Identification of Potato Virus X (PVX) and Potato Leafroll Virus (PLRV) Using Molecular Detection...

1

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

2

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


(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)



Sabaragamuwa University of Sri Lanka.

Date: ……………………………

………………………………

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

(External Supervisor)

Deputy Director

Plant Virus Indexing Centre (PVIC)

Gabadawaththa, Homagama.

Date: …………………………

…………………………………

Dr. G.D. Kapila Kumara

Head of the department



Sabaragamuwa University of Sri Lanka.

Date: ………………………….

3

Dedication

Affectionately dedicated

To

My ever loving

Parents

And

Teachers…..

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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 24

0C (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


0C (minimum). Then 5 leaf stage

tomato plants were inoculated under the temperature between 300C (maximum) and 22

0C

(minimum). PLRV was inoculated potato plants using the grafting method and the vector

methods under the temperature between 380C (maximum) and 24

0C (minimum). Both

PVX and PLRV inoculated plants were subjected to DAS-ELISA tests. Virus infected

samples were collected from farmer‟s 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 µL of 10 X PCR buffer (with MgCl2), 2.5 µL of dNTP‟s

(10 mM), 1 µL of Sense primer, 1 µL of Antisense primer, 0.3 µL of Taq polymerase,

16.8 µL of Deionized water, and 3 µL of cDNA template. Suitable annealing temperature

is 520C for 30 second.

Keywords: - RT-PCR, PCR buffer, dNTP‟s, primer, annealing temperature

ii

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.

iii

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

iv

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 24

0C (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 24

0C (minimum). .................................................................... 35

4.1.3 The detection of PVX from tomato samples which inoculated at 2-3 leaf stage under the


0C (minimum). .................................................................... 36

4.1.4 The detection of PVX from tomato samples which inoculated at 5 leaf stage under the


0C (minimum). .................................................................... 37

4.2 The detection of PLRV from potato samples which inoculated under the temperature

between 380C (maximum) and 24

0C (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

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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

vi

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




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LIST OF ABRIVIATIONS

DAS-ELISA- Double Antibody Sandwich ELISA

DNA- Deoxyribonucleic acid

dNTP‟s- 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

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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.

2

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

3

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.

4

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 world‟s 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 was-

5

-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.

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

050,000

100,000150,000

M

t

Year

Figure 2.1: Potato importation of Sri Lanka

Figure 1Potato importation of Sri Lanka

6

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), PVY

N (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 PVY

O produced PVY

NTN

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.

7

The symptoms are different according to the PVY strains. Symptoms of PVYO and PVY

C

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, PVY

C and PVY

N 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).

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.

Figure 2.2:PVYNTN

infected tuber Figure 2.3: Leaf mottling or yellowing

http://viralzone.expasy.org/all_by_species/738.html

http://www.dpvweb.net/dpv/showrefs.php?dpvno=354#25



8

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).

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.

Figure 2.4 :PVX -Electron microscope view

9

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

10

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.

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

Figure 2.6 :PLRV - Electron microscope view

11

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 non-

infectious for several hours. After the virus become infective and persist throughout the

aphid‟s life.

Infected plant Phloem Digestive system of aphid enters into nomocel

Enter into new plant when feed 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.

12

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


13

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.

14

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 immune-

blotting) 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).

15

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

16

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).

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.

Figure 2.8 :Five steps in an ELISA procedure

Figure 2.9 :Double antibody sandwich (DAS-) ELISA

17

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 (dNTP‟s) 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.

18

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-95

oC. 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.

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

Figure 2.11 :Schematic of the classical PCR.

19

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 µL 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 dNTP‟s concentration range

is between 20 and 200µM each result in the optimal balance among yield, specificity, and

fidelity. Low dNTP‟s 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 dNTP‟s

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 μM 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).

20

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.

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

Figure 2.12: Reverse transcription polymerase chain reaction

21

(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-PCR‟s 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.

22

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- 1000µL, 100 µL, 10 µL, 2 µL ( 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)

23

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.

24

3.5.2 Inoculation procedure of PVX

To prepare inoculum, 400 µL 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 Temperature

Potato

27/01/2014

-first

inoculation

Maximum temperature- 38 oC

Minimum temperature – 24oC

Green house conditions

Pot no. 2 Plant 1 (PVX

inoculated)

Plant2 (PVX

inoculated)

Pot no. 3 Pant 1 (PVX

inoculated)

Plant 2 (PVX

inoculated)

Pot no. 5 Plant 1 (PVX

inoculated)

---------

Pot-

control

Control (not inoculated)

Table 3.2 PVX inoculation for Solanum lycopersicum at 5 leaf stage


Tomato (cultivar Thilina)

Pot no. 1 Plant 1 (PVX inoculated)

13/02/2014 -

first

inoculation


Minimum temperature – 24oC


Plant 2 (PVX inoculated)

Pot no.2 Plant 1 (PVX inoculated)





---------


25


19/02/2014 -

second

inoculation


---------





Control Control (not inoculated)

Table 3.3: PVX inoculation for Solanum lycopersicum at 2-3 leaf stage



21/02/2014 -

first

inoculation


Minimum temperature -24oC

Green house conditions Pot no. 1 ~ (3 plants PVX inoculated)

Pot no. 2 ~ (4 plants PVX inoculated)

Pot no. 3 ~ (4 plants PVX inoculated)

Pot no. 4~ (4 plants PVX inoculated)

Figure 3.1: PVX inoculated potato plants

Figure 3.2: PVX inoculated tomato plants

5 leaf stage

2-3 leaf stage

26

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 22

0C (minimum) [In the molecular laboratory].

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.



Pot no. 1 Plant 1 (PVX inoculated)

05/03/2014

-first

inoculation

12/03/2014

-second

inoculation


Minimum temperature - 22oC

In the molecular laboratory







---------




---------





Pot no 9 Plant 1 (PVX inoculated)


pot no

10



Control Control (not inoculated)

27

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 24

0C (minimum) [greenhouse conditions].

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


Potato (granola)

27/01/2014 –

grafting

method

28/01/2014-

Aphids


Minimum temperature-

24oC


Pot no.

6

Grafted/Aphids

Pot no.

7

Grafted/Aphids

Pot no.

8

Grafted/Aphids

Pot no.

9

Grafted/Aphids

Pot

control

Control (not

inoculated)

Grafted part/branch

Figure 3.3 : Grafted potato plant

28

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.

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

100µL 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 100µL aliquots of the tested samples were

added to the wells. Then the plate was by 8 quick time washes.

Sample Sample size Date

01 Potato plants which PVX inoculated

under greenhouse conditions

5

2014/02/10

02 Tomato plants which PVX inoculated at 5

leaf stage under greenhouse conditions

14 2014/02/25

03 Tomato plants which PVX inoculated at 5

leaf stage under molecular virology

laboratory conditions

10 2014/03/17

29

Enzyme conjugate was diluted [1:400] with ECI buffer (appendix A2.3). Then 100µL 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 100µL 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

200µL was added to each well of a microtitre plate and incubated at 37oC 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.


(appendixA2.5) using mortars and pestles. Then the mixture was transferred to eppendorf



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 200µL 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 200µL 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.

30

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

Table 3.7: Detection of PLRV by serological method

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

100µL 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.


(appendix) using mortars and pestles. Then the mixture was transferred to eppendorf



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 100µL 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 100µL 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.

Sample Sample size Date

Potato plants which inoculated under

greenhouse conditions

4

2014/02/10

31

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 10µL 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 500µL sterile distilled water was added to

the tube. Then it was inverted and centrifuged at 5000rpm for 1minutes and water was

removed completely. Then 20µL 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 µL 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 For one

PCR tube

5X reverse transcriptase buffer (M-MuLV) with MgCl2 5 µL

dNTP‟s (10mM) 0.8 µL

Antisense primer C 2 1 µL

32

RNAsin Ribonuclease inhibitor 0.1 µL

M-MuLV reverse transcriptase 0.25 µL

Water 4.85 µL

RNA template 8 µL

Total 20 µL

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, 53.4

0C, 55.2

0C, 57

0C and 58

0C. 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 0C

52.3 0C

52.6 0C

53.4 0C

54.3 0C

55.2 0C

55.6 0C

56.6 0C

57 0C

57.6 0C

57.8 0C

58 0C

PCR

tube

01

PCR

tube

02

PCR

tube

03

PCR

tube

04

PCR

tube

05

PCR

tube

06

PCR

tube

07

PCR

tube

08

PCR

tube

09

PCR

tube

10

PCR

tube

11

PCR

tube

12

PCR

tube

13

PCR

tube

14

PCR

tube

15

PCR

tube

16

PCR

tube

17

PCR

tube

18

PCR

tube

19

PCR

tube

20

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 Primer sequence Product size

PLRVv1 5` GTNCARCCNGTNGTNATGGTNAC 3‟ 400bp

PLRVc2 5` RTGCCAYTCNACNCCRTTDATCAT 3‟

33

Table 3.11: PCR mixture

Chemicals For one PCR tube

10 X PCR buffer with MgCl2 2.5 µL

dNTP‟s (10mM) 0.4 µL

Sense primer V 1 1 µL

Antisense primer C 2 1 µL

Taq polymerase 0.3 µL

Deionized water 16.8 µL

cDNA template 3 µL

Total 22 µL

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, 53.4

0C, 55.2

0C, 57

0C and 58

0C) 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 1.2µL 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.

34

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


0C (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. Sender‟s 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 potato- plot 02- 02 0.010 0.009 negative

S15 potato- plot 03 -01 0.003 0.007 negative

S16 potato- plot 03 -02 -0.026 -0.004 negative

S17 potato- plot 05 - 01 -0.022 0.001 negative

BA -0.002 -0.001

THV1 PVX(-) 0.020 0.028

D1 Positive control(kit) 0.050 0.058 positive

35

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-27

0C maximum and 10

0C -12

0C minimum

temperatures.

4.1.2 The detection of PVX from tomato samples which inoculated at 5 leaf stage


0C (minimum).



2. Date : 2014/02/25

3. Crop and No of samples : Tomato -14


5. Identification test : PVX




(1 hr)

Results Values

(3hr)

Results

S1 Tomato- plot 01-plant 01 -0.022 0.000 negative





S6 Tomato- plot 03-plant 04 0.014 0.012 negative


36

S8 Tomato- plot 05-plant 01 -0.027 -0.005 negative





S13 Tomato- plot 07-plant 02 0.003 0.013 negative


BA 0.076 0.068

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 24

0C

(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


0C (minimum).

Two to three leaf stage tomato plants (cultivar Thilina) were inoculated under the

temperature between 38 0C (maximum) and 24

0C (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 18

0C. 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

37

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 24

0C) than summer season (temperature range between 22

0C 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


0C (minimum).



2. Date : 2014/03/17

3. Crop and No of samples : Tomato -10


5. Identification test : PVX – DAS ELISA




(1 hr)

Results Values

(overnig

ht)

Results

S1 Pot - 1 -0.002 0.001 negative

S2 Pot – 2 0.002 0.009 negative

S3 Pot – 3 -0.001 0.007 negative

S4 Pot – 4 -0.002 0.006 negative

S5 Pot – 5 -0.003 0.000 negative

38

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

BA 0.054 0.054

THV1 Tomato(PVIC) 0.000 0.016

D1 Positive control (kit) 0.013 0.017 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 22

0C (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


0C (minimum).


1. Reference No: PVIC/SEROLOGY/ELISA/2014/26

2. Date : 2014/02/10

3. Crop and No of samples : Potato -04


5. Identification test : PLRV



Sample

Sample detail Values

(1 hr)

Results Values

(3hr)

Results

S1 Potato-pot 06 -0.009 -0.004 negative

S2 Potato-pot 07 -0.011 0.005 negative


39


BA 0.065 0.065

THV1 PLRV(-) 2012/10/22 0.024 0.012

D1 Positive control(kit) 0.061 0.065 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

RT–PCR 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

40

within short time. Boom et al., (1990) and Zacharzewska et al., (2014) have been used

similar silica captured method for extract potato virus‟s 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 20°C – 25°C 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 (54

0C

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 53.4

0C

form extracted viral RNA. At all other annealing temperatures (55.20C, 57

0C and 58

0C)

products were not amplified successfully.

Lane 1= 520C, Lane 2=53.4

0C, Lane 3= Molecular size marker (2000bp DNA ladder),

Lane 4=55.20C, Lane 5=57

0C, Lane 6=58

0C, Lane 7= Negative control

400bp

500bp

400bp

1 2 3 4 5 6 7

Figure 4.1 Amplified products of PLRV using different annealing temperature

41

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).

Figure 4.2 Amplified products of PLRV annealing temperature at 52oC

500bp

400bp

400bp

42

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 µL of 10

X PCR buffer (with MgCl2), 2.5 µL of dNTP‟s (10 mM), 1 µL of Sense primer, 1 µL of

Antisense primer, 0.3 µL of Taq polymerase, 16.8 µL of Deionized water, and 3 µL 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

Extension at 720C 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 30

0C) 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.

30 cycles

43

References

Ahouee,K. H. Habibi, M.K and Mosahebi,G. H., 2010. Detection of potato leafroll virus

isolated from potato fields in Tehran province in aphids by immunocapture reverse

transcription polymerase chain reaction. African Journal of Biotechnology , 9, pp.2349-

2352.

Alan Brunt , Karen Crabtree , Michael Dallwitz , Adrian Gibbs , Leslie Watson , and Eric

Zurcher, 1996. Plant Viruses Online. [Online] Available at: http://pvo.bio-

mirror.cn/descr651.htm [Accessed 11 March 2014].

Alan Brunt, Karen Crabtree , Michael Dallwitz , Adrian Gibbs , Leslie Watson , and Eric

Zurcher, 1997. Plant Viruses Online. [Online] Available at:

http://sdb.im.ac.cn/vide/descr644.htm [Accessed 02 March 2014].

Anon., n.d. alawathugoda.com. [Online] Available at:

http://potatoes.alawathugoda.com/history.html [Accessed 14 March 2014].

Anon., n.d. Department of agriculture Sri Lanka. [Online] Available at:

http://www.agridept.gov.lk/images/stories/site/PDF/Publication/English/BOOK/proposed

plan.pdf [Accessed 15 March 2014].

Anon., n.d. Extension Plant Pathology. [Online] Available at:

http://www.plantpath.ksu.edu/pages/extension [Accessed 20= February 2014].

Anon., n.d. FAO Statistical Yearbook 2013. [Online] Available at:

http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor [Accessed 30

March 2014].

Anon., n.d. Freegardeningplants.com. [Online] Available at:

http://www.freegardeningplants.com/Freegardeningplants.com.html [Accessed 02 March

2014].

Anon., n.d. infonet-biovision. [Online] Available at: http://www.infonet-

biovision.org/res/res/files/1057.400x400.jpeg [Accessed 12 March 2014].

Anon., n.d. micronotes. [Online] Available at:

http://www.microrao.com/micronotes/pcr.pdf [Accessed 03 April 2014].

Anon., n.d. Pest and diseases and control measures. [Online] Available at:

http://www.navagoviya.org/index.php?option=com_content&view=article&id=17&lang=

en [Accessed 15 March 2014].

Anon., n.d. Potato Consumption Per Capita. [Online] Available at:

http://www.helgilibrary.com/indicators/index/potato-consumption-per-capita [Accessed

10 February 2014].

Anon., n.d. Potato world. [Online] Available at: http://www.fao.org/potato-

2008/en/world/index.html [Accessed 05 March 2014].

http://pvo.bio-mirror.cn/descr651.htm

http://pvo.bio-mirror.cn/descr651.htm

http://sdb.im.ac.cn/vide/descr644.htm

http://potatoes.alawathugoda.com/history.html

http://www.agridept.gov.lk/images/stories/site/PDF/Publication/English/BOOK/proposedplan.pdf

http://www.agridept.gov.lk/images/stories/site/PDF/Publication/English/BOOK/proposedplan.pdf

http://www.plantpath.ksu.edu/pages/extension

http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor

http://www.freegardeningplants.com/Freegardeningplants.com%20.html

http://www.infonet-biovision.org/res/res/files/1057.400x400.jpeg

http://www.infonet-biovision.org/res/res/files/1057.400x400.jpeg

http://www.microrao.com/micronotes/pcr.pdf

http://www.navagoviya.org/index.php?option=com_content&view=article&id=17&lang=en

http://www.navagoviya.org/index.php?option=com_content&view=article&id=17&lang=en

http://www.helgilibrary.com/indicators/index/potato-consumption-per-capita

http://www.fao.org/potato-2008/en/world/index.html

http://www.fao.org/potato-2008/en/world/index.html

44

Anon., n.d. Reverse transcription polymerase chain reaction. [Online] Available at:

http://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction [Accessed

01 March 2014].

Anon., n.d. www.potato.org.uk. [Online] Available at: http://www.potato.org.uk/media-

gallery/detail/13214/2925 [Accessed 20 February 2014].

Awan, A. Rul Haq,I. Babar,M E. and Nasir,I A, 2010. Molecular Detection Of Potato

Leaf Roll Polerovirus Through Reverse Transcription Polymerase Chain Reaction In

Dormant Potato Tubers. Pakistan Journal of Botany, 42(5), pp. 3299-3306.

Ayoni V.D.N, Karunagoda.K, Waranakulasooriya, 2009. Potato Production of Sri Lanka.

Annual Symposium of Department of Agriculture (ASDA), 1, pp.9-12.

Balogun, O.S., XU, L., Teraoka, T., & Hosokawa, D., 2000. Effects of single and double

infections with Potato virus X and Tobacco mosaic virus on disease development, plant

growth and virus accumulation in tomato. Fitopatologia Brasileira , 27, pp.241-248.

Balogun,O.S and Teraoka,T., 2004. Time-course analysis of the accumulation of phenols

in tomato seedlings infected with Potato Virus X and Tobacco mosaic virus.

BIOKEMISTRI , 16(2), pp.112-20.

Balogun, O.S., 2009. Comparative Pathogenic Responses Of Winter And Summer

Tomato Crops To Infection With Potato Virus X And The OM Strain Of TMV. World

Journal Of Agricultural Sciences, 5(S), pp.856-862.

Bercks, R., 1970. Descriptions of Plant viruses. Potato virus X. Scotland: Culross and

Son Ltd.

Bercks, R., 1989. discription of Plant Virus. [Online] Available at:

http://www.dpvweb.net/dpv/showdpv.php?dpvno=354 [Accessed 15 March 2014].

Boom,R Sol,C J. Salimans,M M Jansen,C L. Wertheim-van Dillen, P M and van der

Noordaa,J., 1990. Rapid and Simple Method for Purification of Nucleic Acids. JOURNAL

OF CLINICAL MICROBIOLOGY, Mar. 1990, pp.495-503.

Cerkauskas, R., 2005. Tomato Diseases -Potato Virus X. Shanhua; Taiwan : AVRDC –

The World Vegetable Center.

Cerkauskas, R., 2005. Tomato Diseases -Potato Virus X. In Cerkauskas, R. Tomato

Diseases. Shanhua; Taiwan : The World Vegetable Cente. pp.3-4.

Cieslinska, M.., 2004. Detection of strawberry mottle virus (SMoV) using RT-PCR-

comparison of two RNA extraction methods. ournal of Fruit and Ornamental Plant

Research, 12, pp.17-22.

http://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction

http://www.potato.org.uk/media-gallery/detail/13214/2925

http://www.potato.org.uk/media-gallery/detail/13214/2925

http://www.dpvweb.net/dpv/showdpv.php?dpvno=354

45

Derrick, P.M and Barher, H , 1997. Short and long distance spread of potato leafroll

luteovirus: effects of host genes and transgenes conferring resistance to virus

accumulation in potato. Journal of General Virology, 78, pp.243–251.

EL-Sway,A and Hadidi,A, 2000. Detection of Potato Leafroll Virus in tissue culture-

derived potato plantlets. Arab Journal of Biotech, 3(2), pp.199-200.

Eskandari, F., Sylvester, E. S. and Richardson, J., 1978. Evidence for lack of propagation

of potato leaf roll virus in its aphid vector, Myzus persicae. Phytopathology , 69, pp.45-

47.

Fernando KM.E.P and Premasiri H.M.R., 2006. Evaluation of productivity of potatoes in

Nuwara Eliya and Badulla districts with the aid of GIS techniques. Vidyodaya J of SCI,

13, pp. 49-64.

Frances c, mellor and Richard, S.S, 1971. Heat stable strains of potato leaf roll virus.

PHYTOOPATHOLOGY , 61, pp.246-247.

Gleason, M.L., 2002. Plant Disease. [Online] Available at:

http://dx.doi.org/10.1094/PDIS.2002.86.5.561A [Accessed 03 March 2014].

Gray, S., De Boer, S., Lorenzen, J., Karasev, A., Whitworth, J., Nolte, P., 2010. Potato

virus Y: an evolving concern for potato crops in the United States and Canada. Plant

Diseases, 94, pp.1384–1397.

Gumus, M and Paylan I. C, 2013. Detection of viruses in seeds of some vegetables by

reverse transcriptase polymerase chain reaction (RT-PCR). African Journal of

Biotechnology, pp.3892-3897.

Gunasinghe,W.A.D.S.K, Dassanayake, E.M and Ubesyerara, N.M, 2009. uccessful

detection of RNA viruses by Reverse transcription polymerase reaction chain reactions.

Annual Symposium of Department of Agriculture (ASDA), 11, pp.57-62.

Haididi A, Montessori M.S, Levy L, Goth R.W, Converse R.H, Madkour MA,

Skrzeckowski L J, 1993. Detection of potato leaf roll and strawberry mild-yellow-edge

luteoviruses by reverse transcriptase polymerase chain reaction amplification. Plant

Disisease, pp.77-79.

Hooker, W.J., 1982. Virus disease of potato-Technical information Bulletin 19. Peru:

International Potato Center Lima.

Hossain,M. B. Nasir, I. A.Tabassum B. and Husnain T., 2013. Molecular characterization,

cloning and sequencing of coat protein gene of a Pakistani potato leaf roll virus isolate

and its phylogenetic analysis. African Journal of Biotechnology, 12(11), pp.1196-1202.

Huisman, Linthorst, B and Cornelissen, 1988. The Complete Nncleotide Sequence of

Potato Virus X and Its Homologies at the Amino Acid Level with Various Plus-stranded

RNA Viruses. Journal of General Virology. , 69, pp. 1789-1988.

http://dx.doi.org/10.1094/PDIS.2002.86.5.561A

46

Innis, M. A., K. B. Myambo, D. H. Gelfand, and M. A. D. Brow., 1988. DNA sequencing

with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain

reaction. USA: Natl. Acad. Sci.

Jayasinghe, U., 1988. Potato leafroll virus; PLRV Technical Information Bulletin 22.

Peru: Internajional potato Center.

Joseph, M. B. Garnsey, S. M, Gonsalves, D. Moscovitz,M. Purcifull, D. E. Clark, M. F.

and Loebenstein, G., 1978. The Use of Enzyme-Linked Immunosorbent Assay for

Detection of Citrus Tristeza Virus. The American Phytopathological Society. , 69, pp.190-

194.

Karpova, O. V. Zayakina, O. V. Arkhipenko, M. V. Sheval, E. V. Kiselyova, O. I.

Poljakov, V. Yu. Yaminsky, I. V. and Rodionova N. P. , 2006. Potato virus X RNA-

mediated assembly of singletailed ternary „coat protein–RNA–movement protein‟

complexes. Journal of General Virology , 87, pp.2731–2740.

Kassanis, B., 1950. Heat inactivation of leaf roll virus in potato tubers. Ann.appl.Biol, 37,

pp.339-341.

Kawchuk, L. M. Martin, R. R. Rochon, D. M. and Mcpherson J., 1989. Identification and

Characterization of the Potato Leafroll Virus Putative Coat Protein Gene. Journal of

General Virology, 70, pp.783-788.

Kumar P.L, Jones,A.T and Waliyar,F, 2004. Serological and Nucleic Acid Based Method

For the Detection of Plant Viruses. patancheru: virology Unit, ICRISAT.

Lauffer, M., and T. E. Cartwright., 1952. Ultracentrifugation studies on potato latent

mosaic virus. Arch. Biochem. Biophys, 33, pp.371-375.

Lizarraga,aR. Panta,A. Jayasinge,V and Dodds,J., 1991. Tissue culture for elimination of

pathogen-CIP research guide3. Peru: International potato center(CIP).

Loebenstein, G., 1976. Potato leaf roll virus. In M.J. O'Brien & A.E. Rich, eds. Potato

diseases. Washington: U.S. Dept. of Agriculture. pp.69-75.

MacKenzie D.J., McLean M.A., Mukerij S., Green M., 1997. Improved RNA extraction

from woody plants for the detection of viral pathogens by reverse transcriptase-

polymerase.chain reaction. Plant Disease, pp222-226 .

Marianne J. Huisman, HUUB J. M. Linthorst, John F. Bol and Ben J. C. Cornelissen.,

1989. The Complete Nncleotide Sequence of Potato Virus X and Its Homologies at the

Amino Acid Level with Various Plus-stranded RNA Viruses. Journal of General

Virology, 69, pp.1789-98.

Mullis, K., E Faloona, S. Scharf, R. Saiki, G. Horn, and Erlich, H., 1986. Specific

enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring

Harbor Symposia on quantative bilogy, LI, pp.263-273.

47

Muthoni, J.Shimelis,H and Melis, R., 2012. Management of Bacterial Wilt of Potatoes in

Kenya. Journal of Agricultural Science, 9, pp.64-78.

Naidu, R.A. and Hughes, J.D.A, 2001. Methods for the detection of plant virus diseases.

Book of Abstracts of the First conference on Plant virology in sub-Saharan Africa, 4:2,

pp.233-260.

Natti, Kirkpatrick & Ross, 1953. Host range of Potato leafroll virus. American jornal of

Potato, 30, pp.55-64.

Peiman,M and Xie,C, 2006. Sensitive detection of potato viruses PVX, PLRV and PVS

by RT-PCR in potato leaf and tuber. Australasian Plant Disease Notes, 1, pp.41–46.

Price, M., 1992. Examination of potato virus X proteins synthesized in infected tobacco

plants. Journal of virology, 66(9), pp.56-58.

Qamar,N and Khan,K.M, 2003. Relationship of Enviromental conditions Conditions

Conductive for potato Virus X(PVX) Disease Development on six varieties/advanced

lines of potato. online journal of biological sciences, 3(2), pp.247-252.

Querci,M. Baulcombe,D.C. Golbdach,R.W and Salazar,L.F., 1995. Analysis of the

Resistance-Breaking Determinants of Potato Virus X (PVX) strain HB on Different

Potato Genotypes expressing Extreme Resistance to PVX. Molecular Plant Pathology,

85.no 9, pp.1003-1010.

Sathiamoorthy, K. Prange, R. Mapplebeck, L. and Haliburton, T., 1985. Potato

Production in Sri Lanka. American Journal of Potato Research, 62, pp.555-564.

Sathiamoorthy, K. Prange, R. Mapplebeck, L. and Haliburton, T, 1985. New and

Reviews. Potato Production in Sri Lanka. American Journal of Potato Research, 62,

pp.555-564.

Shi, J. Choi, D. Kim, B.D. and Kang, B.C., 2008. Study on Inheritance of Potato virus X

Resistance in Capsicum annuum. The Plant Pathology Journal, 24(4), pp.433-438.

Singh, R.S., 1998. Virus disease of potato , Seventh Edition. New Delhi: Oxford and IBH

Publishing Co.PVT.Ltd.

Sipahioğlu, H.M., Usta, M. and Ocak, M., 2006. Use of dried highphenolic laden host

leaves for virus and viroid preservation and detection by PCR methods. Journal of

Virological Methods, pp.120-124.

Stols, A. L. H., HILL-VAN Der meulen, G. W. & Toen, M. K. I., 1970. Electron

microscopy of Nicotianaglutinosa leaf cells infected with potato virus X. Virology 40,

pp.168-170.

48

Richard Tarn, 1999. The Potato. [Online] Available at:

http://www.elements.nb.ca/theme/agriculture/richard/richard.htm [Accessed 02 April

2014].

Voller, A.Bidwell, D. & Bartlett, A., 1976. Microplate immunoassay for the

immunodiagnosis of virus infections. In Friedman, N.R.R.a.H.H. Handbook of Clinical

Immunology. Washington, D.C.: American Society for Microbiology. pp.506-512.

Wilcockson,J and Hull,R., 1973. The Rapid Isolation of Plant Virus RNAs using Sodium

Perchlorate. Journal of Virology, pp.23: 107-111.

Zacharzewska, B. Przewodowska, A and Treder, K, 2014. The Adaptation of Silica

Capture RT-PCR for the Detection of Potato Virus Y. American Journal of Potato

Research, pp.528-532

http://www.elements.nb.ca/theme/agriculture/richard/richard.htm

49

Appendix

A1 Buffers used for RT-PCR and PCR process

A1.1 5x TAE

Tris base - (40mM tris base) 24.25g

GA - 5.7ml

0.5 M EDTA- 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 50µL

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

50

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

Adjust pH to 9.6 stores at 4 oC.

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.

51

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)

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Identification of Potato Virus X (PVX) and Potato Leafroll Virus (PLRV) Using Molecular Detection...

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