pea early browning virus national diagnostic protocol

Pea Early Browning Virus

National Diagnostic Protocol

Angela Freeman

Department of Primary Industries

Primary Industries Research Victoria, Horsham.

December 2006

ACKNOWLEDGMENTS

Plant Health Australia funded the project to develop this manual as part of their National Diagnostic

Protocols Initiative. Dr Merrin Spackman, DPI-Victoria, Horsham, supplied the PCR protocols

described in this manual.

DISCLAIMER

The scientific and technical content of this document is current to the date published and all efforts

were made to obtain relevant and published information on the pest. New information will be included

as it becomes available, or when the document is reviewed. The material contained in this publication

is produced for general information only. It is not intended as professional advice on any particular

matter. No person should act or fail to act on the basis of any material contained in this publication

without first obtaining specific, independent professional advice. Plant Health Australia and all persons

acting for Plant Health Australia in preparing this publication, expressly disclaim all and any liability to

any persons in respect of anything done by any such person in reliance, whether in whole or in part,

on this publication. The views expressed in this publication are not necessarily those of Plant Health

Australia.

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Contents

1.0 Introduction

2.0 National Diagnostic Protocol Procedure

2.1 Purpose and scope of diagnostic protocol

2.2 Responsibility

2.3 Procedure

2.4 Documentation

2.5 Records

3.0 Pest Risk Analysis

3.1 Background

3.2 Species name

3.3 Synonyms

3.4 Common names

3.5 Host Range

3.6 Distribution

3.6.1 Australian status

3.6.2 Current distribution

3.6.3 Potential distribution in Australia

3.7 Plant parts affected

3.7.1 Vegetative

3.7.2 Seedborne

3.8 Disease features

3.9 Biology

3.9.1 Identification

3.9.2 Virus strains

3.9.3 Serological relationships

3.9.4 Symptoms

3.9.5 Disease cycle

3.9.6 Dispersal

3.10 Assessment of likelihood

3.10.1 Entry potential

3.10.2 Establishment potential

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3.10.3 Host potential

3.10.4 Spread potential

3.11 Overall entry, establishment and spread potential

3.12 Assessment of consequences

3.12.1 Economic impact

3.12.2 Environmental impact

3.12.3 Social impact

3.13 Combination of likelihood and consequences to assess risks

3.14 Surveillance

3.15 Diagnostics

3.16 Training

3.17 References

4.0 Diagnostic protocol

4.1 The diagnostic test/s and diagnostic sequence

4.2 The initial samples

4.2.1 Sample handling and subsampling

4.2.2 Sample storage

4.2.3 Visual symptoms

4.2.4 Documentation

4.3 Further samples

4.3.1 Sample collection, transport and storage

4.3.2 Sample locations

4.4 Confirmation of diagnosis

5.0 Identification of pathogen (primary diagnostic test)

5.1 Reverse transcriptase polymerase chain reaction assay (RT-PCR)

5.1.1 Introduction

5.1.2 RNA extraction method

5.1.2.1 General items required

5.1.2.2 RNA extraction

5.1.3 PCR protocol


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5.1.3.2 PCR primers

5.1.3.3 PCR controls

5.1.3.4 PCR reagents and Master mixes

5.1.3.5 PCR programs

5.1.3.6 Electrophoresis for standard PCR gel analysis

5.1.3.7 Real-time PCR analysis

5.1.4 Results

5.1.5 Reagents

5.1.6 Recipes

5.1.7 Ordering information


6.1 Electron microscopy

6.1.1 Introduction

6.1.2 General items required

6.1.3 Sap dip (negative staining) method

6.2 Indicator plants

6.2.1 Introduction


6.2.3 Method

6.2.4 Buffer recipes

6.2.4.1 Inoculation buffer

6.2.5 Indicator plant species and reactions

7.0 Images

7.1 PEBV symptoms on host plants

Figure 6. PEBV symptoms on pea




Figure 10. PEBV symptoms on lucerne

Figure 11. PEBV symptoms on Nicotiana clevelandii

Figure 12. PEBV symptoms on Chenopodium amaranticolor

7.2 PEBV symptoms on host seed

Figure 13. Symptoms of PEBV on pea seed

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7.3 Electron micrographs of PEBV

Figure 14. PEBV virus particles

Figure 15. PEBV virus particles

8.0 References and websites

8.1 References

8.2 Websites

9.0 Appendices

Appendix 1 Preliminary Information Data Sheet (Plantplan, 2004)

Appendix 2 Personal Hygiene

Appendix 3 Harvest Machinery

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List of Figures

Figure 1. Flow chart of the basic procedures and responsibilities.

Figure 2. Flow chart of protocols for the diagnosis of suspect PEBV-infected

plants.

Figure 3. Potential distribution of PEBV in Australia.

Figure 4. The expected PCR product amplified from PEBV.

Figure 5. Typical Melt curve analysis for real time PCR of PEBV samples using

SYBR green chemistry.

Figure 6. PEBV symptoms on pea.




Figure 10. PEBV symptoms on lucerne.

Figure 11. PEBV symptoms on Nicotiana clevelandii.

Figure 12. PEBV symptoms on Chenopodium amaranticolor.

Figure 13. Symptoms of PEBV on pea seed.

Figure 14. PEBV virus particles.

Figure 15. PEBV virus particles.

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1.0 Introduction

Pea early browning virus (PEBV) (Tobravirus, family unassigned) is one of only three

confirmed tobraviruses, all of which are characterised by bipartate genomes, separately

encapsidated, and transmitted by soilborne trichodorid nematodes. PEBV is one of a number

of viruses which are seedborne in a range of temperate pulses and has been found in

Europe and North Africa. The virus is highly seedborne in Pisum sativum (field pea) and

Vicia faba (faba bean, broad bean, tick bean) (Boulton RE 1996).

PEBV was first described by Bos and van der Want (1962) as a disease of peas in the

Netherlands and had been observed on crops for many years. PEBV causes large necrotic

segments to develop in the leaves and stipules and sometimes on the stem and pods of

peas. Brown necrotic patches develop in the crop and infected plants may die, particularly if

infection occurs early in the season. PEBV has since been found in Great Britain, Europe

and north Africa, not only in peas but in faba beans, French beans, lupins, lucerne and some

clovers and medics. However, serious disease symptoms are generally only reported for

peas and in some cultivars of other hosts PEBV does not become systemic.

Three distinct strain groups of PEBV have been described. The Dutch type strain is prevalent

in the Netherlands but not found in Britain (Harrison BD 1973). There is also a Dutch apical

necrosis strain (Hubberling N, Hijberts F 1968). The British strain, which has two host range

variants, is only distantly serologically related to the Dutch type strain (Harrison BD 1964,

1966). Swedish and Moroccan isolates of PEBV differ slightly from those of the Dutch and

British strains in their reactions on indicator plants (Gerhardson B, Ryden K, 1979, Lockhart

BEL, Fischer HU, 1976). A third strain group, known as the broad bean yellow band serotype

was originally thought to be a distinct species (Russo et al. 1984) but sequencing showed

that it was a new serotype of PEBV (Robinson DJ, Harrison BD 1985a). A deviant isolate of

this strain was also found in Algeria (Mahir et al. 1992).

PEBV has a bipartate genome, with each particle separately encapsidated. Infectious virus

particles can exist without a coat protein and cause disease expression in hosts. Robinson

and Harrison (1985a) were able to show that PEBV was able to form pseudorecombinants by

mixing RNA-1 from one isolate with RNA-2 from another isolate, These factors make

serological methods for the detection of PEBV unreliable, particularly in a quarantine

situation. Therefore assays based on detection of nucleic acid such as PCR or nucleic acid

hybridisation are the preferred detection methods. PCR has been used successfully for the

8

detection of all three strain groups in the DPI-Horsham pulse post-entry quarantine testing

program for ten years.

Control of PEBV in pulses is undertaken by sowing healthy seed and undertaking crop

management practices which reduce alternate sources of the virus (eg weed control,

proximity to other crops, etc) and avoiding areas where the disease has been found and soil

contains infectious nematode vectors.

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2.0 National Diagnostic Protocol Procedure

2.1 Purpose and scope of diagnostic protocol

The purpose of this manual is to provide a nationally accepted, standardised protocol for the

accurate detection of pea early browning virus (PEBV) in temperate pulses. PEBV is a

quarantinable pathogen in Australia and is routinely tested for in the post-entry quarantine

program at the DPI Temperate Pulse Quarantine Station, Horsham, Victoria, using PCR.

The manual is designed for easy access to the relevant sections required to identify the

pathogen. The manual contains the Pest Risk Analysis for PEBV for Australia, the primary

diagnostic protocols (PCR) and secondary confirmatory methods (electron microscopy,

indicator plant tests), images of virus symptoms on host plants and seeds, and references

and appendices.

2.2 Responsibility

Figure 1 shows a flow diagram of the responsibilities and procedures required when a

suspect sample is received. The responsibilities are also listed quite clearly in the

following points:

A: State/territory agriculture departments receiving suspect plant sample:

Receiving scientists will record details of the sample so that a trace back can occur if

required.

Receiving scientists will examine the sample and provide diagnostic services (in this

case, conducting the PCR test) to identify the pathogen.

Receiving scientists will notify the State Quarantine Authority (eg. DPI-Victoria Plant

Standards Branch) of the suspect sample.

The State Quarantine Authority will examine the evidence and inform the Office of the

Chief Plant Protection Officer (OCPPO) and AQIS and advise scientists of required action.

The State Quarantine Authority will participate in the Consultative Committee on Exotic

Plant Pests and Diseases (CCEPPD), chaired by the Chief Plant Protection Officer and

decisions made and actions required will be passed onto state scientists for action.

Scientists may be requested to provide expert advice to the CCEPPD.

Scientists will conduct a second type of diagnostic test (secondary confirmatory test) as

advised by the State Authority.

Scientists will send part of the sample to the interstate confirmatory laboratories for

repeat of the primary diagnostic test as advised by the State Authority.

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Under direction from the State Authority, state scientists will undertake delimiting surveys

if required and undertake diagnostics on survey samples.

The State Authority will liaise with industry representatives.

The State Authority will develop communication strategies in conjunction with the

CCEPPD.

The State Authority will report to all interested parties (OCPPO, CCEPPD, AQIS, national

bodies and industry) as required.

The State Authority will keep up to date with the processing of the suspect sample and

will notify the clients of the final result and the corresponding decision for that result.

The State Authority will handle all correspondence with clients. This is very important

and is to be made clear to other personnel involved with handling the sample that they are

not to correspond with the client.

B: Interstate agriculture departments

Scientists will re-examine the suspect sample.

Scientists will repeat diagnostic tests and confirm diagnosis.

Scientists may be requested to provide expert advice to the CCEPPD.

State Quarantine Authority will inform the Chief Plant Protection Officer and the CCEPPD

and will implement their decisions.

C: Office of the Chief Plant Protection Officer (OCPPO)

OCPPO will convene the CCEPPD and all decisions regarding the steps involved in

handling and diagnosing the original sample will be made by the committee.

The CCEPPD will determine whether or not the incursion requires a national response or

involves only one state and will determine the need for delimiting surveys.

Information from each state will be provided to the CCEPPD to enable national decisions

to be made.

OCPPO will provide media releases to the public and interested parties.

OCPPO and the CCEPPD will determine whether or not the pathogen can be eradicated,

contained or will be declared endemic.

2.3 Procedure

Figure 2 shows the order of steps /procedures to be undertaken in the diagnostic process in

a flow diagram.

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2.4 Documentation

An electronic and a hard copy of this manual are maintained by the Senior Virologist, Primary

Industries Research Victoria (PIRVic), Dept. of Primary Industries-Horsham, Victoria and

Plant Health Australia.

2.5 Records

The Recording sheets contained in Appendix 1 must be copied and filled in as appropriate

for each sample received and kept together in a file marked “Suspect pea early browning

virus samples”.

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Sample received at State Agriculture Department

Figure 1. Flow chart of the basic procedure and responsibilities of the relevant Departments if a

suspect sample is received.

Sample logged into relevant diagnostic system and details

recorded

Pathologist tests sample using defined

protocols

Pathologist notifies State Quarantine

Authority

State Quarantine Authority informs

OCPPO and advises Pathologist Pathologist repeats tests and confirms by

a second method

Pathologist sends subsample to

confirmatory lab for testing using the

defined protocols

CCEPPD meets and discusses results

and advises scientists of further action

Plantplan used to develop emergency

response plan Further sampling or elimiting survey

may be undertaken

OCPPO and State Authorities develop

a communication strategy OCPPO and CCEPPD decide on

feasibility of eradication or containment

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Collect samples from crop (shoot or leaf) or seed sample as appropriate

Divide sample into 3 subsamples

Long term storage

Freeze or dry Store sample at 4ºC

until processed Sample sent to

confirmatory lab

Identify initial samples using PCR

Confirm diagnosis using EM

Test survey samples using PCR

Figure 2. Flow chart of protocols for the diagnosis of suspect PEBV-infected plants

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3.0 Pest Risk Analysis

3.1 Background

Pea early browning virus is listed on the Australian Quarantine and Inspection Service

(AQIS) ICON Import Conditions database as a quarantinable pathogen in Australia. PEBV is

tested for in post-entry quarantine in all hosts in which it is seedborne (Pisum and Vicia

species) as required in the regulations listed on ICON.

3.2 Species name

Pea early browning virus (Genus Tobravirus, family unclassified).

3.3 Synonyms

Broad bean yellow band virus.

3.4 Common names

Pea early browning virus.

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3.5 Host range

Host Reference Fabaceae:

Pisum sativum (field pea) Bos L, van der Want JPH (1962), Gibbs AJ,

Harrison BD (1964), Harrison BC (1966),

Verhoyen M, Goethals M (1967), Lockhart

BEL, Fischer HU (1976), Gerhardson B,

Ryden K (1979), Edwardson JR, Christie RG

(1991), Mahir et al. (1992), Bos et al. (1993)

Brunt et al. (1997), Boulton RE (1996),

Wang-Dao et al. (1997), Abraham A,

Makkouk KM (2002).

Vicia faba (broad bean, faba bean) Bos L, van der Want JPH (1962), Lockhart

BEL, Fischer HU (1976), Cockbain et al.

(1983), Russo et al. (1984) Fortass M, Bos L

(1991), Mahir et al. (1992), Bos et al. (1993),

Boulton RE (1996).

Vicia faba var minor (tick bean) Fiedorow ZG (1983), Edwardson JR, Christie

RG (1991).

Phaseolus vulgaris (French bean) Bos L, van der Want JPH (1962),

Gerhardson B, Ryden K (1979), Lockhart

BEL, Fischer HU (1976), Mahir et al. (1992),

Boulton RE (1996).

Lupinus luteus (yellow lupin) Popieszny H, Frencel I (1985), Boulton RE

(1996).

Medicago sativa (lucerne) Bos L, van der Want JPH (1962), Gibbs AJ,

Harrison BD (1964), Boulton RE (1996).

Medicago lupulina (black medic) Bos L, van der Want JPH (1962).

Trifolium incarnatum (crimson clover) Schmidt HE (1977).

Trifolium pratense (red clover) Bos L, van der Want JPH (1962).

Compositae:

Callistephus chinensis (common China aster) Schmidt HE (1977).

Zinnia elegans (common zinnia) Bos L, van der Want JPH (1962).

Cruciferae:

Capsella bella-pastoris (shepherd's purse) Bos L, van der Want JPH (1962).

16

Solanaceae:

Solanum nigrum (black nightshade) Bos L, van der Want JPH (1962).

Tropaeolaceae:

Tropaeolum majus (common nasturtium) Bos L, van der Want JPH (1962).

3.6 Distribution

3.6.1 Australian status

Exotic.

3.6.2 Current distribution

Regions: Africa, Europe Jones RAC, McLean GD (1989), Brunt et al.

(1997), CAB International (1999). Countries:

Algeria Mahir et al. (1992).

Belgium Verhoyen M, Goethals M (1967).

Ethiopia Abraham A, Makkouk KM (2002).

Great Britain Gibbs AJ, Harrison BD (1964), Harrison BC

(1966), Hughes et al. (1986), Boulton RE

(1996).

Italy Russo et al. (1984), Conti M (1984),

Robinson DJ, Harrison BD (1985).

Libya Bos et al. (1993).

Morocco Lockhart BEL, Fischer HU (1976), Fischer

HU (1979), Fortass M, Bos L (1991).

Netherlands Bos L, van der Want JPH (1962), Van Hoof

HA (1969), Bos et al. (1993), Boulton RE

(1996).

North Africa Mahir et al. (1992).

Poland Fiederow ZG (1980), Fiederow ZG (1983).

Sweden Gerhardson B, Ryden K (1979).

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3.6.3 Potential distribution in Australia

As per host plants- temperate and sub tropical grain belt (See Figure 3 below).

Figure 3. Potential distribution of PEBV in Australia

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3.7 Plant parts affected

3.7.1 Vegetative

All.

3.7.2 Seedborne

Pisum sativum (field pea) Bos L, van der Want JPH (1962), Cockbain

et al. (1983), Agarwal VK, Sinclair JB (1987),

Johnstone GR (1989), Jones RAC, McLean

GD (1989), Richardson MJ (1990),

Edwardson JR, Christie RG (1991), Brunt et

al. (1997), Boulton RE (1996), Schmitt et al.

(1998).

Vicia faba (broad bean) Rothamsted Experimental Station (1982),

Cockbain et al. (1983), Johnstone GR

(1989), Frison et al. (1990), Mahir et al.

(1992), Boulton RE (1996).

Vicia faba var minor (tick bean) Fiedorow ZG (1983), Edwardson JR, Christie

RG (1991).

3.8 Disease features

PEBV was first described by Bos and van der Want (1962) as a disease of peas in the

Netherlands and had been observed on crops for many years. PEBV causes large necrotic

segments to develop in the leaves and stipules and sometimes on the stem and pods of

peas. Brown necrotic patches develop in the crop and infected plants may die, particularly if

infection occurs early in the season. The virus is seedborne in some pea varieties and is

spread from plant to plant by soilborne trichodorid nematodes. PEBV has since been found

in Great Britain, Europe and north Africa, not only in peas but in faba beans, French beans,

lupins, lucerne and some clovers and medics. However, serious disease symptoms are

generally only reported for peas and in some cultivars of other hosts, PEBV does not

become systemic. Interactions between PEBV, bean leafroll virus (BLRV) and pea enation

mosaic virus (PEMV) have been found to cause serious disease in faba beans.

PEBV has a bipartate genome, with each particle separately encapsidated. Infectious virus

particles can exist without a coat protein and cause disease expression in hosts. This makes

19

serological methods for the detection of PEBV unreliable, particularly in a quarantine

situation. Therefore assays based on detection of nucleic acid such as PCR or nucleic acid

hybridisation are the preferred detection methods.

3.9 Biology

3.9.1 Identification

Identification is based on PCR. A reverse transcriptase PCR test (RT-PCR test), which

detects all strains of PEBV, has been developed in our laboratory and used as a diagnostic

test in post-entry quarantine for ten years (Freeman et al. 1998). The PCR test is able to

detect all three strain groups of PEBV. Robinson (1992) developed an assay for tobacco

rattle tobravirus (TRV), involving reverse transcription and PCR for detection of TRV RNA.

He found that the sensitivity of the method was sufficient to enable detection of 10 ng total

nucleic acid from an infected plant or in a relatively crude nucleic acid preparation from 60

mg of infected leaf tissue. He was able to detect a wide range of serological variants and

non-particle producing types.

Serological tests are considered unreliable for the detection of tobraviruses due to their

ability to infect hosts and cause disease expression when the particles are unencapsidated

(Robinson DJ, Harrison BD 1985a, Boulton 1996). Although the risk of encountering MN-type

isolates (RNA-1 only) may be low in nature, it is unaccaptable in a quarantine or biosecurity

situation. In the Netherlands, ELISA has been used for the large scale, routine indexing of

pea seeds for PEBV (Vuurde JWL, Maat DZ 1985).

3.9.2 Virus strains

A number of strains of PEBV have been described. The Dutch type strain is prevalent in the

Netherlands but not found in Britain (Harrison BD 1973). There is also a Dutch apical

necrosis strain (Hubberling N, Hijberts F 1968). The British strain, which has two host range

variants is only distantly serologically related to the Dutch type strain and is spread

predominantly by different trichodorid nematode species (Harrison BD 1964, 1966). Swedish

and Moroccan isolates of PEBV differ slightly from those of the Dutch and British strains in

their reactions on indicator plants (Gerhardson B, Ryden K, 1979, Lockhart BEL, Fischer HU,

1976). A third strain group, known as the broad bean yellow band serotype was originally

thought to be a distinct species (Russo et al. 1984) but sequencing showed that it was a new

serotype of PEBV (Robinson DJ, Harrison BD 1985a). A deviant isolate of this strain was

also found in Algeria (Mahir et al. 1992).

20

PEBV has a genome consisting of two single-stranded RNA molecules, each separately

encapsidated in a protein coat. The larger RNA-1 molecules (known as L particles) can

replicate and spread throughout the plant in the absence of the smaller RNA-2 molecules

(known as S particles) and cause typical disease symptoms in the host. As it is the RNA-2

which encodes the coat proteins, the RNA-1 is spreading and replicating in an

unencapsidated form. RNA-2 particles are not infective on their own (Boulton RE 1996).

Virus isolates with RNA-1 and RNA-2 present and encapsidated, which are easily

mechanically transmitted, have been termed M-type isolates. Those non-encapsidated

isolates consisting of RNA-1, which are difficult to transmit mechanically, have been termed

MN-type isolates (Robinson JD, Harrison BD 1985a). Robinson and Harrison (1985a) were

able to show that PEBV was able to form pseudorecombinants by mixing RNA-1 from one

isolate with RNA-2 from another isolate, inoculating indicator plants and counting local

lesions and testing for the presence of nucleoprotein.

Other studies have shown that viable recombinants between tobravirus species can be

produced in the laboratory, for example by replacing the coat protein gene of PEBV with that

of TRV (MacFarlane et al. 1994). Anomalous tobravirus isolates have also been found in

nature. Robinson et al. (1987) found two naturally occurring tobravirus isolates (TRV I6 and

N5) which give symptoms of tobacco rattle virus (TRV), but which react serologically with

antisera to PEBV, not TRV. Goulden et al. (1991) also described an anomalous TRV isolate

(TCM) whose RNA-2 molecule contains sequence that appears to have come from PEBV

RNA-2.

Robinson and Harrison (1985b) studied 14 tobravirus isolates and from the pattern of

sequence homologies and serological and biological properties, concluded that they formed

three distinct virus species, namely TRV, PEBV and pepper ringspot virus (PRV). Robinson

and Harrison considered the three viruses to have distinct gene pools, within, but not

between which genomic RNA molecules are freely compatible. The TRV isolates I6 and N5

are considered anomalous because they appear to be exceptions to this rule (Robinson et al.

1987).

3.93 Serological relationships

Robinson and Harrison (1985b) considered that the genomic RNA molecules within the gene

pool of each tobravirus species were freely compatible. Robinson and Harrison (1985a) were


isolate with RNA-2 from another isolate, and that RNA-1 from an isolate could cause typical

21

host infections in an unencapsidated form, without the presence of RNA-2. These

characteristics of the tobraviruses make serological detection and identification unreliable. A

positive serological test for PEBV will indicate the presence of virus but will not distinguish

pseudorecombinants and therefore may not identify the isolate correctly, based on its

biological or disease symptoms. A serological test will not detect any MN-type PEBV isolates

(RNA-1 only).

The serological relationship between tobravirus isolates has been used as one of the

characteristics studied to determine relationships within and between species. Robinson and

Harrison (1985b) used hybridization experiments with complementary DNA copies, serology

and biological properties to group 14 tobravirus isolates into three virus species, TRV, PEBV

and PRV. Various studies have found differing degrees of serological relatedness between

the three viruses, which reflects the variability and relatedness of the RNA-2 of the three

viruses. Maat (1963) studied the serological properties of PEBV and TRV and concluded that

they were distinct but serologically related viruses. Gibbs and Harrison (1964), in their study

of the British isolate of PEBV, found no serological relationship between the Dutch and

British isolates and any other virus but commented that their antisera was less antigenic than

that of Maat (1963). Allen (1967) found that there was a serological relationship between the

Oregon strain of tobacco rattle virus and pea early browning virus. Kurppa et al. (1981) found

that there was no relationship between spinach yellow mottle virus, a distinct strain of

tobacco rattle virus, and PEBV in gel diffusion or electron microscope serological tests but

they found a distant relationship in micro-precipitin tests.

3.9.4 Symptoms

The symptoms of PEBV in peas vary from symptomless to brown patches on plants to plant

death (Bos L, van der Want JPH 1962, Harrison 1965, Lockhart BEL, Fischer HU 1976,

Mahir et al. 1992). Bos and van der Want (1962) described the symptoms in peas as

appearing about two months after sowing, beginning as a vascular necrosis which extends

into surrounding tissue causing purplish-brown discolorations. These are irregularly

distributed across the stems, leaves and petioles of the plant and result in necrosis, wilting

and death of the tissue. Often pods develop fleck and ring-like purplish-brown necrotic

patterns and seeds of infected pods may show faint chlorotic and finely wrinkled spots.

Bos and van der Want (1962) carried out field tests, sowing a range of plant species,

including a number of legumes, in soil which had lead to severe infections of PEBV in peas

the previous year. They found that other than peas, no naturally infected species (including

22

yellow lupins, French beans, faba beans, clovers and medics) showed any symptoms of

PEBV. Of particular note was the fact that faba bean plants contained large numbers of virus

particles but appeared to be symptomless carriers.

Most other reports of PEBV in faba beans describe symptomless infections (eg. Cockbain et

al., 1983, Fiedorow ZG 1980, 1983) or a mild mosaic (Lockhart BEL, Fischer HU 1976).

Russo et al. (1984) described a possible new tobravirus from faba beans in Italy, which

caused yellow rings, line patterns or yellow vein banding on leaves and malformations and

necrosis of pods. They named the virus Broad bean yellow band virus (BBYBV) but

Robinson and Harrison (1985) established it as a new serotype of PEBV. Another isolate of

PEBV was described in Algeria, which gave similar reactions on faba beans to the BBYB

serotype.

Cockbain et al. (1983) found that necrosis occurred in faba beans as a result of interactions

between PEBV and Bean leafroll virus (BLRV). In glasshouse experiments, infection of faba

bean plants with one of the viruses did not cause necrosis but a mixed infection induced both

stem and leaf necrosis and sometimes, early death.

Although Bos and van der Want (1962) had reported symptomless infection of French beans

with PEBV in their field trials, most mechanical transmission experiments have resulted in

striking local lesions and only occasional systemic infection (eg. Bos L, van der Want JPH

1962, Lockhart BEL, Fischer HU 1976, Gibbs AJ, Harrison BD 1964). Gerhardson and

Ryden (1979) reported the first natural infection of French bean with PEBV from an island in

Sweden. Field symptoms were characterised by growth reductions and slightly deformed

leaves with mosaic and necrotic spots or ring formations.

Pospieszny and Francel (1985) reported the first natural infection of yellow lupins with PEBV

in crops from Central Europe. The plants were also infected with Cucumber mosaic virus

(CMV). The symptoms of retarded growth and shoot death are typical of CMV and it was not

clear whether or not any symptoms could be attributed to PEBV.

Symptoms on pasture legumes are sometimes found and Gibbs and Harrison (1964)

reported chlorotic chevrons and necrotic line patterns on naturally infected lucerne in

England.

PEBV has been reported to be seed transmitted in field peas, (1-61%), broad bean (9-45%)

23

and in tick bean (0.3-8%) (Bos L, van der Want JPH 1962, Edwardson JR, Christie RG 1991,

Mahir MAM, Fortass M, Bos L 1992).

3.9.5 Disease cycle

PEBV is an obligate plant pathogen. It survives between growing seasons of the primary

pulse host in an alternative host or in infected seed. The natural host range includes many

food and pasture legumes and natural hosts in eight families (Edwardson JR, Christie RG

1991). Carryover in infected seed is a likely method of survival of the virus as it can be highly

seedborne in peas and faba beans. Bos and van der Want (1962) found that the trichodorid

nematode vectors of PEBV retain their infectivity between seasons and can spread PEBV to

a new crop sown on the site of an infected crop from the previous year.

PEBV is transmitted by nematodes of the Trichodorus and Paratrichodorus genus. Van Hoof

(1962) established that the Dutch strains of PEBV were transmitted by P. teres and P.

pachydermus, which only occur in sandy soils. Gibbs and Harrison (1964) and found that P.

teres, the most common vector in the Netherlands only occurred at a limited number of sites

in England where PEBV infected crops occurred and established that T. viruliferus was the

main vector. Harrison (1966) established that T. primitivus and T. anenomes were also

vectors of PEBV.

3.9.6 Dispersal

PEBV has been reported to be transmitted by the nematodes Paratrichodorus anemones, P.

teres, P. pachydermis, Trichodorus primitivus and T. viruliferous (Edwardson JR, Christie RG

1991). Different nematode species have been shown to transmit isolates of PEBV from

England and the Netherlands (Harrison BD 1966). The nematode vectors can spread the

virus over relatively short distances and from season to season in areas that have had an

infected crop the previous year. Long distance spread depends on the virus being seed

transmitted.

3.10 Assessment of likelihood

3.10.1 Entry potential

HIGH: PEBV is seedborne in Pisum sativum (field pea) and Vicia faba (broad bean) (Bos L,

van der Want JPH 1962, Cockbain AJ et al. 1983).

Seed transmission of PEBV was first reported by Bos and van der Want (1962) in the pea

cultivar Rondo at an average rate of 37% from infected seeds to seedlings under a range of

24

conditions. Lockhart and Fischer (1976) also reported seed transmission of PEBV of 30% in

peas by testing pea seed from infected plants by inoculation. Cockbain et al. (1983) reported

that no transmission of PEBV was detected in seed harvested from an infected pea crop but

5% transmission was detected from experimentally infected plants. Fiederow (1983) and

Pospieszny and Frencel (1985) reported 61% and 25% seed transmission rates respectively

through pea seed.

Mahir et al. (1992) reported seed transmission rates of PEBV in faba beans at rates of 9% for

the Algerian isolate and over 45% for one of the Dutch type strain isolates. They also

reported seed transmission in Nicotiana rustica of 4%. Bos and van der Want (1962) found

that seed from sap inoculated and systemically infected French bean plants (Phaseolus

vulgaris) did not contain virus.

3.10.2 Establishment potential

HIGH: PEBV has t he pot ent ial t o survive and becom e est ab lished in m ost o f it s

legum inous host s. Dist r ibu t ion is no t lim it ed by environm ent al cond it io ns t hat

p revail in Aust ralia. Based on it s cur rent w or ld d ist r ibu t ion and know n

cond it ions o f survival, i t is likely t o survive w herever m ajor host s are grow n.

PEBV is seedborne in its main hosts, peas and faba beans, and these crops are well

established in Australia and their cropping areas are suitable for PEBV to establish. The

natural host range of PEBV is listed in Table 3.5 and includes 20 species in 15 genera of 7

families (Edwardson JR, Christie RG 1991). However, experimental transmissions have

shown that at least 30 species in at least ten families have been infected by sap inoculation,

although many do not become systemically infected.

High seed transmission rates have been reported for PEBV in field pea (up to 61%) and

broad bean (greater than 45%) (Bos L, van der Want JPH 1962, Cockbain AJ et al. 1983,

Popieszny H, Frencel I 1985, Mahir et al. 1992).

PEBV infections in peas may give characteristic symptoms or be symptomless. In other

hosts such as faba beans, PEBV infections are often symptomless, therefore it is possible

that the virus will not be detected immediately. However, unless a vector was found to co-

exist with the virus, widespread establishment could not occur, as the virus will not spread in

the field. A maximum rate of seed transmission of 61% has been reported for peas,

suggesting that in the absence of field spread, the proportion of infected seed will decrease

25

each year.

3.10.3 Host potential

HIGH: PEBV has been reported in natural infections in 20 species in 15 genera of 7 families

(Edwardson JR, Christie RG 1991). Legume hosts include field peas, faba beans, French

beans, yellow lupins, lucerne and medics and clovers. It also infects members of the

Compositae, Cruciferae, Linaceae, Papaveraceae, Solanaceae and Tropaeolaceae.

3.10.4 Spread potential

LOW: The nematode vectors are not present in Australia. The main source of introducing this

virus into disease-free areas is through infected seed. It is expected that only localised

infection would occur where infected seed is sown, with no secondary spread in the field.

Further spread would depend on the rate of seed transmission and distribution of the

harvested seed.

3.11 Overall entry, establishment and spread potential

The overall pest rating is MEDIUM (ratings based on PHA Industry Biosecurity Planning

Guide) or LOW based on Biosecurity Australia ratings.

3.12 Assessment of consequences

3.12.1 Economic impact

LOW: The economic impact is likely to be low due to the absence of the known vectors in

Australia. If the vector also entered Australia with the virus and both became established,

then the economic impact would be greatly increased.

3.12.2 Environmental impact

NEGLIGIBLE: there is no potential to degrade the environment or otherwise alter the

ecosystems by affecting species composition or reducing the longevity or competitiveness of

wild hosts. It has no effect on human or animal health.

3.12.3 Social impact

NEGLIGIBLE: there is no potential to affect the social environment.

3.13 Combination of likelihood and consequences to assess risks

The pest risk is MEDIUM/LOW, the economic impact is LOW, the environmental and social

impacts are NEGLIGIBLE. Therefore the economic risk rating is MEDIUM/LOW, the

26

environmental risk rating is LOW and the social risk rating is LOW (Risk ratings based on

PHA Industry Biosecurity Planning Guide).

3.14 Surveillance

PEBV is a quarantinable pathogen and is actively tested for in post-entry quarantine. Regular

surveys of pulse crops for endemic viruses are undertaken and tested using ELISA or TBIA.

Samples could also have RNA extracted and be tested for PEBV by PCR.

3.15 Diagnostics

Samples suspected of being infected with PEBV would need to be identified quickly and

accurately. The accompanying report describes methods for sampling and diagnosing PEBV

using PCR and other confirmatory methods. The described PCR procedure for PEBV

detection is used on a regular basis at the DPI Post-entry Quarantine Station for temperate

Pulses at DPI-Horsham, Victoria, and could be undertaken by any trained virologist. The

initial diagnosis would need to be confirmed by another virologist and by a second method.

3.16 Training

There is a general need for industry training in biosecurity and awareness of the potential

impact of exotic diseases. Due to the similarity of symptoms of a range of pulse viruses on

pulse hosts, training and education need to be of a general nature. Training in the recognition

of seed symptoms is likely to maximise the likelihood of early detection.

3.17 References

See Section 8.

27

4.0 Diagnostic protocol

4.1 The diagnostic test/s and diagnostic sequence

Robinson and Harrison (1985b) considered that the genomic RNA molecules within the gene

pool of each tobravirus species were freely compatible. Robinson and Harrison (1985a) were


isolate with RNA-2 from another isolate, and that RNA-1 from an isolate could cause typical

host infections in an unencapsidated form, without the presence of RNA-2. These

characteristics of the tobraviruses make serological detection and identification unreliable. A

positive serological test for PEBV will indicate the presence of virus but will not distinguish

pseudorecombinants and therefore may not identify the isolate correctly, based on its

biological or disease symptoms. A serological test will not detect any MN-type PEBV isolates

(RNA-1 only). Although the risk of encountering an MN-type of isolate may be considered low

in a country where PEBV is endemic and is tested for in routine screening, the risk is

unacceptable in a quarantine or biosecurity situation such as in Australia where PEBV is an

exotic pathogen. PEBV is a single-stranded, positive sense RNA virus and an RT-PCR test

has been developed at DPI-Horsham and used for ten years for the routine detection of

PEBV in the pulse post-entry quarantine virus-testing program. The test was found to be

satisfactory for detecting all three strain groups of PEBV (Dutch, British, BBYB groups). In

the post-entry quarantine program, 100 leaf samples are pooled, extracted and subsampled

for testing. PEBV is readily detected in a pooled sample containing one out of a hundred

PEBV-infected leaves. Recently the PEBV RT-PCR test has been adapted for use as real-

time fluorescent RT-PCR test.

Robinson (1992) developed an assay for tobacco rattle tobravirus (TRV), involving reverse

transcription and PCR for detection of TRV RNA. He found that the sensitivity of the method

was sufficient to enable detection of 10 ng total nucleic acid from an infected plant or in a

relatively crude nucleic acid preparation from 60 mg of infected leaf tissue. He was able to

detect a wide range of serological variants and non-particle producing types. A number of

other RT-PCR tests for the detection of TRV have been published (eg. Martin J 1998,

Crosslin et al. 1999), including real-time flourescent RT-PCR (Mumford et al. 2000) but none

for PEBV.

28

4.2 The initial samples

4.2.1 Sample handling and subsampling

It is important that the samples are entered onto sample reference sheets (Appendix 1)

which contain sufficient information to enable revisiting of the site, describe symptoms and

other relevant information and recording of diagnostic test results. It is vital that information is

provided here to ensure that samples are handled correctly, that sub-samples are taken as

reference samples and so that material can be sent to other experts for confirmation.

4.2.2 Sample storage

As soon as the diagnostician becomes aware that the sample submitted for diagnosis may

be an exotic or emergency pathogen, the diagnostician has the responsibility to seek expert

advice from State Plant Standards or equivalent or AQIS or the Office of the Chief Plant

protection officer (OCPPO) on the appropriate manner/location in which the sample should

be stored and appropriate further testing/action. It is not appropriate for the diagnostician to

continue tests without informing the proper authorities. In Victoria, suspected PEBV-infected

plants can be stored in the DPI pulse quarantine station's AQIS registered storage area for

quarantine samples. Reference material from the original sample should always be kept: for

virus samples, material should be dried and/or frozen, and if possible nucleic acid extractions

conducted.

4.2.3 Visual symptoms

Visual symptoms should be recorded and photos taken where possible.

4.2.4 Documentation

It is important to note that proper documentation of samples and diagnostic procedures and

results is initiated at this stage.

4.3 Further samples

It is important to note that proper documentation of samples and diagnostic procedures and

results is initiated at this stage.

4.3.1 Sample collection, transport and storage

It is important that samples are collected and stored correctly as deteriorating plant samples

may be unsuitable for diagnostic tests. Leaf samples should be placed in labelled sealed

plastic bags and stored in the field in a cooled, insulated container (Esky). Samples should

then be transferred to a refrigerator if they are to be tested within a week of collection. If

29

there are to be delays in testing, then samples for use in PCR tests should be frozen or

dried. This practice is not recommended as it is likely to reduce the sensitivity of the test and

should only be resorted to if there is some impediment to rapid receival and processing of a

batch of samples. If samples are to be used in electron microscopy tests (confirmatory tests)

then fresh samples should be used to prepare microscope slides and the remaining tissue

frozen or dried. Frozen or dried tissue is satisfactory for inoculating indicator plants. If a

survey is being conducted and a team of people is assembled to assist, pictures of plants

with virus symptoms will help in sample selection. Advice on phytosanitary measures

required to prevent disease spread in the field should be provided (Appendix 2).

4.3.2 Sample locations

It is important to record the precise location of all samples collected, preferably using GPS,

or if this is not available, map references including longitude and latitude and road names

should be recorded.


It is important that all diagnoses of suspected exotic and emergency pathogens are

undertaken according to the following parameters: the diagnostician has expertise in this

form of diagnosis, the test is undertaken as described in this manual, the results are

confirmed by diagnosis in another recognised laboratory or another diagnostician and where

possible diagnosis is confirmed by a second method. Methods suitable for confirming the

primary diagnosis are described in Section 6 (eg electron microscopy to confirm presence of

the correct size virus particle).

30

5.0 Identification of pathogen (primary diagnostic test)

5.1 Reverse transcriptase polymerase chain reaction assay (RT-PCR)

5.1.1 Introduction

A PEBV RT-PCR test was developed at DPI-Horsham for post-entry quarantine indexing of

pulses and has been used successfully for ten years. The test is suitable for detection of all

three strain groups of PEBV and has been adapted for use as real-time fluorescent RT-PCR

test. Both methods are described below.

5.1.2 RNA extraction method

5.1. 2.1 General items required

1. Samples - infected/suspect plant tissue, healthy controls.

2. 2-20 l pipettes, 20-200 l pipettes, 200-1000 l pipettes, and sterile tips.

3. Balance (that weighs to at least two decimal places) and weighboats.

4. Resealable plastic bags.

5. Rolling pin.

6. Disposable gloves.

7. Microcentrifuge.

8. 1.5 ml and 2 ml sterile microcentrifuge tubes.

9. 70°C incubator.

10. Fume hood.

11. Sterile distilled water

12. Rneasy RNA isolation kit (Qiagen).

5.1.2.2 RNA extraction

1. Add 19.8 ml of Mackenzie extraction buffer to a 2 g leaf tissue sample and grind in a

resealable plastic bag with a rolling pin.

2. Transfer 990 l of the sample to a 2 ml tube.

3. Add 10 l β-mercaptoethanol and shake to mix.

4. Add 100 l 20% Na-lauryl-sarkosyl and shake to mix.

5. Incubate at 70oC for 15 min or until plant material is a brown colour.

6. Centrifuge for 30 sec at 14,000 rpm to remove leaf material.

7. Pipette 450 l of supernatant onto the QIA shedder spin columns (RNeasy RNA isolation

kit – Qiagen cat #74904).

8. Centrifuge for 2 min at maximum speed.

31

9. Transfer supernatant of flow-through fraction to fresh microcentrifuge tube without

disturbing the cell-debris.

10. Add 0.5 volume 100% ethanol and mix.

11. Add sample to RNeasy mini column and place in a 2 ml collection tube.

12. Close lid and centrifuge 15 sec at 1,000 rpm. Discard flow through.

13. Add 700 l buffer RW1 to the RNeasy column. Close lid and centrifuge 15 sec at 1,000

rpm. Discard flow through.

14. Pipette 500 l of buffer RPE onto RNeasy column. Close lid and centrifuge for 15 sec at

1,000 rpm to wash column. Discard flow through.

15. Pipette 500 l of buffer RPE onto RNeasy column. Close lid and centrifuge for 2 min at

1,000 rpm. Discard flow through.

16. To elute, transfer column to new 1.5ml collection tube. Pipette 30-50 l directly onto the

RNeasy silica membrane. Close tube and centrifuge for 1 min at 10,000 rpm.

5.1.3 PCR protocol


1. 0-2 l, 2-20 l, 20-200 l, and 200-1000 l pipettes and sterile tips.

2. 0.2 ml sterile PCR tubes.

3. Microcentrifuge.

4. Disposable gloves.

5. Cooler racks.

6. Thermocycler.

7. DNA Molecular Weight markers (hyperladder IV, Bioline®).

8. Vertical gel electrophoresis tanks and rigs (CBS Scientific Co. model DASG-400-50).

9. Power pack.

10. UV transilluminator with camera.

5.1.3.2 PCR primers RNA extracts should be assayed with the following primer pair (396 bp product):

1588 – 5‟ GGA TTT GAA AAT TGA TTG GAG GC 3‟. 1589 – 5‟ GGG CGT AAT AAC GCT TAC GTA G 3‟.

5.1.3.3 PCR controls

1) Positive control, ie. an RNA extract from plant tissue infected with PEBV.

2) Uninfected plant control ie. an RNA extract from uninfected plant tissue of the same

species as that used for the positive control.

32

3) Non-template control ie. an aliquot of the PCR master mix without RNA template.

5.1.3.4 PCR reagents and Master mixes

Reverse transcribe PEBV RNA and amplify cDNA using Invitrogen Superscript III one step

(cat # 11732020) with the following protocol:

1) Master mix for standard PCR protocol (gel analysis):

Superscript III / Platinum Taq Mix 0.5 L

2x Reaction Mix 12.5 L

Upstream Primer 1588 (10 M) 1 L

Downstream Primer 1589 (10 M) 1 L

Nuclease free Water 9 L

RNA template 1 L

Total 25 L

2) Master mix for real-time PCR protocol (fluorescence):

Platinum SYBR Green SuperMix UDG 12.5 L

ROX Reference Dye (optional) 0.5 L

Upstream Primer 1588 (10 M) 1 L

Downstream Primer 1589 (10 M) 1 L

RO Water 8 L

DNA template product from above protocol 2 L

Total 25 L

5.1.3.5 PCR programs

1) For gel analysis use program:

1. 48 o C 15 mins.

2. 95 o C 2 mins.

3. 95 o C 15 sec.

4. 60 o C 30 sec.

5. Repeat steps 3-4 50 times.

6. 25 o C Hold.

2) For real-time analysis use program:

1. 50 oC Hold 2 mins.

2. 95 oC Hold 2 mins.

3. 95 oC Hold 15 sec.

33

4. 55 oC Hold 30 sec

5. 72 oC Hold 30 sec – acquiring to Cycling A (FAM/Sybr).

Repeat steps 3-5 40 times.

6. Melt – ramp from 72 oC to 95 oC, hold 45 secs on the first step, hold 5 secs on next steps.

Melt A (FAM/Sybr, ROX).

5.1.3.6 Electrophoresis (for standard PCR gel analysis):

1. Add 2μl loading dye to PCR reaction product.

2. Run 5μl PCR reaction product and 5μl size marker (hyperladder IV), on horizontal 1.5%

agarose + Ethidium bromide in 1x TAE at 80V for 2hrs.

3. Visualise on UV transilluminator.

5.1.3.7 Real-time analysis (using Corbett-Research Rotor-Gene)

Melt Analysis

Cycle samples on Corbett-Research Rotor Gene 3000 cycler with a final melt ramp

step as indicated in section 5.1.3.5.

„Melt curve‟ analysis is used to distinguish the primer dimer from the diagnostic band

especially in samples with low concentrations of virus.

„Melt curve‟ analyses the derivative of the raw data after smoothing. Peaks in the

curve are grouped into „bin‟ groups and all peaks below the threshold are discarded.

For the pea early browning virus real time PCR, using Invitrogen reverse

transcriptase and PCR master mix, the product peak/bin is defined at 81.5oC (this

temperature will vary with different chemistries so melt temperature of the product

must be determined empirically by agarose gel analysis).

The temperature threshold is set at 79.0oC. Any peak below this threshold is

classified as primer/dimer.

To detect low levels of virus the dF/dT can be defined as low as 0.2.

34

5.1.4 Results

1) Agarose gel

Figure 4: The expected 396 bp product amplified from pea early browning virus isolates

using primers 1588 and 1589. lane 1: hyperladder IV (Bioline); lanes 2-5: Algerian isolate.

lanes 6-9: Dutch isolate; lanes 10-1:3 Lybian isolate; lanes 14-17: Healthy tissue. lanes 18-

21: No template control.

2) Melt curve

Figure 5: Typical Melt curve analysis for real time PCR of PEBV samples using SYBR green

chemistry. Positive samples fall into Bin A set at 83oC. The peaks occuring at around 75 oC

reflect primer/dimmer.

35

5.1.5 Reagents

Invitrogen Superscript III one step (cat # 11732020).

5.1.6 Recipes

1. MacKenzie Extraction Buffer

200 ml final

Guanidine Thiocyanate 94.56 g 4 M

3 M Sodium Acetate 13.33 ml 0.2 M

0.5 M EDTA pH 7.0 10 ml 25 mM

Polyvinyl pyrolidone 40 5 g 2.5% W/V

β-mercaptoethanol 10 μl 1%

Dissolve solids and mix solutions in 200 ml sterile RO water.

Add β-mercaptoethanol in step 3 of protocol.

Store at room temperature.

NB. Β-Mercaptoethanol Is a toxic substance. Use only in fumehood. Appropriate gloves, safety glasses and laboratory coats must be worn at all times. EDTA is a hazardous substance. Read MSDSs before use.

2. 10 x TAE Buffer

1 L

Tris base 48.4 g

Glacial Acetic acid 11.42 g

0.5 M EDTA pH 8.0 20.0 ml

Dissolve components in 1 L sterile RO water.

Store at room temperature.

Dilute to 1X concentration for use.

3. 1.5% Agarose with ethidium bromide

100ml

DNA grade agarose 1.5 g 1.5%

10 x TAE 20 ml 1x

RO water 80 ml

10mg/ml Ethidium bromide 3 l 0.3 mg/ml

Dissolve agarose in TAE by heating in microwave for 1 min 20 sec.

Mix by shaking.

Cool to hand temperature and add ethidium bromide, mix.

Pour into get tray with comb to set

36

4. 6x Loading dye

50 mL

Ficoll type 4000

bromophenol blue

xylene cyanoll FF*

7.5 g

0.125 g

0.125 g

Dissolve in to 50ml RO water

5.1.7 Ordering Information

1. Suppliers and catalogue numbers

Chemical Supplier Catalogue #

Agarose Progen 2000011

Bromophenol blue Sigma B-5525

EDTA Sigma 43178-8

Ethanol Merck 10476.9020

Ethidium bromide Merck 443922U

Tris base Merck 108382

Xylene cyanol FF Aldrich 33594-0

2. PCR requirements

Consumable Supplier Catalogue #

PCR Master Mix Promega M7501

Hyperladder IV Bioline BIO-33030

37


6.1 Electron microscopy

6.1.1 Introduction

PEBV is one of three recognised members of the tobravirus genus, all of which have

distinctive, rigid rod-shaped particles in two lengths with an obvious axial canal. Estimates of

particle length for PEBV range from 193-225 nm for the long particles and 54-105 nm for the

short particles, with particle diameters being approximately 21-22 nm (Edwardson JR,

Christie RG 1991).

Key references: Noordham D (1973), Ball EM (1974), Milne RG (1986), Roberts IM (1986).


1. Samples - leaves, shoots or washed roots.

2. Electron microscope grids: copper 400 mesh, Formvar coated, then carbon coated.

3. Glass microscope slides, waxed glass microscope slides, plastic wells, pasteur pipettes,

filter papers, fine forceps.

4. Distilled water, 0.1M sodium phosphate buffer, pH 7.0.

5. Freshly prepared stains: 2% phosphotungstic acid (PTA) and 2% uranyl acetate (UA)

dissolved in distilled water, adjusted to pH 7.0 with NH3.

6.1.3 Sap dip (negative staining) method

1. Using the scalpel blade, cut approximately 3 mm2 of the test plant material and place it

on a clean microscope slide (if the test material has any suspicious virus symptoms, take

the tissue from this area).

2. Place a 3 mm diameter drop of PTA next to the piece of plant material and thoroughly

crush the plant material into the PTA. If necessary add an extra drop of PTA.

3. Pick up a coated grid with forceps and touch it, coated-side down, onto the drop of PTA

and plant sap mixture.

4. After 2-3 seconds, drain the excess droplet of the grid by touching its edge with a piece of

torn filter paper.

5. Allow the grid to dry for approximately 2 minutes then observe grids for virus particles

using an electron microscope.

38

6.2 Indicator plant tests

6.2.1 Introduction

PEBV has a wide host range and it has been reported in natural infections in 20 species in

15 genera of 7 families (Edwardson JR, Christie RG 1991). Legume hosts include field peas,

faba beans, French beans, yellow lupins, lucerne and medics and clovers. It also infects

members of the Compositae, Cruciferae, Linaceae, Papaveraceae, Solanaceae and

Tropaeolaceae.

6.2.2 General Items required

1. Samples from PEBV-suspect plants, normally fully expanded young leaves or dried or

frozen leaf tissue.

2. PEBV-infected samples and healthy host samples as positive and negative controls.

3. Selected indicator plant species (See 6.3.5), 3-6 plants of each species for each test

sample. (NB. It is probably simpler to grow the indicator plants with 3-6 plants per large pot).

4. Mortars and pestles.

5. Sponges, tags for pots, marker pen, wash bottle.

6. Phosphate buffer (See 6.2.4.1), and fine carborundum powder.

6.2.3 Method

1. Collect control samples and test samples (young fully expanded leaves, dried leaves,

frozen leaves). Keep fresh material on ice in an Esky.

2. On each indicator plant, sprinkle carborundum powder onto the four youngest fully

expanded leaves.

3. In clean mortar, grind up a small quantity of test plant material with a pestle, using about 5-

10 volumes of 0.05 M phosphate buffer, depending on type of plant material.

4. Using a clean foam square, dip into ground up sap mixture and gently wipe onto leaves of

a set of indicator plants that have been sprinkled with carborundum powder.

5. Leave for a few minutes then wash off powder and buffer mixture from the leaves using a

wash bottle.

6. Label tag with sample identity and date and place in pot.

39

7. Using new foam square each time, repeat above steps for each sample and controls, as

required.

8. Soak mortar and pestle in bucket with bleach overnight and wash hands with soap.

9. Observe and record indicator plant symptoms regularly over a 4-6 week period.

6.2.4 Buffer recipes

6.2.4.1 Inoculation Buffer: 0.05M Phosphate buffer, pH 7.5 with 2% PVP

1 l

NaH2PO4.2H2O

Na2HPO4

PVP

1.25g

5.96g

20g

Method : Add 1.25g of NaH2PO4.2H2O and 5.96g Na2HPO4 to 900ml of distilled water and

dissolve. If necessary, adjust to pH 7.5 with 1M NaOH. Add 20g of PVP and dissolve. Make

up to 1 litre with distilled water.

40

6.2.5 Indicator plant species and reaction to PEBV

Indicator Species Symptoms Reference

Chenopodium amaranticolor

Small yellow or necrotic local lesions. Some virus isolates tend to produce scattered necrotic lesions in systemically infected leaves but most isolates do not become systemic.

Brunt et al. (1997), Bos L, van der Want JPH (1962).

Chenopodium quinoa

Necrotic and chlorotic local lesions, sometimes systemic causing chlorotic spots and malformation.

Brunt et al. (1997).

Cucumis sativa Necrotic local lesions, not systemic. Brunt et al. (1997).

Gomphrena globosa

Small white local lesions, not systemic. Brunt et al. (1997).

Nicotiana tabacum

Latent systemic infection. Brunt et al. (1997).

Nicotiana glutinosa

Diffuse local lesions, then occasional systemic mosaic and chlorotic line patterns.

Brunt et al. (1997).

Nicotiana clevlandii

Diffuse chlorotic or necrotic local lesions in inoculated leaves, then faint systemic mosaic. The first leaves to be invaded systemically may develop necrotic markings but later-formed leaves are nearly symptomless although infected.

Brunt et al. (1997),

Bos L, van der Want JPH (1962).

Phaseolus vulgaris

Necrotic local lesions or rings, then systemic mosaic and leaf malformation.

cv. The Prince: Necrotic lesions up to 3 mm diameter in inoculated primary leaves . Rarely infected systemically, developing sporadic necrotic lesions.



Pisum sativum Necrotic or chlorotic local lesions, then systemic infection. Systemically infected leaves are stunted



Tetragonia tetragonioides

Local and systemic necrotic concentric rings. Brunt et al. (1997).

41

7.0 Images

7.1 PEBV symptoms on host plants

Figure 6. Symptoms of PEBV on pea, showing necrosis of leaves and stem.

(CAB International, 2006. Crop Protection Compendium. Wallingford, UK: CAB International.)

42

Figure 7. Symptoms of PEBV on pea, showing browning and death of leaves.


43

Figure 8. Symptoms of PEBV on pea, showing necrosis of leaves and stem.


44

Figure 9. Veinal necrosis on leaves and stipules of pea cv. Emblem naturally infected with

PEMV.

(Harrison B (1973). CMI/AAB Description of Plant Viruses No. 120.)

http://www.dpvweb.net/dpv/showdpv.php?dpvno=257

45

Figure 10. Necrotic line patterns on leaf of lucerne naturally infected with PEMV.


Figure 11. Nicotiana clevelandii leaf systemically infected with PEMV showing necrotic

markings.




46

Figure 12. Chlorotic local lesions on a Chenopodium amaranticolor leaf inoculated with

PEBV.


7.2 Symptoms on host seed


47

Figure 13. Seed of pea cv. Rondo, showing severe wrinkling caused by infection with PEBV

(left) compared with healthy seed (right).


7.3 Electron micrographs of PEBV

Figure 14. Virus particles in phosphotungstate. Bar represents 100 nm.



48

Figure 15. L and S particles of a British PEBV isolate.


49

8.0 References and websites

8.1 References

Abraham A, Makkouk KM (2002). The incidence and distribution of seed-transmitted viruses

in pea and lentil seed lots in Ethiopia. Seed Science and Technology 30: 567-574.

Agarwal VK, Sinclair JB (1987). Principles of seed pathology (2 vols.) (CRC Press: Boca

Raton, Florida).

Allen TC (1967). Serological relationship between the Oregon strain of tobacco rattle virus

and pea early browning virus. Phytopathology 57: 97.

Ball EM (1974). Serological tests for the identification of plant viruses. American Phytopathological Society. 31 p.

Boulton RE (1996). Pea early-browning tobravirus Plant Pathology 45:13-28.

Bos L, Mahir MAM, Makkouk KM (1993). Some properties of pea early-browning tobravirus

from faba bean (Vicia faba L.) in Libya. Phytopathologia Mediterranea 32: 7-13.

Bos L, van der Want JPH (1962). Early browning of pea, a disease caused by a soil and

seed-borne virus. Tijdschrift Plantenziektin 68: 368-390.

Boulton RE (1996). Pea early-browning tobravirus. Plant Pathology 45:13-28.

Brunt AA, Crabtree K, Dallwitz MJ, Gibbs AJ, Watson L, Zurcher EJ (Eds) Version: 16 th

January 1997. Plant viruses online: Descriptions and lists from the VIDE database.

http://biology.anu.edu.au/Groups/MES/vide/ (1996 onwards).

CAB International (1999). Crop Protection Compendium. Global Module. CAB International.

Cockbain AJ, Woods RD, Calilung VCJ (1983). Necrosis in field beans (Vicia faba) induced

by interactions between bean leaf roll, pea early-browning and pea enation mosaic viruses.

Annals of Applied Biology 102: 495-499.

Conti M (1984). Tobraviruses. Informatore Fitopatologico 34 :65-69.

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Cooper JI, Mayo MA (1972). Some Properties of the particles of three tobravirus isolates.

Journal of General Virology 16: 285-297.

Crossslin JM, Thomas PE, Brown CR (1999). Distribution of tobacco rattle virus in tubers of

resistant and susceptible potatoes and systemic movement of virus into daughter plants.

American Journal of potato research 76: 191-197.

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199.

Edwardson JR, Christie RG (1991). Handbook of viruses infecting legumes (CRC Press,

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Fiederow ZG (1980). Some virus diseases of horse bean, Tagungs-Bericht Akademie der

landwirtschaftswissenschaften der Deutschen Demokratischen Republik, Berlin 184 361-6.

Fiedorow ZG (1983). Pea early browning virus on horse bean (Vicia faba L. ssp. minor).

Zeszyty Problemowe Postepow Nauk Rolniczych 291: 97-110.

Fischer Hu (1979). The identification and differentiation of virus infections of broad bean.

Awamia 57: 41-72.

Fortass M, Bos L (1991). Survey of faba bean (Vicia faba L) for viruses in Morocco.

Netherlands Journal of Plant Pathology 97: 369-380.

Freeman A, Davidson A, Eastwood R (1998). Detection of pea early browning virus in

imported Vicia and Pisum germplasm using a polymerase chain reaction (PCR) test. In:

Proceedings of the 12th Biennial Australasian Plant Pathology Society Conference, Perth,

Sept., 2005.

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Gerhardson B, Ryden K (1979). An Isolate of pea early browning virus from field-grown

Phaseolus vulgaris. Phytopathologische Zeitschrift 95: 93-96.

Goulden MG, Lomonossoff GP, Wood KR, Davies JW (1991). A model for the generation of

tobacco rattle virus (TRV) anomalous isoalates: pea early browning virus RNA-2 acquires

TRV sequences from both RNA-1 and RNA-2. Journal of general Virology 72: 1751-1754.

Harrison BD (1966). Further studies on a British form of pea early-browning virus. Annals of

Applied Biology 57: 121-9.

Harrison BD (1967). UK, Rothamsted Experimental Station: Report for 1966: 115.

Hubbeling N, Hijberts F (1968). Jversl. Inst. plziektenk. Onderz. 1967: 131.

Hughes G, Davies JW, Wood KR (1986). In vitro translation of the bipartite genomic RNA of

pea early browning virus. Journal of General Virology 67: 2125-2133.

Johnstone GR (1989). Workshop on production, maintenance and exchange of healthy

legume germplasm, May 15-16, 1990, Burnley, Victorian Department of Agriculture and

Rural Affairs. Report of the Proceedings and Recommendations of the Workshop for

Standing Committee on Agriculture.

Jones RAC, McLean GD (1989). Virus diseases of lupins. Annals of Applied Biology 114:

609-637.

Kurppa A, Jones AT, Harrison BD, Bailiss KW (1981). Properties of spinach yellow mottle, a

distinctive strain of tobacco rattle virus. Annals of Applied Biology 98: 243-254.

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occurring in Morocco. Phytopathology 66: 1391-1394.

Maat DZ (1963). Pea early-browning virus and tobacco rattle virus - two different but

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Mahir MAM, Fortass M, Bos L (1992). Identification and properties of a deviant isolate of the

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Switzerland).

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Robinson DJ, Hamilton WDO, Harrison BD, Baulcombe DC (1987). Two anomalous

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68: 2551-2561.

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Robinson DJ, Harrison BD (1985a). Evidence that broad bean yellow band virus is a new

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66: 2003-2009.

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tobraviruses and evidence for the existence of three separate viruses. Journal of General

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du pois (pea early browning virus). Parasitica 23: 128-131.

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8.2 Websites

AAB-CMI Descriptions of plant viruses http://www.dpvweb.net/index.php

All the virology on the WWW http://www.virology.net/garryfavwebplant.html

CABI Crop Protection Compendium http://www.cabicompendium.org/cpc/home.asp

http://www.dpvweb.net/index.php

http://www.virology.net/garryfavwebplant.html

http://www.cabicompendium.org/cpc/home.asp

54

International Committee on Taxonomy of Viruses (ICTV) db Descriptions

http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm

Plant viruses online- descriptions and lists from the VIDE database

http://image.fs.uidaho.edu/vide/refs.htm

http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm

http://image.fs.uidaho.edu/vide/refs.htm

55

Appendix 1. Preliminary Information Data Sheet (Plantplan, 2004).

Date: / /

SUBJECT:

Site details:

Ownership:

Location:

Map (lat. & long.):

GPS identifier:

Host plant location (clearly mark plant if necessary):

HOST DETAILS:

Species and variety:

Age:

Developmental stage:

DAMAGE:

Description of symptoms:

Part of host affected:

Percent incidence:

Percent severity:

DETAILS OF WHEN AND WHERE THE PEST WAS FIRST NOTICED:

RECORDS OF PRODUCT MOVEMENT ON AND OFF DETECTION SITE:

SYMPTOMS / PHOTOGRAPHS:

FURTHER DETAILS OR COMMENTS:

56

Appendix 2. Personnel Hygiene

On entering the paddock, personnel must :

Wear protective overalls and rubber boots.

Prepare footbath of bleach, and spray bottles of methylated spirits brew (95% metho, 5%

water) for use following completion of the inspection.

Conduct inspections by foot (refer to Appendix 3 Machinery Hygiene for vehicle

access).

On leaving the paddock, personnel must :

Wash boots in footbath of disinfectant (solution of household bleach 10%) and remove

adhering material, ie soil, with a suitable brush (ie domestic scrubbing brush).

Spray boots with methylated sprits brew until soaked .

Remove overalls and place into a bag and seal.

Exterior of sample bags to be sprayed/swabbed with methylated spirits brew.

Spray hands with methylated spirits brew irrespective of whether disposable gloves have

been worn.

You must decontaminate before leaving the paddock always.

Overalls must be washed and allowed to completely dry before being used again. If

disposable overalls are used, they can be either washed, or if disposed, sent to land fill or

burnt.

57

Appendix 3. Machinery Hygiene

No machinery, including vehicles, are to enter paddock without prior approval from the

applicant. Approval to use vehicles in paddock must be included with the application for

access.

Decontamination procedures must be followed immediately before leaving the site at

the area identified for decontamination.

Decontaminate the machinery by removing all visible lupin trash and wash down with a

high pressure spray using detergent, paying particular attention to the underside, axles,

wheels and tyres. This also includes all hand held tools such as hoes and shovels.

Personal decontamination procedures must follow the decontamination of machinery.

It is recommended that any machinery or vehicle that has entered the paddock is not to be

taken into another green lupin crop this season.

Harvest Machinery

In addition to the above requirements, machinery will be cleaned of all seed and trash

remaining. This material will be destroyed in a manner approved by the relevant State

Authority (ie, landfill within quarantine boundary or similar).

pea early browning virus national diagnostic protocol

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