scholarly journals Detection of Poinsettia mosaic virus by RT-PCR in Euphorbia spp. in New Zealand

Plant Disease ◽  
2007 ◽  
Vol 91 (1) ◽  
pp. 110-110 ◽  
Author(s):  
B. S. M. Lebas ◽  
F. M. Ochoa-Corona ◽  
D. R. Elliott ◽  
J. Z. Tang ◽  
B. J. R. Alexander
Keyword(s):  
New Zealand ◽  
Mosaic Virus ◽  
Rna Extraction ◽  
Virus Detection ◽  
Amino Acid Identity ◽  
Rt Pcr ◽  
Taq Polymerase ◽  
Pcr Products ◽  
Area North ◽  
Das Elisa

Euphorbia pulcherrima (poinsettias) are commonly infected with Poinsettia mosaic virus (PnMV), which resembles the Tymovirus genus in its morphology and viral properties (2) but is closer to the Marafivirus genus at the sequence level (1). Symptoms induced by PnMV range from leaf mottling and bract distortion to symptomless (2). The presence of PnMV in plants imported into New Zealand had never been proven. Leaves of 10 E. pulcherrima samples and six samples from other Euphorbia spp. (E. atropurpurea, E. lambii, E. leuconeura, E. mellifera, E. milii, and E. piscatorial) were collected in the Auckland area, North Island in 2002. Isometric particles of 26 to 30 nm in diameter were observed with electron microscopy in 3 of 10 E. pulcherrima samples. These three samples produced systemic chlorosis and crinkling symptoms on mechanically inoculated Nicotiana benthamiana, which tested PnMV positive by double-antibody sandwich (DAS)-ELISA (Agdia, Elkart, IN). No particles or symptoms on N. benthamiana were observed with the other Euphorbia spp., which were also PnMV-negative by DAS-ELISA. A reverse transcription-polymerase chain reaction (RT-PCR) was developed to further characterize PnMV. Specific primers were designed from the PnMV complete genome sequence (Genbank Accession No. AJ271595) using the Primer3 web-based software (4). Primer PnMV-F1 (5′-CCTGTATTGTCTCTTGCCGTCC-3′) and primer PnMV-R1 (5′-AGAGGAAAGGAAAAGGTGGAGG-3′) amplified a 764-bp product from nt 5291 of the 5′-end RNA polymerase gene to nt 6082 of the 3′-untranslated region (UTR). Total RNA was extracted from leaf samples using the Qiagen Plant RNeasy Kit (Qiagen Inc., Chastworth, CA). RT was carried out by using PnMV-R1 primer and MMLV reverse transcriptase (Promega, Madison, WI). The PCR was performed in a 20-μl volume reaction containing 2 μl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM PnMV-F1 primer, and 1 U of Taq polymerase (Promega) with a denaturation step (94°C for 5 min), 30 amplification cycles (94°C for 30 s; 55°C for 30 s; 72°C for 1 min), and a final elongation (72°C for 5 min). The sequence of the RT-PCR product (Genbank Accession No. DQ462438) had 98.7% amino acid identity to PnMV. PCR products were obtained from two of three PnMV ELISA-positive E. pulcherrima and three of three PnMV ELISA-positive symptomatic N. benthamiana. The failure to amplify the fragment from all ELISA-positive PnMV is likely because of the presence of inhibitors and latex in E. pulcherrima (3) that make the RNA extraction difficult. Thus, while RT-PCR may be useful for further characterizing PnMV isolate sequences, ELISA may be more reliable for virus detection. In conclusion, to our knowledge, this is the first report of PnMV in E. pulcherrima but not in other Euphorbia spp. in New Zealand. E. pulcherrima plants have been imported into New Zealand for nearly 40 years, and the virus is probably widespread throughout the country via retail nursery trading. References: (1) B. G. Bradel et al. Virology 271:289, 2000. (2) R. W. Fulton and J. L. Fulton. Phytopathology 70:321, 1980. (3) D.-E. Lesemann et al. Phytopathol. Z. 107:250, 1983. (4) S. Rozen and S. Skaletsky. Page 365 in: Bioinformatics Methods and Protocols: Methods in Molecular Biology. S. Krawetz and S. Misener, eds. Humana Press, Totowa, NJ, 2000.

scholarly journals The spreading of Alfalfa mosaic virus in lavandin in Croatia

2014 ◽  
Vol 29 (2) ◽  
pp. 115-122 ◽  
Author(s):  
Ivana Stankovic ◽  
Karolina Vrandecic ◽  
Jasenka Cosic ◽  
Katarina Milojevic ◽  
Aleksandra Bulajic ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Chenopodium Quinoa ◽  
Alfalfa Mosaic Virus ◽  
Amino Acid Identity ◽  
Acid Identity ◽  
Lavandula Stoechas ◽  
Pcr Products ◽  
Test Plants ◽  
Das Elisa

A survey was conducted in 2012 and 2013 to detect the presence and distribution of Alfalfa mosaic virus (AMV) in lavandin crops growing in continental parts of Croatia. A total of 73 lavandin samples from six crops in different localities were collected and analyzed for the presence of AMV and Cucumber mosaic virus (CMV) using commercial double-antibody sandwich (DAS)-ELISA kits. AMV was detected serologically in 62 samples collected at three different localities, and none of the samples tested positive for CMV. For further analyses, six selected samples of naturally infected lavandin plants originating from different localities were mechanically transmitted to test plants: Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana and Ocimum basilicum, confirming the infectious nature of the disease. Molecular detection was performed by amplification of a 751 bp fragment in all tested samples, using the specific primers CP AMV1/CP AMV2 that amplify the part of the coat protein (CP) gene and 3?-UTR. The RT-PCR products derived from the isolates 371-13 and 373-13 were sequenced (KJ504107 and KJ504108, respectively) and compared with the AMV sequences available in GenBank. CP sequence analysis, conducted using the MEGA5 software, revealed that the isolate 371-13 had the highest nucleotide identity of 99.5% (100% amino acid identity) with an isolate from Argentina originating from Medicago sativa (KC881010), while the sequence of isolate 373-13 had the highest identity with an Italian AMV isolate from Lavandula stoechas (FN667967) of 98.6% (99% amino acid identity). Phylogenetic analysis revealed the clustering of selected isolates into four molecular groups and the lavandin AMV isolates from Croatia grouped into two distinct groups, implying a significant variability within the AMV lavandin population.

scholarly journals First Report of Tomato Brown Rugose Fruit Virus on Tomato in Syria

Plant Disease ◽  
2021 ◽  
Author(s):  
Ziad M Hasan ◽  
Nidà Mohammed Salem ◽  
Imad D. Ismail ◽  
Insaf Akel ◽  
Ahmad Y Ahmad
Keyword(s):  
Mosaic Virus ◽  
Rna Extraction ◽  
Coastal Region ◽  
Rt Pcr ◽  
Specific Primers ◽  
Total Rna ◽  
First Report ◽  
Species Specific ◽  
Das Elisa ◽  
Total Rna Extraction

Tomato (Solanum lycopersicum L.) is an important vegetable crop worldwide. In spring and autumn 2017, virus-like symptoms were observed on greenhouse grown tomato plants in the east of Akkar plain (south of coastal region, Tartous governorate, Syria). These symptoms were: mild to severe mosaic on the apical leaves, brown necrosis on sepals, receptacle and flower’s cluster carrier, and severe symptoms of brown rugose and discoloration on fruit. During next growing seasons, disease spread was observed in most of Syrian coastal region with disease incidence ranged from 40% to 70% by 2020. Tomato brown rugose fruit virus (ToBRFV) was suspected as a main causal agent of the disease, especially since its first report in Jordan, a neighboring country (Salem et al. 2016), Palestine (Alkowni et al. 2019), Turkey (Fidan et al. 2019), Germany (Menzel et al. 2019), Italy (Panno et al. 2019), America (Camacho-Beltrán et al. 2019), Egypt (Amer and Mahmoud, 2020), and recently in Spain (Alfaro-Fernandez et al. 2021). In November and December 2020, seventy-one leaf samples from symptomatic plants (59 from Tartous and 12 from Lattakia governorates) and seven from asymptomatic ones (5 from Tartous and 2 from Lattakia) were collected and tested for the presence of ToBRFV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using ToBRFV-commercial kit (LOEWE® Biochemia, Germany) following the manufacturer’s instructions. Results showed, forty-three of symptomatic samples reacted positively (38 in Tartous and 5 in Lattakia) and none of asymptomatic ones. On the other hand, sap mechanical inoculation of 10 tomato cv. Mandaloun F1 (Enza Zaden, the Netherlands) plants using a positive tomato isolate gave systemic mosaic symptoms in all plants identical to those observed in the original plants in the field, after 13 days of inoculation, and necrotic local lesions on 10 plants of Nicotiana tabacum after 5 days, indicating the presence of a tobamovirus in general. ToBRFV infection was confirmed in all mechanically-inoculated plants by DAS-ELISA. Further tests were necessary to investigate ToBRFV presence, because of its serological relationships with another tobamoviruses. Six representative symptomatic samples (ELISA-positive) and two asymptomatic (ELISA-negative) samples were subjected to total RNA extraction using the SV-Total RNA Extraction kit (Promega, U.S.A.) following the manufacturer’s instructions. The samples were tested by two-step reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers and protocols for most common tomato-infecting viruses, including: tomato chlorosis virus and tomato infectious chlorosis virus (Dovas et al. 2002), pepino mosaic virus (PepMV) and tomato torrado virus (Wieczorek et al. 2013), alfalfa mosaic virus (Parrella et al. 2000), tomato spotted wilt virus (Salem et al. 2012) and a pair of primers: ToBRFV-F2 (5’-CATATCTCTCGACACCAGTAAAAGGACCCG-3’) and ToBRFV-R2 (5’-TCCGAGTATAGGAAGACTCTGGTTGGTC-3’) targeting a region of the RNA dependent RNA polymerase (RdRp), of the ToBRFV genome (KT383474; Salem et al. 2016). First-strand cDNA synthesis was carried out using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Promega) and random primer according to the manufacturer's protocol, then followed by PCR with the seven species-specific primers. Only ToBRFV was detected among all tested viruses in symptomatic samples (ELISA-positive), and none of the tested viruses was detected in the asymptomatic plants. To confirm the presence of ToBRFV, two selected RdRp-specific PCR amplicons (872 bp) were purified and ligated into pGEM T-Easy Vector (Promega), and three clones were sequenced (GenBank accession nos. MZ447794 to 96). BLASTn analysis showed that the nucleotide sequences are 99.77-100% identical and shared around 99% identity to RdRp of ToBRFV isolate (MT118666) from Turkey available in the GenBank. Accordingly, the presence of ToBRFV was confirmed by bioassays on indicator plants, DAS-ELISA, RT-PCR, and further sequencing. To our knowledge, this is the first report of ToBRFV infecting tomato in Syria, and this requires special emphasis for further investigations because of the virus severity, easy transmission ability and absent of commercial resistance varieties till now.

scholarly journals First Report of Wheat streak mosaic virus on Wheat in New Zealand

Plant Disease ◽  
2009 ◽  
Vol 93 (4) ◽  
pp. 430-430 ◽  
Author(s):  
B. S. M. Lebas ◽  
F. M. Ochoa-Corona ◽  
B. J. R. Alexander ◽  
R. A. Lister ◽  
J. D. F. Fletcher ◽  
...  
Keyword(s):  
New Zealand ◽  
Mosaic Virus ◽  
Total Yield ◽  
The United States ◽  
Plant Viruses ◽  
Wheat Streak Mosaic Virus ◽  
Rt Pcr ◽  
Wheat Curl Mite ◽  
Experimental Trials ◽  
Das Elisa

In August of 2005, seeds of wheat (Triticum aestivum) breeding line 6065.3 tested positive for Wheat streak mosaic virus (WSMV; genus Tritimovirus) by a WSMV-specific reverse transcription (RT)-PCR assay (2). The sequence of the 200-bp amplicon (GenBank Accession No. FJ434246) was 99% identical with WSMV isolates from Turkey and the United States (GenBank Accession Nos. AF454455 and AF057533) and 96 to 97% identical to isolates from Australia (GenBank Accession Nos. DQ888801 to DQ888805 and DQ462279), which belong to the subclade D (1). As a result, an extensive survey of three cereal experimental trials and 105 commercial wheat crops grown on the South Island of New Zealand was conducted during the 2005–2006 summer to determine the distribution of WSMV. Wherever possible, only symptomatic plants were collected. Symptoms on wheat leaf samples ranged from very mild mosaic to symptomless. In total, 591 leaf samples suspected to be symptomatic were tested for WSMV by a double-antibody sandwich (DAS)-ELISA (DSMZ, Braunschweig, Germany). Of the 591 symptomatic samples, 81 tested positive. ELISA results were confirmed by RT-PCR with novel forward (WSMV-F1; 5′-TTGAGGATTTGGAGGAAGGT-3′) and reverse (WSMV-R1; 5′-GGATGTTGCCGAGTTGATTT-3′) primers designed to amplify a 391-nt fragment encoding a region of the P3 and CI proteins. Total RNA was extracted from the 81 ELISA-positive leaf samples using the Plant RNeasy Kit (Qiagen Inc., Chatsworth, CA). The expected size fragment was amplified from each of the 81 ELISA-positive samples. The positive samples represent 30 of 56 wheat cultivars (54%) collected from 28 of 108 sites (26%) sampled in the growing regions from mid-Canterbury to North Otago. These results suggest that WSMV is widespread in New Zealand both geographically and within cultivars. WSMV is transmitted by the wheat curl mite (Aceria tosichella) (3), which had not been detected in New Zealand despite repeated and targeted surveys. WSMV is of great economic importance in some countries, where the disease has been reported to cause total yield loss (3). Although WSMV is transmitted by seeds at low rates (0.1 to 0.2%) (4), it is the most likely explanation of the spread of the disease in New Zealand. References: (1) G. I. Dwyer et al. Plant Dis. 91:164, 2007. (2) R. French and N. L. Robertson. J. Virol. Methods 49:93, 1994. (3) R. French and D. C. Stenger. Descriptions of Plant Viruses. Online publication. No. 393, 2002. (4) R. A. C. Jones et al. Plant Dis. 89:1048, 2005.

scholarly journals First Report of Cucumber mosaic virus in Saintpaulia ionantha in Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (4) ◽  
pp. 573-573 ◽  
Author(s):  
J. Y. Yoon ◽  
G. S. Choi ◽  
I. S. Cho ◽  
S. K. Choi
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
First Report ◽  
African Violet ◽  
Saintpaulia Ionantha ◽  
Local Lesions ◽  
Pcr Products ◽  
Das Elisa

African violet (Saintpaulia ionantha) is an ornamental species of the family Gesneriaceae and is characterized by fleshy leaves and colorful flowers. This popular, exotic ornamental, originally from Kenya and Tanzania, is vegetatively produced from cutting and tissue culture (1). In May 2013, virus-like foliar symptoms, including a mosaic with dark green islands and chlorosis surrounding the veins, were observed on an African violet plant in a greenhouse located in Icheon, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with a commercial immunostrip kit (Agdia, Elkhart, IN). The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-AV1) caused necrotic local lesions on Chenopodium amaranticolor at 5 days post-inoculation (dpi), while mild to severe mosaic was observed in Nicotiana glutinosa, N. tabacum ‘Samsun NN,’ Cucurbita pepo ‘Super-Top,’ Physalis angulate, and Solanum lycopersicum ‘Unicorn’ 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ∼28 nm in diameter, respectively. To verify whether CMV-AV1 is the cause of disease symptoms observed in African violet, virus-free African violet (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-AV1. At 8 weeks after inoculation, all plants produced systemic mosaic and chlorosis surrounding veins, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated African violet plants remained symptomless and virus-free. The presence of CMV-AV1 in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CPTALL3 and CPTALL5 (2), amplifying the entire CP gene and part of an intergenic region and 3′-noncoding region of CMV RNA3. RT-PCR products (960 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-AV1 CP sequence (Accession No. AB842275) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-AV1 infection. CMV may pose a major threat for production of African violet since the farming of African violet plants is performed using the vegetative propagation of the African violet leaves in Korea. In particular, mosaic and chlorosis symptoms in African violet cause damage to ornamental quality of African violet. To our knowledge, this is the first report of CMV infection of African violet in the world. References: (1) S. T. Baatvik. Fragm. Flor. Geobot. Suppl. 2:97, 1993. (2) S. K. Choi et al. J. Virol. Methods 83:67, 1999.

scholarly journals First Report of Zantedeschia mosaic virus Infecting a Zantedeschia sp. in New Zealand

Plant Disease ◽  
2008 ◽  
Vol 92 (8) ◽  
pp. 1253-1253 ◽  
Author(s):  
T. Wei ◽  
M. N. Pearson ◽  
D. Cohen ◽  
J. Z. Tang ◽  
G. R. G. Clover
Keyword(s):  
New Zealand ◽  
Amino Acid ◽  
Mosaic Virus ◽  
Amino Acid Identity ◽  
Cut Flowers ◽  
Rt Pcr ◽  
Acid Identity ◽  
Virus Particles ◽  
Blast Search ◽  
Cp Gene

In February 2004, leaf yellowing, mottling, and mosaics were observed on a few plants of a Zantedeschia sp. (calla lily) growing in Rangiora, Canterbury, New Zealand. Zantedeschia spp. are known to be susceptible to at least 13 virus species (1). No symptoms were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis, or N. tabacum when inoculated with sap from symptomatic plants. However, electron microscopy of crude sap preparations from a symptomatic Zantedeschia sp. and inoculated N. clevelandii plants revealed the presence of flexuous, filamentous virus particles approximately 700 nm long and 12 nm wide. No virus particles were seen in the other inoculated indicator species. Nucleic acid was extracted from leaves of the infected Zantedeschia sp. and N. clevelandii plants and tested in reverse transcription (RT)-PCR using published potyvirus-specific primers (4). PCR amplicons of the expected size (327 bp) were obtained from both plant species and sequenced directly. The products were identical, and a BLAST search in GenBank showed 99% nucleotide identity with a Taiwanese isolate of the species Zantedeschia mosaic virus (ZaMV) (GenBank Accession No. AY026463). A product of 1,531 bp (GenBank Accession No. EU544542) was amplified from symptomatic Zantedeschia by RT-PCR using novel forward (5′-GCACGGCAGATAAACACGAC-3′) and reverse (5′-GTGGGCAACCTTCAACTGTG-3′) primers designed to amplify the 3′ untranslated region (3′UTR), coat protein (CP), and partial nuclear inclusion b protein (NIb) genes. The product was sequenced and had 94% nucleotide identity with a South Korean ZaMV isolate (GenBank Accession No. AB081519), with 95% nucleotide (97% amino acid) identity in the CP gene. A second crop of Zantedeschia spp. in Tauranga, New Zealand (approximately 700 km north of Rangiora) was observed to have similar disease symptoms. Symptomatic plants tested positive in ELISA using a potyvirus-specific monoclonal antibody (Agdia Inc., Elkhart, IN). Nucleic acid was extracted from leaves of symptomatic plants and tested in RT-PCR using potyvirus-specific primer pairs, PV2I/T7 and D335 and U335 and PV1/SP6, which amplify overlapping regions within the 3′UTR, CP, and NIb genes (2,3). The products were sequenced and a consensus sequence of 1,793 bp was generated (GenBank Accession No. EU532065). A BLAST search showed that the sequence had 78% nucleotide (88% amino acid) identity with Zantedeschia mild mosaic virus (ZaMMV) (GenBank Accession No. AY626825). However, the sequences had only 73% nucleotide (79% amino acid) identity in the CP gene, and therefore, this second virus may be a distinct species. To our knowledge, this is the first report of ZaMV in New Zealand. Cut flowers are an increasingly important commodity in New Zealand and Zantedeschia is one of the most important crops; in 2005, exports of rhizomes and cut flowers of the genus were worth NZ$10.9 million. These viral diseases may require management to ensure that the quality of production is maintained. References: (1) C. H. Huang et al. Plant Pathol. 56:183, 2007. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) A. M. Mackenzie et al. Arch. Virol. 143:903, 1998. (4) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000.

scholarly journals First Report of Cucumber mosaic virus in Catharanthus roseus in Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1283-1283
Author(s):  
S.-K. Choi ◽  
I.-S. Cho ◽  
G.-S. Choi ◽  
J.-Y. Yoon
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Catharanthus Roseus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
First Report ◽  
Bedding Plant ◽  
Local Lesions ◽  
Pcr Products ◽  
Das Elisa

Catharanthus roseus, commonly known as Madagascar rosy periwinkle (also called vinca), is a tropical perennial herb of the family Apocyanaceae. Periwinkle is a bedding plant widely used in Korea because of its drought tolerance, low maintenance, and varied flower colors. In May 2013, virus-like foliar symptoms, including a mosaic with malformation of leaves, were observed on a periwinkle plant in a greenhouse located in Chonbuk Province, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with an immune-strip kit developed by our laboratory. The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia, Elkhart, IN). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-Vin) caused necrotic local lesions on Chenopodium amaranticolor at 5 days-post-inoculation (dpi), while mild to severe mosaic was observed in Capsicum annuum, Cucumis sativus, Cucurbita pepo ‘Cheonggobong,’ Nicotiana glutinosa, N. tabacum‘Samsun NN,’ Physalis angulate, and Solanum lycopersicum ‘Pink-Top’ 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ~28 nm in diameter, respectively. To verify whether CMV was the causal agent for the disease symptoms observed in naturally infected periwinkle, virus-free periwinkle (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-Vin. At 6 weeks after inoculation, all plants produced systemic mosaic and distortion of leaves, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated periwinkle plants remained symptomless and virus-free. The presence of CMV-Vin in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with a RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CMVCPFor and CMVCPRev (1), which amplified the entire CP gene. RT-PCR products (657 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from a positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-Vin CP sequence (Accession No. AB910598) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-Vin infection. Being perennial, periwinkle plants could serve as a reservoir for CMV to infect other ornamentals and cultivated crops (2). To our knowledge, this is the first report of CMV infection on periwinkle in Korea. References: (1) S. K. Choi et al. Virus Res. 158:271, 2011. (2) P. Palukaitis et al. Adv. Virus. Res. 41:281, 1992.

scholarly journals Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants

2021 ◽  
Author(s):  
Katarzyna Trzmiel
Keyword(s):  
Mosaic Virus ◽  
Simultaneous Detection ◽  
Brome Mosaic Virus ◽  
Mottle Virus ◽  
Grass Species ◽  
Replicase Gene ◽  
Rt Pcr ◽  
Pcr Products ◽  
Cereal Plants ◽  
Cocksfoot Mottle Virus

AbstractBrome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

scholarly journals Isolado do vírus do mosaico do pepino obtido de bananeira no Estado de São Paulo pertence ao subgrupo Ia

2001 ◽  
Vol 26 (1) ◽  
pp. 53-59 ◽  
Author(s):  
MARCELO EIRAS ◽  
ADDOLORATA COLARICCIO ◽  
ALEXANDRE L.R. CHAVES
Keyword(s):  
Mosaic Virus ◽  
Vigna Unguiculata ◽  
Sao Paulo ◽  
São Paulo ◽  
Rt Pcr ◽  
Nicotiana Glutinosa ◽  
Musa Sp ◽  
De Se ◽  
Pcr Rflp ◽  
Das Elisa

Em 1996, foi feita a caracterização parcial de um isolado do vírus do mosaico do pepino (Cucumis mosaic virus, CMV) obtido de bananeira (Musa sp.) proveniente do município de Miracatu, SP. Com o objetivo de se determinar o subgrupo do isolado de CMV, recorreu-se às técnicas de ELISA, RT-PCR, RFLP e seqüenciamento de fragmentos de RNA genômico. Amostras de folhas infetadas, desidratadas com cloreto de cálcio e armazenadas à -20 °C desde 1994 na viroteca do Laboratório de Fitovirologia e Fisiopatologia, foram inoculadas em plantas de Nicotiana glutinosa. Dez dias após a inoculação, folhas apresentando mosaico foram utilizadas para DAS-ELISA e extração de RNAs totais. Em ELISA, houve reação apenas contra o anti-soro específico para CMV subgrupo I. Através de RT-PCR com primers desenhados para anelar em regiões conservadas da porção terminal 3' do gene da capa protéica, foi amplificado um fragmento de DNA com 486 pares de bases. O produto obtido via RT-PCR foi submetido à digestão com as enzimas EcoRI, HindIII, BamHI e MspI, obtendo-se um padrão de restrição esperado para o subgrupo I. Estes resultados foram confirmados através do seqüenciamento do produto de PCR, o qual apresentou homologia de 96% a 98% com os isolados do CMV pertencentes ao subgrupo I. Pelos sintomas observados na hospedeira diferencial Vigna unguiculata, o isolado foi confirmado como sendo do subgrupo Ia.

scholarly journals Variabilidade genética de Sugarcane mosaic virus, causando mosaico em milho no Brasil

2011 ◽  
Vol 46 (4) ◽  
pp. 362-369 ◽  
Author(s):  
Marcos Cesar Gonçalves ◽  
Diogo Manzano Galdeano ◽  
Ivan de Godoy Maia ◽  
César Martins Chagas
Keyword(s):  
Mosaic Virus ◽  
Sao Paulo ◽  
Sugarcane Mosaic Virus ◽  
Maize Dwarf Mosaic Virus ◽  
São Paulo ◽  
Rt Pcr ◽  
Das Elisa

O objetivo deste trabalho foi caracterizar biológica e molecularmente três isolados de Sugarcane mosaic virus (SCMV) de lavouras de milho, analisá-los filogeneticamente e discriminar polimorfismos do genoma. Plantas com sintomas de mosaico e nanismo foram coletadas em lavouras de milho, no Estado de São Paulo e no Município de Rio Verde, GO, e seus extratos foliares foram inoculados em plantas indicadoras e submetidos à análise sorológica com antissoros contra o SCMV, contra o Maize dwarf mosaic virus (MDMV) e contra o Johnsongrass mosaic virus (JGMV). Mudas de sorgo 'Rio' e 'TX 2786' apresentaram sintomas de mosaico após a inoculação dos três isolados, e o DAS-ELISA confirmou a infecção pelo SCMV. O RNA total foi extraído e usado para amplificação por transcriptase reversa seguida de reação em cadeia de polimerase (RT-PCR). Fragmentos específicos foram amplificados, submetidos à análise por polimorfismo de comprimento de fragmento de restrição (RFLP) e sequenciados. Foi possível discriminar os genótipos de SCMV isolados de milho de outros isolados brasileiros do vírus. Alinhamentos múltiplos e análises dos perfis filogenéticos corroboram esses dados e mostram diversidade nas sequências de nucleotídeos que codificam para a proteína capsidial, o que explica o agrupamento separado desses isolados e sugere sua classificação como estirpes distintas, em lugar de simples isolados geográficos.

scholarly journals Short communication: Pepino mosaic virus, a new threat for Serbia’s tomatoes

2020 ◽  
Vol 18 (4) ◽  
pp. e10SC05
Author(s):  
Ivana Stankovic ◽  
Ana Vucurovic ◽  
Katarina Zecevic ◽  
Branka Petrovic ◽  
Danijela Ristic ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rt Pcr ◽  
Tomato Production ◽  
Pepino Mosaic Virus ◽  
Tomato Plants ◽  
Rapid Spread ◽  
Infected Tomato ◽  
Dark Green ◽  
Relationship Of ◽  
Das Elisa

Aim of study: To report the occurrence of Pepino mosaic virus (PepMV) on tomato in Serbia and to genetically characterize Serbian PepMV isolates.Area of study: Tomato samples showing virus-like symptoms were collected in the Bogojevce locality (Jablanica District, Serbia).Material and methods: Collected tomato samples were assayed by DAS-ELISA using antisera against eight economically important or quarantine tomato viruses. Three selected isolates of naturally infected tomato plants were mechanically transmitted to tomato ‘Novosadski jabučar’ seedlings. For confirmation of PepMV infection, RT-PCR was performed using specific primers PepMV TGB F/PepMV UTR R. Maximum-likelihood phylogenetic tree was constructed with 47 complete CP gene sequences of PepMV to determine the genetic relationship of Serbian PepMV isolates with those from other parts of the world.Main results: The results of DAS-ELISA indicated the presence of PepMV in all tested samples. Mechanically inoculated ‘Novosadski jabučar’ seedlings expressed yellow spots and light and dark green patches, bubbling, and curled leaves. All tested tomato plants were RT-PCR positive for the presence of PepMV. The CP sequence analysis revealed that the Serbian PepMV isolates were completely identical among themselves and shared the highest nucleotide identity of 95.1% (99.2% aa identity) with isolate from Spain (FJ263341). Phylogenetic analysis showed clustering of the Serbian PepMV isolates into CH2 strain, but they formed separate subgroup within CH2 strain.Research highlights: This is the first data of the presence of PepMV in protected tomato production in Serbia. Considering increased incidence and rapid spread in Europe, the presence of PepMV on tomato could therefore represent serious threat to this valuable crop in Serbia.

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