3’ NON-CODING REGION RT-PCR DETECTION AND MOLECULAR HYBRIDIZATION OF PLUM POX VIRUS IN ANTHERS OF INFECTED STONE FRUIT

1995 ◽  
pp. 331-339 ◽  
Author(s):  
L. Levy ◽  
A. Hadidi ◽  
M. Kolber ◽  
G. Tokes ◽  
M. Nemeth
Keyword(s):  
Pcr Detection ◽  
Molecular Hybridization ◽  
Stone Fruit ◽  
Plum Pox Virus ◽  
Coding Region ◽  
Rt Pcr

scholarly journals Occurrence of Stone Fruit Viruses in Plum Orchards in Latvia

2013 ◽  
Vol 67 (2) ◽  
pp. 116-123
Author(s):  
Alina Gospodaryk ◽  
Inga Moročko-Bičevska ◽  
Neda Pūpola ◽  
Anna Kāle
Keyword(s):  
Mosaic Virus ◽  
Large Scale ◽  
Ringspot Virus ◽  
Stone Fruit ◽  
Detection Methods ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
Detection Rates ◽  
Arabis Mosaic Virus ◽  
Das Elisa

To evaluate the occurrence of nine viruses infecting Prunus a large-scale survey and sampling in Latvian plum orchards was carried out. Occurrence of Apple mosaic virus (ApMV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), and Plum pox virus (PPV) was investigated by RT-PCR and DAS ELISA detection methods. The detection rates of both methods were compared. Screening of occurrence of Strawberry latent ringspot virus (SLRSV), Arabis mosaic virus (ArMV), Tomato ringspot virus (ToRSV) and Petunia asteroid mosaic virus (PeAMV) was performed by DAS-ELISA. In total, 38% of the tested trees by RT-PCR were infected at least with one of the analysed viruses. Among those 30.7% were infected with PNRSV and 16.4% with PDV, while ApMV, ACLSV and PPV were detected in few samples. The most widespread mixed infection was the combination of PDV+PNRSV. Observed symptoms characteristic for PPV were confirmed with RT-PCR and D strain was detected. Comparative analyses showed that detection rates by RT-PCR and DAS ELISA in plums depended on the particular virus tested. The results obtained in this study revealed that commonly grown plum cultivars in Latvia are infected with economically important stone fruit viruses and highlight the need to implement a programme to produce and propagate virus-free planting material.

scholarly journals Detection and Partial Molecular Characterization of Two Plum pox virus Isolates from Plum and Wild Apricot in Southeast Kazakhstan

Plant Disease ◽  
2004 ◽  
Vol 88 (9) ◽  
pp. 973-979 ◽  
Author(s):  
S. Spiegel ◽  
E. M. Kovalenko ◽  
A. Varga ◽  
D. James
Keyword(s):  
Nucleotide Sequence Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plum Pox Virus ◽  
Polymorphism Analysis ◽  
Coding Region ◽  
Rt Pcr ◽  
Nucleotide Substitutions ◽  
Western Blot Assay ◽  
C Terminus ◽  
Wild Apricot

Plum pox virus (PPV) was detected in wild apricot and cultivated plum maintained in a germ plasm collection in Kazakhstan. Both isolates were typed as D strain, with no evidence of recombination. The virus was detected by triple-antibody sandwich enzyme-linked immunosorbent assay (ELISA) utilizing the universal PPV-specific monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) assay using primers that amplified a 243-bp fragment in the C-terminus of the coat protein (CP) coding region. Immunocapture (IC) RT-PCR was used to detect PPV in nine wild apricot accessions, including eight ELISA-negative and one ELISA-positive. The plum and apricot isolates reacted positively in Western blot assay with the universal MAb 5B, and negatively with the strain-M-specific MAb-AL. Restriction fragment length polymorphism analysis applied to the amplified 243-bp fragment showed that restriction sites for AluI and RsaI were present in the were present in the plum and apricot samples. An amplified 836-bp cDNA fragment derived from the P3-6K1 coding region of both isolates had restriction profiles typical for strain D. Nucleotide identities of 99 to 100% were observed for the 243-bp fragments of the Kazakhstan isolates when compared with the corresponding regions of strain D, and 94 to 95% identity with strain M. Nucleotide sequence analysis of the entire CP coding region of the plum and apricotisolates resulted in the identification of a unique deletion of six nucleotides (two deduced proline amino acid residues) in the N-terminal region in the plum isolate. This is the first deletion of this nature observed among PPV isolates. The DAG motif was present in both isolates. Several nucleotide substitutions in the CP coding region were common to the plum and apricot isolates and appear to be unique to the Kazakstan isolates. This suggests a close relationship between the isolates.

scholarly journals Detection of a New and Unusual Isolate of Plum pox virus in Plum (Prunus domestica)

Plant Disease ◽  
2003 ◽  
Vol 87 (9) ◽  
pp. 1119-1124 ◽  
Author(s):  
D. James ◽  
A. Varga ◽  
D. Thompson ◽  
S. Hayes
Keyword(s):  
Western Blot ◽  
Immunosorbent Assay ◽  
Molecular Techniques ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plum Pox Virus ◽  
Polymorphism Analysis ◽  
Coding Region ◽  
Rt Pcr ◽  
Western Blot Assay ◽  
Restriction Sites

Plum pox virus (PPV) isolate 3174-01 was detected by triple-antibody sandwich enzyme-linked immunosorbent assay using the universal PPV monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) using primers that amplify a 243-bp fragment in the C-terminus of the coat protein coding region. The restriction sites RsaI and AluI were absent from this fragment, which is a feature unique to PPV-C isolates. The restriction sites in 3174-01 were replaced by GTAA/GTGA and GGCA, respectively. There was 95 to 99, 94, 91, and 92 to 94% identity of the 243-bp fragment of 3174-01 with the corresponding region of the strains C, D, EA, and M, respectively. Attempts to detect the virus by RT-PCR using strain C-specific primers in three different approaches were unsuccessful. All molecular techniques assessed in attempting to strain type isolate 3174-01 gave negative results, or results inconsistent for D or M in the case of P3-6K1 restriction fragment length polymorphism analysis. Isolate 3174-01 reacted in Western blot assay with MAb 5B, with an estimated molecular mass of 32 kDa. No reaction was observed with D-, M-, EA-, or C-specific monoclonal antibodies in Western blot or enzyme-linked immunosorbent assay. The molecular and serological data seem to indicate that PPV isolate 3174-01 does not belong to any of the recognized strains of PPV.

scholarly journals First Report of Peach latent mosaic viroid in Peach Trees in Montenegro

Plant Disease ◽  
2012 ◽  
Vol 96 (1) ◽  
pp. 150-150 ◽  
Author(s):  
I. Mavric Pleško ◽  
M. Viršcek Marn ◽  
Z. Miladinovic ◽  
J. Zindovic
Keyword(s):  
Prunus Persica ◽  
Stone Fruit ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
First Report ◽  
Fruit Species ◽  
Hop Stunt Viroid ◽  
Viroid Infection ◽  
Fruit Samples ◽  
Peach Trees

Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) are known to infect stone fruit species worldwide. The viroid infection can be latent or induce a variety of disease symptoms. Stone fruit samples were collected in Montenegro for a Plum pox virus (PPV) survey in 2007. Thirteen samples infected with PPV, taken from 12-year-old peach trees (Prunus persica (L.) Batsch, cv. Elegant Lady) in the area of Cemovsko field, were tested for the presence of PLMVd and HSVd by reverse transcription (RT)-PCR. Mild or severe mosaic, chlorotic rings, and fruit deformations were observed on some trees. Total RNA was extracted from all samples with a RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) and RT-PCR was performed. Samples were tested for HSVd and PLMVd infection using primer pairs RF-43/RF-44 for PLMVd (1) and VP-19/VP-20 for HSVd (2). Amplification products of approximately 348 bp were obtained from nine samples with PLMVd primers. Amplification products from seven samples were successfully cloned into pGEM-T Easy Vector (Promega, Madison, WI) and used for transformation of Escherichia coli. At least four clones of each sample were sequenced. Obtained sequences were 337 and 338 nucleotides long and shared 90.3 to 100% identity. Consensus sequences of each sample were deposited in GenBank under Accession Nos. JF927892–JF927898. They showed 92.6 to 97.9% identity among each other, 94 to 98% identity with the PLMVd isolate G sequence (Accession No. EF591868) and 91.8 to 94.4% identity with PLMVd sequence M83545. HSVd was not detected in analyzed samples. PLMVd infections were found on peach trees in an area where approximately 40% of the peach production is located. Therefore, PLMVd infections can pose a threat to peach production in Montenegro. To our knowledge this is the first report of PLMVd infection of peach in Montenegro. References: (1) S. Ambrós et al. J. Virol. 72:7397, 1998. (2) S. A. Kofalvi et al. J. Gen. Virol. 78:3177, 1997.

scholarly journals Reaction of transgenic plum cv. HoneySweet to the Plum pox virus after a severe infection of Monilinia sp. – short communication

2018 ◽  
Vol 55 (No. 1) ◽  
pp. 8-10 ◽  
Author(s):  
Polák Jaroslav ◽  
Neubauerová Tereza ◽  
Komínek Petr ◽  
Kundu Jiban Kumar
Keyword(s):  
Czech Republic ◽  
Short Communication ◽  
Severe Infection ◽  
Pcr Detection ◽  
Plum Pox Virus ◽  
Prunus Domestica ◽  
Rt Pcr ◽  
The Czech Republic ◽  
Das Elisa ◽  
Year 2013

Resistance to Plum pox virus (PPV) in transgenic Prunus domestica L., clone C5 (cv. HoneySweet) was evaluated in a regulated field in the Czech Republic for fifteen years (2002–2016). PPV mild symptoms appeared in C5 trees only in several leaves situated close to the point of inoculum grafting up to 2010. No symptoms of PPV were observed in the years 2011–2013 and results of ELISA and RT-PCR detection tests were negative. In the twelfth year (2013), there was a severe unusual natural attack of plum trees by Monilinia sp. This Monilinia sp. attack occurred only one time – in 2013. There was no Monilinia sp. infection in 2002–2012 and in 2014–2016. Mild PPV symptoms reappeared in several leaves of transgenic plum trees in the next two years (2014–2015) and the presence of PPV was proved by DAS-ELISA and confirmed by RT-PCR.

scholarly journals Simultaneous Detection of the Three Ilarviruses Affecting Stone Fruit Trees by Nonisotopic Molecular Hybridization and Multiplex Reverse-Transcription Polymerase Chain Reaction

2000 ◽  
Vol 90 (12) ◽  
pp. 1330-1336 ◽  
Author(s):  
M. Saade ◽  
F. Aparicio ◽  
J. A. Sánchez-Navarro ◽  
M. C. Herranz ◽  
A. Myrta ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Fruit Trees ◽  
Plant Viruses ◽  
Molecular Hybridization ◽  
Stone Fruit ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

scholarly journals Involvement of RpoN in Regulating Bacterial Arsenite Oxidation

2012 ◽  
Vol 78 (16) ◽  
pp. 5638-5645 ◽  
Author(s):  
Yoon-Suk Kang ◽  
Brian Bothner ◽  
Christopher Rensing ◽  
Timothy R. McDermott
Keyword(s):  
Binding Site ◽  
Sigma Factor ◽  
Model Organism ◽  
Coding Region ◽  
Rt Pcr ◽  
Obvious Effect ◽  
Content Type ◽  
Definitive Evidence ◽  
Relative Contribution ◽  
Insertional Inactivation

ABSTRACTIn this study with the model organismAgrobacterium tumefaciens, we used a combination oflacZgene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of AsIIIoxidation. A deletion mutation that removed the RpoN binding site from theaioBApromoter and anaacC3(gentamicin resistance) cassette insertional inactivation of therpoNcoding region eliminatedaioBAexpression and AsIIIoxidation, althoughrpoNexpression was not related to cell exposure to AsIII. Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters foraioB,phoB1,pstS1,dctA,glnA,glnB, andflgBthat were examined by using qualitative RT-PCR andlacZreporter fusions to assess the relative contribution of RpoN to their transcription. The expressions ofaioBanddctAin the wild-type strain were considerably enhanced in cells exposed to AsIII, and both genes were silent in therpoN::aacC3mutant regardless of AsIII. The expression level ofglnAwas not influenced by AsIIIbut was reduced (but not silent) in therpoN::aacC3mutant and further reduced in the mutant under N starvation conditions. TherpoN::aacC3mutation had no obvious effect on the expression ofglnB,pstS1,phoB1, orflgB. These experiments provide definitive evidence to document the requirement of RpoN for AsIIIoxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by AsIIIor by this sigma factor.

Simultaneous multiplex RT-PCR detection of four viruses associated with maize lethal necrosis disease

2021 ◽  
pp. 114286
Author(s):  
Xiaoqin Li ◽  
Yu Li ◽  
Wenli Hu ◽  
Yingjuan Li ◽  
Yan Li ◽  
...  
Keyword(s):  
Pcr Detection ◽  
Rt Pcr

scholarly journals RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro

2006 ◽  
Vol 80 (20) ◽  
pp. 10055-10063 ◽  
Author(s):  
Adrian Valli ◽  
Ana Montserrat Martín-Hernández ◽  
Juan José López-Moya ◽  
Juan Antonio García
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Plum Pox Virus ◽  
Coding Region ◽  
Long Distance ◽  
Systemic Spread ◽  
The Family ◽  
Silencing Suppression ◽  
Green Fluorescent

ABSTRACT The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.

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