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IDENTIFICATION OF POTATO VIRUS X (PVX) AND POTATO

LEAFROLL VIRUS (PLRV) USING MOLECULAR DETECTION


TECHNIQUE

W.W.M.B.P.B.Weerasinghe
08/AG/026

Department of Export Agriculture


Faculty of Agricultural Sciences
Sabaragamuwa University of Sri Lanka
Belihuloya
2014

IDENTIFICATION OF POTATO VIRUS X (PVX) AND POTATO


LEAFROLL VIRUS (PLRV) USING MOLECULAR DETECTION
TECHNIQUE

W.W.M.B.P.B.Weerasinghe
(08/AG/026)

This report is submitted in partial fulfilment of the degree of


B.Sc. Agricultural Sciences and Management
Department of Export Agriculture
(Specialization in Horticulture)
Faculty of Agricultural Science,
Sabaragamuwa University of Sri Lanka,
2014.
Approved By
..
Dr. M. L. M. Chandrika Dissanayake
(Internal Supervisor)
Senior Lecturer (Gr. I)
Department of Export Agriculture
Faculty of Agricultural Sciences
Sabaragamuwa University of Sri Lanka.

Dr. (Mrs) B.M.V.S.Basnayake


(External Supervisor)
Deputy Director
Plant Virus Indexing Centre (PVIC)
Gabadawaththa, Homagama.

Date:

Date:

Dr. G.D. Kapila Kumara


Head of the department
Department of Export Agriculture
Faculty of Agricultural Sciences
Sabaragamuwa University of Sri Lanka.
Date: .
2

Dedication
Affectionately dedicated
To

My ever loving
Parents
And
Teachers..

ABSTRACT
Potato (Solanum tuberosum) is an economically important and highly demanded tuber
crop in Sri Lanka. Various diseases of potato are the major obstacles to improve potato
production in Sri Lanka. Among of them Potato Leafroll Virus causes to 90% production
loss and Potato Virus X causes to 15% production loss. Early detection of these viruses
using a reliable technique is the most economical control measure. Reverse transcriptase
polymerase chain reaction (RT-PCR) is a reliable, sensitive and common method for
detection and identification of plant viruses. But the PCR conditions should be optimized
for a better result. Improperly optimized PCR assays cause for mispriming and produce
nonspecific products. The objective of this study was to optimize RT-PCR conditions for
detecting PVX and PLRV. Initially PVX and PLRV were inoculated to host plants at
Plant Virus Indexing Centre. PVX was mechanically inoculated to young potato plants
under the temperature between 380C (maximum) and 240C (minimum). Due to
unfavourable climatic conditions for potato, tomato (cultivar Thilina) was chosen as host
plants. PVX was inoculated to 2-3 leaf stage tomato plants and 5 leaf stage tomato plants
under the temperature between 380C (maximum) and 240C (minimum). Then 5 leaf stage
tomato plants were inoculated under the temperature between 300C (maximum) and 220C
(minimum). PLRV was inoculated potato plants using the grafting method and the vector
methods under the temperature between 380C (maximum) and 240C (minimum). Both
PVX and PLRV inoculated plants were subjected to DAS-ELISA tests. Virus infected
samples were collected from farmers fields in Nuwara Eliya district due to the
unsuccessful virus inoculation process at the Plant Virus Indexing Centre. PLRV infected
potato samples were collected from Meepilimana seed potato farm. Size fractionated
silica extraction method was used to extract RNA. PLRVv1 and PLRVc2 were used as
specific primers for PLRV optimization. Expected band size (400bp) was observed from
locally isolated PLRV. The study revealed that PCR amplification of PLRV isolated from
Nuwara Eliya district is possible with specific primer PLRVv1 and primer PLRVc2 and
optimum PCR conditions are 2.5 L of 10 X PCR buffer (with MgCl2), 2.5 L of dNTPs
(10 mM), 1 L of Sense primer, 1 L of Antisense primer, 0.3 L of Taq polymerase,
16.8 L of Deionized water, and 3 L of cDNA template. Suitable annealing temperature
is 520C for 30 second.
Keywords: - RT-PCR, PCR buffer, dNTPs, primer, annealing temperature

ACKNOWLEDGEMENT
Foremost, I would like to convey my earnest gratitude to my internal supervisor Dr. M. L.
M. Chandrika Dissanayake Senior Lecturer, Department of Export Agriculture, Faculty of
Agricultural sciences, Sabaragamuwa University of Sri Lanka, for her excellent
supervision, encouragement and helpful criticism to make this effort a success.
I wish to express my deepest gratitude to my external supervisor Dr. (Mrs)
B.M.V.S.Basnayake, Deputy Director, Plant Virus Indexing Centre (PVIC) for her
inspiring guidance and great help that in correcting manuscript and giving constructive
advices in numerous ways to make this effort a success.
I wish to extend my sincere thanks to Dr. A.D. Ampitiyawaththa, Dean, Faculty of
Agricultural Sciences, Sabaragamuwa University of Sri Lanka for his valuable assistance
given me to complete the project successfully.
I wish to express my special thanks to Dr. Manel Dissanayake, Director of Plant Virus
Indexing Centre (PVIC) for facilitating me to carry out my investigation and guiding me
devoting their valuable time. I offer my sincere thanks to the entire staff of Plant virus
indexing center, for helping me in numerous ways in making my investigation a success.
Especially My deepest gratitude and grateful thanks are due to Mrs Nadeeka (Research
Officer), Mrs Damayanthi Dissanayake (Research Assistant), Mr Lasantha Hettiarachi
(Agricultural Instructor) and Mrs Ruchira Ekanayake (Agricultural Instructor) for their
assistance during my research project.
It is with a warm heart that I express my deepest gratitude to my parents for the
understanding and constant encouragement. I would wish to express my indebted thanks
to all my fellow workers who were constantly behind my success.
Finally, I wish to thank not only those who were mentioned individually, but also who
have contributed directly or indirectly to complete this research program successfully.

ii

TABLE OF CONTENTS
Contents
ABSTRACT .......................................................................................................................i
ACKNOWLEDGEMENT ................................................................................................. ii
TABLE OF CONTENTS ................................................................................................. iii
LIST OF FIGURES ......................................................................................................... v
LIST OF TABLES ........................................................................................................... vi
LIST OF ABRIVIATIONS ............................................................................................. vii
CHAPTER 1 .....................................................................................................................1
INTRODUCTION................................................................................................................... 1
Objectives ..................................................................................................................................................... 3

CHAPTER 2 .....................................................................................................................4
LITERATURE REVIEW ....................................................................................................... 4
2.1 Potato production and consumption of the world .................................................................................... 4
2.2 Potato production and consumption of the Sri Lanka .............................................................................. 4
2.3 Economic significance of potato in Sri Lanka ......................................................................................... 5
2.4 Virus disease of potato in Sri Lanka ........................................................................................................ 6
2.4.1 Potato virus Y (PVY) ............................................................................................................................ 6
2.5 Potato virus X (PVX) ............................................................................................................................... 7
2.5.1 Nucleic acid composition of PVX ........................................................................................................ 8
2.5.2 Detection methods of PVX ................................................................................................................... 9
2.5.3 Natural host range of PVX .................................................................................................................... 9
2.5.4 Test plant of PVX ................................................................................................................................. 9
2.5.4.1 Symptoms of Solanum lycopersicum ............................................................................................... 10
2.6 Potato leaf roll virus (PLRV) ................................................................................................................. 10
2.6.1 Nucleic acid composition of PLRV .................................................................................................... 11
2.6.2 Detection methods of PLRV ............................................................................................................... 12
2.6.3 Natural host range of PLRV ............................................................................................................... 12
2.6.4 Test plant of PLRV ............................................................................................................................. 12
2.7 Potato virus Transmission methods ....................................................................................................... 13
2.7.1 Transmission of PVX.......................................................................................................................... 13
2.7.2 Transmission of PLRV ....................................................................................................................... 14
2.8 Serological methods ............................................................................................................................... 14
2.8.1 Enzyme linked immunosorbent assay (ELISA) .................................................................................. 15
2.8.1.1 Double antibody sandwich (DAS) ELISA ....................................................................................... 16
2.8.1.2 Triple antibody sandwich (TAS) ELISA ......................................................................................... 16
2.9 Polymerase chain reaction (PCR) .......................................................................................................... 17
2.9.1 PCR conditions optimization .............................................................................................................. 19
2.9.2 Reverse transcription polymerase chain reaction (RT-PCR) .............................................................. 19
2.10 RNA extraction methods ..................................................................................................................... 20

iii

CHAPTER 3 ................................................................................................................... 22
Material and methods ........................................................................................................... 22
3.1 Equipment and tools used ...................................................................................................................... 22
3.2 Experimental sites .................................................................................................................................. 23
3.3 Agro ecological conditions of PVIC ...................................................................................................... 23
3.4 Experimental environment ..................................................................................................................... 23
3.5 Inoculation of PVX ................................................................................................................................ 23
3.5.1 Preparation of Phosphate buffer .......................................................................................................... 23
3.5.2 Inoculation procedure of PVX ............................................................................................................ 24
3.6 Inoculation of PLRV .............................................................................................................................. 26
3.6.1 Grafting method .................................................................................................................................. 27
3.7 Detection of PVX and PLRV by serological method(DAS-ELISA) ..................................................... 28
3.7.1 Detection of PVX by DAS-ELISA ..................................................................................................... 28
3.7.1.1 Detection of PVX by DAS-ELISA, commercial kit agdia ............................................................... 28
3.7.1.2 Detection of PVX by DAS-ELISA, commercial kit DSMZ ............................................................ 29
3.7.2 Detection of PLRV by DAS-ELISA, commercial kit agdia ............................................................... 30
3.8 Optimization of PCR conditions for PLRV ........................................................................................... 31
3.8.1 RNA extraction PLRV infected leaves ............................................................................................... 31
3.8.2 Reverse transcription and cDNA synthesis ......................................................................................... 31
3.8.2 Determination of annealing temperature ............................................................................................. 32
3.8.3 Analyses of PCR products .................................................................................................................. 33
3.8.4 Testing the reproducibility .................................................................................................................. 33

CHAPTER 4 ................................................................................................................... 34
RESULTS AND DISCUSSION ............................................................................................. 34
4.1 The detection of PVX using the DAS- ELISA tests .......................................................... 34
4.1.1 The detection of PVX from potato samples which inoculated under the temperature between
380C (maximum) and 240C (minimum). ...................................................................................................... 34
4.1.2 The detection of PVX from tomato samples which inoculated at 5 leaf stage under the
temperature between 380C (maximum) and 240C (minimum)..................................................................... 35
4.1.3 The detection of PVX from tomato samples which inoculated at 2-3 leaf stage under the
temperature between 380C (maximum) and 240C (minimum)..................................................................... 36
4.1.4 The detection of PVX from tomato samples which inoculated at 5 leaf stage under the
temperature between 300C (maximum) and 220C (minimum)..................................................................... 37

4.2 The detection of PLRV from potato samples which inoculated under the temperature
between 380C (maximum) and 240C (minimum).................................................................... 38
4.3 Determination of annealing temperature for PLRV ........................................................ 39

Chapter 5........................................................................................................................ 42
Conclusion and suggestions ................................................................................................... 42
6.1 Conclusion ............................................................................................................................................. 42
6.2 Suggestions ............................................................................................................................................ 42

References ...................................................................................................................... 43
Appendix ........................................................................................................................ 49

iv

LIST OF FIGURES
Figure 2.1 Potato importation of Sri Lanka ............................................................................... 5
Figure 2.2 PVYNTN infected tuber ............................................................................................. 7
Figure 2.3 Leaf mottling or yellowing ....................................................................................... 7
Figure 2.4 PVX -Electron microscope view .............................................................................. 8
Figure 2.5 Genome of PVX ....................................................................................................... 9
Figure 2.6 PLRV - Electron microscope view ......................................................................... 10
Figure 2.7 Genome of PLRV ................................................................................................... 12
Figure 2.8 Five steps in an ELISA procedure .......................................................................... 16
Figure 2.9 Double antibody sandwich (DAS-) ELISA ............................................................ 16
Figure 2.10 Triple antibody sandwich (TAS) ELISA .............................................................. 17
Figure 2.11 Schematic of the classical PCR. ........................................................................... 18
Figure 2.12 Reverse transcription polymerase chain reaction ................................................. 20
Figure 3.1 PVX inoculated potato plants ................................................................................. 25
Figure 3.2 PVX inoculated tomato plants ................................................................................ 25
Figure 3.3 Grafted potato plant ............................................................................................... 27
Figure 4.1 Amplified products of PLRV using different annealing temperature .................... 40
Figure 4.2 Amplified products of PLRV annealing temperature at 52oC ................................ 41

LIST OF TABLES
Table 3.1 PVX inoculation for Solanum tuberosum ................................................................ 24
Table 3.2 PVX inoculation for Solanum lycopersicum at 5 leaf stage .................................... 24
Table 3.3 PVX inoculation for Solanum lycopersicum at 2-3 leaf stage ................................. 25
Table 3.4 PVX inoculations for Solanum lycopersicum at 5 leaf stage in the molecular
laboratory ................................................................................................................................. 26
Table 3.5 PLRV inoculation of Solanum tuberosum ............................................................... 27
Table 3.6 Detection of PVX by serological method ................................................................ 28
Table 3.7 Detection of PLRV by serological method .............................................................. 30
Table 3.8 Reverse transcription reaction mixture .................................................................... 31
Table 3.9 PCR annealing temperature gradient ....................................................................... 32
Table 3.10 specific primers for detecting of PLRV, and the size of amplified products......... 32
Table 3.11 PCR mixture .......................................................................................................... 33
Table 4.1 Test Results .............................................................................................................. 34
Table 4.2 Test Results .............................................................................................................. 35
Table 4.3 Test Results .............................................................................................................. 37
Table 4.4 Test Results .............................................................................................................. 38

vi

LIST OF ABRIVIATIONS
DAS-ELISA- Double Antibody Sandwich ELISA
DNA- Deoxyribonucleic acid
dNTPs- Deoxynucleotide triphosphates
ECI- Enzyme Conjugate Immunoassay
ELISA- Enzyme linked immunosorbent assay
EtBr- Ethium Bromide
PBS-T-Phosphate buffer saline-Tween
PCR- Polymerase Chain reaction
PLRV- Potato Leafroll Virus
PNP- p-nitrophenol
PVIC- Plant Virus Indexing Centre
PVX- Potato Virus X
RNA- Ribonucleic acid
RNAsin- Ribonuclease inhibitor
Rtase- Reverse transcriptase
RT-PCR- Reverse transcriptase PCR

vii

CHAPTER 1
INTRODUCTION
Potato (Solanum tuberosum) is the most famous tuber crop in Sri Lanka, even though it is
not native to Sri Lanka and restricted few districts due to climatic conditions. According
to data of department of census and statistic of Sri Lanka (2012), total area of potato
cultivation is 4,847 Ha. The major potato growing seasons in Nuwara Eliya district is
Yala (February-May) and minor season is Maha (September - December). It also
widely grows in the Badulla district in paddy fields and high land during Yala and
Maha seasons respectively. Poor storage conditions, unavailability of suitable potato
seeds and susceptibility to diseases are the major problems of potato production of Sri
Lanka (Sathiamoorthy et al., 1985).
Potato virus X (PVX), potato virus Y (PVY) and potato leafroll virus (PLRV) are the
most prominent viruses in Sri Lankan potato cultivation. Potato virus X (PVX) is the
most distributed potato viruses due to latent infections of some strains. The potato mild
mosaic virus is another synonym for PVX. It infects many species in the Solanaceae and
15 other families of the plant kingdom. PVX was first reported by Smith in the United
Kingdom in 1931 (http://pvo.bio-mirror.cn/descr651.htm). PVX can be easily transmitted
by mechanical inoculation and natural contact between plants. There are four PVX
strains. These different PVX strains cause different symptoms in potato cultivation
(Bercks, 1970). Symptoms produced by PVX are variable, depending on the strain
present. In some strain, no visible symptoms are produced. But some strains show mild
mosaic, mottling distortion of leaves and plant stunning symptoms. Especially PVX
causes for a significant yield reduction (Shi et al., 2008). PVX shows more severe
symptoms combined with the PVY (Bercks, 1970). Also in tobacco it causes mottling or
necrotic spotting, in tomato it causes mosaic and slight stunting.
Potato leafroll virus (PLRV) is one of the most important viruses of potato cultivation. It
was first described by Quanjer et al., (1916). Potato phloem necrosis, Potato Virus 1 and
Solanum Virus 14 are the other synonyms for this virus. PLRV affect to reduce the
quality and quantity of the potato yield. PLRV is a phloem restricted virus and blocks
starch transportation from the leaves to the tubers.

PLRV transmits through tubers and via green perch aphid (Eskandari et al., 1978). There
are two kinds of symptoms in PLRV. Plants that become infected in the current growing
season show primary symptoms. Symptoms that appear on the plants growing from
infected seed tubers are called secondary symptoms (Jayasinghe, 1988). Primary
symptoms change according to the time of infection, potato verity and environmental
conditions. Primary symptoms are younger leaves become upward rolling, leaf margin
become necrotic and with the time leaf rolling may extend to older leaves. But symptoms
are

not

visible

in

potatoes

which

are

infected

late

in

the

season

(http://www.plantpath.ksu.edu/pages/ extension). Secondary symptoms are severely


rolling of lower leaves, young leaves become pale colour, leathery texture of leaves and
reduce the growth of plants. Also, some certain potato varieties (Russell, Burbank and
Green Mountain) show net necrosis of tubers (Jayasinghe, 1988).
Many methods have been developed for identifying and detect plant viruses. Such as
symptomatology, transmission tests, microscopy, detection methods based on viral coat
protein and detection methods based on virus nucleic acid. Detection methods based on
viral coat protein (serological or immunological assay) are one of the famous methods for
detecting plant viruses in plant pathology. Serological tests are broadly divided as liquid
and solid phase tests. The enzyme-linked immunosorbent assay (ELISA) is a one of
popular solid phase heterogeneous immunoassay test. It was introduced by Clark and
Adams (1977). It is usually done in micro titre plates made up of either polystyrene or
polyvinyl chloride. ELISA broadly divides into two categories as direct ELISA procedure
and indirect ELISA procedure. They differ in the way the antigen and antibody complex
is detected. Double antibody sandwich (DAS) ELISA, triple antibody sandwich (TAS)
ELISA, protein A-sandwich (PAS) ELISA and direct antigen-coated (DAC) /antigen
coated plate-trapped antigen (PTA) ELISA are the four types of ELISA used for plant
virus detection. DAS ELISA and TAS ELISA are the most widely used methods for plant
virus detections.
Polymerase chain reaction (PCR) procedure is a one of the most sensitive method for
virus detection (Mullis et al., 1986). PCR is an in vitro method for amplifying targeted
nucleic acid sequence. This procedure is applicable directly to DNA plant viruses. If use
plant viruses with RNA genome First, it has to convert into converted into
complementary DNA (cDNA). This process is called reverse transcription polymerase
2

chain reaction (RT-PCR). Then it can use for normal PCR. After successful amplification,
DNA can be analysed by agarose gel electrophoresis method.
Potato is a one of the most important economical crop in Sri Lanka. There is a
comparatively high cost of production for potato cultivation. Disease management is very
important to generate higher profit. Viral diseases are one of the major problems of potato
production. Because of there are no economically feasible chemical agents against plant
virus. Early identification is more important to take correct management decisions. PCR
is the most suitable methods for identification of plant viruses. But improper optimized
PCR assays cause for reducing the sensitivity and yield. Therefore PCR conditions were
optimized to minimize the failures and improve the performance.
Objectives
1. To develop molecular based detection techniques to identify PVX and PLRV.
2. To develop optimum PCR condition to detect PVX and PLRV accurately.

CHAPTER 2
LITERATURE REVIEW
2.1 Potato production and consumption of the world
Potato is the fifth most important food crop of the world after wheat, corn, rice and sugar
cane. But until the early 1990s, most potatoes were grown and consumed in Europe,
North America and countries of the former Soviet Union. Since then potato production
and consumed was dramatically increased in Asia, Africa and Latin America. But now
China is the biggest potato producer of the world. Asia and Europe are the major potato
production region of the world. Asia consumes almost half of the worlds potato supply.
But comparatively potato production of Africa and Latin America regions is smaller than
Asia and Europe. (http://www.fao.org/potato-2008/en/world/index.html). Europe takes
the first place in terms of per capita consumption of potatoes. It is 34 Kg per year. The per
capita consumption of potato in Asia is about 24 Kg with the highest per-capita in China.
The corresponding figure in Bangladesh and India is about 21.1 Kg, 17 Kg respectively
(http://www.helgilibrary.com/indicators/index/potato-consumption-per-capita).
Potato production and demand of developing countries is still increasing. Because potato
provides a primary source of energy for poor people and produce more food quickly
where land resources is limited. Not only that, but also there is a good potential for further
gains in production and consumption.
2.2 Potato production and consumption of the Sri Lanka
Potato cultivation of Sri Lanka started in 1850 by Sir Samuel Baker (http://
potatoes.alawathugoda.com/history.html). But commercial-scale potato cultivation started
in 1957 from Nuwara-Eliya and Badulla districts. Now it has become one of major crop
of Sri Lanka due to the high return of income. Photo production has now successfully
established in Nuwara-Eliya, Badulla, Jaffna and Puttalum districts. The average potato
yield of Sri Lanka is 16 T/ha but can reach as high as 40 T/ha (Sathiamoorthy et al.,
1985). There is a significant higher productivity in Badulla and Nuwara-Eliya districts
than the others (Ayoni et al., 2009).
According to the census and statistics department of Sri Lanka (2013) the total national
potato production was 75,352 MT in 2011/2012 and extent of potato cultivation was4

-4,847 Ha in 2011/2012. The per capita consumption of potato in Sri Lanka remains low
due to rice being the staple food of Sri Lanka. It was 7 Kg per annum in 2010 (http://
www.agridept.gov.lk/images/stories/site/PDF/Publication/English/BOOK/proposedplan.p
df).
2.3 Economic significance of potato in Sri Lanka
Potato is an economically important crop in Sri Lanka. The potato was named as an
essential food item in 2007 and it was subjected to a Special Commodity Levy (SCL)
under the Special Commodity Act. At present there are about 30,000 growers involve in
potato cultivation, of which 80% occupies less than 0.4 ha. But there has been a reduction
of national potato production due to the rising cost of production (Fernando et al., 2006).
The cost of production is estimated at around Rs. 292,000 per acre. But the average cost
of production of potato greatly varies due to the type of seeds and other agronomic
practices. Pest and disease cause to increase the cost of production of potato (Fernando et
al., 2006).
Selection of disease free seed potato is very important to get higher profit. Locally
produced and imported seed potatoes are used to cultivate potatoes. The price of good
quality imported seeds is 30-35% higher than the locally produced seeds. Sri Lankan
potato farmers spend 55%-60% of the total cost. Most of the farmers therefore tend to use
the locally produced seeds or the seeds obtained from the previous harvest. These seed
potato quality is lower and lead to reduce the production. Sri Lanka imports potato from
other countries such as Pakistan, India and China. It has gradually increased during the
past few years. The quantity imported in 2009 was more than twofold compared to that of
2005.
150,000
100,000
M 50,000
0
t

Year

Figure 2.1: Potato importation of Sri Lanka


Figure 1Potato importation of Sri Lanka

Source: - Census and statistics department of Sri Lanka (2013)

Potato importation varies within the year, according to local production. The import is
high in January, June and July as the domestic production remained low during these
months. But within the potato season potato importation is reduced by the government not
only that increased the tax for import potato to protect local potato farmers. For example a
levy of Rs. 20 per kilogram was then imposed on the imported potato. Also the imported
potato was subjected to Custom Duty of Rs. 20 per Kilogram and all other taxes such as
PAL (Port and Airport Development Levy), SRL (Social Responsibility Levy) and VAT
(Value Added Tax).
2.4 Virus disease of potato in Sri Lanka
More than 25 different potato viruses have been identified. These viruses are often
described by their initials (Hooker, 1982). Potato virus X (PVX), potato virus Y (PVY),
potato virus S (PVS), potato virus M (PVM), potato virus A (PVA) and potato leafroll
virus (PLRV) are the most important viruses when consider their distribution and effect
on yield. These viruses may cause loss of yield, latent infection, changes in leaf colour,
leaf deformation, stunning, and tuber necrosis and leaf deformation (Hooker, 1982).
Potato cultivation of Sri Lanka still not much exposed to all of these viruses. But potato
virus X, potato virus Y and potato leaf roll virus are more common in Sri Lanka. Among
of them PLRV is the most distributed potato virus in Sri Lanka (Potato virus disease
leaflet PVIC).
2.4.1 Potato virus Y (PVY)
Potato virus Y (PVY) is the type member of the Potyviridae. It infects a range of
Solanaceae family crops such as potato, chilli, tomato and tobacco. There are three types
of major strain of PVY. There are PVYC (stipple streak strains), PVYN (tobacco veinal
necrosis strains) and PVYO (common strain). PVY can combine with PVX and causes to
greater loss of yield. Also recombination between PVYN and PVYO produced PVYNTN
which can cause very severe economic damage to yield. PVYO has been distributed in
most in most potato growing countries. PVYC in eastern Australia, South America, New
Zealand, Europe, South Africa and North America; and PVYN in North America, New
Zealand, Europe, Africa and South America ( Gray et al.,2010). PVY can be spread by
mechanical contact and aphids. But the major source of PVY inoculum is infected seed
tubers.
6

The symptoms are different according to the PVY strains. Symptoms of PVYO and PVYC
are leaf mottling or yellowing, leaf deformation, necrotic leaf spots or rings, veinal
necrosis, necrotic stem-streaking, leaf drop and premature death of stems. PVYN causes
milder forms of leaf mottling. PVYO, PVYC and PVYN do not show any symptom from
the tuber. They appear as normal. But PVYNTN shows irregular brownish colour rings,
which turn necrotic and sink into the tuber, forming necrotic arcs in the flesh and cracking
the skin at the surface (http://www.potato.org.uk/media-gallery/detail/13214/2925).

Figure 2.2:PVYNTN infected tuber

Figure 2.3: Leaf mottling or yellowing

Source: - http://www.potato.org.uk/media-gallery/detail/13214/2925
Planting healthy seed potato is the most effective method for control PVY. Insecticides
can be used to control aphids. But the usage of insecticide is largely ineffective in the
control of PVY because they do not act fast enough to kill aphids quickly and thereby
prevent virus spread. Field sanitation and use harvesting methods that minimize skinning
and bruising are the other control methods (Roger et al., 2003).
2.5 Potato virus X (PVX)
PVX is a member of genus potexvirus and family Alphaflexiviridae. It is flexuous rod
shaped virus (Stols et al., 1970) and modal length is about 515 nm and 13.5 nm in
diameter (Karpova et al., 2006). Particle specific volume is 0.73 cm3/g (Lauffer and
Cartwright, 1952). Particle weight is c. 35 x 106 (Huisman et al, 1988).There are four
strains (PVX1, PVX2, PVX3, and PVX4) according to their reactions with genes of
potato cultivars for localized hypersensitivity (Nb and Nx) and extreme resistance (Rx)
(Querci et al., 1995). Group 1 strain induces the hypersensitivity reaction in potatoes
carrying either the Nx or Nb gene. Group 2 strains induce hypersensitivity only in Nb
potatoes.

Group 3 strains induce hypersensitivity only in Nx potatoes. Group 4 strains overcome


both Nx and Nb. Symptoms of PVX are changed according to the strain. Symptoms
quickly develop in growing plants at temperatures above 210C. But no symptoms
development and virus multiplication occur at temperature above 30 oC (Singh, 1998).

Figure 2.4 :PVX -Electron microscope view


Source: - http://ictvdb.bio-mirror.cn/WIntkey/Images/a2.html
2.5.1 Nucleic acid composition of PVX
PVX contains a linear positive sense, single standard RNA. Base composition is 22% G,
32% A, 24% C, and 22% U (the main potato virus of potato crop). Genomic RNA of
PVX has a 5 m7GpppG cap, a 3' poly (A) tail and 5 open reading frames (ORFs) in one
genomic RNA of PVX. Leader region extends from nucleotide 1 to nucleotide 84. The
ORF 1 extends from nucleotide 85 to nucleotide 84. The second ORF start position is
nucleotide 4486 and end position is nucleotide 5164. The ORF 3 starts at nucleotide 5147
and stop nucleotide 5492. ORF 2 and ORF 3 are partly overlapped.
The ORF 4 extends from nucleotide 5427 to nucleotide 5637. Start position of ORF 5 is
nucleotide 6362 and end point is nucleotide 6438 (Price, 1992). ORF 1 involve for RNA
polymerase activity. Products of ORF 2, 3, and 4 are involved in cell to cell transport of
PVX together with a functional coat protein. ORF 5 encodes the coat protein. Encoded
proteins of ORF are Mr 165588 (166K), Mr24622 (25K), Mr 12324 (12K), Mr 7595 (8K)
and 25080 (coat protein) respectively. The ORF 1 product contained domains of similar
to the tobacco mosaic virus 126K and 183K products.

The ORF 2 and 3 products showed similarity with the barley stripe mosaic virus 58K and
14K proteins, the beet necrotic yellow vein virus 42K and 13K products and the white
clover mosaic virus 26K and 13K products, respectively ( Marianne et al., 1988).

Figure 2.5: Genome of PVX

Source: - http://emboj.embopress.org/content/16/12/3675
2.5.2 Detection methods of PVX
PVX identification from field observation is not very much reliable. Because of most of
time symptoms are latent and depend on environmental conditions. The virus is strongly
immunogenic and can be detected by a variety of serological tests. Time-resolved
fluoroimmunoassay is the most sensitive serological test (detection limit 5 pg/ml)
(http://www.dpvweb.net/dpv/showdpv.php?dpvno=354). PVX can be easily detected
through commercial ELISA kits using potato leaves and dormant or sprouted tubers.
Diffusion test of agar can be used both for detection and determining strain relationships.
Immunoelectron microscopy method also can be used to detect PVX. Reverse
transcriptase chain reaction (RT-PCR) is the most sensitive method to detect PVX.
2.5.3 Natural host range of PVX
Natural host range mainly limited to solanaceous species. Main host plants are Brassica
rapa, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosu, Petunia hybrid,
Solanum nigrum and cyphomandra betacea.
2.5.4 Test plant of PVX
Test plants for local symptoms:
1. Chenopodium amaranticolor
2. Chenopodium quinoa
3. Gomphrena globosa
4. Nicotiana hesperis 67A

Test plants for systemic symptoms:


1. Datura stramonium
2. Nicotiana hesperis 67A
3. Nicotiana miersi
4. Solanum lycopersicum
Source: - www. Qba nk .eu / Virus / Biolo MICS . aspx ? Table = Plant % 20
Virus % 20 Species %2 0 Database & Rec= 56 & Fields =All
2.5.4.1 Symptoms of Solanum lycopersicum
PVX infection of tomato usually shows leaf mottling and slight stunting, but in some
cases the foliage may have distinct yellowing depending upon the virus strain. The
mottled areas may have small, brown spots (World vegetable center fact sheet, Tom
Kalb).
2.6 Potato leaf roll virus (PLRV)
Potato leafroll virus is a member of genus Polerovirus and family Luteoviridae. It is a
phloem limited spherical shape virus (Kawachuk et al., 1988). Diameter is 24 nm. New
strains of PLRV have been detected by Webb in 1955. PLRV strains cannot distinguish
from serological methods. These strains have been distinguished by the severity of
symptoms induced in potato or by their ease of transmission by Myzus persicae. PLRV is
heat sensitive and can be eradicated from heat treatments (Frances et al., 1971).PLRV
can be eliminated by keeping tuber under 37.5 0C for 25 days.

Figure 2.6 :PLRV - Electron microscope view


Source: - http://www.dpvweb.net/dpv/showfig.php?dpvno=291&figno=06
Several PLRV transmit aphids have been reported. Both lave and adults can transmit
PLRV virus. Myzus persicae is the most efficient aphid. Macrosiphum euphorbia is
10

another PLRV transmit aphid but it is less effective than Myzus persicae. M.nicotiane
also has been reported as PLRV transmit aphid in Brazil. To successful transmission
aphids must feed on phloem for at least 20 minutes to 30 minutes (Jayasinghe, 1988).
After the virus enters into virus body, during the incubation period virus remain as noninfectious for several hours. After the virus become infective and persist throughout the
aphids life.
Infected plant Phloem

Digestive system of aphid

Enter into new plant when feed

enters into nomocel

Enter into the salivary glands

Source: - http://en.wikipedia.org/wiki/Potato_leafroll_virus
2.6.1 Nucleic acid composition of PLRV
PLRV consists of linear single standard positive sense RNA. The viral genome is
covalently linked to a 7.2 KDa protein at its 5 start and does not contain a poly A
sequence of its 3 terminus. Genomic RNA of PLRV consists of 8 ORF.ORFs of PLRV
are separated by a small intragenic region (non-coding region of 197 nucleotides) into
two gene clusters (ORF 0-2 and ORF 3-7). The first gene cluster is translated directly
from genomic RNA. ORF 0 encode protein 28K which responsible for symptom
development. ORF 1 produces 70K which causes for motifs characteristic of helicases.
ORF 2 encodes 69k which produces polymerases to form part of the viral replicase.
ORF3 (23K) encodes a coat protein gene, and the most conserved coding region, ORF4
(17K), encodes a movement protein, while ORF5 (56K) encodes an aphid transmission
factor. ORF 0 and ORF 1 are overlapped in different frames. The second cluster is
translated from sub genomic RNA (sgRNA) 1 (ORF 3-5) and sub genomic RNA
(sgRNA) 2 (ORF 6-7). sgRNA serve as a mRNA. sgRNA 2 encodes two viral proteins.
ORF 6 encodes 7.1 k and ORF 7 encodes 14k. PLRV genome in the coat protein region
is similar to a beet western yellows virus and barley yellow dwarf virus.

11

Figure 2.7: Genome of PLRV


Source: - http://www.pnas.org/content/100/15/8939/F1.expansion.html
2.6.2 Detection methods of PLRV
ELISA is the only available serological method for detecting PLRV due to low
concentration of infected plant (Jayasinghe, 1988). But Also very low concentration of
PLRV virus, in plants grow at temperatures of 30 0C and older plants may cause to
ELISA detection. Also unsprouted tubers may not detect PLRV. Various methods based
on nucleic acid detection can be used to detect PLRV. Sway and Hadidi (2000) reported
that RT-PCR is more sensitive than ELISA for the detection of PLRV. PLRV
serologically related with Tobacco necrotic dwarf, beet western yellows/beet mild
yellowing, bean leafroll, subterranean clover red leaf and barley yellow dwarf viruses
(http://sdb.im.ac.cn/vide/descr644.htm).
2.6.3 Natural host range of PLRV
Hosts are mainly in the Solanaceae family. But there are some non Solanaceae plants
such as Amaranthus caudatus, Celosia argentea, Gomphrena globosa and Nolana
lanceolata are susceptible (Natti et al., 1953). Earlier, it was considered PLRV has a
narrow host range. But now Twenty-two new Hosts of Potato leafroll virus has been
reported (http://dx.doi.org/10. 1094/PDIS.2002.86.5.561A).
2.6.4 Test plant of PLRV
1. Datura stramonium
2. Physalis floridana
3. Solanum tuberosum ssp. tuberosum
Sources: - http://sdb.im.ac.cn/vide/descr644.htm

12

2.7 Potato virus Transmission methods


Potato virus experimentally can be transmitted from plant to plant by mechanical contact,
grafting, vegetative propagation, seed, pollen, common dodder and vectors. Mechanical
contact needs a wound of plant body and transmission can be done by contact between
potato tubers, between leaves and between the roots. 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) can be transmitted
through mechanical contact. Grafting is considered as a universal method for transmitting
systemic viruses. Virus transmission through grafting will not always successful, if the
virus is unable to cross the grafting union, or if the virus source plant not totally invaded.
Vegetative propagation is an easy method for potato virus transmission. Almost all the
potato virus can be transmitted by this method. Virus transmission through seed is a most
effective method. This method can be used in two ways, through seed cover (externally)
and through the embryo and the endosperm (internally). The PSTVd can be successfully
transmitted through seed under laboratory conditions.
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. PSTVd and PVT are
transmitted by pollen or the ovule of infected plants. 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 Cuscuta
subinclusa. This type of transmission is effective when mechanical inoculation will not
give positive results. The most important viruses transmitted by aphids to potato are
PLRV, PVY and PVA. PLRV is persistent viruses but other viruses are non-persistent.
2.7.1 Transmission of PVX
PVX can be easily transmitted by mechanical inoculation. Also PVX infected seed potato
is another common method for transmit PVX. PVX is not transmitted by aphids but plant
hoppers and other chewing insects can transmit the virus due to mechanical contact when
they feed. PVX can be transmitted through root touching. Also PVX can transmit by
zoospores of fungus Synchytrium endibioticum. Also PVX can experimentally transmit by
Cuscuta campestris.

13

2.7.2 Transmission of PLRV


Grafting is easiest and most common method of PLRV transmission. After the grafting it
takes seven days to virus movement through two graft union. Virus movement occurred
soon after start functional phloem containing across the graft union (Derrick and Barher,
1997). It is also possible to transmit PLRV to plants by agro inoculation. This is a another
form of mechanical inoculation in which plants are injected with agrobacterium cells that
carry a Ti plasmid containing a full length DNA copy of the virus genome ( Mayo et al .,
2000). Aphids (Myzus persicae, Myzus persicae and Myzus nicotiane) can also use for
PLRV transmission.
2.8 Serological methods
In the serological methods specific antibodies are used to detect their respective antigens.
Antibodies are composed of immunoglobulin (Ig) proteins produced in the animal body in
response to the presence of antigen. Antibodies are usually foreign protein, complex
carbohydrates, polynucleotides, or lipopolysaccharides. These antibodies can bind to a
particular antigen. There are two kinds of antibodies, monoclonal antibodies (an antibody
produced by a single clone of cells or cell line and consisting of identical antibody
molecules) and polyclonal antibodies (heterogeneous mixture of antibodies directed
against various epitopes of the same antigen).
There are two types of sociological types, solid phase assays (ELISA, western immuneblotting) and liquid phase assays (agar gel single and double diffusion, ring precipitation
or agglutination) (Naidu and Hughes, 1998).
The main disadvantage of serological methods is based on the antigenic properties of the
virus structural properties. Also serological methods have limited application such as the
detection of viroid, satellite RNAs, viruses which occur as extremely diverse serotypes
and viruses that are poor immunogens or are difficult to purify. But serological methods
are particularly suitable for developing countries due to easier to perform, cost effective
and the require regents are readily available (Kumar et al., 2004).

14

2.8.1 Enzyme linked immunosorbent assay (ELISA)


ELISA is inexpensive, reliable and sensitive assay method for detecting many plant
viruses (Joseph et al., 1978). The basic principle of the ELISA is immobilizing the
antigen onto a solid surface, or capturing antigen by specific antibodies. Then the reaction
is detected by means of enzyme labelled antibodies and substrate. If the reaction is
positive the enzymes converts substrate to product which can be easily recognized by its
colour. Also ELISA can be used to measure the concentration of the antigen, according to
colour severity. This method is very useful than other serological methods for the
identification of most viruses, including PLRV and PVA, which are found only in low
concentrations in infected plants. Also, this method need lower amount of antiserum than
other serological method. But this technique comprises numerous steps than other
serological methods. The micro plate method is the most common method for virus
detection and term ELISA was introduced by voller et al., 1976.
The major disadvantage of this method is the requirement that the pathogen must be
present at high enough concentrations in the tissue for the reaction to proceed to a level
that can be detected. To perform an ELISA test generally following materials is needed.
There are ELISA plate, micropipettes, ELISA plate reader, incubator, refrigerator, light
box, motor and pestles. Also following solutions are needed such as carbonate or coating
buffer, phosphate buffer saline (PBS), phosphate buffer saline tween (PBS-T), antibody
buffer (PBS-TPO) and distilled water.
Generally there are five steps in an ELISA procedure (http:// www. elisa-antibody.com/
ELISA-Introduction/ELISA-Principle)
1. The antibody is allowed to adsorb to the walls of the well
2. The test solution is added and if antigen is present it will bind to the antibody
3. The wells are washed and conjugated antibody is added
4. The wells are washed again and the chromogenic substrate is added
5. If the antigen is present reaction of a substrate with the enzyme to produce a
colored product

15

Figure 2.8 :Five steps in an ELISA procedure

Source: - http://www.elisa-antibody.com/ELISA-Introduction/ELISA-Principle
2.8.1.1 Double antibody sandwich (DAS) ELISA
Virus molecules are sandwiched by two antibody molecules in DAS ELISA procedure. In
this assay polyclonal antibody or IgG extracted from the polyclonal serum are coated onto
the ELISA plate. Then antigen is added. The trapped antigen is detected by the addition
of enzyme labelled antibodies. DAS ELISA is highly strain specific and requires a good
amount of antiserum to prepare enzyme conjugate (Kumar et al., 2004).

Figure 2.9 :Double antibody sandwich (DAS-) ELISA


Source: - R.A Naidu and J.D.A Hughes (1998), Methods for the detection of plant virus
diseases
2.8.1.2 Triple antibody sandwich (TAS) ELISA
Triple antibody sandwich ELISA (TAS ELISA) is similar to the DAS ELISA, but there is
an additional step involved before adding detecting antibody enzyme conjugate.
Monoclonal antibody (MAb) is used in this additional step.

16

Then MAb is detected by adding an enzyme conjugated species that does not react with
the trapping antibody, followed by colour development reagent.

Figure 2.10: Triple antibody sandwich (TAS) ELISA

Source: - R.A Naidu and J.D.A Hughes (1998), Methods for the detection of plant virus
diseases
2.9 Polymerase chain reaction (PCR)
PCR is a technique used for amplification of a specific segment of a nucleic acid. This
technique was developed by Kary mullis in the 1980. Although the PCR is a sensitive
technique for virus detection following conditions can be caused to reduce the sensitivity
of the PCR. There are contaminations from extraneous DNA, cross-contamination
between samples, low Mg2+ concentration or high Mg2+ concentration, non-specific of
primers and primer-primer dimmer formation (Sridhar Rao, 2006).
To perform a PCR test generally following material and solutions are needed. There are
thermal cycler (thermo cycler), UV transillumination, sample dsDNA with a target
sequence, thermo stable DNA polymerase, two oligonucleotide primers, Deoxynucleotide
triphosphates (dNTPs) and reaction buffer (with or without magnesium iron) and
magnesium chloride. These solutions are needed to prepare the PCR mixture. Then, using
thermo cycler amplifies the targeted DNA sequence. PCR is carried out in three major
steps. These steps named as denaturation, primer annealing and primer extension. The
three step cycles are repeated between 30 and 40 cycles in an automated thermal cycler
until sufficient product is produced. The amplified products can be detected using gel
electrophoresis. Ethidium bromide is used for staining and visualization with UV
transillumination.
17

Visualization of a band containing DNA fragments of a particular size can indicate the
presence of the target sequence in the original DNA sample. Absence of a band may
indicate that the target sequence was not present in the original DNA sample.
Denaturation
Double slandered DNA is converted into single slandered DNA due to broken of
hydrogen bounds by heat. Usually denaturation for 0.5-2 minutes at 94oC-95oC. But if the
amplified DNA has a very high GC content, denaturation time may be increased up to 3-4
minutes. Insufficient denaturation of DNA results in the inefficient utilization of template
in the first amplification cycle and in a poor yield of PCR product.
Primer annealing
Primers bind to the DNA in this stage. Annealing temperature is depending on the
nucleotide composition and length of the primer. The optimal annealing temperature is
50C lower than the melting temperature of primer template dsDNA. Typical primer
annealing is 550C for 20 second. However the annealing temperature should be optimized
otherwise the nonspecific PCR products are obtained in addition to the expected product.
Primer extension
The taq polymerase enzymes are involved for the primer extension. The taq polymerase
enzyme extends the primer, adding nucleotides on to primer from 5 to 3.
Finally, two double standard DNA molecules are made from each one double stranded
DNA molecules that the length and concentration was denatured. Typical prime extension
temperature is 720C for 30 seconds. But extension time depends on of the targeted
sequence. The extending time is usually increased by 1 minute for each 1000 bp.

Figure 2.11 :Schematic of the classical PCR.


Source: - http://microfluidics.stanford.edu/Projects/Current/OnChipPCR.html
18

2.9.1 PCR conditions optimization


There are standard PCR protocols. But single protocols are not appropriate to all
situations. Because they are presented only as starting conditions for designing new PCR
applications. Magnesium ion concentration, reaction buffer, enzyme concentration,
primer design, template, cycle parameters and nucleic acid cross contamination are the
factors that influence to the amplification of products during PCR. When developing a
PCR protocol, it is important to consider about reagent concentrations, cycling
temperatures cycle number.
The recommended Taq DNA polymerase concentration range is between 1 and 2.5 units
per 100 L reaction when other parameters are optimized. Requirement of Taq DNA
polymerase is vary with respect to individual target templates or primers. Nonspecific
background products are accumulate if the Taq DNA polymerase concentration high. If
two low insufficient amount of yield is made. Recommended dNTPs concentration range
is between 20 and 200M each result in the optimal balance among yield, specificity, and
fidelity. Low dNTPs concentration cause to minimize mispriming at nontarget sites and
reduce the likelihood of extending misincorporated nucleotides (Innis et al., 1988). Low
Mg2+ concentration cause to low yield and high Mg+ concentration cause to nonspecific
products.

PCRs should contain 0.5 to 2.5 mM magnesium over the total dNTPs

concentration. Optimization of primer annealing temperature is a critical factor.


Increasing the annealing temperature enhances discrimination against incorrectly
annealed primers and reduces misextension of incorrect nucleotides at the 3' end of
primers. Primer concentrations between 0.1 and 0.5 M are generally optimal. Higher
primer concentrations cause to promote mispriming and accumulation of nonspecific
products and may increase the probability of generating a template-independent artifact
termed a primer-dimer.
2.9.2 Reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR is a very sensitive method which uses reverse transcriptase enzymes to make
DNA from an RNA (mRNA) template. PCR mixture which use in RT-PCR is very
similar to normal PCR mixtures. The difference is adding reverse transcriptase enzymes.
This enzyme synthesizes single standard DNA, which called cDNA (DNA that made
from RNA template).
19

Then cDNA undergoes a normal PCR process under the control of DNA polymerases
(Polymerase is an enzyme that helps replicate or repair DNA), resulting in the synthesis
of millions of double-stranded DNA molecules representing the sequence of the original
RNA.

Figure 2.12: Reverse transcription polymerase chain reaction


Source:http://en.wikipedia.org/wiki/File:Reverse_transcription_polymerase_chain_reactio
n.jpg
There are two kinds of RT PCR approaches, one step RT- PCR approach and two steps
RT-PCR approach. In the one step RT-PCR approach, the entire reaction (from cDNA
synthesis to PCR amplification) occurs in a single tube. But in the two step RT-PCR
approach, the reverse transcriptase and PCR amplification are carried out in separate
tubes. There are developed RT-PCR protocols for one step RT- PCR approach and two
steps RT-PCR approach. In the one step RT- PCR approach, the starting RNA templates
are prone to degradation and the use of this approach is not recommended when repeated
assays from the same sample is required. But one step RT-PCR approach causes to
minimize the experimental variation due to all of the enzymatic reactions occurs in a
single tube. However, one-step approach is reported to be less accurate compared to the
two-step approach ( http:// en.wikip edia.org / wiki / Reverse_

transcription_

polymerase _ chain_reaction).
2.10 RNA extraction methods
RNA extraction is the most important step of a RT-PCR assay due to the accuracy and
reliability of RT-PCR test depends on the quality and quantity of total nucleic acids

20

(Sipahioglu et al., 2006). Extracted RNA can be easily degraded due to unfavourable
conditions and the presence of active Ribonuclease (RNases) in the tissues itself.
RNases is a very active enzyme that catalyses the degradation of RNA into smaller
components. Therefore RNA extraction is practically difficult and it is needed sterilized
glassware and plastic ware to avoid RNases contaminations. Phenols, polyphenols and
polysaccharide substances in tissues prevent reverse transcriptase especially in RNA
extraction phase and reduce RT-PCRs reliability (Sipahioglu et al., 2006). RNA
extraction protocols should overcome these problems. There are standard RNA extraction
protocols. Such as acid guanidium-phenol-chloroform extraction, salt or detergent
method, silica captures method, lithium chloride method and citric buffer method (Gumus
and Paylan, 2013). Also, there are lots of commercially available RNA extraction kits.
Phenol, salt and/or a detergent are the most common RNA extraction methods. However,
there are some disadvantages of these methods. Such as the poor yield and purity of
nucleic acid from some viruses, the need for a long time and/or many steps in nucleic acid
preparation, and the UV absorption and the noxious nature of phenol (Wilcockson and
Hull, 1973).Acid guanidium-phenol-chloroform extraction is useful for processing large
number of samples and for isolation of RNA from minimum quantities of cells or tissue
samples. This method based on the lysing and nuclease inactive properties of
guanidinium thiocyanate together with the binding properties of silica partials. The main
advantage of this method is providing a pure preparation of underrated RNA in high yield
and can be completed within 4 hours. The silica capture method can be used to get the
highest concentration of yield and the best purity of total RNA when compare to other
methods such as lithium chloride and citric buffer (Sipahioglu et al., 2006). MacKenzie et
al., (1997) reported that high amounts of nucleic acids can be obtained with the silica
capture method. In 2004, Ciedlinska (2004) performed a study about effective RNA
extraction methods for strawberries and found the lithium chloride method the most
effective of all tried methods.
Gunasinghe et al., 2009 have been developed an RNA extraction method using Guanidine
thiocyanate and fractionated silica. This method is a cost effective, efficient and efficient
method. Also, RNA can be extracted within one hour using this method.

21

CHAPTER 3
Material and methods
3.1 Equipment and tools used
Electronic balance (Adventurer OH AUS)
ELISA plates (Micro plate zero-well)
Incubator (Binde made in Germany ranges 5-3000C)
ELISA plate reader (Anthos 2020)
Forceps and scalpel
Mortars and pestles
Eppendorf tubes
Bench centrifuge (Ice Centra -4B centrifuge)
Micropipettes- 1000L, 100 L, 10 L, 2 L ( Thermo electron corporation and
BRAND)
Constant speed mixer (LAB-LINE)
Dry bath incubator (DB-005 Gemmy Industrial Corp.)
Thermometer
PCR tubes
Themocycler (TECHNE-PROGENE)
Elecrophoresis apparatus (VWR Scientific.)
UV transilluminator (2UV Transilluminator.)
USB Digital web Camera (SPW-2002 Golden Eye)
Microwave oven (Multiwave LG)
Microprocessor pH meter (PH211-HANNA instruments)
Heating magnetic stirrer (VELP Scientifica)

22

3.2 Experimental sites


The experiment was carried out in a greenhouse of PVIC Homagama, Gabadawaththa and
the molecular virology laboratory of PVIC Homagama, Gabadawaththa.
3.3 Agro ecological conditions of PVIC
Homagama (PVIC) is located in the agro ecological zone of WL3
Annual rainfall-1520 mm
Average maximum temperature- 310C
Average minimum temperature-24oC
Relative humidity- 85%
3.4 Experimental environment
The experiment was carried out January to April. The maximum temperature was 38 0C
and minimum temperature was 240C in the green house and the maximum temperature
was 30 0C and minimum temperature was 22 0C in the molecular virology laboratory.
3.5 Inoculation of PVX
Mechanical / sap inoculation method was used to inoculate PVX virus. DSMZ ELISA kit
positive sample was used as a source of inoculum. Disease free Potato tubers and tomato
plants were planted in planting pots in a greenhouse house of PVIC. Plants were irrigated
daily. Albert solution was used as fertilizer.
3.5.1 Preparation of Phosphate buffer
Stock solution A was prepared by dissolving 0.136 g KH2PO4 in 100 ml of distilled
.

water. Stock solution B was prepared by dissolving 0.178 g of Na2HPO4 H2O in 100 ml
of distilled water. 0.05g Na2SO3 was dissolved in 50 ml of distilled water. 49 ml of stock
A and 51 ml of stock B were mixed. pH was adjusted up to 7.

23

3.5.2 Inoculation procedure of PVX


To prepare inoculum, 400 L of phosphate buffer was measured into an eppendorf tube.
Very small amount PVX positive sample was dissolved in the buffer. The one drop of
Na2SO3 was added. Inoculated plants were labelled. Youngest leaf was used for
inoculation. The layer of carborandum dust was applied on the upper surface of the leaf.
The inoculation sap was rubbed gently on the upper surface of the leaf by finger. The
inoculated surfaces were rinsed by sprinkling distilled water. The inoculated plants were
covered with wet newspaper sheet for 24 hours to provide a humid environment and
protect from light and wilting.
Table 3.1: PVX inoculation for Solanum tuberosum
Cultivated date- 07/01/2014
Plant species

Inoculated date

Potato
Pot no. 2 Plant
1
(PVX 27/01/2014
-first
inoculated)
Plant2
(PVX inoculation
inoculated)
Pot no. 3 Pant
1
(PVX
inoculated)
Plant
2
(PVX
inoculated)
Pot no. 5 Plant
1
(PVX
inoculated)
--------PotControl (not inoculated)
control

Temperature
Maximum temperature- 38 oC
Minimum temperature 24oC
Green house conditions

Table 3.2 PVX inoculation for Solanum lycopersicum at 5 leaf stage


Plant species
Tomato (cultivar Thilina)
Pot no. 1 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.2 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.3 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.4 Plant 1 (PVX inoculated)
--------Pot no.5 Plant 1 (PVX inoculated)

Inoculated date

Temperature

Maximum temperature- 38 oC
Minimum temperature 24oC
13/02/2014 first
inoculation

Green house conditions

24

Pot no.8

Plant 2 (PVX inoculated)


Plant 1 (PVX inoculated)
--------Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Plant 1 (PVX inoculated)

Control

Plant 2 (PVX inoculated)


Control (not inoculated)

Pot no.6
Pot no.7

19/02/2014 second
inoculation

Table 3.3: PVX inoculation for Solanum lycopersicum at 2-3 leaf stage
Plant species
Tomato (cultivar Thilina)
Pot no. 1 ~ (3 plants PVX inoculated)
Pot no. 2 ~ (4 plants PVX inoculated)
Pot no. 3 ~ (4 plants PVX inoculated)

Inoculated date
21/02/2014 first
inoculation

Temperature
Maximum temperature- 38 oC
Minimum temperature -24oC
Green house conditions

Pot no. 4~ (4 plants PVX inoculated)

Figure 3.1: PVX inoculated potato plants

5 leaf stage

2-3 leaf stage

Figure 3.2: PVX inoculated tomato plants


25

Table 3.4: PVX inoculations for Solanum lycopersicum at 5 leaf stage


PVX was inoculated to 5 leaf stage tomato plants again under the temperature between
300C (maximum) and 220C (minimum) [In the molecular laboratory].
Plant species
Tomato (cultivar Thilina)
Pot no. 1 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.2 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.3 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.4 Plant 1 (PVX inoculated)
--------Pot no.5 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.6 Plant 1 (PVX inoculated)
--------Pot no.7 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no.8 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Pot no 9 Plant 1 (PVX inoculated)
Plant 2 (PVX inoculated)
Plant 1 (PVX inoculated)
pot no
10
Plant 2 (PVX inoculated)
Control

Inoculated date

Temperature

05/03/2014
-first
inoculation

Maximum temperature- 30 oC
Minimum temperature - 22oC

12/03/2014
-second
inoculation

In the molecular laboratory

Control (not inoculated)

3.6 Inoculation of PLRV


Disease free Potato tubers were planted in pots in a greenhouse of PVIC. Plants were
watered daily. Albert solution was used as fertilizer. Grafting method and vector method
were used to transfer PLRV virus. PLRV infected plants were collected from seed potato
farm, Meeplimana, Nuwara Eliya.

26

3.6.1 Grafting method


Side grafting method was used. Small branches (Scion) with a few leaves from infected
plant were grafted to diseased free potato plants. Then grafted potato plants were covered
with moist plastic bags. Grafted tomato Plants were placed under the temperature
between 380C (maximum) and 240C (minimum) [greenhouse conditions].

Grafted part/branch

Figure 3.3 : Grafted potato plant


3.6.2 Vector transmission method
Aphids were collected from PLRV infected potato plants in the Meeplimana seed potato
farm. Previously grafted plants were used. Then they were taken to PVIC with a moist
tissue paper. Then container was closed and kept it for one day at a cool, shaded place to
starve the aphid. Aphids were placed on young potato leaves and allow them to feed on
leaves. Plants were covered with net/mesh and placed in the greenhouse.
Table 3.5: PLRV inoculation of Solanum tuberosum

Plant species
Potato (granola)
Pot no. Grafted/Aphids
6
Pot no. Grafted/Aphids
7
Pot no. Grafted/Aphids
8
Pot no. Grafted/Aphids
9
Pot
Control
control
inoculated)

Inoculated date
27/01/2014
grafting
method
28/01/2014Aphids

Temperature
Maximum temperature- 38
o
C
Minimum
temperatureo
24 C
Green house conditions

(not

27

3.7 Detection of PVX and PLRV by serological method (DAS-ELISA)


3.7.1 Detection of PVX by DAS-ELISA
Leaves of PVX inoculated plants (potato and tomato) were used to detect virus.
Table 3.6: Detection of PVX by serological method
Agdia and DSMZ commercial ELISA kits were used to detect PVX.

01
02
03

Sample
Sample size
Potato plants which PVX inoculated
5
under greenhouse conditions
Tomato plants which PVX inoculated at 5
14
leaf stage under greenhouse conditions
Tomato plants which PVX inoculated at 5
10
leaf stage under molecular virology
laboratory conditions

Date
2014/02/10
2014/02/25
2014/03/17

3.7.1.1 Detection of PVX by DAS-ELISA, commercial kit agdia


The DAS ELISA procedure was carried out using a commercially available kit (brand
agdia) for first two samples. The positive and negative controls were used from the
ELISA kit.
Capture antibody was diluted in coating buffer [1:400] (appendix A2.1). The volume of
100L was added to each well of a microtitre plate and incubated at room temperature for
four hours. Then the plate was washed with PBST (appendix A2.2) using a wash bottle.
Well were subjected to 3 quick time washes (fill all the wells completely with PBST, and
then quickly empty them again. Repeat 2 times). Then the plate was blotted by tapping
upside down on tissue paper.
0.1g of leaf tissue was crushed separately per sample with 1ml of extraction buffer
(appendix A2.5) using mortars and pestles. Then the mixture was transferred to eppendorf
tubes. Then eppendorf tubes were centrifuged at 5000rpm for 5 minutes. After
centrifugation, the supernatant was saved and 100L aliquots of the tested samples were
added to the wells. Then the plate was by 8 quick time washes.

28

Enzyme conjugate was diluted [1:400] with ECI buffer (appendix A2.3). Then 100L of
the mixture added to each well and incubated at room temperature for two hours. Then
the plate was by 8 quick time washes.
Then 100L aliquots of freshly prepared substrate buffer/ PNP buffer (appendix A2.4)
were added to each well and incubate at room temperature for 60 minutes. The colour
reaction was measured by visual observation and ELISA plate reader.
3.7.1.2 Detection of PVX by DAS-ELISA, commercial kit DSMZ
The DAS ELISA procedure was carried out using a commercially available kit (brand
DSMZ) for third sample. The positive and negative controls were used from the ELISA
kit.
Purified IgG was diluted in coating buffer [1:1000] (appendix A2.1). The volume of
200L was added to each well of a microtitre plate and incubated at 37 oC for four hours.
Then the plate was washed with PBST (appendix A2.2) using a wash bottle. Well were
washed 3 times with 3 minute intervals. Then the plate was blotted by tapping upside
down on tissue paper.
0.1g of leaf tissue was crushed separately per sample with 1ml of extraction buffer
(appendixA2.5) using mortars and pestles. Then the mixture was transferred to eppendorf
tubes. Then eppendorf tubes were centrifuged at 5000rpm for 5 minutes. After
centrifugation, the supernatant was saved and 200L aliquots of the tested samples were
added to the wells. Then the plate was washed three times as in above step.
IgG-AP was diluted [1:1000] with conjugate buffer (appendix A2.6). Then 200L of the
mixture added to each well and incubated at 37oC for four hours. Then the plate was
washed 3 times as in the above procedure.
Then 200L aliquots of freshly prepared substrate buffer (appendix A2.4) were added to
each well and incubate at room temperature for 30-60 minutes. The colour reaction was
measured by visual observation and ELISA plate reader.

29

3.7.2 Detection of PLRV by DAS-ELISA, commercial kit agdia


Table 3.7: Detection of PLRV by serological method

Sample
Potato plants which inoculated under

Sample size
4

Date
2014/02/10

greenhouse conditions

Leaves of PLRV inoculated plants (potato) were used to extract virus RNA.The DAS
ELISA procedure was carried out using a commercially available kit (brand agdia). The
positive and negative controls were used from the ELISA kit.
Capture antibody was diluted in coating buffer [1:400] (appendix A2.1). The volume of
100L was added to each well of a microtitre plate and incubated at room temperature for
four hours. Then the plate was washed with PBST (appendix A2.2) using a wash bottle.
Well were subjected to 3 quick time washes (fill all the wells completely with PBST, and
then quickly empty them again. Repeat 2 times). Then the plate was blotted by tapping
upside down on tissue paper.
0.1g of leaf tissue was crushed separately per sample with 1ml of extraction buffer
(appendix) using mortars and pestles. Then the mixture was transferred to eppendorf
tubes. Then eppendorf tubes were centrifuged at 5000rpm for 5 minutes. After
centrifugation, the supernatant was saved and 100L aliquots of the tested samples were
added to the wells. Then the plate was by 8 quick time washes.
Enzyme conjugate was diluted [1:400] with ECI buffer (appendix A2.3). Then 100L of
the mixture added to each well and incubated at room temperature for two hours. Then
the plate was by 8 quick time washes.
Then 100L aliquots of freshly prepared substrate buffer (appendix A2.5) were added to
each well and incubate at room temperature for 60 minutes. The colour reaction was
measured by visual observation and ELISA plate reader.

30

3.8 Optimization of PCR conditions for PLRV


PLRV infected samples were collected from Meeplimana seed potato farm and collected
samples were used to optimize PCR conditions.
3.8.1 RNA extraction PLRV infected leaves
Size fractionated silica extraction method (Gurusinghe et al., 2009) was used to extract
and purify the viral RNA for RT-PCR assay.
A symptomatic leaf tissue 0.1g was crushed with 1ml of lysis buffer (appendix A1.2)
mortar and pestle. Then lysate was transferred to an eppendorf tube and centrifuged at
5000 rpm for 5 minutes. After centrifugation, the supernatant was saved and 10L of
fractionated slica was added.
Then the mixture was vortex thoroughly and kept for 5 minutes and vortex again. After
that, the mixture was centrifuged at 5000rpm for 1 minutes and the supernatant was
discarded.
Then 1 ml of RNA wash buffer (appendix A1.3) was added and the tube was vortex. The
tube was then centrifuged at 5000rpm for 1 minutes and the supernatant was discarded.
Again, 1 ml of RNA wash buffer was added and the tube was centrifuged at 5000rpm for
1 minutes. The supernatant was discarded and 500L sterile distilled water was added to
the tube. Then it was inverted and centrifuged at 5000rpm for 1minutes and water was
removed completely. Then 20L of sterile distilled water was added to the tube and it
was incubated at 56oC for 1 minute in dry bath. Finally the tube was centrifuged at 5000
rpm for 3 minutes and 14 L of RNA extract was removed to a PCR tube.
3.8.2 Reverse transcription and cDNA synthesis
Extracted PLRV RNA samples were used for synthesis cDNA with PLRV antisense
primer (reverse primer C2)
Table 3.8: Reverse transcription reaction mixture
Chemicals
5X reverse transcriptase buffer (M-MuLV) with MgCl2
dNTPs (10mM)
Antisense primer C 2

For
one
PCR tube
5 L
0.8 L
1 L
31

RNAsin Ribonuclease inhibitor


M-MuLV reverse transcriptase
Water
RNA template

0.1 L
0.25 L
4.85 L
8 L
20 L

Total

In thermocycler first strand synthesis was done at 37oC for 45 minutes, followed by
inactivation at 95oC for 15 minutes.
3.8.2 Determination of annealing temperature
20 PCR reactions were carried out for five different annealing temperatures starting from
520C, 53.40C, 55.20C, 570C and 580C. These annealing temperatures were based on
previously published values (Peiman and Xie 2006, Ahouee et al., 2010, Hossain et al.,
2013 and Awan et al., 2010).
Table 3.9: PCR annealing temperature gradient
52
C
PCR
tube
01
PCR
tube
06
PCR
tube
11
PCR
tube
16
0

52.3
0
C

52.6
0
C

53.4
0
C
PCR
tube
02
PCR
tube
07
PCR
tube
12
PCR
tube
17

54.3
0
C

55.2
0
C
PCR
tube
03
PCR
tube
08
PCR
tube
13
PCR
tube
18

55.6
0
C

56.6
0
C

57
C
PCR
tube
04
PCR
tube
09
PCR
tube
14
PCR
tube
19
0

57.6
0
C

57.8
0
C

58
C
PCR
tube
05
PCR
tube
10
PCR
tube
15
PCR
tube
20
0

The Synthesized PLRV cDNA were amplified with virus specific primer pairs, which
were specific for the coat protein gene of PLRV.
Table 3.10: specific primers for detecting of PLRV and the size of amplified
products
Primer
PLRVv1
PLRVc2

Primer sequence
5` GTNCARCCNGTNGTNATGGTNAC 3
5` RTGCCAYTCNACNCCRTTDATCAT 3

Product size
400bp

32

Table 3.11: PCR mixture


Chemicals
10 X PCR buffer with MgCl2
dNTPs (10mM)
Sense primer V 1
Antisense primer C 2
Taq polymerase
Deionized water
cDNA template
Total

For one PCR tube


2.5 L
0.4 L
1 L
1 L
0.3 L
16.8 L
3 L
22 L

The thermo cycler conditions were initial denaturation 940C for 1 minute, followed by 30
cycles, denaturation at 940C 1 minute, temperature gradient was given for annealing
temperature (520C, 53.40C, 55.20C, 570C and 580C) for 30 seconds, extension 72 0C for
40 second with final extension 72 0C for 10 minutes.
3.8.3 Analyses of PCR products
The PCR product was separated by gel electrophoresis on a 2% agarose gel in 0.5 X TEA
buffers (appendix A1.1) containing 1.2L of EtBr (Ethium Bromide).
The 2000bp DNA ladder (gene script crop) was used as the size marker. After
electrophoresis, amplified products were viewed under UV transilluminatier (2UV
transilluminator) and the gel was photographed by web camera (SPW-2002 Golden Eye).
Presence of amplified products at 400bp position was recorded by comparing the position
of the DNA molecular marker. Then it was compared with the expected PCR product size
specific for PLRV.
3.8.4 Testing the reproducibility
The new potato samples were collected from the former field of Nuwara- Eliya and they
were retested with the above procedure to determine the reproducibility of the optimized
conditions.

33

CHAPTER 4
RESULTS AND DISCUSSION
This experiment was carried out to optimize the conditions of the RT - PCR process to
detect PVX and PLRV. Because molecular based identification systems are more reliable
and sensitive than other methods. Initially there were two major steps in this experiment,
inoculation the viruses and optimization the PCR conditions through inoculated samples.
4.1 The detection of PVX using the DAS- ELISA tests
4.1.1 The detection of PVX from potato samples which inoculated under the
temperature between 380C (maximum) and 240C (minimum).
ELISA TEST REPORT DETAIL
1. Reference No

: PVIC/SEROLOGY/ELISA/2014/22

1. Date

: 2014/02/10

2. Crop and No of samples

: Potato-5

3. Senders Address

: Bhashana

4. Identification test

: PVX

5. Antiserum

: Antiserum1:400 , Conjugate 1:400

Table 4.1: Test Results

Sample

Sample detail

Values
(1 hr)

Results

Values
(3hr)

Results

S13

potato- plot 02 -01

0.001

0.002

negative

S14
S15

potato- plot 02- 02


potato- plot 03 -01

0.010
0.003

0.009
0.007

negative
negative

S16

potato- plot 03 -02

-0.026

-0.004

negative

S17

potato- plot 05 - 01

-0.022

0.001

negative

-0.002

-0.001

BA
THV1

PVX(-)

0.020

0.028

D1

Positive control(kit)

0.050

0.058

positive

34

First potato plants were used to inoculate PVX under the temperature between 380C
(maximum) and 240C (minimum). Most of inoculated potato plants were not survived due
to bacterial wilt infection and unfavourable climatic conditions for potato cultivation. Any
symptoms of PVX were not observed. However, Querci et al., 1995 have reported
Solanum sucrense exhibit symptoms 10 days after inoculation and Gomphrena globosa
exhibit symptoms 6-12 days after inoculation. Survived potato plants were subjected to
DAS- ELISA tests. But all the ELISA tests were negative. High temperature may be the
reason for unsuccessful of inoculation. Qamar and Khan (2003) have reported the
affection of temperature for PVX disease development in six potato varieties (FSD-red,
Sante, Desree, Kiran, Diamont and SH-5). They have reported the temperature play a
vital role in the development of the disease on these varieties. Maximum PVX disease
severity was recorded by them at 250C-270C maximum and 100C -120C minimum
temperatures.
4.1.2 The detection of PVX from tomato samples which inoculated at 5 leaf stage
under the temperature between 380C (maximum) and 240C (minimum).
ELISA TEST REPORT DETAIL
1. Reference No

: PVIC/SEROLOGY/ELISA/2014/26

2. Date

: 2014/02/25

3. Crop and No of samples

: Tomato -14

4. Senders Address

: Bhashana

5. Identification test

: PVX

6. Antiserum

: Antiserum1:400 , Conjugate 1:400

Table 4.2: Test Results


Sample

Sample detail

S1

Tomato- plot 01-plant 01

Values
(1 hr)
-0.022

Results

Values
(3hr)
0.000

Results
negative

S2
S3

Tomato- plot 01-plant 02


Tomato- plot 02-plant 01

-0.014
-0.025

0.005
0.000

negative
negative

S4

Tomato- plot 02-plant 03

-0.020

0.004

negative

S5

Tomato- plot 03-plant 01

-0.001

0.006

negative

S6

Tomato- plot 03-plant 04

0.014

0.012

negative

S7

Tomato- plot 04-plant 01

-0.009

0.010

negative

35

S8

Tomato- plot 05-plant 01

-0.027

-0.005

negative

S9

Tomato- plot 05-plant 02

-0.022

0.002

negative

S10

Tomato- plot 06-plant 01

-0.018

0.002

negative

S11

Tomato- plot 06-plant 02

-0.021

0.012

negative

S12

Tomato- plot 07-plant 01

-0.017

0.015

negative

S13

Tomato- plot 07-plant 02

0.003

0.013

negative

S14

Tomato- plot 08-plant 01

-0.006

0.008

negative

0.076

0.068

BA
THV1

PVX(-)

-0.048

0.012

D1

Positive control(kit)

0.229

0.637

Positive

Then, tomato (cultivar Thilina) was selected as the host plant due to bacterial wilt
infection and unfavourable climatic conditions for potato cultivation. 5 leaf stage tomato
plants were inoculated under the temperature between 380C (maximum) and 240C
(minimum). But the ELISA result was negative and any symptoms of PVX were not
observed.
4.1.3 The detection of PVX from tomato samples which inoculated at 2-3 leaf stage
under the temperature between 380C (maximum) and 240C (minimum).
Two to three leaf stage tomato plants (cultivar Thilina) were inoculated under the
temperature between 38 0C (maximum) and 240C (minimum). But similarly, any
symptoms of PVX were not observed. Due to negative results of 5 leaf stage tomato plant
inoculation (above mention), an ELISA test was not carried out for this inoculation.
Inoculation of PVX for tomato plants were based on previous two investigations. 5 leaf
stage Fukuju no 2 tomato cultivar had been successfully inoculated by Balogun et al.,
2000 under maximum temperature 300C and minimum temperature 180C. Similarly, 2-3
leaf stage Fukuju no 2 tomato cultivar had been successfully inoculated by Balogun and
Teraoka, 2004.
Tomato cultivar or inoculation temperature can be the reason for unsuccessful inoculation
of PVX for tomato plants. Because Balogun and Teraoka, (2004) reported that PVX
infection not only affected the quantity, but may also have altered the type of phenol

36

components of the infected tomato plant. Therefore tomato cultivar (Thilina) which used
may be a reason for unsuccessful of inoculation.
Some publication revealed the interaction between temperature and PVX inoculation.
Higher temperature causes for symptomless infection (Loebenstein, 1976). Symptoms are
expressed in growing plants at temperatures above 210C and no symptom development
and virus multiplication occurs at temperatures above 300C (Singh, 1998). According to
the investigation of Balogun et al., (2000), the PVX symptom expression is quicker in
winter season (5 days) than the summer season (10 days). PVX infectivity is retained at
200C for several weeks (Loebenstein, 1976). Balogun, (2009) revealed that PVX virus
accumulation in the tomato plant (Fukuju no 2) is more in winter season (temperature
range between 160C and 240C) than summer season (temperature range between 220C and
320C) and PVX symptoms shown moderate severity in summer. Therefore, unfavourable
temperature also can be a reason for unsuccessful inoculation.
4.1.4 The detection of PVX from tomato samples which inoculated at 5 leaf stage
under the temperature between 300C (maximum) and 220C (minimum).
ELISA TEST REPORT DETAIL
1. Reference No

: PVIC/SEROLOGY/ELISA/2014/33

2. Date

: 2014/03/17

3. Crop and No of samples

: Tomato -10

4. Senders Address

: Bhashana

5. Identification test

: PVX DAS ELISA

6. Antiserum

: Antiserum1:1000 , Conjugate 1:1000

Table 4.3: Test Results


Sample

Sample detail

Values
(1 hr)

Results

S1

Pot - 1

-0.002

Values
(overnig
ht)
0.001

Results

S2
S3

Pot 2
Pot 3

0.002
-0.001

0.009
0.007

negative
negative

S4

Pot 4

-0.002

0.006

negative

S5

Pot 5

-0.003

0.000

negative

negative

37

S6

Pot - 6

-0.003

0.009

negative

S7

Pot-7

-0.001

0.008

negative

S8

Pot-8

-0.004

0.006

negative

S9

Pot 9

-0.004

0.004

negative

S10

Pot - 10

-0.004

-0.002

negative

0.054

0.054

Tomato(PVIC)

0.000

0.016

Positive control (kit)

0.013

0.017

BA
THV1
D1

Positive

Then again PVX was inoculated to 5 leaf stage tomato plants. But inoculation
temperature was changed this time to reduce the temperature effect. The inoculation
temperature range was from 300C (maximum) to 220C (minimum). But it was not able to
maintain temperature range below 30 0C and any symptoms of PVX were not observed
and ELISA test was negative.
4.2 The detection of PLRV from potato samples which inoculated under the
temperature between 380C (maximum) and 240C (minimum).
ELISA TEST REPORT DETAIL
1. Reference No: PVIC/SEROLOGY/ELISA/2014/26
2. Date

: 2014/02/10

3. Crop and No of samples

: Potato -04

4. Senders Address

: Bhashana

5. Identification test

: PLRV

6. Antiserum

: Antiserum1:400 , Conjugate 1:400

Table 4.4: Test Results


Sample detail

Values
(1 hr)

S1

Potato-pot 06

S2
S3

Potato-pot 07
Potato-pot 08

Sample

Results

Values
(3hr)

Results

-0.009

-0.004

negative

-0.011
-0.010

0.005
0.004

negative
negative

38

S4

Potato-pot 09

BA

-0.005

0.008

0.065

0.065

THV1

PLRV(-) 2012/10/22

0.024

0.012

D1

Positive control(kit)

0.061

0.065

negative

Positive

Grafting method and vector transmission method (aphids) were used to inoculate PLRV.
Potato plants were not successfully grafted and vector transmission also was not
successful. High/unsuitable temperature may be the reason for unsuccessful inoculation.
Because of PLRV is a heat sensitive virus (Frances et al., 1971). PLRV can be eliminated
by keeping tuber under 37.5 0C for 25 days (Kassanis, 1950 and Lizarraga et al., 1991).
ELISA is the only serological method to identify PLRV due to low concentration of
PLRV in infected plants (Jayasinghe, 1988). But Plants that are grown at temperatures
300C and older plant may not always detect infection through ELISA (Loebenstein,
1976).
Due to PVX and PLRV inoculations were unsuccessful experimental procedure was
changed. Next procedure was collecting PLRV and PVX infected samples from potato
field and optimizes the PCR conditions through collected samples. Samples had to collect
early stage in the potato season. Therefore PLRV disease incident was not common, and
PVX infected samples were not able to collect. Therefore, only PCR optimization was
carried out for PLRV.
4.3 Determination of annealing temperature for PLRV
RTPCR is a one of most reliable technique to detect PLRV. Various investigations have
been carried out to establish optimized RT-PCR method for PLRV. But there was no
previously published RT-PCR optimized system to identify PLRV in Sri Lanka.
Size fractionated silica extraction method (Gurusinghe et al., 2009) was applied to extract
RNA in this study. Because Size fractionated silica extraction method is the RNA
extraction method which uses in PVIC. In this method, RNA was extracted by adsorption
on to the silica particles after guanidium buffer treatment. This is a cost effective method
as well as RNA can be extracted within one hour. Therefore more samples can be indexed

39

within short time. Boom et al., (1990) and Zacharzewska et al., (2014) have been used
similar silica captured method for extract potato viruss RNA.
In this study PLRV infected leaves were applied to extract RNA. But other investigations
have used not just leaves, but also dormant tubers and aphids have been applied to extract
RNA. PLRV have been successfully detected using sprouted tubers as well as dormant
tubers stored at 20C 25C for 4 months (Awan et al., 2010). PLRV infected aphids
have been successfully applied to detect PLRV by Ahouee et al., 2010. Hadidi et al.,
1993 have reported that the detection of PLRV from aphids by RT-PCR and it is more
sensible than other methods.
Determination of annealing temperature was based on few already published RT-PCR
systems. There are Peiman and Xie 2006 (530C for 45 second), Ahouee et al., 2010 (540C
for 45 seconds), Hossain et al., 2013 (550C for 1 minutes) and Awan et al., 2010 (58 0C
for 1 minutes). PCR gradient was set up by using these annealing temperatures. These
RT-RCR systems have been used different RNA extraction methods, PCR mixtures and
primer pairs.
Amplified products (400bp) were obtained at annealing temperature 520C and 53.40C
form extracted viral RNA. At all other annealing temperatures (55.20C, 570C and 580C)
products were not amplified successfully.

1 2 3 4

5 6 7
500bp

400bp

400bp

Figure 4.1 Amplified products of PLRV using different annealing temperature


Lane 1= 520C, Lane 2=53.40C, Lane 3= Molecular size marker (2000bp DNA ladder),
Lane 4=55.20C, Lane 5=570C, Lane 6=580C, Lane 7= Negative control

40

500bp
400bp

400bp

Figure 4.2 Amplified products of PLRV annealing temperature at 52oC


The most prominent band was observed at annealing temperature 520C. Therefore, it was
selected as the suitable annealing temperature at PVIC under their primer pairs, chemical
concentrations and RNA extraction method (Size fractionated silica extraction method).

41

Chapter 5
Conclusion and suggestions
6.1 Conclusion
PCR amplification of PLRV that isolates of Sri Lanka is possible with specific primer
PLRVv1

(5`GTNCARCCNGTNGTNATGGTNAC3)

and

primer

PLRVc2

(5`

RTGCCAYTCNACNCCRTTDATCAT 3). Optimum PCR conditions are 2.5 L of 10


X PCR buffer (with MgCl2), 2.5 L of dNTPs (10 mM), 1 L of Sense primer, 1 L of
Antisense primer, 0.3 L of Taq polymerase, 16.8 L of Deionized water, and 3 L of
cDNA template.
Thermal cycle parameters are,
Initial denaturation at 940C for 1 minute
Denaturation at 940C for 1 minute
Annealing temperature at 52 0C for 30 second

30 cycles

Extension at 72 C for 40 second


Final extension at 720C for 10 minutes
6.2 Suggestions
This experiment was carried out in a greenhouse of PVIC which maximum temperature
usually reaches more than 300C in day time. There are evidences about the interaction
between environmental temperature and the inoculation of PVX and PLRV. Thus, it is
important to inoculate PVX and PLRV under controlled environment condition (between
180C and 300C) or avoid the higher temperature seasons.
Recommended host plants (e.g.: - Datura stramonium) should be used to inoculate Potato
viruses under the climatic condition of WL3 area. Because of climatic conditions are not
suited for cultivation Potato. If use potato as host plant it is recommended to cultivate on
sterilize soils to avoid bacteria wilt. When inoculate PVX, it is significant to consider
about host plant cultivar and virus strain. Because of PVX symptoms can be varied
according to strains and cultivars.
Future studies should be focused to develop a multiple RT-PCR assay for PLRV and
PVX instead of mono specific/ uniplex RT- PCR assays. Monospecific / uniplex RT-PCR
assays requiring separate amplification of each virus. It is potentially expensive and
resource intensive.
42

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48

Appendix
A1 Buffers used for RT-PCR and PCR process
A1.1 5x TAE
Tris base - (40mM tris base)
GA

24.25g

0.5 M EDTA-

5.7ml
10ml

All components mixed together in to prepare 1L


A1.2 Lysis buffer (pH 6.4) for 100ml
40 mM tris for1.14) PH 6.4

0.484g

17mM EDTA

0.632g

4M GUSCN

47.264g

1% Triton x100

1ml

Tris ----Trisma base , minimum 99.9% titration C4H11NO3


EDTA--- Ethyenediamine N,N,N,N Tera active acid C4H11NO3
GUSCN ---- Guanidin Thiocyanate
1% Triton x100 ------ t- octyiphenoxypolyethxyethanol
A1.3 Wash buffer (RNA) (pH 7.4) for 100ml

50% absoulute ethanol

50L

10mM tris PH -7.4 (121.14)

0.121g

1mM EDTA (9372.2)

0.037g

60mM Nacl (58.44)

0.35g

Double autoclaved distilled water


First dislove Tris , EDTA and Nacl
Finally add absolulte ethanol

49

A2 Buffers formation for ELISA tests


A2.1Carbonate coating buffer (1X)
Dissolve in distilled water to 1000ml
Sodium carbonates (anhydrous)

1.59g

Sodium bicarbonate

2.93g

Sodium azide

0.2g

Adjust pH to 9.6 stores at 4 oC

A2.2 PBST buffer (Wash buffer) (1X)


Dissolve in distilled water to 1000ml
Sodium Chloride

8.0g

Sodium Phosphate (anhydrous)

1.15g

Potassium Phosphate, monobasic (anhydrous)

0.2g

Potassium chloride

0.2g

Tween-20

0.2g

Adjust pH to 7.4

A2.3 ECI buffer (1X)


Add to 1000ml 1X PBST
Bovine serum albumin (BSA)

2.0g

Polyvinaylpyrrolidone (PVP) MW 24-4000

20.0g

Sodium azide

0.2g
o

Adjust pH to 9.6 stores at 4 C.

A2.4 Substrate buffer /PNP buffer (1X)


Dissolve in 800ml distilled water
Magnesium chloride hexahydrayte

0.1g

Sodium azide

0.2g

Diethanolamine

9.0g

Adjust pH to 9.8 with hydrochloric acid; adjust final volume to 1000ml with distilled
water, store at 4oC.

50

A2.5 General Extraction buffer (GEB 1X)


Dissolve in 1000ml of 1X PBST
Sodium sulfite (anhydrous)
Polyvinaylpyrrolidone (PVP) MW 24-4000

20.0g

Sodium azide

0.2g

Powdered egg (chicken) albumin, grade 11

2.0g

Tween-20

20g

Adjust pH to 9.6 stores at 4 oC

A2. 6 Conjugate buffer


PBST buffer
2% PVP
0.2% egg albumin (stigma A-5253)

51