TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.2 DETECTION/Indicator Plants

Section 2.2.2
Disinfecting Soil
Basic potato seed and virus indicator plants can be produced in the
greenhouse using a substrate mix consisting of soil, peat moss, and
sand. Disinfest the mix to prevent tuber and plant contamination by soil-
borne pests and pathogens.

Disinfesting through biological, chemical, or physical methods is usually

expensive and requires careful handling, especially when using highly
toxic chemicals.

In the greenhouse, pathogen-free in vitro plantlets, seeds, cuttings or

tuberlets are planted in a disinfested substrate to ensure that tuber
production is free from fungi, bacteria, viruses, or nematodes.
Disinfestation should also include the elimination of insects and weed
seed that affect the production of high-quality seed and vigorous plants.

Disinfestation with Methyl Bromide

Several commercial products are available for disinfesting substrate, but
their application may require several days. However, in spite of its high
toxicity, methyl bromide gas can be easily applied in a few days and is
highly effective.
 1. Treatment chamber
Figures 1 and 2 show the materials needed and their proportions. Walls
should be built on a concrete base using concrete covered bricks.
Chamber capacity must be 3 m3, with loading and unloading facilities for
the mix or substrate. The chamber must be 3.60 m long, 1.20 m wide,
and 0.70 m deep.

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 Along the upper edge of the walls of the chamber, make a ditch 5 cm
wide and 2 cm deep around the chamber (A).

Use clear plastic sheets (0.8–1 mm thick) as a cover (B). This sheeting
should be between A and B, so that it may be used more than once.

Construct a 1/4" x 1 and 3/4" laminated iron frame (C) that fits into the
ditch of the container. Include handles on this frame to ease lifting and
lowering it on and off the treatment chamber. The function of this frame is
to hold the plastic sheet in place. The sheet, in turn, seals the container
before treatment, thereby preventing any gas escape.

Insert a tube (D) capped with a nut through one of the walls at 65 cm
(Figure 2). The tube should be slightly above the level of the soil to allow
the gas to flow into the chamber freely. Connect the methyl bromide gas
to the chamber. There are two ways of doing this:

a) With a nut (E) that connects a large (92-kg or 200-lb) cylinder to

the tube by means of a duct. Gas is released by means of a
control tap on the cylinder.

b) With an applicator tube, specially designed for 460-g cylinders

connected directly to the tube in the chamber. This type of
applicator has a clamp that, when tightened, causes the gas
cylinder top to be perforated and a hole to be formed, allowing the
gas to escape down the tube and into the chamber.

Make holes (Figure 3) in the damp soil (F) to facilitate the flow of the gas
into the chamber. The holes should have a 2-cm diameter made by a
wooden stick of that size. Make sure they reach the bottom of the
chamber and are 15–20 cm apart.

To seal the container, cover it with the thick plastic, and place the metal
frame on top, ensuring that it sits in the rim of the chamber and effectively
seals it. To make the container totally airtight, spread damp soil or sand
on top of the frame.

Note: During application and when opening the chamber, use gloves, gas
mask, etc. Close the disinfestation chamber tightly to avoid gas leaks.

2. Dosage
The amount of gas (m3) used will vary from 1–1.5 lbs, depending on the
type of soil mixture. Heavy or highly infested soils will need higher doses.
After the first application of a given dose, it is advisable to verify if there
are still fungi, bacteria, or nematodes at the applied dose.

3. Treatment period
The treatment period for methyl bromide is 72 hours. After this treatment
period, uncover the chamber, turn the soil, and let it ventilate for 72
hours. After this, the soil may be used.

As a control, to ensure that there are no toxic residues, put some of the
soil and a lettuce plant into a pot: if the plant suffers no changes, the soil
is safe to use.
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 4. Sealing the container
An alternative method of sealing the chamber (rather than making a rim)
is to attach 10 bolts to the top edge of the chamber, construct the frame,
and cover as shown in Figure 4.

This cover is a wooden frame lined with 4-mm plywood (G). For the
frame, use 3/4" lathes for a final depth of 2 cm (1" rough = approx. 3/4"
planed). Perforate holes along the edge of the cover. These holes must
coincide with the positions of the bolts around the chamber. Between the
wooden cover and the chamber, place a sheet of rubber or canvas as a
seal, remembering to make 10 holes in this as well, for the bolts.

Seal the container securely, capping the bolts with

butterfly clasps

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 5. Methods for Applying the Gas
The alternative methods for applying the methyl bromide gas are shown
in Figures 5 and 6.

a) Gas under high pressure from a 92-kg tank

Build the disinfestation chamber (Figure 5, H). Place a tube through the
wall and connect it by using a conducting hose to the 92-kg (200 lb)
methyl bromide cylinder. Inside the chamber, attach the tube to another
plastic tube with small holes at equal distances from each other along its
length. Lay this along the bottom of the chamber to allow for equal
distribution of the gas. The gas flows because it is under pressure in the
cylinder. Before treatment, make the holes in the damp soil, as described
in Figure 3.

Because it is heavier than air, methyl bromide gas rises quickly, then
flows down through the holes into the soil. When a large gas cylinder is
used, place it on a scale to determine, by checking weight loss, the
quantity of gas used in relation to the volume of soil being treated.

b) Gas in a small cylinder

The same type of disinfection chamber is used (Figure 5, I). When a 460-
g cylinder is used, apply the gas using the nozzle connected to the pipe,
which passes through the chamber wall. The gas flows out over the
surface of the soil, then sinks into the holes made in the soil and spreads.

c) Without applicator nozzles

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 If there is no applicator nozzle on either the 460-g or 92-kg cylinder, use
the same type of chamber with a plastic cover (as shown in Figure 6, J).

A piece of wood pierced by a nail should then be attached to the top of

the small 460-g cylinder. Place the cylinder in the chamber and cover with
plastic, as described earlier. Put pressure on the wood through the plastic
cover; this will perforate the cylinder and allow the gas to escape and
move through the soil as described.

d) When no chamber is available

If neither a chamber nor applicator nozzle are available (Figure 6, K), pile
the soil mixture to be treated on plastic sheeting. Place a small 460-g gas
cylinder and the wooden piece containing the piercing device on top of
the pile. Cover completely with more plastic sheeting and seal the edges
by piling damp sand or soil around the edge of the plastic. Press the
wood through the plastic so that the piercing device makes a hole in the
cylinder and allows the gas to escape and spread through the soil.

Disinfection Using the Solarization Method

Pathogen eradication from both soil and substrate can be achieved using
physical means such as dry heat, steam, hot water, solar radiation, or low
temperatures.

Using solarization, it is possible to eliminate fungi, nematodes, and other

pathogens, as well as weed seeds in the substrate. In addition, this
method permits the use of complementary fungicides, nematicides, etc.,
in much lower-than-recommended doses.

Solarization involves the use of solar radiation to control insects and

pathogens, using transparent plastic sheets to cover the substrate, which
intensifies the effects of sunlight.

The substrate to be disinfected is a mixture of 2 parts peat moss, 1 part

soil, and 1 part sand (2:1:1), with a moisture content equivalent to that of
field capacity.

Solarization is performed outside the greenhouse in a yard or plot where

the substrate can be exposed to the sun. A transparent plastic sheet
0.05–0.50 cm thick is used. A thicker sheet is recommended, because it
is more durable and maintains the temperature better. The transparent
plastic sheet lets sunlight pass through but keeps it from refracting. This
traps all solar radiation with higher temperatures during the hours of
greater insolation.

Disinfection is based on the physical process of alternating high and low

temperatures. The moisture in the substrate plays a major role due to the
production of mist during the hottest hours of the day. This steam
condenses when the temperature goes down at night. Thus, a
pasteurization process takes place throughout the treatment. Fluctuations
between daytime and nighttime temperatures easily break the biological
cycles of pathogens present in the substrate.

1. Making the outdoor treatment structure

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 In a cleared area, level a space 1-m wide and the required length, and
cut a piece of plastic sheeting 1.5-m wide and 0.5-m longer than the
space. Take two boards (10" x 8" x 1") and arrange them perpendicularly
within the cleared area (see Figure 7). Use bricks at each end to support
the wooden boards. Place plastic inside the wooden bed and up over the
edges.

Pile the substrate on the plastic sheet up to 20–30 cm high (Figure 8).
Using a rake, spread it uniformly over the entire prepared surface. Add
water until the normal field capacity is reached, and shape the pile by
folding in the four edges. The bed should now be about 1-m wide x 20-30
cm x the chosen length (Figure 9). After the bed is formed and covered
with plastic, remove the bricks and lumber pieces used to shape it.

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 Cover the top with a second plastic sheet (1.5 m x same length). The
edges should extend beyond the bed to facilitate the hermetic sealing
(pile wet sand around the edge as shown in Figure 10). Insert a
thermometer through the plastic into the substrate to register the
temperature during solarization (Figure 10). This will record when the
highest temperature was reached, according to the incidence of solar
radiation.

Immediately after sealing, construct three arches to support a plastic

cover for the bed. The arch (Figure 11) can be made of plastic, iron,
branches, plastic pipe, or other materials. The different arch materials are
secured using stakes in the following manner (see Figure 12):

--plastic or iron rods inserted into a wooden stake (A),

--green bamboo branches tied to a wooden stake (B),
--green tree branches tied to a wooden stake (C),
--plastic pipes (those used for electric cables) placed over an iron stake
(D).

Arrange the arches over the substrate bed, 1 m apart. The distance
between the stakes at the base of the arches should be approximately
1.4 m, depending on the thickness of the bed. Be sure to leave a space
of at least 20 cm between the arches and the bed (Figure 12).

Place a third plastic sheet over the arches to cover the entire chamber.
Use two plastic sheets, if one is not wide enough. Join the plastic using
two wooden sticks 1 cm x 2 cm the length of the bed. Place one stick
under the plastic sheets and the other on top, joining the two sheets
between the sticks (Figure 13). Nail together using 3/4" nails. Carefully
place the plastic sheets over the anchored arches with the joined edges
of the plastic in the center (Figure 14).

Cover the edges of the plastic that are in contact with the ground with
sand to hermetically seal the chamber (Figure 14).

2. Alternative methods using the solarization treatment

An alternate method is to place the substrate in transparent polyethylene

sleeves or tubes. These can be made by using two wooden strips and
nails, or by folding the opening of the tube inward to form a bag (Figure
15).

Bags prepared in this way do not need to be covered with arches or extra
plastic sheets. Trials using this system have demonstrated that the
substrate reached 45o–55o C during daylight hours when the sun's rays
fell perpendicularly (maximum radiation). However, when they were put
under arches and an extra plastic sheet (Figure 16), the temperature rose
to 74oC under the solar radiation conditions (La Molina, Lima, Peru).

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 3. Duration of treatment

The solarization process takes 4–6 weeks. Tests should be done to

determine how long the process takes in a particular location.

Depending on the number of hours of exposure to sun and the intensity

of solar radiation, a well-disinfested substrate can be obtained in a short
time.

4. Potential range of plant pests and pathogens present in the

substrate subject to elimination

Substrate holds a wide range of pathogenic microorganisms, nematodes,

and insects. They usually attack roots and stems near the soil line,
producing considerable losses in greenhouse propagation beds.

Fungi

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 According to Pullman (1981), solarization reduces the populations of
several fungus species present in the planting substrate, such as
Verticillium dahliae, Pythium sp., Rhizoctonia solani, and Thielaviopsis
basicola.

Bacteria

Many of the bacteria affecting the growth of potatoes are eliminated using
this method. Some researchers have found that solarization-treated soils
presented fewer bacteria and fungus colonies on PDA (potato dextrose
agar), thus confirming the efficiency of solarization treatment.

Nematodes

Solarization eliminates all nematodes present in treated substrate.

Meloidogyne incognita is efficiently eliminated. Globodera spp.,
Pratylenchus, Paratrichodorus, Criconemella, Xiphinema, and Pratylenchus
spp. are reduced between 42% and 100%.

Insects

Some insect larvae and pupae that attack potatoes can be eliminated.

5. Other effects of solarization

Nutrient availability

Solarized soil shows a definite increase in organic matter (OM) and mineral
content. Concentrated NO3 increases. Other ions such as NH , K+, Ca+, Mg+,
4
and Cl+ also increase.

The substrate is usually prepared using a mixture of peat moss, sand, and
soil. Thus, it has a high organic matter content. When subject to variations in
temperature and moisture content, the mineralization process of organic
matter is accelerated. However, in certain stages of plant development,
symptoms of minor deficiencies may be present, but this may be due to
destruction of beneficial soil flora. The deficiencies can be easily overcome
by applying chelated foliage fertilizer.

Weed seeds

Up to 100% of weed seeds and vegetatively-propagated organisms present

in planting substrate (i.e. rhizomes and bulbs) are eliminated with this
treatment.

The high temperatures obtained during solarization effectively destroy those

kinds of propagative organisms in substrates that are used to produce basic
seed in greenhouses and virus indicator plants.

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 Recommended Literature
Aguilar, J.; Vitorelli, C. 1987. Desinfeste el substrato de siembra con
bromuro de metilo para producir "semilla basica" de papa en
invernadero. INIPA/COTESU, Centro Internacional de la Papa (CIP),
Lima, Peru.
Aguilar, J. et al. 1989. Desinfeste el substrato de siembra por el método
de solarizacion. INIPA/COTESU/CIP, Centro Internacional de la
Papa (CIP), Lima, Peru. Pullman (1981) 12pp.

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