TECNIQUES IN PLANT VIROLOGYCIP Training Manual1.0 TRANSMISSION

Section 1.1Transmission ofPlant VirusesPlant viruses do not penetrate the intact plant cuticle. For this reason,viruses are not disseminated as such by wind or water, and even whenthey are carried in plant sap or debris they generally do not causeinfections unless they come in contact with the contents of woundedliving cell. Viruses, however, are transmitted from plant to plant in anumber of ways, including:

1. Mechanical contact

2. Grafting

3. Vegetative propagation

4. Botanical (sexual) seed

5. Pollen

6. Common dodder

7. Vectors

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1. Mechanical contactMechanical transmission requires the existence of a wound in the plantand subsequent contact of a healthy cell with infected cellular sap. Thewound could be as small as one resulting from the splitting of plant hairs.Under natural conditions, transmission takes place through contact ofleaves. In nature, only a few viruses are disseminated by contact: tomatomosaic virus (TMV), potato virus X (PVX), potato virus S (PVS), Andeanpotato mottle virus (APMV), Andean potato latent virus (APLV), andpotato spindle tuber viroid (PSTVd).

A. Transmission by contact between potato tubers ortuber sprouts.Some viruses may spread when healthy tubers, particularly at thesprouting 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 andbetween roots.Under field conditions, PVX and PSTVd can be easily transmitted tohealthy plants that are in contact with diseased plants. Blowing windpromotes contact between plants and increases the probability of virusdissemination (rubbing effect).

This type of dissemination also depends on such factors as cultivarsusceptibility, virulence of the virus strain, soil fertility, row spacing, anddistance between plants.

Transmission by contact appears to be more frequent in greenhousesthan under field conditions. This may depend on the physiological state ofthe plants, which are more succulent and susceptible under greenhouseconditions. Transmission of PVX through roots touching has beenobserved, but there is no similar evidence for other potato viruses.

The most common mechanical transmission of viruses is by the sap froman 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-infectedplant tissue, and inoculation buffer solution.

Place the plant tissue in the mortar and add 1 to 10 ml of buffer solutionor distilled water per gram of tissue. The most commonly used virus-stabilization buffer solutions are phosphate, citrate, and borate atconcentrations 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 theleaf surface of young plants that have been dusted with a 600-meshabrasive, such as silicon carbide (carborundum), aluminum oxide, ordiatomaceous earth (Celite). The abrasive can be applied using a testtube with a piece of net attached at one end, or an atomizer. Spread theinoculum softly onto the leaves with a cotton swab or with your finger.The inoculated leaves are then rinsed with water and placed in thegreenhouse. 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 inan oven at 150OC for 3 hours.

Symptoms on inoculated leaves usually appear after 3 to 7 days. Thenumber 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 thensystemic symptoms.

Mechanical inoculation by high-pressure spraying has proven to beeffective for several viruses. This technique is useful when a largenumber of plants must be inoculated with a particular virus. To use thistechnique, add approximately 1% fine carborundum to the inoculum andshake this suspension continuously during inoculation. Spraying must bedone at a pressure between 50 and 75 psi. Keep the nozzle 2 to 5 cmfrom the surface of the leaf. This technique is used in screening forresistance to PVX and PVY.

Some substances present in plant tissues, such as infection inhibitorsand inactivators of virus particles, may prevent successful virustransmission:

a) Infection inhibitorsInhibitors are found in many plants, and they diminish infection ininoculated plants. Grinding plants liberates sap and other substances,such as the inhibitors, contained in the cell vacuole. These substancesare 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 usingsubstances that capture or interfere with the inhibitor. This last procedureis difficult to implement, because in many instances the precise nature ofthe inhibitor is unknown. When infected sap is diluted, the virus tends toremain effective after the inhibitor effect has disappeared.

Chemically extracting the inhibitor from the sap results in the re-establishment of the infection. Therefore, the inhibitor has no effect onthe 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 locallesions produced on indicator plants.

b) Virus particle inactivatorsThese compounds (quinones) are formed by the oxidation of polyphenols(found in the vacuoles of plant cells) as they come into contact withcytoplasmic 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 theribonucleases (very common plant enzymes). A single rupture in the virusnucleic acid abolishes virus infectivity. The inactivator acts on the virusitself (by permanently inactivating it) and its effect will not disappear withdilution. 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, thusreducing the quantity of quinones. Some commonly usedantioxidizers are 2-mercaptoethanol, ascorbic acid, cysteine, sodiumbisulfite, 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 duringthe virus purification process to prevent inactivation of virus particles.

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Figure 2. Production of quinones by oxidation of polyphenols in cellvacuole.

2. GraftingGrafting is a technique through which cut tissue surfaces of differentplants 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-restrictedviruses that cannot be transmitted mechanically and viruses whosevectors remain unknown, and for detecting viruses found in lowconcentrations.

Virus transmission by grafting may not be 100%-effective if the virus isunable to cross the graft union, or if the virus source plant was not totallyinvaded and the portion used was virus-free due to irregular virusdistribution.

Successful virus transmission depends on the characteristics of the virusand on the union achieved through grafting. A virus such as potatoleafroll virus (PLRV), which is restricted to the vascular system, can betransmitted only when vascular tissues are united. On the other hand,viruses affecting the parenchyma can be transmitted more easilybecause the transmission depends solely on the union of the cortex orthe medula.

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There are several types of grafts: side grafts, wedge grafts, approachgrafts, bud grafts, or cylinder insertion in potato tubers (heart grafts).

3. Vegetative propagationThis is the most important means of transmission among plants thatpropagate vegetatively by gemmation, grafting, cuttings, tubers, corms,bulbs, or rhizomes. An infected mother plant of this type will yield infectedprogeny.

4. Botanical (sexual) seedApproximately 100 viruses are transmitted through sexual seed(commonly called botanical seed, or "true seed" in the case of potato). Invirology, this type of transmission is known as vertical transmission.

Transmission by seeds is the most serious when a vector is present. Forexample, bean common mosaic virus shows a very high percentage oftransmission through seed and has a very efficient (aphid) vector. Wheneven a small amount of infected seed is planted, aphids spread theinfection to healthy plants.

Viruses can be transmitted by seed in one of two ways:

• Externally, on the seed cover. This happens through contamination ofthe 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 bedisinfected with chemicals such as HCI (37%) diluted to 1/20 for 4 to8 hours.

• Internally, in the embryo and the endosperm. In this case, the viruscannot be eliminated by disinfection with chemical products orotherwise. The virus remains inside the seed for a long time, andtherefore long-distance dissemination of the virus (e.g., beancommon mosaic, tobacco ringspot) is more likely to occur.

Several factors influence the percentage of transmission of virus bysexual seed:

• Virus type or strain (100% for tobacco ringspot; 1% for APLV).

• Species and variety of host plant. Nepoviruses are transmitted inmany host plants (barley stripe virus from 0 to 75%, depending onthe variety).

• Stage of plant development. Younger infected plants usually show ahigher 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 decreasesvery rapidly with storage, and can be lost before the seed loses itscapacity to germinate. The cherry necrotic ringspot virus disappearsafter 6 years. Some viruses can be detected during the seedformation stage, but will disappear at maturity. This happens becausemature or germinating seeds contain inactivators that are absent inyoung seeds. In most seed-transmitted viruses, the virus apparently

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comes from the ovule of the infected plant. However, in severalreported cases, the virus found in the seed seems to come just asfrequently from the pollen fertilizing the flower.

The most important seed-transmitted potato agent is the spindle tuberviroid (PSTVd), whose transmission efficiency can reach 100%. Inlaboratory experiments, PVT has also shown a high degree oftransmissibility (65%).

To test virus transmission by seed, collected seeds of infected plants areplanted to observe the percentage of infected plantlets. Seeds fromhealthy plants of the same variety are simultaneously planted as acontrol. A few hundred seeds may suffice to determine the percentage ofseed transmission for viruses showing a high degree of transmissibility. Iftransmissibility is low, however, several thousand seeds may benecessary.

5. PollenViruses transmitted by pollen do not only infect the seed and plantlets.They can also propagate through the fecundated flower and infect themother plant. Although flower pollination with virus-infected pollen canlead to a lower fruit yield, compared with yields obtained with virus-freepollen, transmission through pollen seems to be extremely rare, or occurswith only a few viruses.

Both PSTVd and PVT are transmitted by pollen or the ovule of infectedplants, but infection of the mother plant via infected pollen has not beendemonstrated.

6. Common dodderMany viruses can be transmitted between plants from families sotaxonomically different that transmission by grafting is impossible due totissue incompatibility.

The common dodder (Cuscuta sp.) is a parasitic plant that absorbs sapand viruses, if present, through its haustorium. The two species mostfrequently used in transmission tests are Cuscuta campestris and C.subinclusa. This type of transmission is effective when mechanicalinoculation will not give positive results.

For this type of transmission, the common dodder is usually grownparasitically on plants with viral symptoms. Once new stems haveformed, test plants are placed close to the infected plant, which will beparasitized by the common dodder, thus creating a bridge of living tissuefor the transmission of viruses. Alternatively, sprouts cut from a dodderplant growing on a diseased plant are wound around the stem and leavesof the healthy plant.

The virus may multiply in the tissue of the dodder plant during theprocess of transmission. This characteristic can be used to isolate thevirus.

If infected and healthy test plants are grown under a light source, thetransmission 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, therate of transmission is higher (10 out of 10 plants). This is due to thehigher translocation of nutrients and viruses through the dodder, from theplant under the light source to the plant growing in the shade.

7. VectorsMany viruses are transmitted naturally by vectors. A vector is adisseminating agent that carries virus particles from sick plants to healthyplants. 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 ofthese vectors are sucking insects. Aphids transmit more viruses than anyother insect group, followed by leafhoppers, whiteflies, thrips, plant lice,and beetles. Mites, though not actually insects, are included in thiscategory 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 thevirus 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, todevelop the capacity to transmit the virus.

• Period of inoculation (PI) is the time the insect needs to transmit thevirus once it is on the healthy plant.

• Period of retention or infectivity (PR) is the period during which theinsect remains viruliferous.

There are three known types of virus transmission by insects:

1. Non-persistent transmission. Viruses are acquired and transmitted inless 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 approximately15 minutes and transmitted for up to 2 days (e.g., the citrus "tristeza"virus).

3. Persistent transmission. Viruses are acquired and transmitted overlonger periods. A latency or incubation period is needed betweenacquisition and inoculation of the virus, which can persist throughoutthe insect's life (e.g., PLRV).

Other differences between non-persistent and persistent transmissionare as follows.

Non-persistent transmission takes place exclusively through aphids. Theacquisition and inoculation periods go from 5 seconds to 5 minutes, andthere is no latency period. The retention or infectivity period varies from afew 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 virusand its vector; therefore the same virus may be transmitted by severalaphid species. Fasting increases the efficiency of transmission. This typeof transmission is very stable, and viruses transmitted in this way mayreach very high concentrations in the plant tissue. They can also betransmitted mechanically, as is PVY.

Persistent transmission can take place through vectors such as aphids,leafhoppers, whiteflies, thrips, and beetles. The acquisition andincubation periods vary from 10 to 60 minutes, while the latency periodlasts from 12 hours to several days. The virus persists throughout theinsect's life. This is a more specific type of transmission because someviruses are transmitted by a certain vector only. Fasting does notincrease the efficiency of transmission. Viruses transmitted in thismanner are usually restricted to the phloem of the host plant andtherefore cannot be transmitted mechanically (e.g., PLRV).

Viruses transmitted non-persistently are known as non-circulatingviruses, whereas those transmitted persistently are known as propagativeor circulating viruses. This difference depends on whether or not theymultiply in the vector.

B. Transmission by nematodes

Viruses transmitted by nematodes have a wide range of hosts and mayalso be transmitted though seed or pollen.

Approximately a dozen viruses that infect plants are transmitted by one ormore species of the three ectoparasitic genera of nematodes (thosefeeding on the external part of the root). The genera Xiphinema andLongidorus are vectors of the spherical-particle viruses known asNepoviruses, such as tobacco and tomato ringspot viruses or the fanleafvirus of grapes. Nematode genus Trichodorus transmits two tubular-particle viruses belonging to the group Tobravirus: tobacco rattle virusand greenpea early browning virus.

Adults of nematode species Xiphinema and Longidorus measure from 3to 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. Trichodorusspecies 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 onthe epidermal cells of the root tip.

Virus transmission takes place when the nematodes feed on an infectedplant and then move to the roots of healthy plants. Under favorableconditions, nematodes can acquire a virus in 15–60 minutes. A similarperiod is required for transmission to a healthy plant. The virus particlesadhere to the walls of the stylet when the nematode feeds in the plant.The particles are liberated as saliva flows in the opposite direction towardthe cells of the plant on which the nematode is feeding.

The virus may be retained in different parts of the nematode's bodydepending on the genus and, occasionally, on the species. For example,Longidorus retains the virus in the foresection of the probe, whileXiphinema and Trichodorus retain the virus inside the probe and on thewalls of the esophagus. A difference in retention location determines the

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period during which the nematode remains infective. Xiphinema andTrichodorus remain infective for a longer period (up to 10 months) thanLongidorus (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 anematode'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 areobligate parasites consisting of one or a few cells that form thick-walled,resting sporangia and that may survive in dry soil for long periods. Soil-borne vectors can be identified by determining whether the pathogen istransmitted from dessicated soil to host plants, because virus-transmitting nematodes cannot survive in dry soil at the normalenvironment temperature.

Two of the four known genera of virus-transmitting fungi, Olpidium andSynchytrium, belong to the order Chytridiales, while the other two,Polymyxa and Spongospora, belong to the Plasmodiophorales. Olpidiumbrassicae transmits tobacco necrosis, lettuce big vein, and tobacco stuntviruses; O. cucurbitacearum transmits cucumber necrosis virus; andPolymyxa graminis transmits wheat mosaic and other soil-transmittedgrain viruses. Spongospora subterranea is the vector of potato mop-topvirus (PMTV). Synchytrium endobioticum has been reported to be one ofthe vectors of PVX, but this has not been confirmed under fieldconditions.

All these fungi are intracellular, that is, they penetrate root cell, and theyproduce zoospores (flagellated mobile spores). Thus, transmissionoccurs when the fungus produces resting sporangia in the root tissue ofthe cultivated plant near the end of the season. These sporangiadifferentiate by forming zoospores that escape and swim through thedamp soil after heavy rainfall or irrigation.

These zoospores reach the infected plant, form a cyst by losing theflagellum, and eject a germinative tube inside the root cells. The fungusgrows in the cytoplasm of the root cells of the host plant, and, if virusparticles 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 theyinject 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 virusparticles that are free in the soil. The virus can adhere to the surface andflagellum of the zoospore. When the zoospore forms a cyst, the flagellumis absorbed into its cytoplasm, and when the cytoplasm is injected intothe root cells, the virus particles from the flagellum are injected as well.This type of transmission occurs with Olpidium and tobacco necrosis andcucumber 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 infectedplants in the field because of the slow horizontal dissemination of theagent.

Recommended LiteratureBoxk, 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 investigaciónfitopatológica. 1ra. edición. IICA, Costa Rica. 289 p.

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.