scholarly journals Virus Detection and Production of Virus Free Plant Materials from the Lily by Selecting Basal Filament Flower as Explants

2021 ◽  
pp. 94-103
Author(s):  
Yanjie Lv ◽  
Yajun Dou ◽  
Halizeremu Saidahemaiti ◽  
Xiangfeng He ◽  
Xiangxun Zhao ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Economic Value ◽  
Virus Detection ◽  
Pcr Detection ◽  
Economic Losses ◽  
Rt Pcr ◽  
Plant Materials ◽  
Lily Symptomless Virus ◽  
Virus Removal ◽  
Das Elisa

Lilium is a perennial bulbous flower of Lily family Liliaceae, with high ornamental and economic value. However, Lily is vulnerable to virus infection, which seriously affects the yield and quality of Lily, and poses a great threat to the production, sales, especially export of Lily, and has caused huge economic losses to the related industries. Therefore, the research on lily virus removal methods and virus detection technology has important practical significance to improve the ornamental value and economic value of lily. In this study, the filaments of four susceptible lily varieties,' Valdisole' (A),'Adoration'(LA),' Ice Cube'(OT) and ‘Zantriana’ (O), were used as explants. The filaments of lily were divided into three parts, namely, top, middle, and base. In this paper, the virus detection of tissue culture seedlings induced by lily filaments was carried out by using DAS-ELISA and RT-PCR, and the removal effects of Cucumber mosaic virus,(CMV) and lily symptomless virus (LSV), two common viruses in lily, were explored, and the two detection technologies were compared. The results showed that the success rate of tissue culture seedlings induced by filament base was the highest, and CMV virus could be basically removed. RT-PCR detection is more sensitive than DAS-ELISA detection, but RT-PCR detection requires higher test conditions and technology. Therefore, appropriate virus detection methods can be selected according to actual conditions and severity.

scholarly journals An Evidence-Based Approach to Plum Pox Virus Detection by DASI-ELISA and RT-PCR in Dormant Period

2008 ◽  
Vol 1 ◽  
pp. VRT.S495 ◽  
Author(s):  
Antonio Olmos ◽  
Edson Bertolini ◽  
Nieves Capote ◽  
Mariano Cambra
Keyword(s):  
Virus Detection ◽  
Screening Tests ◽  
Plum Pox Virus ◽  
Evidence Based ◽  
Rt Pcr ◽  
Likelihood Ratios ◽  
Dormant Period ◽  
Plant Materials ◽  
Calculation Of Parameters ◽  
Post Test

An evidence-based approach, such as those developed in clinical and veterinary medicine, was applied to the detection of Plum pox virus (PPV) during the dormant period. A standardized methodology was used for the calculation of parameters of the operational capacity of DASI-ELISA and RT-PCR in wintertime. These methods are routinely handled to test the sanitary status of plants in national or international trading and in those cases concerning export-import of plant materials. Diagnosis often has to be performed during the dormant period, when plant material is commercialized. Some guidelines to interpret diagnostic results of wintertime are provided in an attempt to minimize risks associated with the methods and over-reliance on the binary outcome of a single assay. In order to evaluate if a complementary test increased the confidence of PPV diagnosis when discordant results between DASI-ELISA and RT-PCR are obtained, NASBA-FH also was included. Likelihood ratios of each method were estimated based on the sensitivity and specificity obtained in wintertime. Subsequently, a Bayesian approach was performed to calculate post-test probability of PPV infection in spring. Results of evidence-based approach show that different PPV prevalences require different screening tests. Thus, at very low PPV prevalence levels DASI-ELISA should be used as the election method, whilst at the highest PPV prevalence levels RT-PCR should be performed. NASBA-FH could be used at medium prevalences to clarify discordances between DASI-ELISA and RT-PCR.

scholarly journals Real-time RT-PCR detection of Citrus bark cracking viroid (CBCVd) in hops including an mRNA-based internal positive control

2020 ◽  
Vol 127 (6) ◽  
pp. 763-767
Author(s):  
Luitgardis Seigner ◽  
Marion Liebrecht ◽  
Linda Keckel ◽  
Katharina Einberger ◽  
Carolin Absmeier
Keyword(s):  
Real Time ◽  
Detection Method ◽  
Plant Protection ◽  
False Negative ◽  
Positive Control ◽  
Pcr Detection ◽  
Economic Losses ◽  
Rt Pcr ◽  
Validation Data ◽  
Internal Positive Control

Abstract Citrus bark cracking viroid (CBCVd), formerly known as pathogen in the genus Citrus and first detected in Slovenian hops in 2014, threatens hop production as it leads to important economic losses. Reduction in yield and quality and even death of the infected plants within a few years are typical observations due to CBCVd infections of hops. The viroid is easily transmitted and spreads rapidly. As it cannot be controlled by plant protection measures, avoiding its introduction into hop gardens and eradicating first centres of infection are of utmost importance. An indispensable prerequisite is a reliable detection method suitable for large-scale routine testing. In this study, the development of primers and probe for real-time RT-PCR for sensitive CBCVd detection is described. To exclude “false negative” results, a nad5 mRNA-based internal positive control was included. To our knowledge, this is the first time such a duplex real-time RT-PCR detection method for CBCVd at least in hops is described. In addition, first method validation data are presented.

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 First Report of Cichorium endivia (Asteraceae) as a Natural Host of Groundnut ringspot orthotospovirus in Brazil.

Plant Disease ◽  
2020 ◽  
Author(s):  
Tiago Silva Jorge ◽  
Mirtes Freitas Lima ◽  
Leonardo Silva Boiteux ◽  
Maria Esther N. Fonseca ◽  
Elliot W. Kitajima
Keyword(s):  
Untranslated Region ◽  
Natural Infection ◽  
Plant Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nucleotide Sequencing ◽  
Rt Pcr ◽  
Plant Materials ◽  
First Report ◽  
Cichorium Endivia ◽  
Das Elisa

Endive (Cichorium endivia L.) is a very important cash crop for small farmers in Brazil. During inspections conducted in the summer season of 2019–2020, leaf samples of C. endivia ‘La Spezia’ seedlings exhibiting typical symptoms of orthotospoviruses infection (viz. concentric chlorotic spots and apical leaf deformation; ≈ 10%) were collected in commercial greenhouses in Brasília–DF, Central Brazil. Leaves of one healthy and three symptomatic plants were initially evaluated via double antibody sandwich enzyme-linked immunosorbent assay (DAS–ELISA) with polyclonal antibodies (produced at CNPH) raised against the nucleoprotein of the three major orthotospoviruses: tomato spotted wilt orthotospovirus (TSWV), groundnut ringspot orthotospovirus (GRSV) and tomato chlorotic spot orthotospovirus (TCSV). Strong serological reactions were observed only against GRSV antibodies exclusively in extracts from symptomatic samples. In order to confirm the causal agent of those symptoms, total RNA was extracted (Trizol®; Sigma) from infected leaf samples and used in a two-step reverse transcriptase polymerase chain reaction (RT–PCR) approach. Synthesis of the cDNA was carried out with the J13 primer (5’–CCC GGA TCC AGA GCA AT–3’) (Cortez et al., 2001) followed by PCR assays with the primer pair BR60 (5’–AGA GCA ATC GTG TCA–3`) and BR65 (5’–ATC AAG CCT TCT GAA AGT CAT–3’) (Eiras et al., 2001). This primer set amplifies a fragment of 453 bp including the untranslated region at the 3’ terminus of the small RNA and the protein N–coding gene of at least five orthotospoviruses: TSWV, GRSV, TCSV, chrysanthemum stem necrosis orthotospovirus (CSNV) and zucchini lethal chlorosis orthotospovirus (ZLCV) (Eiras et al., 2001). The obtained amplicons (≈ 432 bp) were subsequently subjected to Sanger dideoxy nucleotide sequencing at CNPH. BLASTn analysis showed >99% identity with a wide array of GRSV isolates available in the GenBank. The nucleotide sequence of Tospo #1 (MT215222) and Tospo #3 (MT215224) isolates displayed 100% identity between them, whereas the Tospo #2 (MT215223) isolate displayed one non–synonymous point mutation in the 3’ untranslated region in comparison with the former two isolates. Three plants of C. endivia, Capsicum annuum L. cv. Ikeda, tomato (Solanum lycopersicum L.) cv. Santa Clara and its isoline ‘LAM–147’ (with the Sw–5 resistance gene), Nicotiana rustica L., Lactuca sativa L. (‘Vanda’ and ‘PI-342444’) and Gomphrena globosa L. were mechanically inoculated individually with each GRSV isolate in order to confirm their pathogenicity. Chlorotic lesions and mosaic were observed seven days after inoculation of all plant materials, except the tomato inbred line ‘LAM–147’, which has the Sw-5 gene that confers broad–spectrum resistance to all Brazilian orthotospoviruses (Boiteux and Giordano, 1993). The GRSV infection was confirmed via DAS–ELISA and RT–PCR 15 days after inoculation, using the same set of antibodies and the primer pair BR60 / BR65. Transmission electron microscopy of ultrathin sections from symptomatic leaf tissues, both from field–infected and experimentally inoculated endive revealed the presence of typical orthotospovirus particles, within endoplasmic reticulum cisternae. Natural infection of endive by TSWV has been reported in Greece (Chatzivassiliou et al., 2000) and by TCSV in São Paulo State, Brazil and in Florida, USA (Subramanya Sastry et al., 2019). To our knowledge, it is the first report of GRSV naturally infecting this Asteraceae species in Brazil. Confirmation of GRSV infection of C. endivia plants is a relevant piece of information aiming to design effective disease management strategies. References: Boiteux, L.S. and Giordano, L. B. 1993. Euphytica 71: 151. Eiras, M. et al. 2001. Fitopatol. Bras. 26: 170. Chatzivassiliou, E.K. et al. 2000 Ann. Appl. Biol. 137: 127. Cortez, I., et al. 2001. Arch. Virol. 146: 265. Subramanya Sastry, K., et al. 2019. Encyclopedia of plant viruses and viroids. Springer, New Delhi. https://doi.org/10.1007/978-81-322-3912-3.

scholarly journals Diagnostics of Grapevine fanleaf virus

Biljni lekar ◽  
2021 ◽  
Vol 49 (1) ◽  
pp. 54-64
Author(s):  
Đina Konstantin ◽  
Goran Barać ◽  
Renata Iličić ◽  
Ferenc Bagi
Keyword(s):  
Visual Inspection ◽  
Virus Detection ◽  
Plant Viruses ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Economic Losses ◽  
Rt Pcr ◽  
Grapevine Fanleaf Virus ◽  
Qrt Pcr ◽  
Propagation Material

Plant viruses cause considerable economic losses and are a threat for sustainable agriculture. Due to the multiple possibilities of infection, they have become widespread. The use of healthy propagation material, free of viroids, viruses and bacteria, is an important strategy in disease control in viticulture. The early and accurate detection of plant viruses is an essential component of their control. Due to the widespread of Grapevine fanleaf virus (GFLV) and its devastating potential, various diagnostic methods are being used. GFLV detection methods based on the specificity of the protein cover (ELISA) and nucleic acid-based virus detection methods (RT-PCR, qRT-PCR). Symptoms of viral diseases are often not distinct and can be confused with those of abiotic stresses, so visual inspection is not reliable enough.

scholarly journals Effect of Antivirals on the Elimination of Lily Symptomless Virus in Lilium Oriental Hybrid `Casa Blanca'

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 822E-823
Author(s):  
Jin Ha Woo* ◽  
Hyo Hoon Nam ◽  
Hyun Suk Lee ◽  
Kyeong Bae Choi ◽  
Jea Tak Yoon ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Survival Rate ◽  
Complete Elimination ◽  
Plant Materials ◽  
Culture In Vitro ◽  
Lily Symptomless Virus ◽  
Lower Survival Rate ◽  
Lower Survival ◽  
Oriental Lily

This study was conducted to find out the effect of antivirals during tissue culture for lily symptomless virus (LSV) free stock production of Oriental lily. Scales and bulblets of LSV-infected Lilium Oriental hybrids `Casa Blanca' were used as plant materials. Scales and bulblets were cultured in Murashige and Skoog (MS, 1962) medium added 20 μM of virazole, and 0, 20, 40, and 80 μM of azauracil or thiouracil for 12 weeks prior to LSV detection to examine the effect of antivirals. The survival rate in scaling was observed to be lower than bulblet culture in vitro and higher concentration of azauracil and thiouracil also effected on lower survival rate. The enlargement of bulblet tended to be promoted in low concentration without regard to kinds of antivirals, and especially most effective in 20 μM of azauracil. Elimenation rate of LSV was 0% in control, on the contrary, antivirals treatment which resulted in complete elimination of LSV.

scholarly journals First Report of Soybean dwarf virus on Soybean in Wisconsin

Plant Disease ◽  
2004 ◽  
Vol 88 (11) ◽  
pp. 1285-1285 ◽  
Author(s):  
A. Phibbs ◽  
A. Barta ◽  
L. L. Domier
Keyword(s):  
Leaf Tissue ◽  
Dwarf Virus ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Elisa Testing ◽  
Soybean Plants ◽  
Soybean Dwarf Virus ◽  
Das Elisa

Soybean dwarf virus (SbDV) causes widespread economic losses on soybean (Glycine max (L.) Merr.) in Japan (4), and has been reported on soybean in Virginia (2), in various legumes in the southeastern United States (1), and in peas in California (3). During late July and early August of 2003, soybean plants in Wisconsin were surveyed for SbDV. In 286 soybean fields at the R2-R4 growth stage, the uppermost fully unfurled leaf was collected from 10 plants at each of five sites. Samples were collected at random without regard to symptoms. SbDV symptom information was not recorded. Samples were stored on ice until frozen at -80°C. Five fields in four Wisconsin counties (Columbia, Lafayette, Sauk, and Waushara) tested positive for SbDV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). DAS-ELISA testing was conducted with reagents from Agdia, Inc (Elkhart, IN) following the manufacturer's protocol. Absorbance was read at 405 nm with a Stat Fax 2100 microplate reader (Awareness Technology, Inc., Palm City, FL) or visually evaluated. DAS-ELISA did not discriminate between strains of SbDV. The presence of SbDV was confirmed, and strain identity was inferred as dwarfing strain using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from homogenized leaf tissue, reverse transcribed, and amplified with the SuperScript One Step RT-PCR System (Invitrogen, Carlsbad, CA) and SbDV-specific primers (5′-CTGCTTCTGGTGATTACACTGCCG-3′ and 5′-CGCTTTCATTTAACGYCATCAAAGGG-3′). Size of the RT-PCR products (110 bp) was consistent with the dwarfing strain, SbDV-D. All locations that tested positive for SbDV showed soybean aphids, Aphis glycines Matsumura (Homoptera: Aphididae), on 100% of soybean plants. Several aphid species have been reported to vector SbDV, but at this time, vector relations in the Wisconsin infections are unknown. To our knowledge, this is the first report of SbDV infecting soybean in Wisconsin. References: (1) V. D. Damsteegt et al. Plant Dis. 79:48, 1995. (2) A. Fayad et al. Phytopathology (Abstr.) 90(Suppl.):S132, 2000. (3) G. R. Johnstone et al. Phytopathology (Abstr.) 74:795(A43), 1984. (4) T. Tamada et al. Ann. Phytopathol. Soc. Jpn. 35:282, 1969.

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 Detection and Frequency of Lily Viruses in Argentina

Plant Disease ◽  
2010 ◽  
Vol 94 (10) ◽  
pp. 1188-1194 ◽  
Author(s):  
S. C. Chinestra ◽  
C. Facchinetti ◽  
N. R. Curvetto ◽  
P. A. Marinangeli
Keyword(s):  
Virus Infection ◽  
Virus Detection ◽  
Lilium Longiflorum ◽  
Polyclonal Antiserum ◽  
Control Measures ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mixed Infections ◽  
Lily Symptomless Virus ◽  
Virus Incidence ◽  
Das Elisa

In a survey of lily growing fields in various regions of Argentina, three viruses, Lily symptomless virus (LSV), Lily mottle virus (LMoV), and Cucumber mosaic virus (CMV), were found in Longiflorum, Asiatic, Oriental, Longiflorum × Asiatic (LA), and Oriental × Trumpet (OT) hybrids. The areas surveyed were between latitude 26° 56′ S and 43° 03′ S, and longitude 65° 21′ W and 71° 29′ W. Virus detection was performed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal antiserum. In infected samples, viruses detected in decreasing order were LSV (60.5%), LMoV (51.0%), and CMV (28.7%) present in single or mixed infections. Virus infection varied among tested hybrids from 36.0% (Oriental Montecristo) to 94.7% (Lilium longiflorum Avita) in 2006 and from 38.9% (OT Yelloween) to 82.1% (LO Triumphator) in 2007, with an overall incidence of 64.1 and 70.7% in 2006 and 2007, respectively. A variation in virus incidence among localities was also observed. The highest virus incidence (89.6 and 87.6% in 2006 and 2007, respectively) was observed in Bahía Blanca (38° 44′ S, 62° 16′ W). The lowest virus incidences, detected in Trevellin (43° 03′ S, 71° 29′ W) and in Malargüe (35° 28′ S, 69° 35′ W), were 47.4 and 48.6% in 2006 and 2007, respectively. Moreover, a different distribution of each virus was observed between localities. The high occurrence of viruses infecting lily crops in Argentina could be due to both the use of infected bulbs for propagation and the lack of preventive virus vector control measures.

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