scholarly journals IDENTIFIKASI TOMATO INFECTIOUS CHLOROSIS VIRUS PENYEBAB PENYAKIT KLOROSIS PADA TANAMAN TOMAT DI CIPANAS JAWA BARAT MELALUI PERUNUTAN NUKLEOTIDA GEN PROTEIN SELUBUNG UTAMA

2015 ◽  
Vol 15 (1) ◽  
pp. 33
Author(s):  
Fitrianingrum Kurniawati ◽  
Gede Suastika ◽  
Giyanto .
Keyword(s):  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Rna Extraction ◽  
Dna Amplification ◽  
Causal Agent ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Polymerase Chain ◽  
Tomato Infectious Chlorosis Virus

Identification of tomato infectious chlorosis virus, the causal agent of chlorosis disease on tomato in Cipanas West Java by sequencing of main coat protein gene nucleotide. Tomato infectious chlorosis virus (TICV) causes chlorosis on tomato. Tomatoes infected by this virus shows interveinal yellowing, necrotic, bronzing, brittleness, and declining in productivity. This study aims to identify the causal agent of chlorotic disease on tomato by sequencing the coat protein gene. The methods involve collecting infected plants, total RNA extraction, cDNA synthesis, DNA amplification, visualization of the results of reverse transcription polymerase chain reaction (PCR), and phylogenetic analysis using BLAST, clustal w, Bioedit v 7.0.5.3, MEGA v 6:06. RT-PCR using spesific primers (CP-F TICV Bam and TICV R-Hind) amplified a DNA band of 792 bp, which has been successfully sequenced and identified as TICV. Nucleotide sequences homology analysis showed that TICV Indonesia_TWJ isolate Cipanas is the same strain as TICV from other countries (99.4 – 100%), such as Spain, Greece, USA, France, and Italy.

scholarly journals Presence and molecular characterization of alfalfa mosaic virus on tobacco in Serbia

2011 ◽  
Vol 26 (3) ◽  
pp. 229-243 ◽  
Author(s):  
Ivana Stankovic ◽  
Ana Vucurovic ◽  
Aleksandra Bulajic ◽  
Danijela Ristic ◽  
Janos Berenji ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Coat Protein ◽  
Mosaic Virus ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Rna Extraction ◽  
Alfalfa Mosaic Virus ◽  
Lower Percentage ◽  
Rt Pcr ◽  
Plant Host

Three-year investigation of the presence and distribution of tobacco viruses in Serbia revealed that Alfalfa mosaic virus (AMV) appeared every year with different frequency in tobacco crops. During 2008, the presence of AMV was detected in most of the tested samples (58.82%) and it was the second most common compared to all other viruses which presence was confirmed in Serbia. In 2006 and 2007, AMV was detected in a significantly lower percentage (2.80% and 13.64%, respectively). This study showed that Alfalfa mosaic virus was more commonly found in multiple infections with two, three or even four detected viruses. Single infections were detected only in 2006, in one tobacco field in the locality of Futog. During this investigation, a rapid and simple protocol was optimized and developed for molecular detection of AMV in tobacco leaves, using primers CPAMV1/CPAMV2 and commercially available kits for total RNA extraction as well as for RT-PCR (reverse transcription - polymerase chain reaction). Using RT-PCR and these primers that flank the AMV coat protein gene, a DNA fragment of 751 bp was amplified, sequenced, and compared with the sequences available in GenBank database. The sequence of isolate 196-08 (GenBank Acc. No. FJ527749) proved to be identical at the nucleotide level of 99 to 93% with those from other parts of the world. Phylogenetic analysis of 27 isolates based on 528 bp sequences of the coat protein gene did not show correlation of the isolates with their geographic origin or plant host and showed that these isolates fall into four molecular groups of strains. Serbian AMV isolate from tobacco belongs to group IV, the group that includes most of the isolates selected for phylogenetic analysis.

scholarly journals EKSPRESI GEN PROTEIN SELUBUNG TOMATO INFECTIOUS CHLOROSIS VIRUS PADA ESCHERICHIA COLI

2016 ◽  
Vol 15 (2) ◽  
pp. 114
Author(s):  
Fitrianingrum Kurniawati ◽  
Gede Suastika ◽  
Giyanto .
Keyword(s):  
Escherichia Coli ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Serological Test ◽  
Coli Strain ◽  
Rt Pcr ◽  
Cp Gene ◽  
Sds Page ◽  
Tomato Infectious Chlorosis Virus

Expression of tomato infectious chlorosis virus coat protein gene on Escherichia coli. Tomato infectious chlorosis virus (TICV) is the causal agent of chlorotic disease of tomato. Detection of TICV can be carried out by RT-PCR and serological test. Titer of TICV in plant tissue is very low because TICV is limited to phloem. Serological detection of TICV requires antiserum which is not available in Indonesia. Producing antibody through cloning and coat protein gene (TICV CP gene) expression is a promising approach in producing antiserum. The objective of this study was to express TICV CP gene as antigen for antiserum production. TICV CP gene was amplified using RT-PCR from total RNA extracted from TICV infected leaves collected from Cipanas, Cianjur, West Java. The amplified CP gene was then sequenced and sub-cloned into pET 21b expression vector, transformed into Escherichia coli strain BL21 DE3(pLysS) and induced expression using IPTG 1 mM overnight at 37 °C. CP that contains 6xhistag was purified using NiNTAspin column and then confirmed by SDS-PAGE. The size of TICV CP gene was 750 bp and the gene was expressed on pET 21 b vector and SDS-PAGE showed a 29 kDa band.

scholarly journals Reverse-Transcription Polymerase Chain Reaction Detection of Citrus Tristeza Virus in Aphids

Plant Disease ◽  
1997 ◽  
Vol 81 (9) ◽  
pp. 1066-1069 ◽  
Author(s):  
Prem Mehta ◽  
R. H. Brlansky ◽  
S. Gowda ◽  
R. K. Yokomi
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Citrus Tristeza Virus ◽  
Protein Gene ◽  
Aphid Species ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tristeza Virus

A rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection of citrus tristeza virus (CTV) in three aphid species. Seven CTV isolates from a worldwide isolate collection were used for aphid acquisition feeding by three aphid species. These included the most efficient CTV vector, the brown citrus aphid, Toxoptera citricida; the melon aphid, Aphis gossypii; and the green peach aphid, Myzus persicae, a non-vector for CTV. A short procedure for nucleic acid extraction from single or groups of aphids was developed. Nucleic acid extracts from 1, 3, 5, and 10 aphids with acquisition-access periods of 24 and 48 h were reverse transcribed and amplified using primers for the coat protein gene of the Florida B3 (T-36) isolate of CTV. PCR-amplified fragments of approximately 670 bp were obtained from all the isolates tested and the amplified product from the aphids fed on citrus infected with isolate B3 was confirmed as the CTV coat protein gene by digesting with various restriction enzymes. This technique will be useful in investigations of CTV-vector-plant interactions and CTV epidemiology.

scholarly journals First Report of Cherry virus A and Little cherry virus-1 in Poland

Plant Disease ◽  
2004 ◽  
Vol 88 (8) ◽  
pp. 909-909 ◽  
Author(s):  
B. Komorowska ◽  
M. Cieślińska
Keyword(s):  
Nucleotide Sequence ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Germplasm Collection ◽  
Mottle Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Primer Sets

Cherry virus A (CVA), a member of the genus Capillovirus, has been reported in sweet cherry in Germany, Canada, and Great Britain. No data are available on the effects of CVA on fruit quality and yield of infected trees. Little cherry disease (LChD) occurs in most cherry growing areas of the world. Symptoms on sensitive cultivars include discolored fruit that remain small, pointed in shape, and tasteless. Three Closterovirus spp. associated with LChD have been described (Little cherry virus-1 [LChV-1], LChV-2, and LChV-3). Diseased local and commercial cultivars of sour cherry trees were found in a Prunus sp. germplasm collection and orchards in Poland during the 2003 growing season. The foliar symptoms included irregular, chlorotic mottling, distortion, and premature falling of leaves. Some of the diseased trees developed rosette as a result of decreased growth and shortened internodes. Severely infected branches exhibited dieback symptoms. Because the symptoms were suggestive of a possible virus infection, leaf samples were collected from 38 trees and assayed for Prune dwarf virus and Prunus necrotic ringspot virus using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). RNA extracted from leaves was used in a reverse transcription-polymerase chain reaction (RT-PCR) with the One-Step RT-PCR with Platinum Taq (Invitrogen Life Technologies) and primer sets specific for CVA (1), LChV-1 (3), and LChV-2 (3). The RNA samples were also tested using RT-PCR for detection of Cherry mottle leaf virus (CMLV), Cherry necrotic rusty mottle virus (CNRMV), and Cherry green ring mottle virus (CGRMV) with specific primer sets (2). Amplification of a 397-bp coat protein gene product confirmed infection of 15 trees with CVA. A 419-bp fragment corresponding to the coat protein gene of LChV-1 was amplified from cv. Gisela rootstock and local cv. WVIII/1. To confirm RT-PCR results, CVA amplification products from local cv. WX/5 and LChV-1 from cvs. Gisela and WVIII/1 were cloned in bacterial vector pCR 2.1-TOPO and then sequenced. The sequences were analyzed with the Lasergene (DNASTAR, Madison, WI) computer program. The alignment indicated that the nucleotide sequence of cv. WX/5 was closely related to the published sequences of CVA (Genbank Accession No. NC_003689) and had an 89% homology to the corresponding region. The nucleotide sequence similarity between the 419-bp fragment obtained from cvs. Gisela and WVIII/1 was 87% and 91%, respectively, compared with the reference isolate of LChV-1 (Genbank Accession No. NC_001836). The sampled trees tested negative for LChV-2, CGRMV, CMLV, and CNRMV using RT-PCR. Some trees tested positive for PNRSV and PDV. To our knowledge, this is the first report of CVA and LChV-1 in Poland. References: (1) D. James and W. Jelkmann. Acta Hortic. 472:299, 1998. (2) M. E. Rott and W. Jelkmann. Eur. J. Plant Pathol. 107:411,2001. (3) M. E. Rott and W. Jelkmann. Phytopathology. 91:61, 2001.

scholarly journals Detection and Confirmation of Potato mop-top virus in Potatoes Produced in the United States and Canada

Plant Disease ◽  
2004 ◽  
Vol 88 (4) ◽  
pp. 363-367 ◽  
Author(s):  
H. Xu ◽  
T.-L. DeHaan ◽  
S. H. De Boer
Keyword(s):  
United States ◽  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
The North ◽  
Potato Mop Top Virus ◽  
Pcr Targeting

Potato mop-top virus (PMTV) was detected in potatoes grown in the United States and Canada during surveillance testing by a reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene in RNA3. Out of 3,221 lots of seed and ware potatoes that were tested, 4.3% were positive for PMTV. The reliability of the survey results was confirmed by reextraction of selected samples and additional RT-PCR tests using two primer sets targeting gene segments in RNA2 and RNA3. Amplicons generated from RNA2 and RNA3 were identified by analysis of fragment length polymorphisms after digestion with BamHI and HindIII, respectively. PMTV was further identified by enzyme-linked immunosorbent assay, bioassay on Nicotiana debneyi, and transmission electron microscopy. Sequencing of a portion of the coat protein gene revealed near 100% identity among isolates from the United States and Canada and >97% homology of the North American isolates with European isolates.

scholarly journals First Report of Cucurbit yellow stunting disorder virus on Melon in China

Plant Disease ◽  
2010 ◽  
Vol 94 (4) ◽  
pp. 485-485 ◽  
Author(s):  
L. Z. Liu ◽  
Y. Y. Chen ◽  
W. M. Zhu
Keyword(s):  
Coat Protein ◽  
Coat Protein Gene ◽  
Fragment Length ◽  
Protein Gene ◽  
Disease Incidence ◽  
Rt Pcr ◽  
First Report ◽  
Genes Encoding ◽  
Pcr Products ◽  
Symptomatic Leaf

Melon (Cucumis melo L.) plants in commercial fields in Shanghai, Jiangsu, and Zhejiang exhibited stunting, deformation, interveinal chlorosis, and leaf mottling in the spring of 2008. In addition, adult and immature whiteflies (Bemisia tabaci biotype B) were present in these melon fields. Thirty-two symptomatic leaf samples were collected from these fields for further analysis (9 from Nanhui County in Shanghai, 11 from Fengxian County in Shanghai, 6 from Kunshan County of Jiangsu, and 6 from Jiashan County of Zhejiang). Total RNA was extracted from these samples along with asymptomatic control plants and screened for the presence of Cucurbit yellow stunting disorder virus (CYSDV) by using primers specific to genes encoding coat protein (2) and HSP70h (1) of CYSDV through reverse transcription (RT)-PCR methods. RNA was successfully extracted from 31 of 32 symptomatic samples. All 31 symptomatic leaf samples tested with coat protein primers were positive for CYSDV and yielded the expected fragment length of 394 bp. The RT-PCR products of the coat protein gene from all 31 isolates were cloned and found to be identical in sequence. Thus, only one was deposited in GenBank (No. GU189240). The submitted sequence of the amplified part of the coat protein gene was 99% identical to the sequence of coat protein gene of CYSDV from Jordan, France, and Florida (GenBank Accession Nos. DQ903107, AY204220, and EU596528, respectively) and 98% identical to that of an isolate from Spain (GenBank Accession No. AJ243000). Similarly, all 31 samples were also positive for CYSDV with the primers specific to HSP70h and yielded the expected fragment length of 175 bp. The RT-PCR products of the HSP70h gene from these isolates were also cloned and found to be identical in sequence. The sequence of the amplified portion of the HSP70h gene was found to be identical to the sequence of HSP70h of CYSDV deposited in GenBank (No. AJ439690.2). CYSDV was noticed in all three surveyed regions and the percentage of disease incidence was approximately 68% in all these regions. The occurrence of CYSDV has been previously reported in Europe (Spain and France), southern Asia (Iran and Jordan), North America (United States and Mexico), and other countries (1). To our knowledge, this is first report of CYSDV in China. References: (1) Y.-W. Kuo et al. Plant Dis. 91:330, 2007. (2) J. E. Polston et al. Plant Dis. 92:1251, 2008.

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