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Transmission of Plant Viruses
Plant viruses do not penetrate the intact plant cuticle. For this reason, viruses are not disseminated as such by wind or water, and even when they are carried in plant sap or debris they generally do not cause infections unless they come in contact with the contents of wounded living cell. Viruses, however, are transmitted from plant to plant in a number of ways, including: 1. 2. 3. 4. 5. 6. 7. Mechanical contact Grafting Vegetative propagation Botanical (sexual) seed Pollen Common dodder Vectors
1. Mechanical contact
Mechanical transmission requires the existence of a wound in the plant and subsequent contact of a healthy cell with infected cellular sap. The wound could be as small as one resulting from the splitting of plant hairs. Under natural conditions, transmission takes place through contact of leaves. In nature, only a few viruses are disseminated by contact: tomato mosaic virus (TMV), potato virus X (PVX), potato virus S (PVS), Andean potato mottle virus (APMV), Andean potato latent virus (APLV), and potato spindle tuber viroid (PSTVd).
A. Transmission by contact between potato tubers or tuber sprouts.
Some viruses may spread when healthy tubers, particularly at the sprouting stage, come into contact with infected tubers in the storehouse. Transmission may also take place when tubers are cut before planting.
B. Transmission by contact between leaves and between roots.
Under field conditions, PVX and PSTVd can be easily transmitted to healthy plants that are in contact with diseased plants. Blowing wind promotes contact between plants and increases the probability of virus dissemination (rubbing effect). This type of dissemination also depends on such factors as cultivar susceptibility, virulence of the virus strain, soil fertility, row spacing, and distance between plants. Transmission by contact appears to be more frequent in greenhouses than under field conditions. This may depend on the physiological state of the plants, which are more succulent and susceptible under greenhouse conditions. Transmission of PVX through roots touching has been observed, but there is no similar evidence for other potato viruses. The most common mechanical transmission of viruses is by the sap from an infected plant rubbing onto the leaves of a healthy plant. To conduct a mechanical transmission test, you need these materials: mortar and pestle, cotton swabs, an abrasive material, virus-infected plant tissue, and inoculation buffer solution. Place the plant tissue in the mortar and add 1 to 10 ml of buffer solution or distilled water per gram of tissue. The most commonly used virusstabilization buffer solutions are phosphate, citrate, and borate at concentrations of 0.01 M to 0.1 M, and pH 6 to 9. Macerate the tissue in the mortar with a pestle. Apply the inoculum to the leaf surface of young plants that have been dusted with a 600-mesh abrasive, such as silicon carbide (carborundum), aluminum oxide, or diatomaceous earth (Celite). The abrasive can be applied using a test tube with a piece of net attached at one end, or an atomizer. Spread the inoculum softly onto the leaves with a cotton swab or with your finger. The inoculated leaves are then rinsed with water and placed in the greenhouse. Before using the mortars again, wash them and place them
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in a 1% sodium hypochlorite (bleach) solution. Rinse again and place in an oven at 150OC for 3 hours. Symptoms on inoculated leaves usually appear after 3 to 7 days. The number of local lesions is proportional to virus concentration in sap. Symptoms may take 10 to14 days or longer to develop on infected hosts. Occasionally, the same plant may first develop local lesions and then systemic symptoms. Mechanical inoculation by high-pressure spraying has proven to be effective for several viruses. This technique is useful when a large number of plants must be inoculated with a particular virus. To use this technique, add approximately 1% fine carborundum to the inoculum and shake this suspension continuously during inoculation. Spraying must be done at a pressure between 50 and 75 psi. Keep the nozzle 2 to 5 cm from the surface of the leaf. This technique is used in screening for resistance to PVX and PVY. Some substances present in plant tissues, such as infection inhibitors and inactivators of virus particles, may prevent successful virus transmission:
a) Infection inhibitors
Inhibitors are found in many plants, and they diminish infection in inoculated plants. Grinding plants liberates sap and other substances, such as the inhibitors, contained in the cell vacuole. These substances are thought to act in one of two ways: • • by reducing the susceptibility of the host plant, or by interfering with the infection process.
The inhibitor effect may be checked by diluting the inoculum or by using substances that capture or interfere with the inhibitor. This last procedure is difficult to implement, because in many instances the precise nature of the inhibitor is unknown. When infected sap is diluted, the virus tends to remain effective after the inhibitor effect has disappeared.
Chemically extracting the inhibitor from the sap results in the reestablishment of the infection. Therefore, the inhibitor has no effect on the virus particle itself, but rather on the infection process.
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Figure 1. Effect of the dilution of infected sap on the number of local lesions produced on indicator plants.
Virus particle inactivators
These compounds (quinones) are formed by the oxidation of polyphenols (found in the vacuoles of plant cells) as they come into contact with cytoplasmic enzymes during tissue maceration (crushing). Quinones are highly toxic compounds that can destroy some proteins. They degrade virus protein subunits, thus exposing the nucleic acid to the ribonucleases (very common plant enzymes). A single rupture in the virus nucleic acid abolishes virus infectivity. The inactivator acts on the virus itself (by permanently inactivating it) and its effect will not disappear with dilution. Quinones provoke chemical reactions that form dark pigments (melanin). The effect of inactivators can be reduced or prevented: • By using antioxidizers that compete for O2 with polyphenols, thus reducing the quantity of quinones. Some commonly used antioxidizers are 2-mercaptoethanol, ascorbic acid, cysteine, sodium bisulfite, and thioglycolic acid, at proportions ranging from 0.1 to 1%. By using polyphenol-capturing substances such as caffeine, nicotine, Al2O3, and PVP (polyvinyl pyrrolidone) at a proportion of 10% (w/w). By using inhibitors of the enzyme polyphenol oxidase such as DIECA (0.001 to 0.01 M), which is a chelating agent that captures copper (Cu) ions.
Some of these products are added to the homogenization buffers during the virus purification process to prevent inactivation of virus particles.
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Figure 2. Production of quinones by oxidation of polyphenols in cell vacuole.
Grafting is a technique through which cut tissue surfaces of different plants are placed in close contact to effect a union. Grafting is considered to be a universal method for transmitting viruses, because systemic viruses can be transmitted by grafting. Some grafts, however, are easier to do than others. Grafting is particularly useful for transmission of phloem-restricted viruses that cannot be transmitted mechanically and viruses whose vectors remain unknown, and for detecting viruses found in low concentrations. Virus transmission by grafting may not be 100%-effective if the virus is unable to cross the graft union, or if the virus source plant was not totally invaded and the portion used was virus-free due to irregular virus distribution. Successful virus transmission depends on the characteristics of the virus and on the union achieved through grafting. A virus such as potato leafroll virus (PLRV), which is restricted to the vascular system, can be transmitted only when vascular tissues are united. On the other hand, viruses affecting the parenchyma can be transmitted more easily because the transmission depends solely on the union of the cortex or the medula.
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There are several types of grafts: side grafts, wedge grafts, approach grafts, bud grafts, or cylinder insertion in potato tubers (heart grafts).
3. Vegetative propagation
This is the most important means of transmission among plants that propagate vegetatively by gemmation, grafting, cuttings, tubers, corms, bulbs, or rhizomes. An infected mother plant of this type will yield infected progeny.
4. Botanical (sexual) seed
Approximately 100 viruses are transmitted through sexual seed (commonly called botanical seed, or "true seed" in the case of potato). In virology, this type of transmission is known as vertical transmission. Transmission by seeds is the most serious when a vector is present. For example, bean common mosaic virus shows a very high percentage of transmission through seed and has a very efficient (aphid) vector. When even a small amount of infected seed is planted, aphids spread the infection to healthy plants. Viruses can be transmitted by seed in one of two ways: • Externally, on the seed cover. This happens through contamination of the pulp of the fruit, which is in contact with the seed cover (e.g., tobacco mosaic virus in hot peppers). In this case, the seed can be disinfected with chemicals such as HCI (37%) diluted to 1/20 for 4 to 8 hours. Internally, in the embryo and the endosperm. In this case, the virus cannot be eliminated by disinfection with chemical products or otherwise. The virus remains inside the seed for a long time, and therefore long-distance dissemination of the virus (e.g., bean common mosaic, tobacco ringspot) is more likely to occur.
Several factors influence the percentage of transmission of virus by sexual seed: • • Virus type or strain (100% for tobacco ringspot; 1% for APLV). Species and variety of host plant. Nepoviruses are transmitted in many host plants (barley stripe virus from 0 to 75%, depending on the variety). Stage of plant development. Younger infected plants usually show a higher probability of virus transmission by sexual seed. In beans, transmission occurs only if the infection takes place before flowering. Age of seed. The infectivity of some viruses in the seed decreases very rapidly with storage, and can be lost before the seed loses its capacity to germinate. The cherry necrotic ringspot virus disappears after 6 years. Some viruses can be detected during the seed formation stage, but will disappear at maturity. This happens because mature or germinating seeds contain inactivators that are absent in young seeds. In most seed-transmitted viruses, the virus apparently
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comes from the ovule of the infected plant. However, in several reported cases, the virus found in the seed seems to come just as frequently from the pollen fertilizing the flower. The most important seed-transmitted potato agent is the spindle tuber viroid (PSTVd), whose transmission efficiency can reach 100%. In laboratory experiments, PVT has also shown a high degree of transmissibility (65%). To test virus transmission by seed, collected seeds of infected plants are planted to observe the percentage of infected plantlets. Seeds from healthy plants of the same variety are simultaneously planted as a control. A few hundred seeds may suffice to determine the percentage of seed transmission for viruses showing a high degree of transmissibility. If transmissibility is low, however, several thousand seeds may be necessary.
Viruses transmitted by pollen do not only infect the seed and plantlets. They can also propagate through the fecundated flower and infect the mother plant. Although flower pollination with virus-infected pollen can lead to a lower fruit yield, compared with yields obtained with virus-free pollen, transmission through pollen seems to be extremely rare, or occurs with only a few viruses. Both PSTVd and PVT are transmitted by pollen or the ovule of infected plants, but infection of the mother plant via infected pollen has not been demonstrated.
6. Common dodder
Many viruses can be transmitted between plants from families so taxonomically different that transmission by grafting is impossible due to tissue incompatibility. The common dodder (Cuscuta sp.) is a parasitic plant that absorbs sap and viruses, if present, through its haustorium. The two species most frequently used in transmission tests are Cuscuta campestris and C. subinclusa. This type of transmission is effective when mechanical inoculation will not give positive results. For this type of transmission, the common dodder is usually grown parasitically on plants with viral symptoms. Once new stems have formed, test plants are placed close to the infected plant, which will be parasitized by the common dodder, thus creating a bridge of living tissue for the transmission of viruses. Alternatively, sprouts cut from a dodder plant growing on a diseased plant are wound around the stem and leaves of the healthy plant. The virus may multiply in the tissue of the dodder plant during the process of transmission. This characteristic can be used to isolate the virus. If infected and healthy test plants are grown under a light source, the transmission rate is low (5 out of 10 plants). However, if the infected plant
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is under a light source and the healthy plant remains in the shade, the rate of transmission is higher (10 out of 10 plants). This is due to the higher translocation of nutrients and viruses through the dodder, from the plant under the light source to the plant growing in the shade.
Many viruses are transmitted naturally by vectors. A vector is a disseminating agent that carries virus particles from sick plants to healthy plants. Insects, nematodes, and fungi are some vectors of plant viruses. A. Transmission by insects
This is the most common virus transmission method in nature. Most of these vectors are sucking insects. Aphids transmit more viruses than any other insect group, followed by leafhoppers, whiteflies, thrips, plant lice, and beetles. Mites, though not actually insects, are included in this category because of their importance as vectors of some viruses. The following definitions must be considered: • • Period of acquisition (PA) is the time the insect needs to acquire the virus from the infected plant. Period of latency or incubation (PL) is the time required by the insect, between the period of acquisition and the period of inoculation, to develop the capacity to transmit the virus. Period of inoculation (PI) is the time the insect needs to transmit the virus once it is on the healthy plant. Period of retention or infectivity (PR) is the period during which the insect remains viruliferous.
There are three known types of virus transmission by insects: 1. Non-persistent transmission. Viruses are acquired and transmitted in less than 5 minutes. These viruses are called "stylet-borne viruses" and are rapidly eliminated by the insect (e.g., PVY). 2. Semi-persistent transmission. Viruses are acquired in approximately 15 minutes and transmitted for up to 2 days (e.g., the citrus "tristeza" virus). 3. Persistent transmission. Viruses are acquired and transmitted over longer periods. A latency or incubation period is needed between acquisition and inoculation of the virus, which can persist throughout the insect's life (e.g., PLRV). Other differences between non-persistent and persistent transmission are as follows. Non-persistent transmission takes place exclusively through aphids. The acquisition and inoculation periods go from 5 seconds to 5 minutes, and there is no latency period. The retention or infectivity period varies from a few minutes to a couple of hours, depending on whether the insect is
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feeding or not. There isn't a high degree of specificity between the virus and its vector; therefore the same virus may be transmitted by several aphid species. Fasting increases the efficiency of transmission. This type of transmission is very stable, and viruses transmitted in this way may reach very high concentrations in the plant tissue. They can also be transmitted mechanically, as is PVY. Persistent transmission can take place through vectors such as aphids, leafhoppers, whiteflies, thrips, and beetles. The acquisition and incubation periods vary from 10 to 60 minutes, while the latency period lasts from 12 hours to several days. The virus persists throughout the insect's life. This is a more specific type of transmission because some viruses are transmitted by a certain vector only. Fasting does not increase the efficiency of transmission. Viruses transmitted in this manner are usually restricted to the phloem of the host plant and therefore cannot be transmitted mechanically (e.g., PLRV). Viruses transmitted non-persistently are known as non-circulating viruses, whereas those transmitted persistently are known as propagative or circulating viruses. This difference depends on whether or not they multiply in the vector. B. Transmission by nematodes
Viruses transmitted by nematodes have a wide range of hosts and may also be transmitted though seed or pollen. Approximately a dozen viruses that infect plants are transmitted by one or more species of the three ectoparasitic genera of nematodes (those feeding on the external part of the root). The genera Xiphinema and Longidorus are vectors of the spherical-particle viruses known as Nepoviruses, such as tobacco and tomato ringspot viruses or the fanleaf virus of grapes. Nematode genus Trichodorus transmits two tubularparticle viruses belonging to the group Tobravirus: tobacco rattle virus and greenpea early browning virus. Adults of nematode species Xiphinema and Longidorus measure from 3 to 10 mm, and have a long, straight probe of approximately 200 m, which is used to feed on the cell content of the plant's root. Trichodorus species are relatively small and thick-bodied, and measure about 1 mm. They have a curved probe about 50 m long, which they use to feed on the epidermal cells of the root tip. Virus transmission takes place when the nematodes feed on an infected plant and then move to the roots of healthy plants. Under favorable conditions, nematodes can acquire a virus in 15–60 minutes. A similar period is required for transmission to a healthy plant. The virus particles adhere to the walls of the stylet when the nematode feeds in the plant. The particles are liberated as saliva flows in the opposite direction toward the cells of the plant on which the nematode is feeding. The virus may be retained in different parts of the nematode's body depending on the genus and, occasionally, on the species. For example, Longidorus retains the virus in the foresection of the probe, while Xiphinema and Trichodorus retain the virus inside the probe and on the walls of the esophagus. A difference in retention location determines the
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period during which the nematode remains infective. Xiphinema and Trichodorus remain infective for a longer period (up to 10 months) than Longidorus (less than 3 months). Viruses are acquired and transmitted by both larvae and adults. Larvae, however, lose their infectivity after molting. Viruses do not multiply in a nematode's tissues, nor are they transmitted to the progeny via eggs. c. Transmission by fungi
There are four known genera of virus-transmitting fungi. All of these are obligate parasites consisting of one or a few cells that form thick-walled, resting sporangia and that may survive in dry soil for long periods. Soilborne vectors can be identified by determining whether the pathogen is transmitted from dessicated soil to host plants, because virustransmitting nematodes cannot survive in dry soil at the normal environment temperature. Two of the four known genera of virus-transmitting fungi, Olpidium and Synchytrium, belong to the order Chytridiales, while the other two, Polymyxa and Spongospora, belong to the Plasmodiophorales. Olpidium brassicae transmits tobacco necrosis, lettuce big vein, and tobacco stunt viruses; O. cucurbitacearum transmits cucumber necrosis virus; and Polymyxa graminis transmits wheat mosaic and other soil-transmitted grain viruses. Spongospora subterranea is the vector of potato mop-top virus (PMTV). Synchytrium endobioticum has been reported to be one of the vectors of PVX, but this has not been confirmed under field conditions. All these fungi are intracellular, that is, they penetrate root cell, and they produce zoospores (flagellated mobile spores). Thus, transmission occurs when the fungus produces resting sporangia in the root tissue of the cultivated plant near the end of the season. These sporangia differentiate by forming zoospores that escape and swim through the damp soil after heavy rainfall or irrigation. These zoospores reach the infected plant, form a cyst by losing the flagellum, and eject a germinative tube inside the root cells. The fungus grows in the cytoplasm of the root cells of the host plant, and, if virus particles are present, they will be incorporated into the fungus cytoplasm. The reproductive cycle of rest sporangia and zoospores is repeated and, if the zoospores find a healthy plant, they will transmit the virus when they inject their cytoplasm into the root cells through the germinative tube. Dissemination of lettuce big vein and tobacco stunt viruses by Polymyxa, Spongospora, and Olpidium is an example of this kind of transmission. Another form of fungal transmission is when the zoospores acquire virus particles that are free in the soil. The virus can adhere to the surface and flagellum of the zoospore. When the zoospore forms a cyst, the flagellum is absorbed into its cytoplasm, and when the cytoplasm is injected into the root cells, the virus particles from the flagellum are injected as well. This type of transmission occurs with Olpidium and tobacco necrosis and cucumber necrosis viruses. Transmission of virus diseases by soil-borne (nematodes and fungi) versus air-borne (insects) agents is easy to identify under field conditions.
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Diseases transmitted by soil-borne agents create patches of infected plants in the field because of the slow horizontal dissemination of the agent.
Recommended Literature Boxk, J.A. (ed.). 1972. Viruses of potatoes and seed potato production. Centre for Agricultural Publishing and Documentation (PUDOC), Wageningen. 233 p. French, R. and T.T. Hebert. 1982. Métodos de fitopatológica. 1ra. edición. IICA, Costa Rica. 289 p. investigación
Gibbs, A. and B. Harrison. 1974. Plant virology: The principles. New York, John Wiley. 292 p. Mathews, R.E.F. 1981. Plant virology. Academic Press, London. 897 p. Noordman, D. 1973. Identification of plant viruses: Methods & experiments. Centre for Agricultural Publishing and Documentation (PUDOC), Wageningen. 207 p.
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