for different viruses, different strains of the same virus, and even for different proteins of the same particle. The complete sequence of amino acids are known only for proteins of TMV and TYMV (turnip yellow mosaic virus). Protein subunit of TMV consists of 158 amino acids in a constant sequence, whereas that of TYMV has 189 amino acids. In TMV, protein subunits are arranged in a helix containing 16 1/3 subunits per turn (49 subunits per three turns). The central hole of the virus particle down the axis has a diam. of 40A, whereas the maximum diam. of the particle is 180 A. Each TMV particle consists of approx. 130 helix turns of protein subunits. The nucleic acid is packed tightly between the helices of protein subunits. In rhabdoviruses the helical nucleoproteins are enveloped in a membrane. In polyhedral viruses the protein subunits are tightly packed, producing 20, or some multiple of 20 facets and form a shell. Within this shell the nucleic acid is folded or otherwise organised.

The nucleic acid of most plant viruses consists of RNA. Only three plant viruses (cauliflower mosaic, dahlia mosaic and carnation etched ring) have been shown to contain DNA. Both DNA and RNA are long chainlike molecules consisting of hundreds or, more often, thousands of units-nucleotides. RNA usually exists as single strands, though in some viruses also as double-stranded.

Protein coat provides protection to nucleic acid. This itself has no infectivity, though its presence generally increases the infectivity of nucleic acid. Nucleic acid alone is responsible for the synthesis and assembly of both the RNA and the protein. Infectivity is strictly the property of nucleic acid which in most plant viruses is RNA. The code for sequence of amino acids consists of codons-each codon is a triplet.(BSc Viruses as Plant Pathogens Notes Study Material)

Replication of Virus (Infection and Virus Synthesis)

Plant viruses enter cells only through wounds made mechanically or by the vectors, or by deposition into an ovule by an infected pollen grain.

The RNA is freed from protein coat. It then induces formation (by the cell) of RNA-polymerases (= RNA-synthetases = RNA-replicases). These, in presence of viral RNA (acting as a template) and nucleotides that compose RNA, produce additional, RNA. The first new RNA produced is not viral but a strand that is a mirror image of the virus, and which, as it is formed, is temporarily connected to the viral strand. Thus, the two form a double-stranded RNA that soon separates to produce the original virus RNA and the mirror image strand, the latter then serving! as a template for more virus RNA synthesis. The replication of some single-stranded RNA viruses that have parts of their RNA in two or more virus particles, of some rhabdoviruses and of some double-stranded RNA viruses differs much from this method of replication. As soon as new viral nucleic acid is produced it induces the host cell to produce the protein subunits that will form the coat of the virus. For TMV, whose RNA consists of 6400 nucleotides and its protein of 158 amino acids, only 474 nucleotides are required to code the sequence of amino acids in protein subunit.

For viral protein synthesis, the part of the viral RNA coding for the viral protein plays the role of m-RNA. The virus utilises the amino acids, ribosomes, and t-RNAs of the host, but it becomes its own m-RNA and the protein formed is for exclusive use by the virus as a coat or other functions.

During virus synthesis, part of its nucleic acid also become involved with synthesis of extra proteins (other than coat). Some of these are enzymes, that may activate or initiate in the cell chemical reactions that may affect the cell physiology. After production of new viral nucleic acid and viral protein subunits, cid appears to organise the protein subunits around it, and the two assembled together to form the complete virus particle, the virion. What are the sites in host cell where viral RNA and protein are synthesised, and the two components assembled? It is known for some viruses.  For TMV, it is shown that free RNA moves into the nucleus and perhaps the nucleolus where it replicates itself. The new viral RNA is then released into the cytoplasm, where it serves as m-RNA, and with the help of ribosomes and t-RNAs, it produces the protein subunits. The assembly of virion occurs also in cytoplasm. In other viruses, all these events occur in the nucleus, from which virions are released into cytoplasm.

The first intact virions appear in plant cells approx. 10 hr. after inoculation The virions may exist singly or in groups and may form amorphous or crystallin inclusion bodies within the cell (cytoplasm, nucleus, nucleolus) in which they are produced.(BSc Viruses as Plant Pathogens Notes Study Material)

Translocation and distribution in plants

The virus moves from one cell to another, and while moving it multiplies in most cells. The viruses move through the plasmodesmata connecting adjacent cells. They infect and multiply in parenchyma cells, and then invade continuously and directly cell-to-cell. In leaf parenchyma cells the virus moves approx. 1 mm or 8 to 10 cells per day. Moreover, besides this cell-to-cell movement, many viruses are rapidly transported over long distances through the phloem, in the sieve tubes moving as rapidly as 15 cm in first 6 min. Once within the phloem, virus moves rapidly in phloem towards apical meristems or other regions as tubers, rhizomes.

The distribution of viruses within plant varies with the virus and the plant. There may be localised distribution to one area of a part or one part of the plant, resulting into local lesions development. In other cases there may be systemic development, when they involve all live cells of the plant.

Symptoms Caused by Plant Viruses

Viruses cause several kinds of symptoms in diseased plants. The viruses may be systemic, associated with each stage of life cycle of plant, or localised, being restricted to some parts of plant only. In almost all viral diseases occurring in the field, the virus is present throughout the plant (systemic infection) and the symptoms are called systemic symptoms. In many artificially inoculated plants and in some natural infections, viruses form local lesions (local infection).

The most common and sometimes the only kind of symptoms is reduced growth rate of the plant. This results into various degrees of dwarfing or stunting of the entire plant. All viral diseases cause some degree of reduction in total yield.

The most obvious symptoms are usually those appearing on the leaf, but some viruses may cause striking symptoms on the stem, roots and fruit with or without symptom development on the leaves. Many viruses may infect some hosts without causing development of visible symptoms. Such viruses are called latent viruses and the hosts are called symptomless carriers. In some cases, there develop symptoms, with some virus remaining temporarily symptomless under certain environmental conditions (high/low temp.). Such symptoms are called masked. Finally, plants may show acute or severe symptoms soon after infection.

The most common types of plant symptoms produced by systemic i infections are mosaics and ringspots. A large number of other less com symptoms are stunt, dwarf, leaf roll, yellows, streak, pox, enation, tumors, pin of stem, pitting of fruit, flattening and distortion of stem etc. These symptoms be accompanied by other symptoms on other parts of the same plant. Brief notes on the different kinds of symptoms now follow:

1. Mosaics. These are characterised by light-green, yellow, or white areas intermingled with the normal green of the leaves or fruit, or of whitish area intermingled with areas of the normal colour of flower or fruit. Depending on the intensity or pattern of discolorations, mosaic-type symptoms may be described as mottling, streak, ring pattern, line pattern, vein-clearing, veinbanding, veinthickening, chlorotic spotting etc.
2. Mottles. It is a kind of mosaic, where on the leaves there develops an irregular pattern of indistinct light and dark areas. Like mosaics, there are green and white or green and yellow areas.
3. Yellows (chlorosis). In extreme cases of mosaics and mottles the leaf may become almost completely yellow due to chlorosis.
4. Vein-clearing. In this case there is chlorosis of leaf tissue in close proximity to the veins. The tissue close to veins turns yellow, remaining area appears green. This is very common in bhindi.
5. Vein-banding. Here the parenchyma close to the veins is green and rest of the lamina surface shows chlorosis i.e. becomes yellow.
6. Ring spots. These are characterised by the appearance of chlorotic or necrotic rings on leaves and sometimes also on fruit and stem.
7. Enations. These are small outgrowths on leaf, stem etc. This is usually associated with mosaics. Ex. tobacco enations.
8. Leaf-curling or leaf-rolling. These are common in papaya, tomato, potato etc., where leaves become curled and rolled to varying extents.
9. Fern-leaf, shoe string. Here leaf lamina is greatly suppressed.
10. Stunting. The general growth of entire plant is affected resulting into unusually shorter size of plant.
11. Virescence. Here entire flower or petals turn to green colour. It is a type of phyllody.
12. Tumors. These are gall-like structures developing on roots or stems. In Fiji disease of sugarcane, elongated galls on leaf are formed.
13. Witche’s broom. Here leaves become very much reduced, internodes are also shortened. There is abnormal growth of leaves turning to a den packed broom-like structure.
14. Little leaf. Here the leaves are reduced in size. In fern-leaf, there is much suppression of the lamina.

Transmission of Plant Viruses

Plant viruses rarely come out of the plant spontaneously. Therefore, viruses are not disseminated by wind or water. Even when they are carried in debris plant sap, they would cause infections only when come in contact with the contents of wounded live cell. Viruses are transmitted from plant to plant (i.e. they actually spread) in a number of ways-vegetative propagation, mechanical transmission through sap, and by seed, pollen, insects, mites, nematodes, fungi dodder etc.

1. Vegetative propagation. Any part of plant used for vegetative propagation will transmit the viruses to the progeny. Thus viruses are transmitted by budding, grafting, cuttings, tubers, corms, bulbs, rhizomes etc. This mode of transmission is most important for ornamental trees and shrubs (propagated by budding, grafting or cuttings) and the field crops like potato and most florist’s crops, that are usually propagated by tubers, corms or cuttings.

Particularly in trees, viruses are transmitted through natural root grafts of adjacent plants. For several tree viruses, natural grafts are the only known means of tree-to-tree spread of the virus.(Viruses as Plant Pathogens Notes Study Material)

2. Mechanical transmission through sap. Under natural conditions direct transfer of sap through contact of one plant with another is uncommon. Such mechanical transmission may take place between closely placed plants. Strong wind may cause the leaves of adjacent plants to rub together, and if wounded, some of their sap is exchanged, thus transmitting any virus present in the sap. Potato virus X (PVX) is most easily transmitted in this way.

Plants are wounded by man during cultural practices in field or greenhouse, and some of the virus-infected sap adhering to tool tally transferred to wounded plants. TMV on tobacco and tomato spreads rapidly in this way. Occasionally virus infected sap is transferred from one plant to another on the mouthparts or body of animals feeding on and moving among the plants. Mechanical transmission is the only authentic means to prove the ability of infection by a virus.(BSc Viruses as Plant Pathogens Notes Study Material)

3. Seed transmission. This is not as common as the abovesaid two methods. More than 100 viruses are reported to be transmitted by seed. However, only a small portion (1-30%) of the seeds derived from virus-infected plants transmit the virus. In some as tobacco ringspot virus in soyabean, almost 100% of seeds of infected plants transmit the virus. Barley stripe mosaic virus also has high transmission of 50-100% seeds. In most cases the virus comes primarily from the ovule of infected plants.(BSc Viruses as Plant Pathogens Notes Study Material)

4. Pollen transmission. Virus transmitted by pollen not only infects the seed and the seedling growing from it, but more important, it can also spread through fertilised flower and down into the mother plant, that thus becomes infected with the virus. This method is important in stone fruit ringspot virus of some plants.

5. Mite transmission. Members of the family Eriophyidae are shown to transmit nine viruses, including wheat streak mosaic, peach mosaic and fig mosaic viruses. These mites have piercing and sucking mouthparts. Mite transmission appears to be specific, since each of the mite species has a restricted host range. In such cases a particular mite is the only known vector for the virus or viruses transmitted by it. Some of mite- transmitted viruses are stylet borne, whereas others are circulatory. One mite (Tetranychidae, spider mites) is known to transmit Potato virus Y.(BSc Viruses as Plant Pathogens Notes Study Material)

6. Nematode transmission. About 12 viruses are shown to be transmitted by one or more species of three genera of soil inhabiting, ectoparasitic nematodes. Members of the genera Longidorus and Xiphinema are vectors of tobacco ringspot, tomato ring spot, raspberry ringspot, cherry leaf roll, tomato black ring, grape fanleaf viruses etc., whereas those of the genus Trichodorus transmit tobacco rattle and pea early browning viruses. Nematodes feed on roots of infected plant and then move on to roots of healthy plants. Larvae as well as adults can aquire the viruses.

7. Fungus transmission. Olpidium, a root-infecting chytrid fungus is known to transmit at least four viruses, tobacco necrosis, cucumber necrosis, lettuce big vein and tobacco stunt viruses. Four other fungi, Synchytrium, Polymyxa, Spongospora and Pythium transmit potato virus X, wheat mosaic virus, potato mop top virus and beet necrotic yellow vein virus respectively. The viruses are present in or on the zoospores and resting spores.

8. Dodder transmission. Several viruses can spread through the bridge formed between two plans by twining stems of the parasitic phanerogam, dodda (Cuscuta sp). Many viruses spread in this way between plants of widely different taxonomic species.(BSc Viruses as Plant Pathogens Notes Study Material)

9. Insect transmission. The most common and economically the most important method of transmission of viruses in the field is by insect vectors. The order Homoptera which includes aphids and leafhoppers contains the largest number and the most important insect vectors of plant viruses. White flies, the mealy bugs, scale insects and the treehoppers, belonging to this order also transmit viruses but are not economically as important as aphids and leafhoppers. Other insect vectors are true bugs (Hemiptera), thrips (Thysanoptera), bettles (Coleoptera) and the grasshoppers (Orthoptera). The most important vectors, i.e. aphids, leafhoppers and other members of Homoptera, as well as true bugs, have piercing and sucking mouthparts; all other vectors have chewing mouthparts and the transmission by the latter is much less common.

On the basis of virus-vector relationship (where virus is carried by vector, and behaviour of virus within the vector), the viruses are also categorised as follows:

(a) Non-persistent or stylet-borne viruses. These are the viruses carried by insects (with sucking mouthparts) on their stylets.

(b) Persistent or circulative viruses. These are the viruses accumulated by the insects (with sucking mouthparts) internally and, after their passage through insect tissues, they are introduced again into plants through the mouthparts.

(c) Propagative viruses. These are some of the circulative viruses that may multiply in their vectors.

The viruses transmitted by insects with chewing mouthparts may also be circulative or they may be carried on the mouthparts (i.e, stylet-borne).

Aphids are the most important insect vectors of plant viruses and transmit the great majority of all stylet-borne viruses. More than 200 species are known as vectors of plant viruses. As a rule several aphid species can transmit the same stylet-borne virus and the same aphid species can transmit several viruses, but in many cases the vector-virus relationship is quite specific. Aphids generally acquire the stylet borne virus after a brief feeding period on the diseased plant, for only a few seconds (30 seconds or less) and can transmit the virus after transfer to and feeding on a healthy plant for a similar brief period. After acquisition, the aphids remain viruliferous from a few minutes to several hours, after which they can no longer transmit the virus. In some cases of aphid transmission of circulative viruses, aphids can not transmit the virus immediately but must wait several hours ager the feeding. However, once they start to transmit the virus, they continue to do so for many days. The viruses in stylet-borne viruses are borne on the tips of the stylets. Several spcies of leafhoppers are involved in transmission of a number of plant viruses. Leafhopper transmitted viruses cause disturbance primarily in the phloem region. All leafhopper transmitted viruses are circulative and several are known to multiply in the vector je. are propagative. Most of leafhoppers require a feeding period (one to several days) before they become viruliferous, but once they acquire the virus they remain viruliferous for the rest of their lives.(BSc Viruses as Plant Pathogens Notes Study Material)

Control of Plant Viruses

Methods to control viral diseases are generally similar to those used for other pathogens, except that as yet chemicals find little application in virus control, although they may be used for vectors (insecticides). Moreover, vectors involved in viral diseases have also complicated the problem in their control. Following methods are used in their control of viral diseases of plants:

1. Exclusion. The best way to control a virus disease is to keep it out of area through quarantine, inspection and certification systems. The existence of symptomless hosts and the absence of obvious symptoms in seeds, tubers, bulbs and nursery stock make quarantines ineffective. Eradication of all sources of inoculum can be effective in annual crops. Diseased plants should be destructed that would eliminate the inoculum from the field.

2. Control of vectors. Plants may be protected against some viruses by protecting them against the vectors. Controlling the insect vectors and removing the weeds which serve as hosts (for vectors) may help in controlling the disease.

3. Use of virus-free planting material. The use of virus-free seed, tubers, budwood, cuttings etc. is the single most important method for control of viruses, especially those lacking insect vectors. Periodic indexing of mother plants producing such planting material is necessary. Several types of inspection and certification programmes are now in effect in different areas producing seeds, tubers and nursery stock for vegetative propagation.

4. Resistant varieties. If adequate resistance genes are found, this is the most satisfactory method. Many plant varieties resistant to certain viruses been developed.(BSc Viruses as Plant Pathogens Notes Study Material)

5. Physical methods. Viruses can be inactivated by heat. Dormant, propagative parts are dipped in hot water (35 to 54°C) for few minutes to hour whereas actively growing plants are kept in greenhouses or growth chambers at 35 to 40°C for several days, weeks or months.

6. Tissue culture. Virus-free plants can also be produced from virus infected plants by culture of short apical meristems (0.1 mm to 1 cm).

7. Modification of cultural practices. Agricultural and horticultural practices may be modified to reduce viral attack, sometimes through their effect on vectors. Sowing date may be adjusted; other practices include reduction of close spacing, mixed cropping pattern (susceptible and non-susceptible plant species) etc, are effective.(BSc Viruses as Plant Pathogens Notes Study Material)

8. Chemical methods. So for no viricides are available. Foliar application of some growth regulators such as gibberellic acid has been effective in stimulating growth of virus suppressed axillary buds in sour cherry yellows. Similar spray with gibberellic acid can overcome the stunting induced by etch virus on tobacco.

Techniques in Plant Virology

As indicated earlier, one has to prove the pathogenicity of the virus. For this the virus particles are to be isolated from the plant, be purified and then inoculated into healthy plant for studying the development of the symptoms.

There are several methods that are used for isolating the virus and purifying it. Study of symptom development is made through mechanical (sap) transmission method. Following are the methods used.

1. Purification of virus. Isolation or as it is usually called, purification of viruses is done through a series of steps. These steps are shown in Figure and are as follows:

(a) Selection of a suitable buffer System. Since viruses are susceptible to small changes in pH, ionic strength and the type of ions, selection of suitable buffer is very important. Since viruses are also susceptible to oxidation, a reducing agent, as ascorbic acid or Na₂SO₃ is also included in the solution used during grinding.

(b) Grinding of infected tissues. Infected tissues could be ground, chopped, homogenized, squeezed or squashed by hand or motor operated devices. After disintegration of the tissue, the juice is passed through several layers of cheese cloth to remove large particles. The crude juice now contains plant cell organelles, dissolved salts, sugars including the viruses.

(c) Removal of extra particles and solutes. The crude juice is treated in various ways and sedimented by centrifugation. This part is also called clarification. The supernatant liquid above the pellet in the centrifuge tube still contains the virus. Sometimes in addition to centrifugation, the crude juice is filtered through any of the filter acids or the virus is precipitated by organic solvents. With this step most of the host contaminants are removed.

(d) Further purification. Actual separation of the virus particles from other macromolecules is done by several methods, that also remove the solutes. The methods are gel filteration, chromatography on sephadex, sepharose or various agar gels, electrophoresis and ultra-centrifugation.

Of these, the most commonly used technique is ultracentrifugation of the plant sap. This involves three to five cycles of alternate high (40,000 to 100,000g or more) and low (3000 to 10,000 g) speeds. This concentrates the virus and separates it from host cell contaminants. Several modifications of the ultra-centrifugation technique, particularly density gradient centrifugation (adapted by M. Brakke of USDA at Lincoln, U.S.A.) are presently employed with excellent results. Sucrose solutions of decreasing concentrations from 60 % – 5% are layered one on top of another in a plastic centrifuge tube, and allowed to spin in nearly horizontal position. The virus band in the gradient can be clearly seen. The band is collected by inserting a syringe into plastic centrifuge tube.

(e) Concentration and storing of the viral fraction. Once the virus is obtained in a relatively pure state, it can be concentrated by (i) removal of most of the solvent from the suspension, or (ii) removal of the virus from the suspension, Absorption of water by dry gels, ultrafiltration and dialysis are common methods to remove the solvent from the virus suspension, ‘whereas ultracentrifugation, electrophoresis, crystallisation. chromatography and microfiltration are the methods to remove virus from the suspension.

The purified viral particles obtained are then used for infection, electron microscopy and serology.(BSc Viruses as Plant Pathogens Notes Study Material)

2. Infection studies. (Mechanical or sap transmission). The purified viral particles are then mechanically deposited into a healthy host to observe the symptom development. For routine studies, the crude or partially purified sap is prepared from infected parts of the plant (young leaves, floral petals) etc. These infected parts are ground with a mortar and pestle or with some other grinder. A buffer solution (usually phosphate buffer) may be added for stabilization of the virus. Expressed sap is strained through cheesecloth and centrifuged at low speeds (to remove tissue fragments) or at alternate low and high speeds (if more purified virus needed). The crude or partially purified sap is then applied to leaf of young plants, previously dusted with an abrasive such as 600-mesh carborundum added to aid in wounding of the cells. Sap is applied by gentle rubbing the leaves with cheesecloth or gauze pad dipped in the sap with the finger, a glass spatula, a brush or small sprayer. Inoculated plants are kept in greenhouse and observed for the development of symptoms (2-21 days).

3. Serology of viruses. Serology has been useful in identification of plant viruses as well as in their control. Purified virus is injected into a mammal (rabbit, mouse, horse) or bird (chicken). The antibodies thus produced and the virus (to be identified) are brought together in many ways. The most common method is precipitin reaction. They are mixed in solution, or they meet at the interface between two solutions, or they diffuse towards each other through an agar gel and meet in a zone in suitable concentrations (Quchterlony test). In all these reactions, there appears either a precipitate at bottom of test tube or a band at the interface where the two meet (antigen, the virus and the antibody).

Tobacco Mosaic Disease

This is the best known of all virus diseases of plants, and worldwide in distribution. This disease affects more than 150 genera of primarily herbaceous. dicotyledonous plants including many vegetables (potato, tomato, cucurbits), flowers and weeds. There are serious losses in yield as well as quality of tobacco, tomato and some other crop plants. It is symptomless on apple and grape. TMV affects plants by damage of leaf, flower and fruit and causes stunting of the plant.

Symptoms

The symptoms include various degrees of chlorosis, curling, mottling, dwarfing, distortion, and blistering dwarfing, distortion and discoloration of flowers, and in some plants even development of necrotic areas on leaf.

The most common symptom on tobacco is the appearance of mottled dark-green and light-green areas on leaves. The dark green areas are thicker light green areas. Stunting of young plants is common, and is accompanied by a slight downward curling and distortion of leaves, that may become narrow and elongated rather than normal oval shape. The petioles may become enlarged (puckered) with enlarged capitate hairs. Old leaves may not show symptoms, young ones develop typical symptoms.

Causal organism

Tobacco Mosaic Virus (TMV) is rod shaped, 300 nm long by 15 nm in diameter. Protein (P) consists of approximately 2130 subunits and each subunit consists of 158 amino acids. The protein subunits are arranged in a helix. The nucleic acid (NA) is single stranded RNA and consists of about 6400 nucleotides. The RNA strand also forms a helix parallel with that of protein and is located on the protein subunits and approx. 208 out from the inner end of the protein subunits. The mol. Wt. of each virus particle is between 39 and 40 million mol. wt. units.

TMV is one of the most thermostable viruses known, the thermal inactivation point of the virus in undiluted plant juice being 93C. However, in dried infected leaves, the virus retains infectivity even when heated at 120°C for 30 mts. Infected plant may contain up to 4g of virus per litre of plant juice and the virus retains infectivity even at dilutions of 1: 1,000,000. In ordinary plant sap, the virus is inactivated in 4-6 weeks, whereas in sterile bacteria free sap the virus may survive for 5 years, and in TMV infected leaves kept dry in the laboratory the virus remains infectious for more than 50 years. TMV is transmitted readily through mechanical sap, grafting and dodder. It is not transmitted by insects, except occasionally through their contaminated feet and jaw. The most common method of transmission of TMV in field and greenhouse is through hands of workers handling infected and healthy plants.(BSc Viruses as Plant Pathogens Notes Study Material)

Disease development

TMV survives in infected leaves and stalks in the soil, on surfaces of contaminated seeds, and on contaminated seedbed cloth, and in natural leaf and manufactured tobacco including cigarettes, cigars etc. The virus initially infects wounded tissues of tobacco seedlings in seedbed or of transplants in the field. Then it spreads in the field throughout the season. TMV in all plants produces systemic infections, invading all parenchyma cells of plant. The virus moves from cell to cell through phloem. In the cytoplasm of cell TMV appears as crystalline aggregates and as amorphous bodies (x-bodies).

Control

(1) Sanitation is the main method. Crop should not be grown at least for two years in seedbeds or fields where diseased crop was grown. Removal of diseased plants and of some solanaceous weeds harboring the virus early in the season helps in reduction and elimination of subsequent spread of the virus.

(2) Chewing and smoking of tobacco during handling of tobacco and other susceptible plants should be avoided.

(3) Workers in the field must wash hands with 3% trisodium phosphate or soap.

(4) Equipment and instruments used in plantations must be sterilised.

(5) TMV-resistant varieties of tobacco must be grown, though these may be of low quality.(BSc Viruses as Plant Pathogens Notes Study Material)

Yellow Vein Mosaic of Bhindi

This disease of bhindi (Okra) has taken a severe form in our country. Infection of four to five week old plants results into unusual retarded growth and only few leaves and fruits are formed on such plants. Such plants result into about 94% loss of the crop yield. The damage is relatively less on old plants.

Symptoms

The chief symptoms develop on leaves. There occurs vein learing and veinal chlorosis of leaves. Infected leaves exhibit a very distinct yellow network of veins along with the thickened veins and veinlets. In severe cases the chlorosis may involve interveinal areas resulting into yellowing of entire leaf. Fun dwarfed malformed, distorted and yellow green in colour.

Causal organism

The Yellow Vein Mosaic Virus (YVMV) of bhindi is not transmitted mechanically through sap. It may artificially be transmitted through grafting. In the field YVMV is transmitted by whiterfly Bremisia tabaci, and perhaps also by bhindi leafhopper, Empoasca devastans. There are several weed hosts of this virus. These are Croton sparsiflora, Malvastrum tricuspidatum and Ageratum sp., growing along the roadside and in wasteland areas.

Control

(1) Protection of crop from white flies and other insects, by spraying with Follidol (0.3%) or other insecticides. Spray must be done early, just after the seedlings come out, say within three weeks after germination.

(2) Four to six sprays of systemic insecticides, ekatox, metasystox, rogor, etc.

(3) One or two applications of thimet or disyston granules to soil.

(4) Eradication of weed hosts.

(5) Disease resistant cultivars. Some wild species of Hibiscus and Abelmoschus have been found good source of resistance.

Leaf Mosaic of Cucurbits

The leaf mosaic virus disease affects a number of cucurbits in almost every area where they are grown. This disease is very common on several cultivated cucurbits including fruit and vegetable crops. It has a wider host range. One of the viruses on cucurbits for example is the cucumber mosaic, that attacks a greater variety of vegetables, ornamentals, and other plants. Among the most important vegetables and ornamentals attacked by this virus are cucumbers, melons, squash, peppers, spinach, tomato, celery, beets, banana, gladiolus, petunias etc.

Symptoms

Young seedlings are never attacked in the field during the first few weeks. Generally 5-6 week old plants are attacked. Young leaves become mottled, distorted and wrinkled, and their edges begin to curl downward. Plants become dwarfed, and they produce few flowers and fruits. The plants develop bushy appearance with leaves forming a rosettelike clump near the ground. Fruits develop pale green or white areas intermingled with dark green, raised areas.

Causal organism

The virus is polyhedral, 30 nm. diam. The virus consists of 180 protein subunits, single stranded RNA and a hollow core. The mol. wt. is 5.8-6.7 million of which 18% is RNA and the rest 82% protein. The thermal inactivation point is about 70°C, while dilution end point is about 1: 10,000. There are many strains of cucumber mosaic virus alone.

The virus is readily transmitted by sap and also by many aphids. The virus survives in many perennial weeds, flowers and crop plants. Insect vectors and cultivation and handling of plants, especially at picking time spread the virus in field to healthy plants. Sometimes the whole field of cucurbits begin to turn yellow with mosaic, immediately after the first pick. The virus produces systemic infection in cucurbits and most other host plants.

Control

(1) Elimination of weed hosts.

(2) Control of insect vectors.

(3) Resistant varieties.

Potato Spindle Tuber

The disease is caused by a viroid, and causes quite severe losses. In some regions it is one of the most destructive diseases of potatoes. It attacks all varieties and spreads rapidly. Though this disease also attack tomato, but is of little economic value in this crop.

Symptoms

Infected potato plants become erect, spindle-shaped and dwarfed. The leaves are small and erect and the leaflets are darker green, occasionally being rolled and twisted. The tubers are elongated with cylindrical middle and tapering ends. The tubers are smoother, with more numerous, conspicuous and shallower eyes. Yield is reduced to about 25% or more.

Causal organism

The Potato Spindle Tuber Viroid (PSTV) is the first recognised viroid. PSTV is an infectious RNA of low mol. wt., approx 80,000 daltons. The RNA is single stranded molecule with extensive regions of base pairing. Under electron microscope, purifed denatured PSTV appears as short strands about 50 nm long with a thickness of a double stranded DNA. Sap from infected plant is infective even at dilutions of 1: 1000 to 1: 10,000. After heating at 75 to 80°C for 10mts. PSTV is quickly inactivated in expressed sap of infected plant. However infectivity remains intact in phenol treated sap. PSTV is mechanically transmissible. It spreads mainly by knives used to cut healthy and infected potato “seed” tubers and during handling and planting of the crop. This viroid is also transmitted by pollen and seed and by several insects including some aphids, grasshoppers, flea beetles and bugs.(BSc Viruses as Plant Pathogens Notes Study Material)

The viroid infects and then spreads systemically throughout the plant. Little or nothing is known on mechanism of spread within the plant, and the development of symptoms.(BSc Viruses as Plant Pathogens Notes Study Material)

Control

Planting of only PSTV free potato tubers in the field.

BSc Viruses as Plant Pathogens Notes Study Material

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