TECNIQUES IN PLANT VIROLOGY

CIP Training Manual

1.0 TRANSMISSION

Section 1.1
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. Mechanical contact
2. Grafting
3. Vegetative propagation
4. Botanical (sexual) seed
5. Pollen
6. Common dodder
7. 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 virus-
stabilization 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 re-
establishment 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.

b) 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.

2. Grafting
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.

5. Pollen
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.

7. Vectors
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 tubular-
particle 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. Soil-
borne vectors can be identified by determining whether the pathogen is
transmitted from dessicated soil to host plants, because virus-
transmitting 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 investigación
fitopatológica. 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.

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