Identification of Maize dwarf mosaic virus in Maize in Poland

From 2005 to 2007 in Southern Wielkopolska, Lower Silesia, and Malopolska regions, maize (Zea mays) plants showing leaf mosaic and stunting symptoms were found. ELISA tests using commercial polyclonal antisera against Maize dwarf mosaic virus (MDMV) obtained from Bioreba (Basel, Switzerland) and Loewe (Munich, Germany) gave positive results in 71 samples. However, the ELISA response for symptomatic plants, in most cases, was low, with OD values ranging from 0.05 to 0.18. Therefore, only eight plants with relatively high virus concentration were chosen for further identification assays. Examination of leaf extracts with an electron microscope revealed the presence of potyvirus-like particles. Symptomatic leaves were positive for MDMV by using immunosorbent electron microscopy (ISEM) with antiserum raised against the Spanish isolate of MDMV (supplied as positive MDMV control from A. Achon, Centre Vdl-Irta, Lleida, Spain). A set of test plants, including sweet corn, dent corn, sorghum (Sorghum vulgare), and true millet (Panicum miliaceum), were mechanically inoculated with extracts from symptomatic plants in 0.05 M phosphate buffer plus 1% β-mercaptoethanol. Inoculated plants developed symptoms typical of MDMV in 2 to 5 weeks (1,2). For further investigations, three virus isolates were chosen. To confirm the identification of MDMV, reverse transcription (RT)-PCR was performed with total RNA isolated from infected plants with primers 3MDF (5′ GAT GAG TTR AAY GTY TAT GCA CGA C 3′), a forward primer in the 3′ region of NIb gene and either 1MDR (5′ RTG CAT RAT TTG TCT GAA AGT TGG 3′) or 3MDR (5′ ACC AVA CCA TYA TWC CAC TC 3′), reverse primers in the 3′ region of the coat protein gene (A. Zare, Shiraz University, personal communication). 3MDF corresponds to nucleotides 8306 to 8330, 3MDR is complementary to nucleotides 8791 to 8813, and 1MDR is complementary to nucleotides 8917 to 8939 of the MDMV genome (GenBank Accession No. AJ001691). The RT-PCR products obtained were analyzed by agarose gel electrophoresis. Amplicons of the expected sizes (635 and 560 bp) were obtained with RNA from symptomatic plants, but not from asymptomatic plants. The sequence of the 576-bp PCR product was deposited in GenBank (Accession No. EU240460). In alignments done with BlastN ( www.ncbi.nlm.nih.gov/blast ), the highest nucleotide sequence identities were 99% with Spanish MDMV isolates (“SP” AM110758, “SP” AJ416645, and “S1” AJ416635), 91% with the Hungarian isolate “Sc/H, sweet corn” AJ542536, 90% with “MDMV-A” U07216, and 87% with an Israeli MDMV (AF395135). On the basis of these findings, the virus isolated from diseased maize plants was identified as MDMV. The significance of MDMV detection is noteworthy because maize has become an important crop in Poland in recent years and acreage is increasing systematically. References: (1) M. A. Achon et al. Eur. J. Plant Pathol. 102:697, 1996. (2) A. J. Gibbs. Maize dwarf mosaic virus. Page 752 in: Viruses of Plants. Descriptions and Lists from the VIDE database. A. A. Brunt et al., eds. CAB International, Wallingford, UK, 1996.

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

Molecular Biology Reports ◽

10.1007/s11033-021-06485-9 ◽

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.

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

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2011000400004 ◽

2011 ◽

Vol 46 (4) ◽

pp. 362-369 ◽

Cited By ~ 10

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.

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First Report of Yam mild mosaic virus in Yam in Guangxi Province, China

Plant Disease ◽

10.1094/pdis-04-11-0350 ◽

2011 ◽

Vol 95 (10) ◽

pp. 1320-1320 ◽

Cited By ~ 4

Author(s):

C. Zou ◽

J. Meng ◽

Z. Li ◽

M. Wei ◽

J. Song ◽

...

Keyword(s):

Mosaic Virus ◽

Viral Genome ◽

Terminal Region ◽

Mild Mosaic ◽

Rt Pcr ◽

Degenerate Primers ◽

Germplasm Exchange ◽

First Report ◽

Pcr Products ◽

Mild Mosaic Virus

Yams (Dioscorea spp.) are widely grown in China as vegetables and herbal medicine. However, studies on viral diseases on yams are still limited. As a pilot project of a government initiative for improving yam productivity, a small study was conducted in Guangxi, a southern province of China, on viral disease in yams. Incidence of virus-like disease for the three extensively grown D. alata cultivars, GH2, GH5, and GH6, were 12 to 40%, 12 to 29%, and 11 to 25%, respectively, as found in a field survey with a five-plot sampling method in 2010. A total of 112 leaf samples showing mosaic or mottling or leaves without symptoms were collected from the cvs. GH2, GH5, GH6, and seven additional cultivars (D. alata cvs. GY2, GY23, GY47, GY69, GY62, GY72, and D. batatas cv. Tiegun). To determine if the symptoms were caused by Yam mild mosaic virus (YMMV; genus Potyvirus, family Potyviridae), total RNA was extracted from leaves with a commercial RNA purification kit (TIANGEN, Beijing, China), and reverse-transcription (RT)-PCR was conducted with a YMMV-specific primer pair (4) that amplifies the 3′-terminal portion of the viral genome. A PCR product with the predicted size of 262 bp was obtained from samples of GH5 (number testing positive of total number of leaves = 5 of 12), GH6 (24 of 42), and GY72 (1 of 1), but not from asymptomatic leaves. PCR products from a GH5 sample (YMMV-Nanning) and a GH6 sample (YMMV-Luzhai) were cloned and sequenced using an ABI PRISM 3770 DNA Sequencer. The two PCR products were 97% identical at nucleotide (nt) level and with the highest homology (89% identity) to a YMMV isolate (GenBank Accession No. AJ305466). To further characterize the isolates, degenerate primers (2) were used to amplify viral genome sequence corresponding to the C-terminal region of the nuclear inclusion protein b (NIb) and the N-terminal region of the coat protein (CP). These 781-nt fragments were sequenced and a new primer, YMMV For1 (5′-TTCATGTCGCACAAAGCAGTTAAG-3′) corresponding to the NIb region, was designed and used together with primer YMMV UTR 1R to amplify a fragment that covers the complete CP region of YMMV by RT-PCR. These 1,278-nt fragments were sequenced (GenBank Accession Nos. JF357962 and JF357963). CP nucleotide sequences of the YMMV-Nanning and YMMV-Luzhai isolates were 94% similar, while amino acid sequences were 99% similar. BLAST searches revealed a nucleotide identity of 82 to 89% and a similarity of 88 to 97% for amino acids to sequences of YMMV isolates (AF548499 and AF548519 and AAQ12304 and BAA82070, respectively) in GenBank. YMMV is known to be prevalent on D. alata in Africa and the South Pacific, and has recently been identified in the Caribbean (1) and Colombia (3). To our knowledge, this is the first report of the natural occurrence of YMMV in China and it may have implications for yam production and germplasm exchange within China. References: (1) M. Bousalem and S. Dallot. Plant Dis. 84:200, 2000. (2) D. Colinet et al. Phytopathology 84:65, 1994. (3) S. Dallot et al. Plant Dis. 85:803, 2001. (4) R. A. Mumford and S. E. Seal. J. Virol. Methods 69:73, 1997.

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Alternanthera mosaic virus Found in Scutellaria, Crossandra, and Portulaca spp. in Florida

Plant Disease ◽

10.1094/pd-90-0833c ◽

2006 ◽

Vol 90 (6) ◽

pp. 833-833 ◽

Cited By ~ 11

Author(s):

C. A. Baker ◽

L. Breman ◽

L. Jones

Keyword(s):

Electron Microscopy ◽

United States ◽

Mosaic Virus ◽

Plant Species ◽

The United States ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Indicator Plants ◽

Partial Identity ◽

Pcr Products

In the fall of 1998, the Division of Plant Industry (DPI) received vegetative propagations of Scutellaria longifolia (skullcap) with symptoms of foliar mosaic, chlorotic/necrotic ringspots, and wavy line patterns from a nursery in Manatee County. Flexuous particles approximately 500 nm long were found with electron microscopy. The plants tested positive for Papaya mosaic virus (PaMV) in an enzyme-linked immunosorbent assay (ELISA) test with antiserum to PaMV (Agdia, Elkhart, IN). However, in immunodiffusion tests (antiserum from D. Purcifull, University of Florida), this virus gave a reaction of partial identity indicating it was related but not identical to PaMV (1). The original infected plants were kept in a greenhouse. In January 2005, a specimen of Crossandra infundibuliformis (firecracker plant) with mosaic symptoms was submitted to the DPI from a nursery in Alachua County. Inclusions found with light microscopy and particles found with electron microscopy indicated that this plant was infected with a potexvirus. This was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) with primers designed to detect members of the virus family Potexviridae (3). These plants reacted positive to PaMV antiserum in ELISA and gave a reaction of partial identity to PaMV in immunodiffusion. A specimen of Portulaca grandiflora (moss rose) with distorted leaves found at a local retail store was also tested and gave the same results. Leaves from each of the three plant species were rubbed onto a set of indicator plants using Carborundum and potassium phosphate buffer. Total RNA was extracted from symptomatic indicator plants of Nicotiana benthamiana. RT-PCR (3) was performed, and PCR products were sequenced directly. Sequences of approximately 700 bp were obtained for all three plant species and showed 98% identity with each other. BLAST search results showed that these sequences were 93% identical to an Alternanthera mosaic virus (AltMV) sequence at the nucleotide level but only 76% identical to PaMV. The amino acid sequences were 98 and 82% identical to AltMV and PaMV, respectively. The PCR products of the virus from Scutellaria sp. were cloned, resequenced, and the sequence was entered into the GenBank (Accession No. DQ393785). The bioassay results matched those found for AltMV in Australia (2) and the northeastern United States (4), except that the Florida viruses infected Datura stramonium and Digitalis purpurea (foxglove). The virus associated with the symptoms of these three plants appears to be AltMV and not PaMV. AltMV has been found in ornamental plants in Australia, Italy, and the United States (Pennsylvania, Maryland, and now Florida). Since this virus is known to infect several plants asymptomatically and can be easily confused with PaMV serologically, it is likely that the distribution of this virus is much wider than is known at this time. References: (1) L. L. Breman. Plant Pathology Circular No. 396. Fla. Dept. Agric. Consum. Serv. DPI, 1999. (2) A. D. W. Geering and J. E. Thomas. Arch Virol 144:577, 1999. (3) A. Gibbs et al. J Virol Methods 74:67, 1998. (4) J. Hammond et al. Arch Virol. 151:477, 2006.

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First Report of Tomato Brown Rugose Fruit Virus Infecting Tomato Crop in Saudi Arabia

Plant Disease ◽

10.1094/pdis-05-21-1065-pdn ◽

2021 ◽

Author(s):

Ahmed Sabra ◽

Mohammed Ali Al Saleh ◽

I. M. Alshahwan ◽

Mahmoud A. Amer

Keyword(s):

Saudi Arabia ◽

Mosaic Virus ◽

Solanum Lycopersicum ◽

Tomato Mosaic Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Pcr Products ◽

Dark Green ◽

Leaf Symptoms

Tomato (Solanum lycopersicum L.) is the most economically important member of family Solanaceae and cultivated worldwide and one of the most important crops in Saudi Arabia. The aim of this study is screening of the most common viruses in Riyadh region and identified the presence of tomato brown rugose fruit virus (ToBRFV) in Saudi Arabia. In January 2021, unusual fruit and leaf symptoms were observed in several greenhouses cultivating tomatoes commercially in Riyadh Region, Saudi Arabia. Fruit symptoms showed irregular brown spots, deformation, and yellowing spots which render the fruits non-marketable, while the leaf symptoms included mottling, mosaic with dark green wrinkled and narrowing. These plants presented the symptoms similar to those described in other studies (Salem et al., 2015, Luria et al., 2017). A total 45 Symptomatic leaf samples were collected and tested serologically against suspected important tomato viruses including: tomato chlorosis virus, tomato spotted wilt virus, tomato yellow leaf curl virus, tomato chlorotic spot virus, tomato aspermy virus, tomato bushy stunt virus, tomato black ring virus, tomato ringspot virus, tomato mosaic virus, pepino mosaic virus and ToBRFV using Enzyme linked immunosorbent assay (ELISA) test (LOEWE®, Biochemica, Germany), according to the manufacturers' instructions. The obtained results showed that 84.4% (38/45) of symptomatic tomato samples were infected with at least one of the detected viruses. The obtained results showed that 55.5% (25/45) of symptomatic tomato samples were found positive to ToBRFV, three out of 25 samples (12%) were singly infected, however 22 out of 45 (48.8%) had mixed infection between ToBRFV and with at least one of tested viruses. A sample with a single infection of ToBRFV was mechanically inoculated into different host range including: Chenopodium amaranticolor, C. quinoa, C. album, C. glaucum, Nicotiana glutinosa, N. benthamiana, N. tabacum, N. occidentalis, Gomphrena globosa, Datura stramonium, Solanum lycopersicum, S. nigrum, petunia hybrida and symptoms were observed weekly and the systemic presence of the ToBRFV was confirmed by RT-PCR and partial nucleotide sequence. A Total RNA was extracted from DAS-ELISA positive samples using Thermo Scientific GeneJET Plant RNA Purification Mini Kit. Reverse transcription-Polymerase chain reaction (RT-PCR) was carried out using specific primers F-3666 (5´-ATGGTACGAACGGCGGCAG-3´) and R-4718 (5´-CAATCCTTGATGTG TTTAGCAC-3´) which amplified a fragment of 1052 bp of Open Reading Frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp). (Luria et al. 2017). RT-PCR products were analyzed using 1.5 % agarose gel electrophoresis. RT-PCR products were sequenced in both directions by Macrogen Inc. Seoul, South Korea. Partial nucleotide sequences obtained from selected samples were submitted to GenBank and assigned the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities ranged between 98.99 % to 99.50 % among them and 98.87-99.87 % identity with ToBRFV isolates from Palestine (MK881101 and MN013187), Turkey (MK888980, MT118666, MN065184, and MT107885), United Kingdom (MN182533), Egypt (MN882030 and MN882031), Jordan (KT383474), USA (MT002973), Mexico (MK273183 and MK273190), Canada (MN549395) and Netherlands (MN882017, MN882018, MN882042, MN882023, MN882024, and MN882045). To our knowledge, this is the first report of occurrence of ToBRFV infecting tomato in Saudi Arabia which suggests its likely introduction by commercial seeds from countries reported this virus and spread in greenhouses through mechanical means. The author(s) declare no conflict of interest. Keywords: Tomato brown rugose fruit virus, tomato, ELISA, RT-PCR, Saudi Arabia References: Luria N, et al., 2017. PLoS ONE 12(1): 1-19. Salem N, et al., 2015. Archives of Virology 161(2): 503-506. Fig. 1. Symptoms caused by ToBRFV showing irregular brown spots, deformation, yellowing spots on fruits (A, B, C) and bubbling and mottling, mosaic with dark green wrinkled and narrowing on leaf (D).

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Status and Epidemiology of Maize Lethal Necrotic Disease in Northern Tanzania

Pathogens ◽

10.3390/pathogens9010004 ◽

2019 ◽

Vol 9 (1) ◽

pp. 4 ◽

Cited By ~ 4

Author(s):

Fatma Hussein Kiruwa ◽

Samuel Mutiga ◽

Joyce Njuguna ◽

Eunice Machuka ◽

Senait Senay ◽

...

Keyword(s):

Mosaic Virus ◽

Plant Diseases ◽

Yellow Dwarf ◽

Dwarf Virus ◽

Maize Dwarf Mosaic Virus ◽

Rt Pcr ◽

Chlorotic Mottle Virus ◽

Maize Chlorotic Mottle Virus ◽

Chlorotic Mottle ◽

Yellow Dwarf Virus

Sustainable control of plant diseases requires a good understanding of the epidemiological aspects such as the biology of the causal pathogens. In the current study, we used RT-PCR and Next Generation Sequencing (NGS) to contribute to the characterization of maize lethal necrotic (MLN) viruses and to identify other possible viruses that could represent a future threat in maize production in Tanzania. RT-PCR screening for Maize Chlorotic Mottle Virus (MCMV) detected the virus in the majority (97%) of the samples (n = 223). Analysis of a subset (n = 48) of the samples using NGS-Illumina Miseq detected MCMV and Sugarcane Mosaic Virus (SCMV) at a co-infection of 62%. The analysis further detected Maize streak virus with an 8% incidence in samples where MCMV and SCMV were also detected. In addition, signatures of Maize dwarf mosaic virus, Sorghum mosaic virus, Maize yellow dwarf virus-RMV and Barley yellow dwarf virus were detected with low coverage. Phylogenetic analysis of the viral coat protein showed that isolates of MCMV and SCMV were similar to those previously reported in East Africa and Hebei, China. Besides characterization, we used farmers’ interviews and direct field observations to give insights into MLN status in different agro-ecological zones (AEZs) in Kilimanjaro, Mayara, and Arusha. Through the survey, we showed that the prevalence of MLN differed across regions (P = 0.0012) and villages (P < 0.0001) but not across AEZs (P > 0.05). The study shows changing MLN dynamics in Tanzania and emphasizes the need for regional scientists to utilize farmers’ awareness in managing the disease.

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AtypicalCTSKTranscripts andARNTTranscription Read-Through intoCTSK

Comparative and Functional Genomics ◽

10.1002/cfg.483 ◽

2005 ◽

Vol 6 (5-6) ◽

pp. 268-276

Author(s):

Fabienne S. Giraudeau ◽

Jean-Philippe Walhin ◽

Paul R. Murdock ◽

Nigel K. Spurr ◽

Ian C. Gray

Keyword(s):

Negative Impact ◽

Cathepsin K ◽

Chromosome 1 ◽

Rt Pcr ◽

Transcription Start ◽

Aryl Hydrocarbon ◽

Pcr Product ◽

Close Proximity ◽

Pcr Products ◽

Human Chromosome 1

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usualCTSKtranscription start site is less than 1.4 kb downstream of the end of the longest reportedARNTtranscript. By generating an RT-PCR product that overlaps both the 3′ end ofARNTand the 5′ end ofCTSK, we show thatARNTtranscripts may extend through theARNT–CTSKintergenic region and progress into theCTSKgene. Furthermore, by using quantitative RT-PCR from several tissues to detect theARNTexpression signature inCTSKintrons, we show thatARNTtranscripts can read through intoCTSKas far asCTSKintron 3, extending approximately 3.7 kb downstream of the end of the longest previously describedARNTmRNA. Given thatARNTandCTSKare expressed in an overlapping range of tissues,ARNTread-through may have a negative impact onCTSKtranscript levels by interfering withCTSKexpression. We also present evidence for novelCTSKtranscripts following sequence analysis ofCTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′ splicing and or 5′ extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the knownCTSKpromoter and possibly in the 3′ UTR ofARNT.

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