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International Plant Protection ConventionDraft Annex to ISPM 27– Begomoviruses transmitted by Bemisia tabaci

2006-023Agenda item 3.3

DRAFT ANNEX to ISPM 27 – Begomoviruses transmitted by Bemisia tabaci (2006-023)

Status box

This is not an official part of the standard and it will be modified by the IPPC Secretariat after adoption.

Date of this document 2016-06-17

Document category Draft annex to ISPM 27 (Diagnostic protocols for regulated pests)

Current document stage To be submitted to TPDP meeting and then Expert consultation

Origin Work programme topic: Viruses and phytoplasmasOriginal subject: Begomoviruses transmitted by Bemisia tabaci (2006-023)

Major stages 2006-05 SC Added topic to work programme

Discipline leads history Mr Brendan RODONI (Discipline lead, AU) Mr Delano JAMES (Referee, CA)

Consultation on technical level

The first draft of this protocol was written by Stephan Winter and Monika Goetz, DSMZ Plant Virus Department

Main discussion points during development of the diagnostic protocol

-

Notes This is a draft document

Adoption

[1] This diagnostic protocol was adopted by the Commission on Phytosanitary Measures in 20--.

[2] The annex is a prescriptive part of ISPM 27 (Diagnostic protocols for regulated pests).

1. PEST INFORMATION[3] The genus Begomovirus (family Geminiviridae) currently involves 288 species (International Committee on

Taxonomy of Viruses, 2014) and thus is the largest genus of plant viruses. New viruses are continuously described and in addition to new species, a very high genetic variation exists within the virus species. Thus a large number of strains and unique genome variants are described and associated with particular disease phenotypes (Kon and Gilbertson, 2012).

[4] Begomoviruses have a broad host range infecting dicotyledonous plants including many species of horticultural and agricultural crops mainly from the Solanaceae, Fabaceae, Cucurbitaceae. The viruses cause serious diseases with enormous yield losses in important world crops including, tomato, cucurbits, cassava and cotton grown in tropical and subtropical regions.

[5] In EPPO region Tomato yellow leaf curl virus and related viruses (A2/182), Squash leaf curl virus (A2/224) are present, the newly emerging Tomato leaf curl New Delhi virus (Alert list) has become a serious problem in zucchini. Bean golden mosaic virus (A1/204) and Tomato mottle virus and other American begomoviruses of capsicum and tomato (A1/225) are quarantine pests for Europe as host plants are widely distributed and virus introduction could result in high economic damage. Further begomoviruses were formerly listed in the EPPO Alert list however are currently not listed as they are covered by the list of Bemisia-transmitted viruses in EU regulations and thus are not considered as an alert situation.

[6] Begomoviruses are transmitted by B. tabaci whiteflies in a circulative persistent manner (Czosnek et al. 2002). These insects are abundant in tropical and subtropical regions of the world and in Mediterranean countries (http://www.cabi.org). B. tabaci is a complex composed of at least 34 cryptic species (Boykin and De Barro, 2014). The B. tabaci Middle East-Asia Minor 1 (MEAM1, former B biotype) and the Mediterranean (MED, former Q biotype) species are invasive and now found in many parts of the world, in Mediterranean Europe, North Africa, USA, China and other Asian countries (http://www.efsa.europa.eu/de/efsajournal/pub/3162).

International Plant Protection Convention of 13

http://www.cabi.org/

2006-023 DRAFT ANNEX TO ISPM 27 – Begomoviruses transmitted by Bemisia tabaci (3.3)

Coinciding the world-wide invasion of this species, begomoviruses are found; the most prevalent and serious are those infecting tomatoes with TYLCV being the most prominent.

[7] Begomoviruses have ssDNA genomes encapsidated in twinned icosahedral nucleocapsids (38 nm long, 22 nm in diameter). They have ether bipartite genomes consisting of two small circular single stranded DNA-A and DNA-B components (~2.7 kb) or a DNA-A like monopartite genome. Many monopartite begomoviruses have associated with beta satellite and alpha satellite molecules also consisting of circular ssDNA of ~1.4 kb. Over 100 beta satellites are currently known (Mansoor et al. 2006; www.dpvweb.net/notes/showallgenusseqs.php?genus=Begomovirus%20betasatellites). They depend on their helper virus for replication and encapsidation, contribute to the systemic spread of the helper virus, interfere with host resistance responses and/or increase virus virulence and thus play an important role in symptom development (Briddon et al 2003, Idris et al, 2005). In some cases systemic symptoms are only expressed when DNA-beta is present (Mansoor et al., 2003; Saeed et al., 2005; Saunders et al., 2000).

[8] Further information on begomoviruses, including a complete list of accepted begomovirus species, the species listed by EPPO and their distribution, distribution of the insect vector B. tabaci can be found at the following websites:

http://www.ictvonline.org/virustaxonomy.asp https://gd.eppo.int/taxon/1BEGOG https://gd.eppo.int/taxon/BEMIAR http://www.cabi.org/isc/datasheet/8925

2. TAXONOMIC INFORMATION [9] Genus name: Begomovirus

[10] Synonyms: none

[11] Taxonomic position: Viruses, group II: ssDNA viruses, Geminiviridae, Begomovirus

[12] Common names:

[13] The taxonomy of the genus Begomovirus which was recently revised is based on the analysis of complete begomovirus genomes or entire DNA-A component sequences for bipartite species in the genus. Pairwise identities of 91 % and 94 % are proposed as the demarcation threshold for begomoviruses belonging to different species and strains, respectively (Brown et al. 2015).

3. VIRUS DETECTION AND IDENTIFICATION[14] Biological assays for virus detection and identification are not feasible as only a few viruses of the genus are

mechanically transmissible and particular indicator plants are not known. Serological tests based on ELISA enable fast and cost effective screening of plant materials for virus presence at large-scale. Antisera and commercial kits are available for the detection of several begomoviruses in diseased plants but are not suitable for virus identification. ELISA testing is feasible for screening purposes and in cases where particular virus species are suspected. Because coat proteins of begomoviruses are highly conserved discrimination between virus species or the resolution of viruses in mixed infections is generally not possible. As such, detection and identification of begomoviruses relies on molecular assays.

[15] This protocol addresses the detection and identification of whitefly-transmitted begomoviruses using molecular methods.

[16] In this diagnostic protocol, methods (including reference to brand names) are described as published, as these defined the original level of sensitivity, specificity and/or reproducibility achieved. The use of names of reagents, chemicals or equipment in these diagnostic protocols implies no approval of them to the exclusion of others that may also be suitable. Laboratory procedures presented in the protocols may be adjusted to the standards of individual laboratories, provided that they are adequately validated

of 13 International Plant Protection Convention

http://www.cabi.org/isc/datasheet/8925

https://gd.eppo.int/taxon/BEMIAR

https://gd.eppo.int/taxon/1BEGOG

http://www.ictvonline.org/virustaxonomy.asp

DRAFT ANNEX TO ISPM 27 – Begomoviruses transmitted by Bemisia tabaci (3.3) 2006-023

3.1 Symptoms[17] Symptoms caused by begomoviruses vary depending on host plant (genus, species, and cultivar) and virus

species/strain. Climatic and environmental conditions and growth stage at the time of infection can also contribute to symptom expression. Symptoms include stunting of the plants, upward and downward curling of leaves, interveinal and marginal chlorosis of leaves, mottling and yellow mosaic, reduction of flower and seed setting. The association of beta satellites and additional begomoviruses or other viruses in mixed infections can enhance disease symptoms

[18] Begomoviruses can be differentiated in those causing typical leaf curl symptoms mostly in young apical parts of the plant like TYLCV (Figure1 left) and those causing mosaic symptoms like VGMV (Figure1 right). However while symptoms can be indicative for begomoviruses, a particular species identification cannot be derived from a phenotype since many diverse species cause very similar disease symptoms. In addition virus tolerant tomato cultivars also show only very mild if any symptoms of infection although virus concentrations can be similar to susceptible cultivars. A proper detection and identification of begomoviruses thus requires the use of diagnostic tools.

3.2 Sampling and sample preparation[19] Appropriate sampling is critical for begomovirus detection. Begomoviruses replicate in plant nuclei and thus virus

replication is highest in meristematic tissues. Thus, young freshly expanding leaves are generally the best tissues used for virus detection. Even if older leaves show more pronounced symptoms, young leaves from the shoot or tillers should be collected for testing.

[20] Molecular detection of begomoviruses in general is based on the amplification of viral genomic DNA and its replication intermediate, dsDNA. This DNA is quite stable and thus leaves sampled for subsequent testing can be dried between filter paper and stored dried at ambient temperatures prior to DNA preparations. However, all other methods, cold storage, freezing etc. to preserve plant materials are equally useful. Since molecular methods are highly sensitive particular care has to be taken to avoid contamination during sampling.



3.3 Molecular detection and identification of begomoviruses [21] In general PCR is used for begomovirus detection and when properly used provides a robust approach to detect

even low concentrations of virus DNA. For the most commonly known begomoviruses specific primer sets and PCR protocols are available to achieve reliable detection of the viruses in infected plant materials. To verify specificity of the PCR, sequencing of the amplicons confirms virus identity.

[22] Quantitative PCR (qPCR) protocols are also developed for the sensitive detection of begomoviruses. The advantage of qPCR is that the methods are generally more sensitive than conventional PCR and there is no post PCR processing required.

[23] A general approach to begomovirus diagnosis is Rolling-circle amplification (RCA). Using phi29 (Φ29) DNA polymerase, DNA including the circular virus DNA is amplified in an isothermal process from random primed DNA. Phi29 polymerase is highly processed and in this unique RCA process, small circular DNA molecules are replicated nearly infinitely. By enzymatic restriction of the RCA products complete virus genome components and including putative satellite DNA molecules can be resolved. Thus the entire begomovirus composition of a plant sample can be analysed.

[24] In this diagnostic protocol, methods (including reference to brand names) are described as these define the original level of sensitivity, specificity and reproducibility achieved. The use of names of reagents, chemicals or equipment in these diagnostic protocols implies no approval of them to the exclusion of others that may also be suitable. Laboratory procedures presented in the protocols may be adjusted to the standards of individual laboratories, provided that they are adequately validated.

3.3.1 Extraction of DNA

[25] Plant material can be ground in liquid nitrogen or frozen at -20°C and crushed using a tissue lyser and steal beads. Homogenization of samples with extraction buffer in plastic bags using a wallpaper roller is also possible.

[26] Several protocols are published for nucleic acid extraction resulting in DNA suitable for all downstream methods listed in this protocol (Lohdi et al. 1994, Dellaporta et al. 1983, Accotto et al. 2000). In addition commercial kits are available for isolation of DNA suitable for PCR. Most extraction methods are well validated for a variety of plant species. The choice of method is dependent on the host being tested and the availability of facilities and equipment. For routine diagnostics it is important to validate an extraction method for a particular host/tissue to ensure reliability.

3.3.2 Virus detection by polymerase chain reaction (PCR)

[27] Various primer sets are available for detection of begomoviruses, groups of viruses, species and strains.

[28] The most commonly used general primers for the genus begomovirus are published by Deng et al. (1994) and for beta satellites from Briddon et al. (2006). For the detection of tomato infecting begomoviruses the primers developed by Rojas et al (1993) and modified by Tsai et al. (2011) proved to be suitable and for the group of begomoviruses infecting legumes (“legumoviruses”) primers developed by Cuong (unpublished information) can be used.

[29] In general DNA extracted by one of the methods described before are suitable for all conventional PCR. However, DNA extracts may contain compounds that are inhibitory to PCR depending on host species, plant organ and age of tissue. Therefore it is important to confirm the PCR competency of the DNA extractions using internal control DNA or control primers. Inhibitory effects by co-extracted compounds can be overcome by further purification, dilution or addition of bovine serum albumin (BSA) to the PCR mixture.

3.3.2.1 Conventional PCR for general detection of begomoviruses [30] The primers Gemini A 146 and Gemini A 672 (Deng et al. 1994) amplify an approx. 500 bp fragment of DNA-A

component (pre coat protein gene, partial coat protein gene) of begomoviruses.

[31] Table x. Primer sequences and their application are described in the table below

Primer name Primer sequence (5’- 3’ orientation) Primer used forPCR Sequencing



Gemini A 146 TAA TAT TAC CKG WKG VCC SC X XGemini A 672 TGG ACY TTR CAW GGB CCT TCA ACA X X

for degenerate primers: K = G/T; R = A/G; S = C/G; W = A/T; Y = C/T; B = C/G/T; V = A/C/G

[32] Table x. Master mix composition and template for PCR and cycling conditions for a final reaction of 25 µl

Reagents Final concentration CommentsPCR grade water N.A. Quantity for total volumePCR buffer 1 XdNTPs 0.2 mM eachMgCl2 2.5 mMGemini A 146 0.2 µMGemini A 672 0.2 µMTaq DNA polymerase 1.25 U Genomic DNA extract 1 µlTotal 25 µlCycling parametersInitial denaturation 94 °C

3 minNumber of cycles 35Denaturation 94 °C

1 minAnnealing 53 °C

1 minElongation 72 °C

1 minFinal elongation 72 °C

10 minExpected amplicons~ 500 bp

3.3.2.2 Conventional PCR for the detection of tomato infecting begomoviruses [33] The primers PAL1v1978B and PAR1c715H (Tsai et al. 2011) amplify an approx. 1.5 kb fragment of DNA-A component (partial

replication associated protein gene, pre coat protein gene, partial coat protein gene).



PAL1v1978B GCA TCT GCA GGC CCA CAT BGT YTT HCC NGT X XPAR1c715H GAT TTC TGC AGT TDA TRT THT CRT CCA TCC A X X

for degenerate primers: N = A/C/G/T; B = C/G/T; D = A/ G/T; H = A/C, T; K = G/T; R = A/ G; S = G/C; Y = C/T; V = G/A/C


Reagents Final concentration CommentsPCR grade water N.A. Quantity for total volumePCR buffer 1 XdNTPs 0.2 mM eachMgCl2 2.5 mMPAL1v1978B 0.2 µMPAR1c715H 0.2 µMTaq DNA polymerase 1.25 U Genomic DNA extract 1 µlTotal 25 µlCycling parametersInitial denaturation 94 °C





10 min



Expected amplicons~ 1.5 kb

3.3.2.3 Conventional PCR for the detection of begomoviruses infecting legumes (“legumoviruses”) [36] The primers LegA cp1F and LegA cp1R (Cuong et al. unpublished) amplify an approx. 280 bp fragment of the

coat protein gene on the DNA-A component.



LegA cp1F GGT CAA GTK TTY AAC ATG TAT GA X XLegA cp1R GCA TGA GTA CAT GCC ATA TAC X X

for degenerate primers: K = G/T; Y = C/T.


Reagents Final concentration CommentsPCR grade water N.A. Quantity for total volumePCR buffer 1 XdNTPs 0.2 mM eachMgCl2 2.5 mMLegA cp1F 0.2 µMLegA cp1R 0.2 µMTaq DNA polymerase 1.25 U Genomic DNA extract (dilution 1:10) 1 µlTotal 25 µlCycling parametersInitial denaturation 94 °C





10 minExpected amplicons~ 280 bp

3.3.2.4 Conventional PCR for the detection of beta satellites [39] The primers Beta01 and Beta02 (Briddon et al. 2002) amplify the complete DNA-beta (minor band) and a 600 bp fragment

(major band).



Beta01 GGT ACC ACT ACG CTA CGC AGC AGC C X XBeta02 GGT ACC TAC CCT CCC AGG GGT ACA C X X

for degenerate primers: K = G/T.




Reagents Final concentration CommentsPCR grade water N.A. Quantity for total volumePCR buffer 1 XdNTPs 0.2 mM eachMgCl2 2.5 mMBeta01 0.2 µMBeta02 0.2 µMTaq DNA polymerase 1.25 U Genomic DNA extract 1 µlTotal 25 µlCycling parametersInitial denaturation 94 °C




1.5 minFinal elongation 72 °C

10 minExpected amplicons~ 1.3 kb fragment and 600-700 bp fragment

3.3.3 Virus detection by rolling-circle amplification (RCA)[42] In case of negative results from other tests and as a generic method for amplification of circular virus genomes,

RCA can be carried out to detect the presence of further/new ssDNA viruses and alpha and beta satellites without prior knowledge of virus sequences. Sample preparation and DNA extraction are identical to other methods. A detailed protocol for a RCA assay published by Knierim et al. (2007). Commercial kits are also available which are applicable for RCA assays if used following the instructions.

[43] RCA generates genome DNA sequences as concatemers and those have to be digested with restriction endonucleases. Restriction enzymes are selected that cut at a single site in the virus genome thus releasing linearised fragments of full-length genome length. Virus identification can then be performed after cloning and sequence analysis. In addition, restriction digests can also be performed with restriction enzymes cutting at multiple sites thus generating fragment patterns. For selected begomoviruses RFLP patterns are published (Haible et al. 2006, Wyant et al. 2012) that can be used to assign to virus species. In silico digestion patterns of published full-length sequences of expected viruses can also be compared. With this approach it is possible to detect unexpected and unknown begomoviruses, mixed infections and satellites. However, for virus identification, only sequence analysis is valid.

[44] Table x. Master mix composition and template for RCA and reaction conditions for a final reaction of 50 µl

Mix I: Reagents for a reaction of 25 µl Final concentration (50 µl)

Comments

PCR grade water N.A. Quantity for total volumedNTPs 0.625 mMRandom hexamer primers 0.25 µMGenomic DNA extract 2.5 µlTotal 25 µlReaction conditionsDenaturation 94 °C

3 minCooling On ice

5 min

Mix II: Reagents for a reaction of 25 µl Final concentration(50µl)

PCR grade water N.A.- Quantity for total volumePCR buffer 1 XPhi29 DNA polymerase 2.5 U Total 25 µl



Combine Mix I and Mix II

Reaction conditionsIsothermal amplification 30 °C

18 hInactivation 65 °C

10 minExpected ampliconconcatemer has to be digested by restriction enzymes

[45] Table x. Master mix composition and template for restriction assay and reaction condition for a final reaction of 10 µl

Reagents Final concentration(10 µl)

Comment

PCR grade water N.A. Quantity for total volumeEnzyme buffer 1 XRestriction enzyme* 5 URCA product 5.0 µlReaction condition 37 °C

1.5 hExpected fragmentsDepending on the chosen enzyme*

* Selection of restriction enzymes depend on the begomovirus expected in the sample. Enzymes that cut at a single site in the virus genome thus releasing linearized fragments of full-length genome length should be preferred. In this case a approx. 2.7 kb fragment is generated.

3.3.4 Controls for molecular tests[46] For a reliable test result to be obtained, the following controls should be considered for each series of nucleic acid

isolations, amplification of the target pest or target nucleic acid depending on the test used and the level of certainty required. As a minimum, for conventional PCR the positive amplification control, internal control and negative amplification control (no template control) should be used.

[47] Positive amplification control (PAC). This is used to monitor the efficiency of the test method (apart from the extraction). Controls containing pre-prepared virus nucleic acid (e.g. cloned virus genomes) and virus containing plant DNA can be used.

[48] Negative amplification control (NAC). This is necessary with conventional PCR to rule out false positives due to contamination during the preparation of the reaction mix. PCR grade water that was used to prepare the reaction mix is added at the amplification stage.

[49] Internal control. For [method name(s)], plant internal controls [name(s) of gene(s) e.g. House Keeper Gene (HKG) such as COX or NAD] should be incorporated into the protocols to eliminate the possibility of PCR false negatives due to extraction failure, nucleic acid degradation or the presence of PCR inhibitors. Preferably the internal control primers should be used [add details, e.g. in a duplex reaction with the pospiviroid/PSTVd primers]. (Add any qualifying information e.g. difficulties that may be encountered, effects on sensitivity, notes on the part of the assay that the gene acts as a control for e.g. with RT-PCR assays. Also examples of successful use of internal controls if known or relevant and not already referred to in the method descriptions in other sections.)

[50] When the internal control [name of gene] is not mentioned in the description of a PCR method, the laboratory should choose an internal control and validate it.

[51] Positive isolation control (PIC). This is used to ensure that nucleic acid from the target is of sufficient quantity and quality and that the target is detected. Nucleic acid is extracted from infected host tissue or healthy plant tissue that has been spiked with the target.

[52] The positive control should be approximately 1/10 of the amount of plant tissue used per plant for the DNA extraction. (Add any other relevant elements, on e.g. adjustments to quantity, amounts of control material to use for different bulking rates etc. and if this control is not Instructions to authors - Diagnostic Protocols (Appendix of 13 International Plant Protection Convention


1) International Plant Protection Convention detected, provide guidance on repeating tests or adjusting the bulking rate until reliable detection is achieved.)

[53] In the case of a high risk of aerosol contamination, and for specific pest, please consider if instructions should be provided to monitor possible cross contamination, e.g. compare the sequences of positive controls and positive samples.

[54] Negative isolation control (NIC). This is used to monitor contamination during nucleic acid extraction and/or cross-reactions with the host tissue. This requires nucleic acid extraction and subsequent amplification of uninfected host tissue. It is recommended to include multiple controls when large numbers of positives are expected.

3.3.5 Interpretation of results[55] In order to assigning results from PCR-based tests the following criteria should be followed:

[56] Verification of the controls NIC and NAC should produce no amplicons. PIC and PAC should produce amplicons of the expected size. When these conditions are met: Tests yielding amplicons of the expected size are considered positive and amplicons can be used for sequencing. Tests yielding no amplicon or amplicons of a different size are considered negative. Tests should be repeated if any contradictory or unclear results are obtained.

4. VIRUS IDENTIFICATION[57] Only molecular methods, also comprising sequence analysis, are appropriate for identification of begomoviruses.

For preliminary identification of begomoviruses, coat protein sequences are generally quite informative. However because recombination is frequent in begomovirus genomes, only the analysis of entire genome/DNA-A component sequences allow unequivocal species and strain assignment. Full-length fragments can be generated by PCR with abutting primers (Briddon et al. 1993) or RCA. The size of amplicons has to be checked by gel electrophoresis. PCR products can be sequenced either directly or by first cloning them into a PCR cloning vector. RCA products have to be cloned after the restriction assay.

[58] Several universal primer sets complementary to conserved sequences in begomovirus genomes are published to test presence/absence of begomoviruses. This “universal” approach is recommended when large sample sizes have to be tested. It has however to be taken into account that some viruses still might escape amplification/detection. PCR products resulting from this “universal begomovirus amplification approach” indicate presence of begomoviruses only. A further proof for virus presence and an indication of its identity is provided by sequencing of the amplicons. This also provides an initial information on the identity of the viruses and permits the design of specific primers for begomovirus detection and/or primers for amplification of the entire DNA component for virus identification by sequencing.

[59] Sequence data can be analyzed using the Basic local Alignment Search Tool, BlastN, available at the National Center for Biotechnology Information (http://ncbi.nlm.nih.fov/). If the sequence of the complete DNA (monopartite) or DNA-A component (bipartite) shares less than 91 % identity with the closest relative the begomovirus is considered to be a new species.

5. RECORDS[60] Records and evidence should be retained as described in section 2.5 of ISPM 27:2006.

[61] In cases where other contracting parties may be affected by the results of the diagnosis, in particular in cases of non-compliance and where begomoviruses are found in an area for the first time, the following records and evidence and additional material should be kept for at least one year in a manner that ensures traceability:

[62] If possible the original sample (labeled appropriately for traceability) should be kept frozen at – 20 °C or at -80 °C.

[63] If relevant, DNA extracts should be kept at– 20 °C or at -80 °C.


http://ncbi.nlm.nih.fov/


6. CONTACT POINTS FOR FURTHER INFORMATION[64] Further information on this protocol can be obtained from:

[65] Plant Virus Department, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell cultures, Messeweg 11-12, 38104 Braunschweig, Germany (Stephan Winter; e-mail: [email protected]. tel.: +49 531 299 3780; fax: +49 531 299 3014).

[66] A request for a revision to a diagnostic protocol may be submitted by national plant protection organizations (NPPOs), regional plant protection organizations (RPPOs) or Commission on Phytosanitary Measures (CPM) subsidiary bodies through the IPPC Secretariat ([email protected]), which will be forwarded it to the Technical Panel on Diagnostic Protocols TPDP.

7. ACKNOWLEDGEMENTS[67] The first draft of this protocol was written by:

Stephan Winter, DSMZ Plant Virus Department (see preceding section)

Monika Goetz, DSMZ Plant Virus Department (see preceding section)

[68] In addition, the following experts were significantly involved in the development of this protocol (to be inserted later)

8. REFERENCES[69] The present standard also refers to other International Standards for Phytosanitary Measures (ISPMs). ISPMs are

available on the IPP at https://www.ippc.int/core-activities/standards-setting/ispms.

[70] Accotto, G.P., Navas-Castillo, J., Noris, E., Moriones, E., Louro, D. 2000. Typing tomato yellow leaf curl viruses in Europe. European Journal of Plant Pathology, 160: 179-186.

[71] Boykin, L.M. & De Barro, P. 2014. A practical guide to identify members of the Bemisia tabaci species complex: and other morphologically identical species. Frontiers in Ecology and Evolution, 2: 1-5; doi: 10.3389/fevo.2014.00045.

[72] Briddon, R.W., Prescott, A.G., Lunness, P., Chamberlin, L.C., Markham, P.G. 1993. Rapid production of full-length, infectious geminivirus clones by abutting primer PCR (AbP-PCR). Journal of Virological Methods, 43:7-20.

[73] Briddon, R.W., Bull, S.E., Amin, I., Idris, A.M., Mansoor, S., Bedford, I.D., Dhawan, P., Rishi, N., Siwatch, S.S., Abdel-Salam, A.M., Brown, J.K., Zafar, Y., Markham, P.G. 2003. Diversity of DNA beta, a satellite molecule associated with some monopartite begomoviruses. Virology, 312: 106–121; doi:10.1016/S0042-6822(03)00200-9.

[74] Brown, J.k., Zerbini, F.M., Navas-Castillo, J., Moriones, E., Ramos-Sobrinho, R., Silva, J.C.F., Fiallo-Olive, E., Briddon, R.W., Hernandez-Zepeda, C., Idris, A., Malathi, V.G., Martin, D.P., Rivera-Bustamante, R., Ueda, S., Varsani, A. 2015. Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Archives of Virology, 160: 1593-1048; doi:10.1007/s00705-015-2398-y.

[75] Czosnek, H., Ghanim, M., Ghanim, M. 2002. The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci – insights from studies with Tomato yellow leaf curl virus. Annals of applied Biology, 140: 215-231.

[76] Dellaporta, S.L., Wood, J., Hicks, J.B. 1983. A plant DNA minipreparation: version II. Plant Molecular Biologiy Reporter, 1:19-21.

[77] Haible, D., Kober, S., Jeske, H. 2006. Rolling cycle amplification revolutionizes diagnosis and genomics of geminiviruses. Journal of Virological Methods, 135: 9-16; doi:10.1016/j.jviromet.2006.01.017.



[78] Idris, A.M., Briddon, R.W., Bull, S.E., Brown, J.K. 2005. Cotton leaf curl Gezira virus-satellite DNAs represent a divergent, geographically isolated Nile Basin lineage: Predictive identification of a satDNA REP-binding motif. Virus Research, 109: 19–32; doi:10.1016/j.virusres.2004.10.002.

[79] International Committee on Taxonomy of Viruses, 2014

[80] Knierim, D. & Maiss, E. 2007. Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus. Archives of Virology, 152: 941-954; doi: 10.1007/s00705-006-0914-9.

[81] Kon, T. & Gilbertson R.L. 2012. Two genetically related begomoviruses causing tomato leaf curl disease in Togo and Nigeria differ in virulence and host range but do not require a beta satellite for induction of disease symptoms. Archives of Virology, 157: 107–120; 10.1007/s00705-011-1139-0.

[82] Lodhi, M.A., Ye, G.-N., Weeden, N.F., Reisch, B. 1994. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Molecular Biology Reporter, 12: 6-13.

[83] Mansoor, S., Zafar, Y., Briddon, R. W. 2006. Geminivirus disease complexes: the threat is spreading. Trends Plant Science, 11: 209–212; doi:10.1016/j.tplants.2006.03.003.

[84] Wyant, P.S., Strohmeier, S., Schäfer, B., Krenz, B., Assunção, I.P., de Lima, G.S., Jeske, H. 2012. Circular DNA genomics (circomics) exemplified for geminiviruses in bean crops and weeds of northeastern Brazil. Virology, 427: 151-157; doi:10.1016/j.virol.2012.02.007.



9. FIGURES

Figure 1: Typical leaf curl symptoms on youngest upmost leaves of tomato and bright yellow mosaic symptoms on Vigna radiata indicate for presence of begomoviruses.



Figure 2: Approaches for detection of begomoviruses and identification of species and strains


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