Thermotherapy for controlling viral diseases

Submitted by: Muhammad Ibrahim Tahir

05-arid-83

Submitted to: Dr. S. M. Mughal

PP-706

Introduction to Plant Virology

Pir Mehr Ali Shah,

ARID AGRICULTURE UNIVERSITY, RAWALPINDI

 Thermotherapy for controlling viral diseases
Pathogen Elimination

Phytopathogens such as nematodes, fungi, bacteria, phytoplasmas viruses and viroids,

can be transmitted from infected to healthy plants. Nevertheless, not all the cells become
infected; sometimes meristematic tissues are free of pathogens, which allow recovery of healthy
plants through techniques of in vitro meristem culture. Thermotherapy is also applied for virus
eradication. This technique has been successfully used for many years in virus eradication for
carnation, geranium, strawberry, and citrus; plants are treated at high temperatures, in screen
houses or growth chambers. Three strategies are employed to obtain free in vitro cultures;
namely, seeking escapes, applying thermotherapy to the stock plant in vitro, meristem culture
and by thermo- and chemo-therapy in vitro. The standard method for virus eradication in many
vegetatively propagated crops is thermotherapy and sometimes combined with meristem culture
for better results. For woody plants, thermotherapy is widely used. The control of viruses
infecting citrus fruits is a challenge. Once a tree is infected with a virus species it remains
infected and after certain period of time, the virus can kill the tree. There is no direct control
method like chemotherapy to cure an infected plant/tree. However, certain alternative measures
such as thermotherapy has been used to eliminate virus from the infected trees and to produce
virus-free bud-wood in citrus fruits.

Thermotherapy

Experiments carried out with viruses and their host plants have shown that when plants
are treated at high temperatures (thermotherapy) the virus concentration is reduced. There are
different explanations for this phenomenon. One explanation is that competition among the
rapidly dividing host cells and the virus particles for the places where nucleic acids and proteins
synthesize results in a change in the balance between the synthesis and degradation of virus
particles. Another explanation is that under high temperatures, the union of the protein subunits
that protect the nucleic acid of the virus becomes weaker and temporal fissures appear, allowing
the attack of nucleases, which inactivate the virus and decreases its concentration.
Thermotherapy has been applied to potato tubers in dormancy. A reduction in virus
concentration has been observed, especially in potato leaf roll virus, which has been successfully
eliminated only with thermotherapy. Thermotherapy applied to the whole plant, as well as to
sprouted tubers, followed by meristems culture, has been successfully used as a standard
procedure for the eradication of many potato viruses. In the standard procedure used at the
International Potato Center (CIP, Lima, Peru), the best results have been obtained when the plant
is cut before being treated with thermotherapy, and the axillary buds continue growing while
receiving high temperature treatment. A regimen of a daily temperature of 36°C for 16 hours,
and 30°C for 8 hours, under a high intensity continued light (5000 lux), improved the score of
virus eradication. The plants are kept under these conditions for four weeks. The meristems,
either of the axillary buds or the topical buds, are isolated or cultivated as shown later.
Thermotherapy can be applied to in vitro plantlets. One-bud nodes (20 nodes/box) are placed in
plastic boxes containing a semisolid propagation media. The boxes are incubated under adequate
conditions and sealed with adhesive tape when the plants reach 3-cm height and have developed
a root system. Then they are treated with thermotherapy as explained before. A month later,
apical meristems are isolated and cultivated in an appropriate culture media.

In an experiment, the combined effect of thermotherapy and tissue culture was evaluated
on Red Globe variety infected with Grapevine leafroll associated virus 2 Red Globe strain
(GLRaV-2-RG). Five plants, derived from a positive mother stock plant to GLRaV-2-RG,
previously verified by RT-PCR were subjected to 20, 40, 60, 80 and 100 days at 38°C and 75%
RH. In each period, shoots were excised from a newly formed shoots and the apical buds of the
new shoots were established in vitro. Thereafter they were subsequently transferred to a rooting
medium and acclimated in a greenhouse. Ten acclimated plants were obtained for each
thermotherapy period and when once the plant canes were lignified, the phloem was used to
carried-out DAS-ELISA test. Plants which resulted negative for this test were submitted to a RT-
PCR test. Shoot growth was observed after 20 days on thermotherapy, and the highest shoot
growth was obtained after 60 days. After this maximum growth, plants began to fade towards at
80 and 100 days heat treatment. After 20 days of thermotherapy, 10% of GLRaV-2-RG-free
plants were recovered and 60% after 40 days. None of the other periods produced healthy plants.
This result shows that not only the exposure time to high temperatures is effective to obtain
GLRaV-2-RG - free plants, but the shoot growth rate is also important. Thus, it becomes evident
that the combination of thermotherapy with tissue in vitro culture is an effective tool to obtain
GLRaV-2-RG-free Red Globe plants

In a study, Virus-free plants were found after both, in vitro and in vivo therapy. Six and
seven positive plants were detected by the ELISA method and the RT-PCR tests, respectively. In
total 122 tests were done. As far as the growth of plants in the course of thermotherapy is
concerned, the best results were obtained on the A medium, where the length of new growth
increments ranged from 37 to 55 mm and the number of dead plants was the lowest. All plants
pro- duced always only one shoot and no multiplication and/or callus formation was observed.
The lowest growth increments were recorded on medium C. On the medium B, all plants died
within the 45 days of thermotherapy; however, this is in contradiction to the results obtained by
Leonhardt et al. (1998), who successfully treated grapevine plants on this rooting medium.
However, these authors used the temperature of 35°C in the course of thermotherapy, i.e. a
temperature that was lower by two degrees than that used in our study, and this could support the
survival of plants. For in vitro thermotherapy process, we recommend to use the medium A.
Rooting media with addition of auxin (media B and C) are, according to the obtained results,
unsuitable because of high mortality of plants. As far as the health status of plants was
concerned, no differences were found between the in vitro and the in vivo therapy. The in vitro
therapy of plant material is recommended by many authors mainly for the possibility to save the
space inside the thermobox. However, the in vivo thermotherapy of plants in the substrate has
several advantages such as a possibility to obtain a higher number of apical segments from one
treated plant and a reduced requirement of in vitro cultures in the course of therapy. Due to this
fact the cost of cultivation media is lower and the numbers of surface-disinfected segments are
reduced as well. A shorter period of sterile cultivation (max. 3 weeks) is another advantage of the
in vivo therapy method because it enables to reduce the risk of somaclonal variability, which
increases in dependence on the number of passages. The length of cultivation period can be
further reduced, for example when the method of micrografting on rooted healthy rootstocks is
used.

It is possible to eliminate PPV in plants using classical thermotherapy in vivo; other

possibilities are thermotherapy or chemotherapy in vitro in cultures of stone fruits. Until now,
only a few original reports dealing with elimination of PPV by in vivo thermotherapy have been
reported

The in vitro maintenance of virus-free plants provides the possibility to maintain a bank

of healthy and more vigorous plants, and with a more accelerated growth, than the infected ones. 
In a seed program, it is essential to initiate this work with virus-free plantlets since this
will affect the seed quality and the yield as well. The virus cleaning procedure is too long and
expensive.
PROCEDURE
1. Approximately 18 to 20 plantlets (virus-infected) are propagated in magentas.
2. After a growth period of 20 to 25 days, or when the plantlets are 4-5 cm high, they are
placed in the thermotherapy chamber. The growth conditions are: 16 hours of light 34 °C;
8 hours of darkness 32 °C
3. The magentas are maintained in the thermotherapy chamber for one month.
4. Afterwards, the magentas are taken out of the chamber, and the outside is cleaned with
98% alcohol. Then they are introduced to the culture room.
5. Meristems are obtained as follows:
Cut the apical portion and remove the leaves that cover the meristem (approximately 3 to
4 leaves); the meristem is observed with a prominent leaf primordium.  Remove the
meristem with part of the leaf primordium; cut only the translucent portion. Use a new
knife. Place the meristem in the culture medium. Be sure that the meristem is in the tube.
6. Evaluate meristem growth and transfer them to fresh media if necessary.
7. Each meristem that originates a plant is called “line”, which will be labeled according to
the accession it belongs to. For example, Yellow Line 1, Yellow Line 2, Yellow Line 3,
etc.
8. Five tubes with several plants are propagated to evaluate the potato virus PSTVd, PVT, to
determine the host range, the morphologic evaluation, and the in vitro maintenance.
 9. The results of each evaluation are obtained, and the infected material is replaced with
clean material.
Temperature
The growth of the plantlets at temperatures higher than 20 °C is accelerated (Do not use
temperatures higher than 30 °C)

The mechanism of virus inactivation with heat therapy is not well understood. However,
in some investigations it has been reported that heat treatment effect viral replication in plant cell
system. Thermotherapy has been extensively used for the elimination of different viruses from
various crop plants and in different viruses/plant infection. Different incubating temperatures and
time period has been used depending upon infected plant species and the infecting virus.
Sustained temperatures of 37°C or above completely inhibit multiplication of many viruses. In
studies with tobacco mosaic virus (TMV), no evidence of viral RNA synthesis was detected
when inoculated plants were maintained at 40°C for several hours. However, when infected
plants were moved from 25°C to 40°C, synthesis of viral dsRNA continued at high temperature
for a period, slowed and then stopped. During the slowing period, if plants were returned to
25°C, synthesis of virus RNA resumed. However, once dsRNA synthesis stopped at 40°C, the
ability to resume viral RNA synthesis at 25°C was temporarily lost. New synthesis occurred only
after a 12 hours lag period. In vivo synthesis of three TMV-specific proteins (160, 110 and 17.5
kDa) was inhibited at 40°C in a pattern similar to high temperature inhibition of dsRNA
synthesis. The studies conducted on cowpea chlorotic mottle virus (CCMV) indicated that RNA
synthesis was inhibited at 40°C. In contrast to TMV where dsRNA synthesis continued only
briefly at 40°C, synthesis of CCMV dsRNA declined gradually. For both TMV and CCMV,
synthesis of viral ssRNA ceased immediately at 40°C but resumed upon return to 25°C. Increase
in temperature above 35°C resulted in inhibition of synthesis of most pre-existing plant proteins
followed by synthesis of a set of a new proteins referred to as heat shock proteins (hsp). In vivo
synthesis of TMV specific protein was found to proceed independently of heat shock effects
(Dawson & Boyd, 1987). When shifted from 40°C to 25°C, host RNA synthesis resumes
immediately. However, there was a 4-8 hour delay before CCMV RNA synthesis resumed and a
16-20 hour delay before TMV RNA synthesis resumed. The exposure of infected plants to high
temperature could eliminate synthesis of both coat protein and movement proteins. This would
likely restrict cell-to-cell movement of pre-existing virus.

CTV was eliminated from infected plants by heat treatment. Preconditioning at 35/30°C
for two weeks followed by another one week at 40/35°C could not eliminate CTV and other
viruses in C. sincencis and C. aurantium. Plants incubated directly at high temperature without
preconditioning were desiccated and did not survive. None of Eureka lemon plant survived
during high temperature treatment (results not shown). However, CTV was successfully
eliminated from citrus bud-wood by incubating citrus plants at 35/30°C for two weeks, followed
by 40/35°C for another one week and finally incubating at 50/40°C for 1 wk in TCC.
Approximately, 50% of plants were lost during one week of incubation at 50/40°C treatment in
TCC. The plants kept at 50/40°C at 35/30°C for two weeks and 40/35°C for another one week
did not survive at high temperature in TCC. CTV was completely eliminated from citrus plants
after one week of incubation period at 50/40°C

References
Infante, R.; Fiore, N. 2006. Combined effect of thermotherapy and in vitro shoot culture on
the Grapevine leafroll associated virus 2 Red Globe strain affecting 'Red Globe' vines.

A.Muhammad, M. Ibrahim, A. Ahmad and Sher Hassan. 2005. Elimination of citrus tristeza
closterovirus from citrus bud-wood through thermotherapy. Pak. J. Bot., 37(2): 423-430, 2005

Virus eradication: tissue culture of meristems, thermotherapy, and chemotherapy. Techniques in

plant virology, CIP training manual

Křižan, B., E. Ondrušiková, V. Holleinová, K. Moravcová, L. Bláhová. 2009. Elimination of

Grapevine fanleaf virus in grapevine by in vivo and in vitro thermotherapy. Hort. Sci . (Prag ue),
36, 2009 (3): 105–108

J. Polák, A. Hauptmanová. 2009. Preliminary results of in vivo thermotherapy of plum, apricot

and peach cultivars artificially infected with PPV-M and PPV-D strains of Plum pox virus. Hort.
Sci. (Prague), 36, 2009 (3): 92–96