Download Pea Early Browning Virus - Plant Biosecurity Toolbox

Diagnostic Methods
for
Pea Early Browning Virus
PEBV
This Diagnostic Protocol can be constantly updated and is only correct at time of printing (Friday
5th of May 2017 at 08:29:18 PM).
The website http://www.padil.gov.au/pbt should be consulted to ensure you have the most
current version before relying on the information contained.
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 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.
Biology
Stages of Development
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.
Host Range
Table 1. Host range of Pea Early Browning Virus.
Host
Fabaceae:
Reference
Medicago lupulina (black medic)
Bos L, van der Want JPH (1962), Gibbs AJ, Harriso
(1964), Harrison BC (1966), Verhoyen M, Goethals
(1967), Lockhart BEL, Fischer HU (1976), Gerhards
B, Ryden K (1979), Edwardson JR, Christie RG (19
Mahir et al. (1992), Bos et al. (1993) Brunt et al. (19
Boulton RE (1996), Wang-Dao et al. (1997), Abraha
Makkouk KM (2002).
Bos L, van der Want JPH (1962), Lockhart BEL, Fis
HU (1976), Cockbain et al. (1983), Russo et al. (198
Fortass M, Bos L (1991), Mahir et al. (1992), Bos et
(1993), Boulton RE (1996).
Fiedorow ZG (1983), Edwardson JR, Christie RG (1
Bos L, van der Want JPH (1962), Gerhardson B, Ry
K (1979), Lockhart BEL, Fischer HU (1976), Mahir
(1992), Boulton RE (1996).
Popieszny H, Frencel I (1985), Boulton RE (1996).
Bos L, van der Want JPH (1962), Gibbs AJ, Harriso
(1964), Boulton RE (1996).
Bos L, van der Want JPH (1962).
Trifolium incarnatum (crimson clover)
Schmidt HE (1977).
Trifolium pratense (red clover)
Compositae:
Bos L, van der Want JPH (1962).
Callistephus chinensis (common China aster)
Schmidt HE (1977).
Zinnia elegans (common zinnia)
Cruciferae:
Bos L, van der Want JPH (1962).
Capsella bella-pastoris (shepherd's purse)
Bos L, van der Want JPH (1962).
Pisum sativum (field pea)
Vicia faba (broad bean, faba bean)
Vicia faba var minor (tick bean)
Phaseolus vulgaris (French bean)
Lupinus luteus (yellow lupin)
Medicago sativa (lucerne)
Solanaceae:
Solanum nigrum (black nightshade)
Tropaeolaceae:
Tropaeolum majus (common nasturtium)
Bos L, van der Want JPH (1962).
Bos L, van der Want JPH (1962).
Distribution
Table 2. Current Distribution of Pea Early Browning Virus.
Regions: Africa, Europe
Jones RAC, McLean GD (1989), Brunt et al.
(1997), CAB International (1999).
Countries:
Algeria
Belgium
Ethiopia
Great Britain
Italy
Libya
Morocco
Netherlands
North Africa
Poland
Sweden
Potential Distribution in Australia
Mahir et al. (1992).
Verhoyen M, Goethals M (1967).
Abraham A, Makkouk KM (2002).
Gibbs AJ, Harrison BD (1964), Harrison BC
(1966), Hughes et al. (1986), Boulton RE
(1996).
Russo et al. (1984), Conti M (1984), Robinson
DJ, Harrison BD (1985).
Bos et al. (1993).
Lockhart BEL, Fischer HU (1976), Fischer HU
(1979), Fortass M, Bos L (1991).
Bos L, van der Want JPH (1962), Van Hoof HA
(1969), Bos et al. (1993), Boulton RE (1996)
Mahir et al. (1992).
Fiederow ZG (1980), Fiederow ZG (1983).
Gerhardson B, Ryden K (1979).
Figure 1. Potential Distribution of PEBV in Australia
Risk Analysis
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
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.
Establishment potential
HIGH
PEBV has the potential to survive and become established in most of its leguminous hosts.
Distribution is not limited by environmental conditions that prevail in Australia. Based on its
current world distribution and known conditions of survival, it is likely to survive wherever
major hosts are grown.
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 coexist 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 each year.
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.
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.
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.
Assessment of consequences
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.
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.
Social impact
NEGLIGIBLE
There is no potential to affect the social environment.
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
environmental risk rating is LOW and the social risk rating is LOW (Risk ratings based on PHA
Industry Biosecurity Planning Guide).
Taxonomy
Name and Synonyms
Species name
Pea early browning virus (Genus Tobravirus, family unclassified).
Synonyms
Broad bean yellow band virus.
Common names
Pea early browning virus.
Taxonomic Description
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).
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).
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
able to show that PEBV was able to form pseudorecombinants by mixing RNA-1 from one
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).
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.
Detection
Detection Method
Responsibility
Figure 2 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.
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.
Figure 2. Flow chart of the basic procedure and responsibilities of the relevant Departments if a
suspect sample is received.
Figure 3. Flow Chart of protocols for the diagnosis of suspect PEBV-infected plants
Procedure
Figure 3 shows the order of steps / procedures to be undertaken in the diagnostic process in a
flow diagram.
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.
Records
The Recording sheets contained in Figure 4 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".
Figure 4. Recording sheet for suspect sample of Pea Early Browning Virus.
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).
The Diagnostic tests and the 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
able to show that PEBV was able to form pseudorecombinants by mixing RNA-1 from one
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 PEBVinfected leaves. Recently the PEBV RT-PCR test has been adapted for use as realtime
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.
The initial samples
Sample handling and subsampling
It is important that the samples are entered onto sample reference sheets 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.
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.
Visual symptoms
Visual symptoms should be recorded and photos taken where possible.
Documentation
It is important to note that proper documentation of samples and diagnostic procedures and
results is initiated at this stage.
Further samples
It is important to note that proper documentation of samples and diagnostic procedures and
results is initiated at this stage.
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 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).
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.
Confirmation of diagnosis
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).
Symptom Description
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
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.
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 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%) 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).
Symptom Images
Figure 5. Symptoms of PEBV on pea, showing necrosis of leaves and stem. (CAB International,
2006. Crop Protection Compendium. Wallingford, UK: CAB International.)
Figure 6. Symptoms of PEBV on pea, showing browning and death of leaves. (CAB
International, 2006. Crop Protection Compendium. Wallingford, UK: CAB International.)
Figure 7. Symptoms of PEBV on pea, showing necrosis of leaves and stem. (CAB International,
2006. Crop Protection Compendium. Wallingford, UK: CAB International.)
Figure 8. 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.)
Figure 9. Necrotic line patterns on leaf of lucerne naturally infected with PEMV. (Harrison B
(1973). CMI/AAB Description of Plant Viruses No. 120.)
♠
Figure 10. Nicotiana clevelandii leaf systemically infected with PEMV showing necrotic
markings. (Harrison B (1973). CMI/AAB Description of Plant Viruses No. 120.)
Figure 11. Chlorotic local lesions on a Chenopodium amaranticolor leaf inoculated with PEBV.
(Harrison B (1973). CMI/AAB Description of Plant Viruses No. 120.)
Figure 12. Seed of pea cv. Rondo, showing severe wrinkling caused by infection with PEBV
(left) compared with healthy seed (right). (CAB International, 2006. Crop Protection
Compendium. Wallingford, UK: CAB International.)
Sites of Infection/Infestation
Plant parts affected
Vegetative: All.
Seedborne:
Pisum sativum (field pea)
Vicia faba (broad bean)
Vicia faba var minor (tick bean)
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).
Rothamsted Experimental Station (1982),
Cockbain et al. (1983), Johnstone GR (1989),
Frison et al. (1990), Mahir et al. (1992),
Boulton RE (1996).
Fiedorow ZG (1983), Edwardson JR, Christie
RG (1991)
Identification
Handling/Preservation
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 Machinery Hygiene (below) 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.
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).
Morphological Methods
Confirmation of diagnosis
Electron microscopy
Figure 13. Virus particles in phosphotungstate. Bar represents 100 nm. (Harrison B (1973).
CMI/AAB Description of Plant Viruses No. 120.)
Figure 14. L and S particles of a British PEBV isolate. (CAB International, 2006. Crop
Protection Compendium. Wallingford, UK: CAB International.)
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).
General items required
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.
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.
Indicator plant tests
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.
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, 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 Buffer recipes below), and fine carborundum powder.
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.
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.
Buffer recipes
Inoculation Buffer: 0.05M Phosphate buffer, pH 7.5 with 2% PVP
NaH2PO4.2H2O
Na2HPO4
1l
1.25g
5.96g
PVP
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.
Indicator plant species and reaction to PEBV
Table 4. Indicator species and reaction to Pea Early Browning Virus.
Indicator Species
Symptoms
Reference
Small yellow or
necrotic local lesions.
Some virus isolates
tend to produce
Brunt et al. (1997),
Chenopodium
scattered necrotic
Bos L, van der
amaranticolor
lesions in systemically Want JPH (1962).
infected leaves but
most isolates do not
become systemic.
Necrotic and chlorotic
local lesions,
Chenopodium quinoa sometimes systemic Brunt et al. (1997).
causing chlorotic spots
and malformation.
Necrotic local lesions,
Cucumis sativa
Brunt et al. (1997).
not systemic.
Small white local
Gomphrena globosa
Brunt et al. (1997).
lesions, not systemic.
Latent systemic
Nicotiana tabacum
Brunt et al. (1997).
infection.
Diffuse local lesions,
then occasional
Nicotiana glutinosa
Brunt et al. (1997).
systemic mosaic and
chlorotic line patterns.
Diffuse chlorotic or
necrotic local lesions
in inoculated leaves,
then faint systemic
Brunt et al. (1997),
mosaic. The first
Nicotiana clevlandii
Bos L, van der
leaves to be invaded
Want JPH (1962).
systemically may
develop necrotic
markings but laterformed leaves are
Phaseolus vulgaris
Pisum sativum
Tetragonia
tetragonioides
nearly symptomless
although infected.
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.
Necrotic or chlorotic
local lesions, then
systemic infection.
Systemically infected
leaves are stunted
Local and systemic
necrotic concentric
rings.
Brunt et al. (1997),
Bos L, van der
Want JPH (1962).
Brunt et al. (1997),
Bos L, van der
Want JPH (1962).
Brunt et al. (1997)
Molecular Methods
Identification of pathogen (primary diagnostic test)
Reverse transcriptase polymerase chain reaction assay (RT-PCR)
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.
RNA extraction method
General items required
1. Samples - infected/suspect plant tissue, healthy controls.
2. 2-20 μl pipettes, 20-200 μl pipettes, 200-1000 μl 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).
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 μl of the sample to a 2 ml tube.
3. Add 10 μl β-mercaptoethanol and shake to mix.
4. Add 100 μl 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 μl of supernatant onto the QIA shedder spin columns (RNeasy RNA isolation kit Qiagen cat #74904).
8. Centrifuge for 2 min at maximum speed.
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 μl buffer RW1 to the RNeasy column. Close lid and centrifuge 15 sec at 1,000 rpm.
Discard flow through.
14. Pipette 500 μl 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 500μl 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 μl directly onto the
RNeasy silica membrane. Close tube and centrifuge for 1 min at 10,000 rpm.
PCR protocol
General items required
1. 0-2 μl, 2-20 μl, 20-200 μl, and 200-1000 μl 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.
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'.
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.
3) Non-template control ie. an aliquot of the PCR master mix without RNA template.
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
2x Reaction Mix
Upstream Primer 1588 (10μM)
Downstream Primer 1589 (10μM)
Nuclease free Water
RNA template
Total
0.5μL
12.5μL
1μL
1μL
9μL
1μL
25μL
2) Master mix for real-time PCR protocol (fluorescence):
Platinum SYBR Green SuperMix UDG
ROX Reference Dye (optional)
Upstream Primer 1588 (10μM)
Downstream Primer 1589 (10μM)
RO Water
DNA template product from above protocol
Total
PCR programs
12.5μL
0.5μL
1μL
1μL
8μL
2μL
25μL
1) For gel analysis use program:
1. 48°C
15 mins.
2. 95°C
2 mins.
3. 95°C
15 sec.
4. 60°C
30 sec.
5. Repeat steps 3-4 50
times.
6. 25°C
Hold.
2) For real-time analysis use program:
1. 50°C
Hold
2. 95°C Hold
3. 95°C Hold
4. 55°C Hold
5. 72°C Hold
2 mins.
2 mins.
15 sec.
30 sec
30 sec - acquiring to Cycling A
(FAM/Sybr).
Repeat steps 3-5 40
times.
6. Melt - ramp from 72°C to 95°C, hold 45 secs on the
first step, hold 5 secs on next steps.
Melt A (FAM/Sybr,
ROX).
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.
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 PCR Programs (above)
'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.
Results
1) Agarose gel
Figure 15. 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 16. Typical Melt curve analysis for real time PCR of PEBV samples using SYBR green
chemistry. Positive samples fall into Bin A set at 83°C. The peaks occuring at around 75°C
reflect primer/dimmer.
Reagents
Invitrogen Superscript III one step (cat # 11732020).
Recipes
1. MacKenzie Extraction Buffer
200 ml
94.56 g
13.33 ml
10 ml
5g
10 μl
Guanidine Thiocyanate
3 M Sodium Acetate
0.5 M EDTA pH 7.0
Polyvinyl pyrolidone 40
β-mercaptoethanol



final
4M
0.2 M
25 mM
2.5% W/V
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
1L
48.4 g
11.42 g
20.0 ml
Tris base
Glacial Acetic acid
0.5 M EDTA pH 8.0



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
DNA grade agarose 10 x
TAE
RO water
100ml
1.5 g 20
ml
80 ml
10mg/ml Ethidium bromide 3 μl
1.5% 1x
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
4. 6x Loading dye
Ficoll type 4000
bromophenol blue
xylene cyanoll FF*
50 mL 7.5 g
0.125 g
0.125 g
• Dissolve in to 50ml RO water
Further Information
Acknowledgements
The information displayed on these Pea Early Browning Virus webpages was sourced from the
National Diagnostic Protocol for PEBV (Freeman, 2006) and was kindly provided by Plant
Health Australia.
Author:
Angela Freeman
Department of Primary Industries
Primary Industries Research Victoria, Horsham.
December 2006
Dr Merrin Spackman, DPI-Victoria, Horsham, supplied the PCR protocols described in this
manual.
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 seedborne 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: 16th
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.
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.
Diseases of grain legumes, 1982. Rothamsted Experimental Station Report for 1981. 1:198-199.
Edwardson JR, Christie RG (1991). Handbook of viruses infecting legumes (CRC Press,
Inc.:Florida).
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.
Frison EA, Bos L, Hamilton RI, Mathur SB, Taylor JD (1990). FAO/IBPGR Technical
Guidelines for the Safe movement of legume germplasm. FAO and IBPGR.
Gibbs AJ, Harrison BD (1964). A form of pea early-browning virus found in Britain. Annals of
Applied Biology 54: 1-11.
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: 609637.
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.
Lockhart BEL, Fischer HU (1976). Some properties of an isolate of pea early-browning virus
occurring in Morocco. Phytopathology 66: 1391-1394.
Maat DZ (1963). Pea early-browning virus and tobacco rattle virus - two different but
serologically related viruses. Netherlands Journal of Plant Pathology 69: 287-293.
MacFarlane SA, Mathis A, Bol JF (1994). Heterologous encapsidation of recombinant pea early
browning virus. Journal of General Virology 75: 1423-1429.
Mahir MAM, Fortass M, Bos L (1992). Identification and properties of a deviant isolate of the
broad bean yellow band serotype of pea early-browning virus from faba bean (Vicia faba) in
Algeria. Netherlands Journal of Plant Pathology 98: 237-252.
Martin J (1998). Use of PCR as a tool for diagnosis of tobacco rattle tobravirus in seed potatoes.
Bulletin OEPP 28: 177-182.
Milne RG (1986). New developments in electron microscope serology and their possible
applications. In: Developments in Applied Biology I: Developments and Applications in Virus
Testing. (eds) R.A.C. Jones and L. Torrance, pp. 179-191. Association of Applied Biologists,
Wellesbourne, Warwicks.
Mumford RA, Walsh K, Barker I, Boonham N (2000). Detection of potato mop-top virus and
tobacco rattle virus using a multiplex real-time fluorescent reverse-transcription polymerase
chain reaction assay. Phytopathology 90: 448-453.
Naumann I (1993).CSIRO Handbook of Australian Insect Names (6th edn). CSIRO Division of
Entomology.
Noordham D (1973). Identification of plant viruses- methods and experiments. Pudoc,
Wageningen. 207 pages.
Popieszny H, Frencel I (1985). Viruses in natural infections of yellow lupin (Lupinus luteus L.)
in Poland. VI. Pea early-browning virus. Acta Phytopathologica Academiae Scientiarum
Hungaricae. 20: 91-5.
Richardson MJ (1990). An annotated list of seed-borne diseases (4th edn) (ISTA: Zurich,
Switzerland).
Roberts IM, (1986). Practical aspects of handling, preparing and staining samples containing
plant virus particles for electron microscopy. In: Developments in Applied Biology I:
Developments and Applications in Virus Testing. (eds) R.A.C. Jones and L. Torrance, pp. 213243. Association of Applied Biologists, Wellesbourne, Warwickshire.
Robinson DJ, Hamilton WDO, Harrison BD, Baulcombe DC (1987). Two anomalous tobravirus
isolates: evidence for RNA recombination in nature. Journal of General Virology 68: 2551-2561.
Robinson DJ, Harrison BD (1985a). Evidence that broad bean yellow band virus is a new
serotype of pea early-browning virus. Scottish Crop Research Institute, Invergowrie, Dundee 66:
2003-2009.
Robinson DJ, Harrison BD (1985b). Unequal variation in the two genome parts of tobraviruses
and evidence for the existence of three separate viruses. Journal of General Virology 66: 171176.
Russo M, Gallitelli D, Vovlas C, Savino V (1984). Properties of broad bean yellow band virus, a
possible new tobravirus. Annals of Applied Biology 105: 223-30.
Schmitt C, Mueller AM, Mooney A, Brown D, MacFarlane S (1998). Immunological detection
and mutational analysis of the RNA2-encoded nematode transmission proteins of pea
earlybrowning virus. Journal of General Virology 79: 281-1288.
Van Hoof HA (1969). Some properties of Dutch pea early-browning virus isolates. Meded.
Rijksfac. Landbwetensch. Gent 34: 888-894.
Verhoyen M, Goethals M (1967). Presence en Belgique du virus du brunissement precoce du
pois (pea early browning virus). Parasitica 23: 128-131.
Vuurde JWL, Maat DZ (1985). Enzyme-linked immunosorbent assay (ELISA) and dispersedye
immuno assay (DIA): comparison of simultaneous and separate incubation of sample and
conjugate for the routine detection of lettuce mosaic virus and pea early-browning virus in seeds.
Netherlands Journal of plant pathology 91: 3-13.
Wang-DaoWen, MacFarlane SA, Maule AJ (1997). Viral determinants of pea early browning
tobravirus seed transmission in pea. Virology (New York) 234: 112-117.
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
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