Broad bean mottle virus DP - Plant Health Australia

Broad Bean Mottle Virus

National Diagnostic Protocol

Angela Freeman

Department of Primary Industries

Primary Industries Research Victoria, Horsham.

May 2006

ACKNOWLEDGMENTS

Plant Health Australia funded the project to develop this manual as part of their National Diagnostic Protocols Initiative. Dr Safaa Kumari (ICARDA) supplied many of the photographs and the protocol for TBIA, which was adapted for 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 Host range potential

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3.10.3 Establishment 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 Enzyme-linked immunosorbent assay

5.1.1 Introduction

5.1.2 General items required

5.1.3 Specific items

5.1.4 Buffer recipes

5.1.4.1 Coating Buffer

5.1.4.2 Phosphate Buffered Saline

5.1.4.3 Wash Buffer

5.1.4.4 Extraction Buffer for DAS ELISA

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5.1.4.5 Conjugate Buffer

5.1.4.6 Substrate Buffer

5.1.4.7 Extraction Buffer for indirect ELISA

5.1.5 Methods

5.1.5.1 Direct double antibody sandwich (DAS) ELISA

5.1.5.2 Indirect ELISA

5.2 Tissue blot immunoassay (TBIA)

5.2.1 Introduction




5.2.5 Methods

5.2.6 Reagent suppliers


6.1 Electron microscopy

6.1.1 Introduction


6.1.3 Methods

6.1.3.1 Sap dip (negative staining) method

6.1.3.2 Immunosorbent electron microscopy

6.1.3.2.1 Trapping method

6.1.3.2.2 Decoration method

6.2 Light microscopy

6.2.1 Introduction


6.2.3 Method

6.3 Indicator plants

6.3.1 Introduction


6.3.3 Method


6.3.4.1 Inoculation buffer

6.3.5 Indicator plant species and reactions

7.0 Images

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7.1 BBMV symptoms on host plants

Figure 3. BBMV symptoms on faba bean




Figure 7. BBMV symptoms on lentil

Figure 8. BBMV symptoms on french bean

7.2 BBMV symptoms on host seed

Figure 9. BBMV symptoms on faba bean seed

7.3 BBMV vectors and vector symptoms

Figure 10. The BBMV vector Sitona lineatus

Figure 11. The BBMV vector Sitona lineatus

8.0 References and websites

8.1 References

9.0 Appendices

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

Appendix 2. Hygiene

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List of Figures

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

relevant Departments if a suspect sample is received.

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

infected samples.

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1.0 Introduction

Broad bean mottle virus (BBMV) (Bromovirus, Bromoviridae) is one of a number of viruses

which are seedborne in a range of temperate pulses and has been found in Africa, Asia,

Europe and the Middle East. The virus is seedborne in Cicer arietinum (chickpea), Pisum

sativum (field pea) and Vicia faba (faba bean, broad bean, tick bean) (Fortass M, Bos L

1992).

BBMV was first described by Bawden et al. (1951) from a severely infected broad bean

(Vicia faba) crop in Nottinghamshire, England. Three quarters of the plants displayed leaf

symptoms, many were dwarfed and produced few or no flowers. Infected plants were in

concentric patches, with the most severely infected in the centre, suggesting the virus was

spreading in the crop from randomly distributed initially infected plants. BBMV was then

found in a faba bean crop in Cambridge in 1957 but was not found elsewhere despite

surveys (Gibbs AJ, 1972). The virus was seen as being of minor importance until reports of

its widespread occurrence began to appear in the 1970s and 1980s. BBMV was reported in

faba bean crops in Portugal (Borges M, Louro D 1974), Sudan (Murant et al. 1974), Morocco

(Assou NM 1978), and Algeria (Ouffroukh A 1985). Makkouk et al. (1988a) then undertook a

regional survey and found BBMV in faba bean crops in Egypt, Morocco, Sudan, Syria and

Tunisia. Fortass and Bos (1991) surveyed faba bean crops in Morocco and found that

luteoviruses and BBMV were the most prevalent viruses. Fortass and Diallo (1993) surveyed

a range of legume crops in Morocco for BBMV and reported natural infection of chickpea,

lentil, pea and common bean. They considered that BBMV was an actual threat to a range of

food legume crops and improvement programs.

BBMV has now been established as having a wide host range among the legumes (Makkouk

et al. 1988a) and is spread by a range of beetle vectors. The symptoms of BBMV can be

confused with other legume viruses which cause mottle/mosaic symptoms eg broad bean

stain virus (BBSV), broad bean true mosaic virus (BBTMV) and bean yellow mosaic virus

(BYMV). Like BBSV, BBMV can affect seed quality by causing necrosis and shrivelling of the

seed. Seed transmission rates in legumes are low and range from 0.1% to 1.4%. Various

strains of BBMV have been distinguished on the basis of slight differences in host range and

symptomology ranging from almost symptomless to severe, but they have generally been

found to be serologically indistinguishable.

BBMV survives between growing seasons of the primary pulse host in an alternative host or

in infected seed. Due to the fact that the natural host range includes at least four families

(Brunt et al. 1997, Makkouk et al. 1988a) and includes many food legumes and non-legume

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wild hosts, it may survive in a range of alternative hosts. Carryover in infected seed is a likely

method of survival of the virus and Makkouk et al. (1988a) and Fortass and Bos (1991)

suggest that seed transmission in faba beans and other legume crops would explain the

widespread occurrence of BBMV in West Asia and North Africa.

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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 broad bean mottle virus (BBMV) in temperate pulses. BBMV 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 ELISA.

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

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

diagnostic protocols (ELISA) and tissue blot immunoassay (TBIA) and secondary

confirmatory methods (light and 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 serological tests ELISA and/or TBIA) 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

PHA.

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 broad bean mottle

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 planFurther sampling or elimiting survey

may be undertaken

OCPPO and State Authorities develop a

communication strategyOCPPO 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 ELISA and/or TBIA

Confirm diagnosis using EM

Test survey samples using ELISA or TBIA

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

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3.0 Pest Risk Analysis

3.1 Background

Broad bean mottle virus (BBMV) is listed on the Australian Quarantine and Inspection

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

BBMV is tested for in post-entry quarantine in all hosts in which it is seedborne (Cicer, Pisum

and Vicia species) as required in the regulations listed on ICON.

3.2 Species name

Broad bean mottle virus.

3.3 Synonyms

None.

3.4 Common names

Broad bean mottle virus.

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3.5 Host range

Host Ref

Cicer arietinum (chickpea) Fortass M, Bos L (1992), Fortass M, Diallo S

(1993), Dafalla G, Hussein M (1994),

Mouhanna et al. (1994), Makkouk et al.

(1995), Ouizbouben A, Fortass M (1997),

Erdiller G, Akbas B (1996), Nene et al.

(1996).

Lens culinaris (lentil) Fortass M, Diallo S (1993), Makkouk KM,

Kumari SG(1995).

Lupinus sp. (lupins) Borges MLV, Sequeira JC (1988).

Pisum sativum (field pea) Edwardson JR, Christie RG (1991), Fortass

M, Bos L (1992), Fortass M, Diallo S (1993).

Phaseolus vulgaris (French bean, red bean) Borges MLV, Sequeira JC (1988), Fortass M,

Bos L (1992), Fortass M, Diallo S (1993),

Mouhanna et al. (1994), Zhao GF (1996).

Vicia faba (broad bean) Fischer HU (1979), Borges MLV (1982),

Botina et al. (1984), Borges MLV, Sequeira

JC (1988), Makkouk et al. (1988a), Sequeira

JC, Borges MLV (1989), Edwardson JR,

Christie RG (1991), Fortass M, Bos L (1992),

Bos et al. (1992), Fortass M, Diallo S (1993),

Mouhanna et al. (1994), Dafalla G, Hussein

M (1994), Makkouk et al. (1995), Brunt et al.

(1997), El Afifi SI, El Dougdoug KA (1997),

CAB International (1999), Najar et al. (2000).

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3.6 Distribution

3.6.1 Australian status

Exotic.

3.6.2 Current distribution

Regions:

Africa, Asia, Europe, Middle East

Edwardson JR, Christie RG (1991), Brunt et

al. (1997), Zhao GF (1996).

Countries:

Algeria Ouffroukh A (1985).

China Zhao GF (1996).

Colombia Botina et al. (1984).

Egypt Makkouk et al. (1988a), El Afifi SI, El

Dougdoug KA (1997).

Great Britain Bawden et al. (1951), Tinsley TW (1957).

Morocco Assou NM (1978), Bourbah M, Fezzaz M

(1979), Fischer HU (1979), Makkouk et al.

(1988a), Fortass M, Bos L (1992), Fortass M,

Diallo S (1993), Ouizbouben A, Fortass M

(1997).

Portugal Borges et al. (1974), Borges MLV (1982),

Borges MLV, Sequeira JC (1988), Sequeira

JC, Borges M de LV (1989).

Sudan Murant et al. (1974), Makkouk et al. (1988a),

Bos et al. (1992), Dafalla G, Hussein M

(1994), Makkouk et al. (1995).

Syria Makkouk et al. (1988a), Mouhanna et al.

(1994).

Tunisia Makkouk et al. (1988a), Najar A, et al.

(2000).

Turkey Erdiller G, Akbas B (1996).

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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 BBMV in Australia

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3.7 Plant parts affected

3.7.1 Vegetative

All.

3.7.2 Seedborne

Cicer arietinum (chickpea) Fortass M, Bos L (1992), Erdiller G, Akbas B

(1996).

Pisum sativum (field pea) Fortass M, Bos L (1992).

Vicia faba (broad bean) Fortass M, Bos L (1992), Makkouk et al.

(1988a).

3.8 Disease features

BBMV is one of a number of seedborne viruses, which infect temperate pulse crops and

cause mottle/mosaic symptoms. Symptoms of BBMV may be difficult to distinguish from

those of broad bean stain virus (BBSV) or broad bean true mosaic virus (BBTMV) on faba

bean. All three viruses can be spread by weevils and a distinguishing feature of infection by

all of them may be chewed leaf margins as a result of the weevil vector feeding (Cockbain et

al., 1975). BBMV and BBSV (but not BBTMV) may cause staining, necrosis or crinkling of

Vicia faba seed. The three viruses are difficult to distinguish on particle morphology as they

all have isometric particles of similar diameter (Makkouk et al. 1988a). BBMV particles are 26

nm diameter and rounded, whilst BBSV and BBTMV have angular, isometric particles of

about 25 nm diameter (Gibbs AJ, Smith HG, 1970). BBMV can be distinguished from BBSV

and BBTMV by its host range, which includes most temperate pulses and extends outside

the Fabaceae (Makkouk et al. 1988a, Fortass M, Diallo S, 1993, Mouhanna et al. 1994, Brunt

et al. 1997). BBSV and BBTMV are characterised by very narrow host ranges, restricted to a

few temperate pulses.

3.9 Biology

3.9.1 Identification

Identification of the virus is based on serological reactions. BBMV can be confused with two

exotic pulse comoviruses, BBSV and BBTMV, which it is difficult to distinguish from by

symptoms and particle morphology, but to which it is serologically unrelated. Sequence data

for BBMV are listed on the Genebank database although there do not appear to be any

published PCR tests for BBMV diagnosis. Indicator plant tests can also be used to identify

the virus and the diagnosis may be confirmed by electron microscopy (Makkouk et al. 1988,

Fischer 1979). BBMV also causes the formation of large characteristic inclusions consisting

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of isometric virus particles in infected plant cells, which are considered diagnostic and are

visible by light microscopy (Makkouk et al. 1988, El-Afifi and El-Dougdoug 1997).

(NB. We are currently developing a PCR test for BBMV detection and this method will be

added to the manual after completion and validation.)

3.9.2 Virus strains

BBMV was first found in England in Nottinghamshire in 1951 in a severely infected faba bean

crop and then in a Cambridge crop in 1957 but was not found elsewhere despite surveys

(Gibbs AJ, 1972). The first report of BBMV outside Great Britain was from the Sudan (Murant

AF, Abu Salih HS, Goold RA (1974) and the Sudan strain was reported as being slightly less

virulent and slightly different antigenically to the English type strain. Botina et al. (1984)

described a strain of BBMV from Columbia, which differed in symptoms and host range from

those described in the literature, but they did not compare strains by serology. Makkouk et al.

(1988a) undertook detailed studies on four isolates of BBMV, one each from Morocco,

Tunisia, Sudan and Syria. They found that although they were indistinguishable serologically

when tested by ELISA and gel diffusion tests, they differed slightly in host range and

symptoms and concluded that they were different strains of BBMV. Sequeira and Borges

(1989) identified isolates of BBMV from faba beans in Portugal which gave very mild

symptoms but found that they could be lethal in peas. The Portugese isolates were found to

be serologically identical to English isolates. Bos et al. (1992) described a new strain of

BBMV from Sudan, which produced a neally symptomless infection in faba beans despite a

high concentration of the virus in infected plants. They reported that it did not differ from

other strains in gel diffusion and ELISA tests, light and electron microscopy, host range and

symptoms on other major hosts. Fortass and Bos (1992) studied ten isolates of BBMV from

Morocco, Algeria, Sudan and Tunisia and were able to group them into three groups based

on pathogenicity (mild, intermediate and severe) towards a number of grain legume

genotypes and Gomphrena globosa but found that they all gave similar reactions to antisera

to a Syrian and and a Moroccan isolate. From their studies, within and between host species,

they concluded that virus isolates differed in virulence rather than aggressiveness and that

hosts differed in sensitivity rather than susceptibility. El-Afifi and El-Dougdoug (1997)

identified three strains of BBMV in Egypt, which differed in their host range and the

localisation of virus particles in infected cells, but found that all strains caused characteristic

inclusion bodies.

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3.9.3 Serological relationships

Rybicki and von Wechmar (1981) studied the serological interrelatedness of the

bromoviruses by determining the relatedness of three group members, BBMV, brome mosaic

virus (BMV) and cowpea chlorotic mottle virus (CCMV). They compared homologous and

heterologous relationships using gel precipitin, "rocket" immunoelectrophoresis and direct,

indirect and double antibody sandwich (DAS) ELISA techniques with native virus and coat

protein under a range of pHs, after fixation with formaldehyde and under capsid swelling

conditions. They concluded that the bromoviruses were serologically related but found that

the relationship depended on antigenic determinants more easily recognised in swollen virus

or free protein than in compact, low pH virus. BMV and CCMV were more closely related to

each other and only distantly related to BBMV. Although the relationship between BMV or

CCMV and BBMV could be demonstrated by manipulation of test conditions, they concluded

that there was no reaction between them under any conditions in the DAS ELISA, which is a

strain-specific means of differentiating viruses. This work confirmed the earlier findings of

Scott and Slack (1971) who found that BMV and CCMV were distantly serologically related to

each other but not to BBMV and Hull (1972), who found that BBMV was less closely related

to BMV and CCMV than they were to each other. Valverde (1985) described a new member

of the bromovirus group, spring beauty latent virus (SBLV), which infected Spring beauty

(Claytonia virginica). Using Ouchterlony double diffusion tests, he concluded that SBLV was

distantly serologically related to BMV and CCMV but not to BBMV. Recent sequence

comparisons between SBLV, BMV, CCMV and BBMV (Fujisaki et al. 2003) demonstrated

that SBLV is closely related to BMV and CCMV but not to BBMV.

3.9.4 Symptoms

Early reports of the symptoms of BBMV on faba beans in England described them as

"characteristic vein-clearing of the youngest leaves, which subsequently faded and was

replaced by a bright interveinal mottle" (Bawden et al. 1951, Walters HJ, Surin P 1973).

Makkouk et al. (1988a) tested 14 faba bean cultivars and nine breeding lines for their

reaction to a Moroccan and a Tunisian strain of BBMV and reported that all genotypes

reacted in about seven days with severe systemic vein chlorosis to chlorotic vein banding,

developing into overall chlorosis in a couple of leaves following the inoculated leaves,

sometimes with remaining interveinal islands of green tissue and in some genotypes with

necrosis along leaf edges (Figs. 3, 4, 5, 6). Plants thereafter more or less recovered

temporarily, with a persistent mottling, marbling or diffuse mosaic recurring in leaves that

developed later. There were some additional or slightly different symptoms in some

genotypes, such as irregular vein chlorosis or vein mosaic and additional necrotic streaks on

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stems. Fortass and Bos (1992) found that faba bean breeding lines all reacted with mottling

symptoms typical of BBMV when inoculated, but varied in the presence or absence of local

symptoms and the degree of stunting that occurred. A different strain of BBMV has been

reported in Sudan, which despite high concentrations of virus in infected plants is neally

symptomless in faba beans, although its host range and symptoms on other major hosts

does not differ from other isolates (Bos et al. 1992). In a yield loss experiment, Makkouk et

al. (1988b) found that infection of faba bean with BBMV 11 weeks (pre-flowering), 15 weeks

(flowering) and 20 weeks (pod setting) after sowing lead to 54%, 84% and 37% yield loss

respectively. BBMV can also cause symptoms on faba bean seed, which resemble those

caused by BBSV and include necrotic patches, staining and shrivelling (Fig. 9).

BBMV has a wide host range and Makkouk et al. (1988a) found that one or more isolate of

BBMV systemically infected 21 species (12 legume and 9 non-legume) out of 27 tested.

They found that infection in several species was symptomless but major legumes such as

chickpea, lentil and especially pea, suffered severely from infection. Fortass and Diallo

(1993) then reported the first natural occurrence of BBMV in chickpea, lentil, pea and

common bean in Morocco. They found the field symptoms on these species difficult to

define, probably due to mixed infections. The symptoms on peas and lentils consisted of a

necrosis of the lower leaves and a mild mosaic on the tip leaves of peas and vein clearing

and mottling on lentils. Chickpeas developed a striking wilt of the lower leaves and a yellow

mosaic of the upper leaves. Common bean leaves developed chlorotic spots. The authors

were unable to determine whether or not these field symptoms were solely caused by BBMV

infection but the symptoms are consistent with those described by other authors and

summarised below.

BBMV infects all pea cultivars and generally causes desiccating, rapidly expanding local

lesions leading to withering of inoculated leaves, followed by systemic stem and apical

necrosis (Bawdwn et al. 1951, Waltters HJ, Surin P 1973, Makkouk et al. 1988a, Bos et al.

1992, Fortass M, Bos L 1992). Fortass and Bos (1992) found that ten pea accessions tested

were vulnerable to BBMV and that some highly vulnerable lines gave additional symptoms of

systemic wilting or necrosis and a few lines gave atypical symptoms of an unusual yellow

mosaic.

Makkouk et al. (1988a) reported differing reactions of two chickpea lines to a Moroccan

isolate of BBMV, one reacting with vein chlorosis followed by mosaic and the other reacting

22

with necrotic stem streaking and tip necrosis. Fortass and Bos (1992) found that nine

chickpea breeding lines tested were all vulnerable to all isolates of BBMV tested.

Makkouk et al. (1988a) found that four lentil genotypes inoculated with the same Moroccan

isolate of BBMV reacted with systemic interveinal chlorosis, mottling or mosaic and

sometimes local necrosis. Fortass and Bos (1992) found that ten lentil lines tested were

mostly but not always susceptible to most BBMV isolates. Symptoms were usually a local

necrotic reaction followed by systemic yellow mosaic and sometimes necrosis (Fig. 7).

Makkouk et al. (1988a) also found that the Moroccan isolate could infect most cultivars of

common bean, usually causing small chlorotic local lesions, usually followed by systemic

infection which may involve yellow stippling or blotching in some lines or interveinal mottle

(Brunt et al. (1997) (Fig. 8).

3.9.5 Disease cycle

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

pulse host in an alternative host or in infected seed. Due to the fact that the natural host

range includes at least four families (Brunt et al. 1997, Makkouk et al. 1988a) and includes

many food legumes and non-legume wild hosts it may survive in a range of alternative hosts.

Carryover in infected seed is a likely method of survival of the virus and Makkouk et al.

(1988a) and Fortass and Bos (1991) suggest that seed transmission in faba beans and other

legume crops would explain the widespread occurrence of BBMV in West Asia and North

Africa. Initial infection of a crop occurs when infected seed is sown or it is spread to the crop

host plants from the alternative host by its beetle vectors. Secondary spread occurs within

the crop during insect vector feeding.

A number of beetle species have been reported as vectors of BBMV, including Acalymma

trivittata (striped cucumber beetle), Diabrotica undecimpunctata (spotted cucumber beetle)

and Colaspis flavida (grape colaspis) (Walters HJ, Surin P 1973). A number of weevil

species are also vectors of BBMV and include Sitona lineatus and Apion arrogans which

have also been reported to transmit BBSV and BBTMV (Cockbain AJ 1971, Rothamstead

Experimental Station Report 1982, Borges M, de LV, Louro D 1974, Edwardson JR, Christie

RG 1991, Makkouk KM, Kumari S 1989,). Ahmed and Eisa (1991) reported that the larvae of

the lesser armyworm (Spodoptera exigua) transmitted BBMV with an efficiency of up to 31%.

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3.9.6 Dispersal

A number of beetle and weevil species have been reported as vectors of BBMV, including

Acalymma trivittata (striped cucumber beetle), Diabrotica undecimpunctata (spotted

cucumber beetle) and Colaspis flavida (grape colaspis), Sitona lineatus and Apion arrogans

(Rothamstead Experimental Station Report 1982, Borges M, de LV, Louro D 1974,

Edwardson JR, Christie RG 1991, Makkouk KK, Kumari S 1989).

Walters and Surin (1973) undertook transmission experiments with five beetle species and

found that Acalymma trivittata (striped cucumber beetle), Diabrotica undecimpunctata

(spotted cucumber beetle) and Colaspis flavida (grape colaspis) all transmitted BBMV at a

transmission rate of 6-10%. Fortass and Diallo (1993) undertook transmission experiments

with curculionid weevils in Morocco and found that Apion radiolus, Hypera variablis (H.

postica), Pachytychius strumarius, Smicronyx cyaneus and Sitona lineatus were all vectors

of BBMV and that S. lineatus was an efficient vector with aquisition and inoculation occurring

at the first bite, 41% transmission rate and virus retention of at least seven days. Makkouk

and Kumari (1995) assessed the ability of four weevil species, naturally occurring on

legumes in Syria, to transmit BBMV and found that Apion arrogans, Sitona limosus and S.

lineatus were able to transmit BBMV but that S. crinita (S. macularius) failed to transmit

BBMV. These findings confirmed their earlier finding of Apion arrogans as a vector of BBMV

(Makkouk KM, Kumari SG, 1989). Borges and de Louro (1974) found that Sitona lineatus

var. viridifrons was abundant in faba bean fields in Portugal and confirmed it as a vector of

BBMV, with a transmission efficiency of about 6%, in glasshouse feeding experiments.

Borges and Sequeira (1988) confirmed it as the vector of BBMV in the field. Ahmed and Eisa

(1991) reported significant crop loss due to BBMV in the Sudan but were unable find any of

the reported vectors infesting affected fields. They observed a relationship between

infestation of the fields with larvae of the lesser armyworm (Spodoptera exigua) and in

glasshouse transmission studies found that the larvae transmitted BBMV with an efficiency of

up to 31%.

3.10 Assessment of likelihood

3.10.1 Entry potential

HIGH BBMV is seedborne in Cicer arietinum (chickpea), Pisum sativum (field pea) and

Vicia faba (broad bean) (Makkouk et al. 1988a, Fortass M, Bos L (1992).

24

A mild strain of BBMV in faba bean in the Sudan was reported to be seed transmitted by

Murant et al. (1974) in conjunction with bean yellow mosaic virus (BYMV). Makkouk et al.

(1988a) then reported experimental seed transmission of BBMV and BYMV in faba bean of

1.37% after inoculating plants in the field. Fortass and Bos (1992) reported the first natural

infections of pea and chickpea with BBMV in Morocco. They undertook host range and seed

transmission experiments in a range of food legumes by inoculating with BBMV. They found

seed transmission in faba bean, pea and chickpea at rates of 1.2%, 0.1% and 1.0%

respectively. They were unable to determine whether seed transmission occurred in lentils,

as infected plants failed to set seed. Erdiller and Akbas (1996) confirmed that BBMV was

seed transmitted in chickpea by growing out and testing farmers' seed in Turkey.

Seed is the only commodity imported for these species, therefore importation of infected

seed may occur. BBMV occurs in most areas where the host species are grown except North

America. Seed of all the above genera are imported for breeding purposes from countries

where BBMV is endemic. The virus would survive intact in seed shipments.

3.10.2 Host range potential

MEDIUM: The natural host range is listed in 3.5 and includes most temperate pulses (faba

bean, field pea, chickpea, lentil, lupin and French or red bean). Experimental transmissions

have shown that many other species in the Fabaceae are susceptible eg. Lathyrus oderatus,

Trifolium incarnatum, T. pratense, T subterraneum, Glycine max (Bawden et al. 1951); L.

oderatus, Lupinus albus, Melilotus officinalis, T. pratense, T. repens (Walters HJ, Surin P

1973); M. albus, Vigna unguiculata (Makkouk et al. 1988a). There are no data on seed

transmission of BBMV in these hosts. BBMV has a wide host range and Makkouk et al.

(1988a) found that one or more isolate of BBMV systemically infected 21 species (12 legume

and 9 non-legume) out of 27 tested. These data suggest that there is potential for BBMV to

establish in species in the Fabaceae beyond its recorded natural host range. Clover pastures

in Australia would be likely potential hosts of BBMV. Susceptible hosts were also identified

by experimental inoculation in the Chenopodiaceae, Cucurbitaceae, Solanaceae and the

Amaranthaceae (Walters HJ, Surin P 1973, Makkouk et al. 1988a, Bos et al. 1992, Brunt et

al. 1997).

3.10.3 Establishment potential

HIGH: BBMV has the potential to survive and become established throughout most or all of

the range of 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.

25

The host crops are well established in Australia and these cropping areas are suitable for

BBMV to establish. The natural host range is listed in Table 3.5, however experimental

transmissions have shown that other species in the Fabaceae (Lathyrus oderatus, Trifolium

incarnatum, T. pratense T. repens, T subterraneum, Melilotus albus, M. officinalis, Lupinus

albus, Glycine max, Vigna unguiculata) are susceptible (Bawden et al. 1951, Walters HJ,

Surin P 1973, Makkouk et al. 1988a). These data suggest that there is potential for BBSV to

establish in species in the Fabaceae beyond its recorded natural host range. Clover pastures

in Australia would be likely potential hosts of BBMV. Susceptible hosts were also identified

by experimental inoculation in the Chenopodiaceae, Cucurbitaceae, Solanaceae and the

Amaranthaceae (Walters HJ, Surin P 1973, Makkouk et al. 1988a, Bos et al. 1992, Brunt et

al. 1997).

Seed to seedling transmission rates are relatively low: faba beans < 1.37%; peas < 0.1%;

chickpeas < 0.9% (Makkouk et al. 1988a, Fortass M, Bos L 1992).

3.10.4 Spread potential

LOW: The pest has potential for natural spread locally. The known vectors are not present in

Australia. The only known way of introducing this virus into disease-free areas is through

infected seed (Naumann, 1993). 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. However, if

the beetle vectors were also brought into Australia, the spread potential would be much

higher. There are no data on the only endemic Sitona species, Sitona discoideus, as a

potential vector of BBMV. The preferred hosts of S. discoideus are lucerne (Medicago sativa)

and burr medic (M. polymorpha). M. sativa is listed as a susceptible experimental host of

BBMV (Brunt et al. 1997). There are no other insect species recorded in Australia of the

same genera as the known BBMV vectors (Naumann 1993).

The symptoms of the virus on the host are not diagnostic and are similar to those caused by

a range of seedborne viruses, therefore it is unlikely that the virus will 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. The rates of seed

transmission are low, suggesting that in the absence of field spread, the proportion of

infected seed will decrease each year.

26

3.12 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.

3.13 Assessment of consequences

3.13.1 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. Crop yields can be severely affected

in areas where the virus and its vector co-exist. Faba bean yield losses have been reported

to range from 37 to 84% (Makkouk et al. 1988b). BBMV is widespread in West Asia and

North Africa and is considered a threat to production (Fortass M, Diallo S 1993) and has

been reported at within crop virus incidences of around 20% (Ouizbouben A, Fortass M

1997, Najar et al. 2000).

Elsheikh and Osman (1995) found that faba bean plants which were inoculated with BBMV

four weeks after sowing showed a 52% reduction in shoot weight and similar reduction in

root weight 12 weeks later. They found that BBMV caused a decrease in the number of pods

(62%), the dry weight of pods (33%), the number of seeds per plant (43%) and the 100-seed

weight (15%). They found that seedborne BBMV caused a decrease in nodulation of 24%

and a decrease in shoot and root dry weight, and the number of nodules and flowers per

plant. Makkouk et al. (1988b) conducted a yield experiment by inoculating faba beans with

BBMV 11 (pre-flowering), 15 (flowering) and 20 (pod setting) weeks after sowing and

recorded 54%, 84% and 37% yield losses respectively.

BBMV can also cause symptoms on faba bean seed, which resemble those caused by BBSV

and include necrotic patches, staining and shrivelling (Fig. 9) which would affect the

marketability of the affected grain.

3.13.2 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.

27

3.13.3 Social impact

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

3.14 Combination of likelihood and consequences to assess risks

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

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

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

PHA Industry Biosecurity Planning Guide).

3.15 Surveillance

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

Regular surveys of pulse crops for endemic viruses could easily be extended to include

screening of samples for BBSV using ELISA or TBIA.

3.16 Diagnostics

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

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

using ELISA and TBIA and other confirmatory methods. The ELISA procedure for BBSV

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. TBIA is

used at the Quarantine Station to confirm positive ELISA results. The initial diagnosis would

need to be confirmed by another virologist and by a second method. BBMV may be been

confused with two exotic pulse comoviruses, BBSV and BBTMV, which are difficult to

distinguish by symptoms and particle morphology but which are serologically distinct

(Cockbain et al., 1975). BBMV is also spread by some of the same vectors as BBSV and

BBTMV. Mixed infections may occur and careful diagnosis is required.

NB. A PCR test for detection of BBMV is currently being developed at DPI-Horsham and will

be added to this manual when completed and validated.

3.17 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,

including BBMV, on pulse hosts, training and education need to be of a general nature.

28

Training in the recognition of seed symptoms is likely to maximise the likelihood of early

detection.

3.18 References

See Section 8.

29

4.0 Diagnostic protocol

4.1 The diagnostic test/s and diagnostic sequence

Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) is the

recommended primary test for detection of BBMV (Clark MF, Adams AN, 1977). There are

published sequence data for BBMV and a PCR test is currently being developed at DPI-

Horsham but is not yet available for routine diagnostics. The method will be added to this

manual following completion and validation. There are extensive published data on

serological detection of BBMV, including the serological relationship between BBMV and

other bromoviruses (Section 3.9.3) and differences between strains of the virus (Section

3.9.2). Tissue-blot immunoassay (TBIA) is included as an alternative primary diagnostic test,

because although less widely used than ELISA it has proved to be the test of choice under

some circumstances (Makkouk KM, Comeau A, 1994). Both ELISA (eg. Makkouk et al.

1988a, Fortass M, Bos L 1992, Bos et al. 1992, Fortass M, Diallo S 1993, Ouizbouben A,

Fortass M 1997, Makkouk et al. 1994) and TBIA (eg. Tadesse et al. 1999, Najar et al. 2000,

El-Muadhidi et al. 2001, Makkouk et al. 1992) have been used widely for the identification of

BBMV from field samples and in surveys.

ELISA is the most commonly used serological method for BBMV detection and its use and

comparisons of various ELISA methods are widely reported in the literature. Rybicki and Von

Wechmar (1981) compared direct, indirect and sandwich ELISA methods for the detection of

three bromoviruses, including BBMV, and found that they were each best suited to a different

application. They concluded that direct ELISA is a sensitive if limited means of showing virus

relationships and that indirect ELISA is an ideal sensitive method for screening large

numbers of antisera for heterospecific reactivity. They found that the addition of one specific

reaction step in the DAS-ELISA, that of adsorbed specific antibody, greatly increased the

specificity and sensitivity of the test, and they recommended DAS-ELISA for specific virus

detection. Salama et al. (2003) compared three ELISA methods for detecting BBMV in faba

bean and found that the most sensitive methods were ELISA with enzyme amplification (EA-

ELISA) and double antibody sandwich ELISA (DAS-ELISA) which both enabled detection of

BBMV in sap diluted to 1/2500. Penicillinase ELISA (PNC-ELISA) was found to be much less

sensitive for BBMV detection.

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

30

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 BBMV-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

there are to be delays in testing then samples for use in ELISA tests should be frozen. This

practice is not recommended as freezing is likely to reduce the sensitivity of the test and

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

31

batch of samples. If samples are to be used in TBIA tests then samples should be blotted

onto multiple nitrocellulose membranes and the remaining tissue frozen or dried. 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).

32

5.0 Identification of pathogen (primary diagnostic test)

5.1 Enzyme-linked immunosorbent assay (ELISA)

5.1.1 Introduction

Direct double antibody sandwich ELISA (DAS-ELISA) (Clark, MF, Adams, AN, 1977) is used

with polyclonal antisera and indirect ELISA (Voller A et al. 1976, Torrance L, Pead MT 1986)

is normally employed with monoclonal antibodies. Both procedures are described below.


1. Samples - leaves, shoots, seedlings (germinated seeds).

2. Microtitre plates (e.g. Nunc maxisorp plate) and lids.

3. Mortars and pestles (sterile and kept cool), or sap extractor machine or plastic sample

bags and rolling pins.

4. 200 L pipettes, 0.5-200 L pipettes, 100-200 L multistepper pipettes and sterile tips.

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

6. pH meter and magnetic stirrer.

7. Plate reader and computer.

8. Incubator kept at 37°C.

9. 1 litre graduated glass bottles with lids, volumetric flasks, flasks, beakers, tube racks

and screw cap 5 ml plastic specimen storage bottles, plastic wash bottles.

10. Disposable gloves, paper towels, plastic reservoir.


1. BBMV antisera and conjugate.

BBMV antisera are not available commercially. The antiserum recommended for this protocol

was produced at the International Centre for Agricultural Research in the Dry Areas

(ICARDA), Aleppo, Syria. It has been used extensively by Dr Khaled Makkouk and Dr Safaa

Kumari (previous and current Manager of the Virology Laboratory, ICARDA, respectively) in

survey and research work throughout the Middle East and North Africa (as cited in this

protocol). It has been used at the DPI pulse quarantine station, DPI, Horsham, Victoria, for

post-entry quarantine testing for BBMV for ten years. It is used successfully in both DAS-

ELISA and TBIA. The BBMV antiserum is conjugated with alkaline phosphatase for use in

DAS-ELISA. 1ml of the ICARDA BBMV antiserum has been purchased and is stored at DPI,

Horsham, for use in an incursion. Dried and frozen BBMV- infected leaf tissue is also stored

in the locked quarantine -18 C storage facility at the quarantine station. A stock of BBMV

antiserum is held at the station for use in post-entry quarantine testing and additional

33

antiserum could be made available for use in an incursion. Additional BBMV- alkaline

phosphatase conjugate would be prepared when required.

NB. If further antisera has to be purchased from overseas an AQIS permit is required. Dr

Angela Freeman, manager DPI pulse quarantine station holds a current permit to import the

antiserum and virus infected tissue.


5.1.4.1 Coating Buffer (pH = 9.6)

1 l 500 ml 250 ml

Na2CO3

NaHCO3

1.59 g

2.93 g

0.79 g

1.46 g

0.39 g

0.73 g

Dissolve in distilled water, adjust pH to 9.6 with HCl and make up to required volume.

5.1.4.2 Phosphate Buffered Saline (pH 7.4) = PBS

5 l

NaCl

KH2PO4

Na2HPO4

KCl

40.00 g

1.00 g

5.75 g

1.00 g

Dissolve in distilled water, adjust pH to 7.4 with NaOH and make up to 5 litres.

5.1.4.3 Wash Buffer: Phosphate Buffered Saline + Tween (pH 7.4) = PBST

5 l

PBS

Tween 20

5 l

25 ml

5.1.4.4 Standard Extraction Buffer: PBST + 2% PVP (pH 7.4)

1 l 500 ml

PBST

PVP-40

(Poly Vinyl

Pyrolidine-40)

1 l

20.00 g

500 ml

10.00 g

Dissolve PVP in PBS solution on a magnetic stirrer.

34

5.1.4.5 Conjugate Buffer: PBST + 2% PVP + 0.2% ovalbumin (pH 7.4)

1 l 500 ml

PBST +2% PVP

Ovalbumin

1 l

2.0 g

500 ml

1.0 g

Dissolve ovalbumin in PBST-PVP solution on a magnetic stirrer.

5.1.4.6 Substrate Buffer (pH = 9.8)

1 l 500 mL 250 mL

Diethanolamine 106.7 g 53.35 g 26.65 g


5.1.4.7 Indirect ELISA Extraction Buffer (pH = 9.6)

1 L 500 mL 250 mL

Na2CO3

NaHCO3

PVP

1.59

2.93

20.00

0.79

1.46

10.00

0.39

0.73

5.00


5.1.5 Methods

5.1.5.1 Direct double antibody sandwich (DAS) ELISA

1. Coat microtitre plate with polyclonal BBMV antibody diluted at recommended rate in

coating buffer using 200 l/well. Coat 60 internal wells only. Put 200 l distilled water in

each of the outside wells.

2. Cover microtitre plate and incubate at 37oC for 4 hours.

3. Extract samples individually in extraction buffer (1:5 or 1:10 w/v) using a fresh mortar and

pestle for each sample, or a sap extractor with crusher rollers that are washed thoroughly

in between each sample or a plastic bag and rolling pin. Collect extract and store in

labelled 5ml plastic specimen bottles.

4. Wash coated plate with wash buffer (PBST) at least three times (3-5 times), using a wash

bottle, allowing a three minutes soak for each wash.

35

5. Pipette sample extracts and control extracts into paired wells (200 l/well) on the

washed, coated plate. Controls should consist of four wells each of extraction buffer

(blank), healthy plant extract (negative) and extracts of at least one known BBMV-

infected plant (positive).

6. Cover microtitre plate and incubate in refrigerator at 4°C overnight. Although overnight

incubation is recommended, 4 hours at 37°C is satisfactory.

7. Remove microtitre plate contents and rinse with PBST, using pressure with the wash

bottle, to remove traces of plant material.

8. Wash plate with wash buffer (PBST) at least three times (3-5 times), using a wash bottle,

allowing a three minutes soak for each wash and ensuring no plant material remains.

9. Dilute conjugate (BBMV antibody conjugated with alkaline phosphatase enzyme) to

recommended rate in conjugate buffer, and add to the 60 inner wells of the plate at 200

l/well. Put 200 l distilled water in each of the outside wells of the plate.

10. Cover microtitre plate and incubate at 37°C for 4 hours.

11. Remove microtitre plate contents. Wash plate with wash buffer (PBST) at least three

times (3-5 times), using a wash bottle, allowing a three minute soak for each wash.

12. Just prior to usage, prepare p-nitrophenyl phosphate (PNP) substrate by dissolving PNP

tablets (Sigma 104-105) in substrate buffer to make a 1 mg/ml solution.

13. Add 200 l/well of freshly prepared PNP substrate to all the wells of the plate. Incubate at

room temperature for 1-2hr or until unambiguous reactions are obtained.

14. Read the absorbance values (OD values) of each well at a wavelength of 405 nm (A405)

using a plate reader after 30-60 minutes and again after 2 hours.

36

5.1.5.2 Indirect ELISA

1. Extract samples individually in indirect-ELISA extraction buffer (1:5 or 1:10 w/v) using a

fresh mortar and pestle for each sample, or a sap extractor with crusher rollers that are

washed thoroughly in between each sample or a plastic bag and rolling pin. Collect

extract and store in labelled 5ml plastic specimen bottles.

2. Pipette sample extracts and control extracts into paired wells (200 l/well) of the plate. Fill

the 60 internal wells only. Put 200 l distilled water in each of the outside wells. Controls

should consist of four wells each of extraction buffer (blank), healthy plant extract

(negative) and extracts of at least one known BBMV-infected plant (positive).

3. Cover microtitre plate and incubate in refrigerator at 4°C overnight.



5. Dilute monoclonal antibody to recommended rate in conjugate buffer, and add to the 60

inner wells of the plate at 200 l/well. Put 200 l distilled water in each of the outside

wells.


7. Remove plate contents. Wash plate with wash buffer (PBST) at least three times (3-5

times), using a wash bottle, allowing a three minute soak for each wash.

8. Dilute anti-species conjugated antibody (eg goat anti-mouse) to the recommended rate in

conjugate buffer and add 200 l/well to the 60 inner wells. Put 200 l distilled water into

each of the outside wells.




37

11. Just prior to usage, prepare p-nitrophenyl phosphate (PNP) substrate by dissolving PNP

tablets (Sigma 104-105) in substrate buffer to make a 1 mg/ml solution.

12. Add 200 l/well PNP substrate to all the wells of the plate.

13. Read the absorbance values (OD values) of each well at a wavelength of 405 nm (A405)

using a plate reader after 30-60 minutes and again after 2 hours.

5.2 Tissue-blot immunoassay (TBIA)

5.2.1 Introduction

Lin et al. (1990) found that TBIA was suitable for detection of viruses in the cucumovirus,

luteovirus, potexvirus, potyvirus and tospovirus groups and from a range of tissue types. Hsu

and Lawson (1991) compared both direct blotting of tomato spotted wilt virus plant tissue

with dot-blot immunoassay and ELISA, both of which involve extracting the virus into buffer.

TBIA was found to be sensitive, reliable and rapid and had the added advantages of

simplicity and convenience. Makkouk et al. (1994) found that TBIA was a simple, sensitive

and quick method for barley yellow dwarf virus detection. Makkouk and Comeau (1994)

looked at a range of modifications to this method and their modifications form the basis of the

method described in this manual.


1. Nitrocellulose membrane (NCM) (CN. 0.45 um) from Schleicher & Schuell, Cat No.

401.196 BA85

2. Goat Anti-Mouse-AP labelled (from Bioreba Cat No. 1031-04 or Sigma Cat No. A-5153)

3. Goat Anti-Rabbit-AP labelled (from Bioreba Cat No. 4050-04 or Sigma Cat No. A-8025)

4. BCIP (from Sigma Cat No. B-8503 or ROCHE Cat no. 1017373)

5. NBT (from Sigma Cat No. N-6876 or ROCHE Cat No. 1087479)

6. Poly Vinyl Alcohol (PVA)

7. Small plastic, flat-bottomed containers with lids (eg empty pipette tip boxes or food storage

boxes).

8. Shaker


1. BBMV polyclonal antiserum form ICARDA (See ELISA section)

38


5.2.4.1 Phosphate Buffered Saline (pH 7.4) = PBS

5 l

NaCl

KH2PO4

Na2HPO4

KCl

40.00 g

1.00 g

5.75 g

1.00 g

Dissolve in distilled water, adjust pH to 7.4 with NaOH and make up to 5 litres.

1.0g NaN3, a carcinogen used as preservative, may be added per 5 litres. As an alternative,

store at – 4 C instead.)

5.2.4.2 Wash Buffer: Phosphate Buffered Saline + Tween (pH 7.4) = PBST

5 l

PBS

Tween 20

5 l

2.5 ml

5.2.4.3 Standard Extraction Buffer: PBST + 2% PVP (pH 7.4)

1 l 500 ml

PBST

PVP-40

(PolyVinyl

Pyrolidine-40)

1 l

20.00 g

500 ml

10.00 g

Dissolve PVP in PBS solution on a magnetic stirrer.

5.2.4.4 Conjugate Buffer: PBST + 2% PVP + 0.2% ovalbumin (pH 7.4)

1 l 500 ml

PBST

Ovalbumin

(Sigma A-5253)

1 l

2.0 g

500 ml

1.0 g

Dissolve ovalbumin in PBST-PVP solution on a magnetic stirrer.

39

5.2.4.5 Substrate Buffer: Tris 0.1 M, NaCl 0.1 M (pH 9.5)

1 l

Tris (0.1 M) 12.1g

NaCl (0.1 M) 5.8g

MgCl2 (5 mM) 0.4g

5.2.4.6 Substrate stock solutions: NBT and BCIP

Tube 1. Prepare a p-Nitro Blue Tetrazolium (NBT) stock solution of 75 mg/ml in 70%

diemethylformamide. Store at -20 C.

Tube 2. Prepare a 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) stock solution of 50

mg/ml in undiluted diemethylformamide. Store at -20 C.

5.2.5 Method for tissue-blot immunoassay (TBIA) on nitrocellulose membranes

1. It is important to select a type of nitrocellolose membrane which will optimise the result.

The preferred membrane is the Schleiger and Schultz Protran 55 m membrane. Do not

use nylon membranes as they give high backgrounds. Prepare nitrocellulose membrane

by stamping a 10 x 10 grid or use a standard 5 x 10 cm sheet and underline every fifth

row of blots to keep straight. Mark top left corner with an asterisk (*). Handle the

membranes with blunt ended forceps. N.B. If blotting faba bean samples, which tend to

oxidise and go brown/ purple on the membrane, pre-treat membrane by first dipping in

1% sodium sulphite.

2. It is much easier to blot petioles than leaves, which have to be rolled into a cigar shape

before blotting, therefore collect leaves with petioles intact if possible. If thin tissues such

as leaves are used, roll them into a tight core or cigar and secure with masking tape.

3. Wearing gloves, hold tissues in one hand and cut petiole or cigar with a scalpel blade

with the other hand to obtain a single plane cut surface. It has been found that it is not

necessary to wash the blade between the cutting of each sample.

40

4. Press the newly cut surface with a firm but gentle force onto one of the cells of the grid on

the nitrocellulose membrane (NCM).

5. Always include blotted positive and negative controls in the test. These can be

blotted at the same time as the samples or a large number of controls can be blotted in

advance and small pieces of NCM with controls can be added to the dish for processing.

6. If TBIA tests are not used regularly it is advisable to run through the procedure outlined

below using only the positive and negative controls to ensure that the test is working well.

If results are not clear, optimise the test by increasing the concentration of antiserum and

checking the viability of the conjugate and possibly increasing its concentration. Some

monoclonal reactions can be improved by incubating overnight in the fridge. This does

not work for polyclonals and gives a high background. The colour development varies

with different conjugates. It may be worth comparing the results with a couple of different

brands of conjugate.

7. Prior to commencing the processing, prepare polyclonal antisera by reacting out

antibodies to healthy plant proteins. First grind 1g fresh healthy host tissue in 20mls PBS-

Tween (or equivalent 1:20 extraction if larger volume needed). Strain to remove

particulate matter. Dilute polyclonal antiserum 1/2000 or as required in the extract and

incubate 2 hrs in the oven at 37 C.

8. Place all membrane/s in one dish by carefully lowering them, making sure the back of the

membranes are wet first. Wash 3 times with PBS-Tween at 5 minute intervals on a small

shaker.

9. Block NCM in 1 µg/ml Polyvinyl Alcohol (PVA) in PBST and incubate for 1 minute at room

temperature. Save PVA as it is reused many times. Mark date on record sheet on back of

bottle

10. Wash 3 times with PBS-Tween at 5 minute intervals.

11. Place membranes into separate, labelled dishes.

12. Remove prepared polyclonal antisera from oven and prepare monoclonal antisera by

diluting 1/2000 or as required in conjugate buffer (PBST +2% PVP + 0.2% ovalbumin).

41

Add diluted antiserum to each dish and incubate for 1 hour at room temperature on a

small shaker. Save antiserum to be reused and mark record sheet on bottle (may be

used up to 10 times).

13. Wash 3 times with PBS-Tween at 5 minute intervals.

14. Add anti-rabbit conjugate (dilution 1/2000 in conjugate buffer) to membranes processed

with polyclonal antisera or anti-mouse conjugate to membranes processed with

monoclonal antibodies and incubate for 1 hour at room temperature on shaker. N.B.

Membranes can be processed together at this stage. Save conjugate to be reused, mark

date on record sheet on back of bottle (may be used up to 10 times).

15. In preparation for Step 16 remove frozen substrate buffer and substrate solutions tube1

and tube 2 from freezer and thaw.

16. Wash as instep 8.

17. Prepare substrate solution. First check pH of substrate buffer- it is essential that the pH is

exactly 9.5. The reaction will be much fainter if the pH is not optimum. For each 5 ml

substrate buffer add 20 µl tube 1 (NBT) and 20 µl tube 2 (BCIP).

18. N.B. The substrate solution must be prepared fresh just prior to usage. Add substate

solution to the dish (all membranes may be processed together as long as there is

sufficient movement between sheets and they do not stick together thus impeding

development). Incubate for 5 minutes on shaker.

19. To stop reaction, wash with deionised H20.

20. Dry membranes and view under a dissecting microscope. Phloem restricted viruses will

show as a clear dark purple staining of the phloem. Viruses which invade the whole plant

will be seen as clear dark purple staining of the whole petiole cross section.

42

5.2.6 Suppliers

Schleicher & Schell

BioScience GmbH,

P.O. Box 4

Hahnestrasse 3

Dassel 1, D-3354

Germany

e-mail: [email protected]

Internet: http//www.s-und-s.de

ROCHE Diagnostics GmbH

Dept. GD-F

68298 Mannheim

Germany

Bioreba AG

Chr. Merian-Ring 7

CH-4153 Reinach BL1

Schweiz

e-mail: [email protected]

mailto:[email protected]

mailto:[email protected]

43


6.1 Electron microscopy

6.1.1 Introduction

BBMV has isometric particles of about 25-26 nm diameter, rounded in profile without

conspicuous capsomere arrangement (Bawden et al. 1951, Gibbs AJ 1972, Brunt et al. 1997,

Makkouk et al. 1988a). Makkouk et al. (1988a) reported that BBMV isometric particles were

readily seen with the electron microscope in crude sap preparations from faba bean field

samples, either fresh or dried, or from faba bean plants inoculated and maintained in the

glasshouse. Virus particles mostly occurred in very high concentrations and a high proportion

of them were penetrated by phosphotungstic acid (PTA) stain.

BBSV and BBTMV also have isometric particles of 25 nm diameter (Gibbs AJ, Smith HG,

1970), therefore direct examination of plant sap using the electron microscope (sap dip) is

only suitable as a confirmatory test for the presence of isometric particles. Immunosorbent

electron microscopy (trapping or decoration) with virus-specific antibodies enables trapping

of particles of the target virus and offers a more definitive test result. However, like ELISA,

this is a serological test, which means that both these forms of diagnosis/virus detection

depend on the quality of the antiserum. It is preferable to use a confirmatory test, which

depends on other properties of the virus.

Key references: Noordham (1973), Ball (1974), Milne (1986), Roberts (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. Freshly prepared dilutions of appropriate virus antibodies in 0.07 M sodium phosphate

buffer, pH 6.5.

44

6.1.3 Method

6.1.3.1 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.

6.1.3.2 Immunosorbent electron microscopy

6.1.3.2.1 Trapping method

1. Pipette 30 l drops of antiserum diluted 1:100, 1:1,000 and 1:10,000 in normal saline

onto waxed glass slides.

2. Float a carbon-coated grid, film-side down, on each drop and incubate for 2-3 hours at

37°C.

3. Wash grids five times in normal saline or place grids in 0.1 M sodium phosphate buffer,

pH 7.0, in plastic wells, agitate at intervals for 10 minutes, transfer to a second plastic

well containing buffer and leave for a further 10 minutes and then drain.

4. Extract plant material in normal saline at 1:5 wt/vol and then centrifuge at 8,000g for 10

minutes.

5. Float grids on 30 l drops of sample sap extract and incubate for 2 hours at room

temperature or for 3-36 hours at 4°C.

6. Wash grids five times in normal saline or place grids in plastic wells, containing normal

saline, agitate at intervals for 10 minutes, transfer to a second plastic well containing

normal saline and leave for a further 10 minutes and then drain.

7. Stain grids with 2% PTA and/or UA by floating grids on the stain for 10 minutes then drain

grids by touching the edge with torn filter paper.

45

8. Observe grids for virus particles using an electron microscope.

6.1.3.2.2 Decoration method

1. As an additional step, just prior to examination of the prepared grid using the electron

microscope, in either of the above procedures, add a drop of suitably diluted antiserum

to the prepared grids, incubate for 3 hours at 37°C and drain.

2. Observe grids using an electron microscope for antibody halos surrounding virus

particles. Such halos indicate the specific binding of the virus-specific antibody to the

trapped virus particles on the grid and therefore provide evidence of the true identity of

the virus, based on the specificity of the antiserum used.

6.2 Light Microscopy

6.2.1 Introduction

Rubio and van Slogteren (1956) found granular inclusion bodies (X-bodies) associated with

cellular infection of BBMV. The X-bodies were visible by light microscopy and electron

microscopy showed that the X-bodies consisted of spherical particles that were identical to

those of BBMV. De Zoeten and Schlegel (1967) studied the ultrastructure of BBMV infected

faba bean leaf cells and found that the mesophyll cells containing virus appeared to be

saturated with virus to the point of crystallisation. In some infected cells, amorphous vesicular

bodies, containing osmiophilic areas and sometimes crystalline virus, could be identified.

They concluded that there was an unusually large amount of virus in the cytoplasm of BBMV

infected cells. More recent authors have used this as a diagnostic characteristic, which

differentiates BBMV from other small, isometric legume viruses (eg. Makkouk et al. 1988a,

Bos et al. 1992, Brunt et al. 1997). However, it should be noted that Comoviruses, such as

BBSV and BBTMV also cause inclusion bodies in infected cells (Brunt et al. 1997). Christie

(1967) developed a rapid staining procedure for differentiating plant virus inclusions in

epidermal strips. This method was used, with minor modifications, by Bos (1969) to observe

inclusion bodies caused by a number of legume viruses and by Makkouk et al. (1988a) to

observe inclusion bodies caused by BBMV.


1. Epidermal strips from stems, petioles or the underside of leaves.

2. Glass microscope slides, petri dishes, forceps.

3. Staining solution made up of 2 volumes ethylene glycol monomethyl ether, 1 volume 95%

ethanol, and 1 volume distilled water.

4. 1% phloxine made up in the above staining solution.

46

5. 1% methylene blue made up in the above staining solution.

6. Light microscope.

6.2.3 Method

1. Place epidermal strips in equal volumes of 1% phloxine solution and 1% methylene blue

solution (made up as described above) in a Petri dish for 15 mins.

2. Rinse strips in water for a few minutes and mount on slides in water.

3. View under light microscope at magnifications of about 500-750X.

Makkouk et al. (1988a) found that infected cells contained enlarged nucleoli and the

cytoplasm contained granular and often vesicular material which was initially widely

distributed but later in the infection often condensed to form granular, partly vacuolated or

vesiculated inclusion bodies, one or more per cell. They were of varying, sometimes globular,

shape and often exceeded the size of the nucleus. They persisted throughout a 30 day

observation period of after inoculation.

6.3 Indicator plant tests

6.3.1 Introduction

Although BBMV has similar particle morphology to BBSV and BBTMV and they all can cause

similar mottle/mosaic symptoms in legume hosts, BBMV differs from BBSV and BBTMV in

that it has a much wider host range and causes local lesion symptoms on a number of

common indicator plants. The host ranges of BBSV and BBTMV are restricted to the

Fabaceae apart from a few hosts experimental hosts of BBSV in the Chenopodiaceae and

Solanaceae. However, these BBSV hosts do not include the common indicator plants

Chenopodium amaranticolor and Nicotiana clevlandii, which are both hosts of BBMV. A

selection of suitable indicator plant species and their reactions to BBMV are given in 6.3.5. A

comprehensive list of indicator plants are given in Walters and Surin (1973) and Makkouk et

al. (1988a).

6.3.2 General Items required

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

frozen leaf tissue.

2. BBMV-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.

47

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

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

6.3.3 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.

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.

48


6.3.4.1 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.

6.3.5 Indicator plant species and reaction to BBMV

Indicator Species Symptoms Time taken for

symptom

expression

Reference

Chenopodium

amaranticolor

Many chlorotic or dry pin-point local lesions 2-5 days Makkouk et al.

(1988a), Bos et al.

1992, Brunt et al.

(1997)

Chenopodium

quinoa

Many chlorotic or dry pin-point local lesions.

Some isolates cause diffuse systemic lesions.

2-5 days Makkouk et al.

(1988a), Bos et al.

(1992)

Cucumis sativa Small chlorotic local lesions, turning dry when

cotyledons turn yellow.

5 days Makkouk et al.

(1988a)

Nicotiana

tabacum

Non-host. Brunt et al. (1997)

Nicotiana

clevlandii

Mild systemic mottle. Gibbs AJ (1972)

Phaseolus

vulgaris

Infects all cultivars causing chlorotic local

lesions, systemic mottle, vein yellowing or

netting

8 days Gibbs AJ (1972)

Pisum sativum Infects all cultivars causing lethal systemic wilt. 2-3 days Gibbs AJ (1972)

Vicia faba Occasionally diffuse chlorotic or necrotic lesions,

veinal chlorosis or banding followed by systemic

blotchy mottle.

2-4days, 7

days, 8 or

more days

Gibbs AJ (1972)

49

7.0 Images

7.1 BBMV symptoms on host plants

Figure 3. BBMV symptoms on faba bean include

mottle/mosaic and veinal chlorosis of the leaves.

50

Figure 4. BBMV symptoms on faba bean include a strong mosaic

resulting in islands of dark and light tissue on the leaves.

(Photo courtesy: Dr S Kumari ICARDA).

Figure 5. BBMV symptoms on faba bean including veinal chlorosis and

mottling of the leaves.

(Photo courtesy: Dr S Kumari, ICARDA).

51

Figure 6. BBMV symptoms on faba bean include interveinal mottle.


Figure 7. BBMV symptoms on lentil (left) include chlorosis, mottling/mosaic and

sometimes local necrosis. Healthy plant (right).


52

Figure 8. BBMV symptoms on french bean include systemic yellow

stippling or blotching or interveinal mottle.


53

7.2 BBMV symptoms on host seed

Figure 9. BBMV symptoms on faba bean seed (right) include necrosis and staining. Healthy

seed (left).

(Photo courtesy: Dr S Kumari, ICARDA)

54

7.3 BBMV vectors and vector symptoms

Figure 10. The BBMV vector, Sitona lineatus, feeding on leaf (note characteristic leaf notching). NB S. lineatus is also a vector of BBSV and BBTMV.

(Photo courtesy: Dr S Kumari, ICARDA, from Agrevo).

55

Figure 11. BBMV vector Sitona lineatus.

(Photo courtesy: Dr S Kumari, ICARDA, from Agrevo).

56

8.0 References and websites

8.1 References

Assou NM (1978). Contribution a l'etude de la feve. Memoire de fin d'etude, Ecole Nationale d'Agriculture de Meknes (Maroc): 34 pp.

Bawden FC, Chauduri RP, Kassanis B (1951). Some properties of broad bean mottle virus. Annals of Applied Biology 38: 774-784.

Borges MLV, Sequeira JC (1988). Protection of legumes in relation to virus infection. Comunicacoes, Jornadas Portuguesas de Proteaginosas: 281-304.

Borges M de LV (1982). Mosaic of faba bean in Portugal. Aetiology and virus-host relations. Garcia de Orta, Estudos Agrononicos 9: 279-284.

Borges M de LV, Louro D (1974). A biting insect as vector of broad bean mottle virus. Agronomia Lusitana 36: 215-216.

Bos L (1969). Inclusion bodies of bean yellow mosaic virus, some less known closely related viruses and beet mosaic virus. . Netherlands Journal of Plant Pathology 75: 137-143.

Bos L, Mahir MAM, Fortass M, Makkouk KM (1992). A mild strain of broad bean mottle virus from faba bean (Vicia faba L.) in the Sudan. Netherlands Journal of Plant Pathology 98: 253-

256.

Bourbah M, Fezzaz M (1979). Contribution a l'etude des viroses de la feve dans la region de Meknes. Memoire de fin d'etude, Ecole Nationale d'Agriculture de Meknes (Maroc): 48 pp.

Botina R, Munoz M de, Guerrero GO (1984). Identification of host plants of broad bean (Vicia faba L.) mottle virus in the Department of Narino. Revista ICA 19: 395-401

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 (BBMV). http://biology.anu.edu.au/Groups/MES/vide/(1997).

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

Christie RG (1967). Rapid staining procedures for differentiating plant virus inclusions in

epidermal strips. Virology 31:268-271.

57

Cockbain AJ (1971). Weevils, aphids and virus diseases of field beans. Report of the Rothamsted Experimental Station for 1970, pp184-186, pub. Harpenden, Herts., UK. Cockbain AJ, Cook SM, Bowen R (1975). Transmission of broad bean stain virus and Echtes Ackerbohnenmosaik virus to field beans (Vicia faba) by weevils. Annals of Applied Biology

81: 331-339.

Dafalla G, Hussein M (1994). Disease and pest outbreaks. Sudan. Identification of a new virus disease affecting legumes in the Sudan. Arab and Near East Plant Protection Newsletter 18: 30.

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

El Afifi SI, El Dougdoug KA (1997). Identification of some mechanically transmitted viruses affecting faba bean plants in Egypt. Annals of Agricultural Science (Cairo) 42: 29-49.

El-Muadhidi MA, Makkouk KM, Kumari SG, Jerjess M, Murad SS, Mustafa RR, Tarik F (2001). Survey for legume and cereal viruses in Iraq. Phytopathologia Mediterranea 40: 224-233.

Erdiller G, Akbas B (1996). Seed transmission of some viruses in chickpea and lentil. Journal of Turkish Phytopathology 25: 93-101.

Fischer HU (1979). The identification and differentiation of virus infections of broad bean. Awamia 57: 41-72.

Fortass M, Bos L (1992). Broad bean mottle virus in Morocco; variability in interaction with food legume species and seed transmission in faba bean, pea and chickpea. Netherlands Journal of Plant Pathology 98: 329-342.

Fortass M, Diallo S (1993). Broad bean mottle virus in Morocco; curculionid vectors, and natural occurence in food legumes other than faba bean (Vicia faba).

Fujisaki K, Hagihara F, Kaido M, Mise K, Okuno T (2003). Complete nucleotide sequence of spring beauty latent virus, a bromovirus infectious to Arabidopsis thaliana. Archives of

Virology: 148: 165-175. Gibbs AJ (1972). Broad bean mottle bromovirus. CMI/AAB Descriptions of Plant Viruses.

No.101. Journal. Gibbs AJ, Smith HG (1970). Broad bean stain virus. CMI/AAB Descriptions of Plant Viruses. No.29. Journal.

Makkouk KM, Dafalla G, Hussein M, Kumari SG (1995). The natural occurrence of chickpea chlorotic dwarf geminivirus in chickpea and faba bean in the Sudan. Journal of Phytopathology 143: 465-466.

58

Makkouk KM, Kumari S (1989). Apion arrogans, a weevil vector of broad bean mottle virus. FABIS Newsletter 24: 26-27.

Makkouk KM, Kumari SG, Al Daoud R (1992). Survey of viruses affecting lentil (Lens culinaris Med.) in Syria. Phytopathologia Meditterranea 31:188-190)

Makkouk KM, Kumari SG (1995). Transmission of broad bean stain comovirus and broad bean mottle bromovirus by weevils in Syria. Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz 102 136-139.

Makkouk KM, Bos L, Rizkallah A, Azzam OI, Katul L (1988a). Broad bean mottle virus: identification, host range, serology, and occurrence on faba bean (Vicia faba) in West Asia

and North Africa. Netherlands Journal of Plant Pathology 94: 95-212.

Makkouk KM, Bos L, Azzam O, Koumari S, Rizkallah A (1988b). Survey of viruses affecting faba bean in six Arab countries. Arab Journal of Plant Protection 6: 61-63. Makkouk KM, Rizkallah L, Madkour M, El-Sherbeeny M, Kumari SG, Amriti AW, Solh MB (1994). Survey of faba bean (Vicia faba L.) for viruses in Egypt. Phytopathologia Meditterranea 33: 207-211.

Mouhanna AM, Makkouk KM, Ismail ID (1994). Survey of virus diseases of wild and cultivated legumes in the coastal region of Syria. Arab Journal of Plant Protection 12: 12-19.

Murant AF, Abu Salih HS, Goold RA (1974). Viruses from broad bean in the Sudan. Annual Report of the Scottish Horticultural Research Institute, 1973: 67.

Najar A, Makkouk KM, Boudhir H, Kumari SG, Zarouk R, Bessai U, Othman FB, (2000). Viral diseases of cultivated legume and cereal crops in Tunisia. Phytopathologia Mediterranea 39: 423-432.

Naumann I (1993). CSIRO Handbook of Australian Insect Names (6th edn). CSIRO Division of Entomology.

Nene YL, Sheila VK, Sharma SB (1996). A world list of chickpea and pigeonpea pathogens. (ICRISAT: India).

Oufffroukh A (1985). Contribution a la connaissance des viroses des plantes en Algerie: inventaire des virus present chez des legumineuses a longue cosse. Etude approfondie de deux maladies isolees de feve et haricot. These de Docteur de 3e cycle, Universite Pierre et Marie Curie, Paris VI: 101 pp.

Ouizbouben A, Fortass M (1997). Survey of chickpea for viruses in Morocco. Bulletin OEPP 27: 249-254.

59

Rothamsted Experimental Station (1983). Report for 1982, Part 1:200-202 pub. Harpenden, Herts., UK. Rybicki EP, Von Wechmar MB (1981). The serology of the Bromoviruses. 1. Serological interrelationships of the Bromoviruses. Virology 109: 391-402.

Scott HA, Slack SA (1971). Serological relationship of brome mosaic and cowpea chlorotic mottle viruses. Virology 46: 490-492.

Sequeira JC, Borges M de LV (1989). Broad bean mottle virus in Portugal, host range, transmission, serology and virion characterisation. Boletim da Sociedade Broteriana 62: 291-303.

Tadesse N, Ali K, Gorfu D, Yusuf A, Abraham A, Ayalew M, Lencho A, Makkouk KM, Kumari SG (1999). Survey for chickpea and lentil virus diseases in Ethiopia. Phytopathologia Meditterranea 38:149-158. Tinsley TW (1957). UK, Rothamsted Experimental Station: Report for 1956, Part 1:98. Valverde RA (1985). Spring beauty latent virus: A new member of the bromovirus group. Phytopathology 75: 395-398.

Voller A, Bartlett A, Bidwell DE, Clark MF, Adams AN (1976). The detection of viruses by enzyme-linked immunosorbent assay (ELISA). Journal of General Virology 33: 165-167.

Walters HJ, Surin P (1973). Transmission and host range studies of broad bean mottle virus. Plant Disease Reporter 57: 833-836.

Zhao GF (1996). Integrated management of rust and virus disease of red bean. Progress of research on plant protection in China. Proceedings of the third national conference of integrated pest management, Beijing, China, 12-15 November 1996: 140-143.

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

Plant viruses online: Descriptions and lists from the VIDE database

http://biology.anu.edu.au/Groups/MES/vide/(1997)

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

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

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

http://biology.anu.edu.au/Groups/MES/vide/(1997

60

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:

61

Appendix 2. Hygiene

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 for vehicle access

On leaving the paddock, personnel must :

wash boots in foot bath 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

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

62

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 NSW

Agriculture (ie, landfill within quarantine boundary or similar)

It is requested that the harvest machinery not be used to harvest lupins again this

season. If this is not possible, it must be used to harvest two crops, other than

lupins, before it can harvest lupins again.

Documents

Broad bean mottle virus DP - Plant Health Australia