Use of crude sap for one-step RT-PCR-based assays of Bean yellow mosaic virus and the utility of this protocol for various plant?virus combinations

Uga, Hiroyuki

doi:10.1007/s10327-004-0166-z

Cited by 6 publications

(6 citation statements)

References 14 publications

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“…Detection of BYMV potyvirus group in infected bean samples using RT-PCR with specific primers showed a fragment of 700 bp. Similar result was mentioned by Vunsh et al (1990), Katoch et al (2002a), Uga (2005) and Kaur et al (2014).…”

Section: Discussionsupporting

confidence: 88%

“…Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was done using Qiagen oneStep RT-PCR enzyme Mix (OneStep RT-PCR kit, Qiagen). Primer BYMV1/ BYMV 2 pair for Bean yellow mosaic virus (BYMV) was used this resulted in an amplification product 700 bp nuclear inclusion body and coat protein gene region primer (5' NIb-CP 3') by Uga (2005). The RT was performed in a thermocyler (Biometra Co.).…”

Section: Rna Extraction and Reverse Transcription-polymerase Chain Rementioning

confidence: 99%

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Nucleotide Sequence of Capsid Protein Gene of Bean yellow mosaic potyvirus in Bean Plants from Al-Makhwah Governorate, Saudi Arabia

Farag¹,

Khattab²,

Al-sham³

2015

International J. of Virology

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The current study represents the first identification of Bean Yellow Mosaic Virus (BYMV) in bean plants (Phaseolus vulgaris) from Al-Makhwah Governorate, Saudi Arabia and nucleotide sequencing of capsid protein gene of BYMV. Thirty plant species and cultivars to twelve different families were mechanically inoculated by BYMV. Seventeen of them showed systemic symptoms mosaic, yellowing, vein clearing, stunting streaking mosaic, malformation and severe mosaic as a result of BYMV infection. Chenopodium amaranticolor and Chinopodium quinoa L. were found to be local lesion host after 4-6 days of inoculation. Two aphids, Myzus persicae Sluz. and Aphis faba were used to study the transmission of BYMV. Aphis faba was found to be the most effective vector with 60% of BYMV transmission. Immunological techniques namely Enzyme Linked Immuno Sorbent Assay (ELISA), Tissue Blot Immuno Binding Assay (TBIA) and Dot Blot Immune Binding Assay (DBIA) were amplified to study BYMV. Positive reaction was obtained and the prevalence of the virus in the flowers and seed parts was confirmed. The RT-PCR products were amplified from total RNA of bean plant tissues using specific primer BYMV1 and BYMV2 (designed on conserved sequences of BYMV NIb-CP). A cDNA fragment of 700 bp nuclear inclusion body and coat protein gene region primer (5'-NIb-CP 3') was amplified. Digoxigenin-labelled BYMV cDNA probe through Southern blot and Dot blot hybridization techniques were employed for the detection of BYMV infected bean plants. A strong positive reaction was observed with bean and faba bean infected with BYMV. A part of the 3' end of Nib-region and the 5' end of the CP region of BYMV Potyvirus Al-Makhwah KSA isolate (700 nt) was sequenced and analyzed (The DNA sequence submitted in GenBank acc.no. LC025531). Identity percentage of Al-Makhwah KSA isolate BYMV Potyvirus (NIb-CP) with a Japanese isolate was 99% and with USA gladiolus isolate and another Japanese isolate were 95% and 93%, respectively confirming that BYMV viral group is diversified mainly in some specific parts of genome, especially in the CP region, whereas NIb gene is very conservative.

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Section: Discussionsupporting

confidence: 88%

Section: Rna Extraction and Reverse Transcription-polymerase Chain Rementioning

confidence: 99%

Nucleotide Sequence of Capsid Protein Gene of Bean yellow mosaic potyvirus in Bean Plants from Al-Makhwah Governorate, Saudi Arabia

Farag¹,

Khattab²,

Al-sham³

2015

International J. of Virology

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“…Two overlapping fragments covering the 5' NIb-CP 3' region of 777 bp to 780 bp, respectively, were obtained with two different primer pair combinations. The first fragment was amplified using the primer pair BYMV1/2 (Hiroyuki Uga, 2005). For the amplification of the second one, the newly designed primer S1CF/S1CR spanning a part of the NIb-CP region was used for sequencing (S1CF: 5'-GATGAGGRGCTTGTGTGTCG-3'; S1CR: 5'-TCACCTGTTCCTCTCCATCC-3').…”

Section: Methodsmentioning

confidence: 99%

“…In contrast to CP, NIb exhibits the highest amino acid sequence homology among the potyviral gene products and acts as an RNA-dependent polymerase (Allison et al, 1986). Therefore, this region is commonly used for designing of BYMV specific primers (Castro et al, 1993;Rosner et al, 1994;Hiroyuki Uga, 2005).…”

Section: Introductionmentioning

confidence: 99%

Variability of Bean yellow mosaic virus isolates in the Czech Republic

Selvaraj¹,

Pokorný²,

Holková³

2009

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Three isolates of Bean yellow mosaic virus (BYMV) from the Czech Republic originating from gladiolus plants were examined according to their biological and molecular characteristics. Partial sequence of coat protein-nuclear inclusion protein b (CP-NIb) coding region (768 bp) of these isolates were determined and compared with the corresponding sequences of different BYMV isolates obtained from GeneBank. Phylogenetic analysis showed that the Czech BYMV isolates were distributed across the three groups of phylogenetic tree. Their placement was not dependent on the geography or host plant.

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“…Among the eight viruses assayed OLYaV was the most predominantly occurring virus in Southern Italy, while SLRSV was detected more frequently in the central Italy (Faggioli et al 2005). Bean yellow mosaic virus was detected by using one step RT-PCR from crude sap of infected dwarf gentian plants (Gentina scabra) at dilutions upto 10 6 -fold indicating that this technique was 100 times more sensitive than indirect ELISA format, and equally sensitive as IC-RT-PCR protocol (Uga 2005).…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Molecular Techniques for Detection of Microbial Pathogens

Narayanasamy

Molecular Biology in Plant Pathogenesis and Disease Management

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Detection, identification and differentiation of microbial plant pathogens -oomycetes, fungi, bacteria, phytoplasmas, viruses and viroids -infecting various crops constitute the basic step for the development of effective crop disease management systems. The conventional cultural methods involving isolation and studying the morphological characteristics using microscope are labor intensive and cumbersome, yielding sometimes, inconclusive results. The molecular techniques, on the other hand, are able to provide precise, reliable and reproducible results rapidly, facilitating early disease management decisions. Biochemical, immunological and nucleic acid-based assays have been preferred, because of the distinct advantages over the conventional methods. The molecular techniques have been very useful in the identification of obligate pathogens causing diseases such as downy mildews, powdery mildews and rusts and also fungi that grow very slowly in the culture media, taking several weeks to produce spore forms that can be used for identification. The availability of antisera, primers and commercial kits has led to widespread application of the molecular techniques for on-site detection during field surveys for assessing the distribution of existing pathogens, occurrence of new/introduced pathogens or strains and for preventing introduction of new pathogens through seeds or planting materials. Furthermore, molecular techniques have been demonstrated to be useful in breeding programs to identify sources of resistance to disease(s). Several protocols for molecular techniques, useful for researchers and students are presented in the Appendix.

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Use of crude sap for one-step RT-PCR-based assays of Bean yellow mosaic virus and the utility of this protocol for various plant?virus combinations

Cited by 6 publications

References 14 publications

Nucleotide Sequence of Capsid Protein Gene of Bean yellow mosaic potyvirus in Bean Plants from Al-Makhwah Governorate, Saudi Arabia

Nucleotide Sequence of Capsid Protein Gene of Bean yellow mosaic potyvirus in Bean Plants from Al-Makhwah Governorate, Saudi Arabia

Variability of Bean yellow mosaic virus isolates in the Czech Republic

Molecular Techniques for Detection of Microbial Pathogens

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