scholarly journals Detection of Banana Mild Mosaic Virus in Musa In Vitro Plants: High-Throughput Sequencing Presents Higher Diagnostic Sensitivity Than (IC)-RT-PCR and Identifies a New Betaflexiviridae Species

Plants ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 226
Author(s):  
Marwa Hanafi ◽  
Wei Rong ◽  
Lucie Tamisier ◽  
Chadi Berhal ◽  
Nicolas Roux ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Diagnostic Sensitivity ◽  
Rapid Screening ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
In Vitro Plants ◽  
Mild Mosaic Virus

: The banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus that infects Musa spp. and has a very wide geographical distribution. The current BanMMV indexing process for an accession requires the testing of no less than four plants cultivated in a greenhouse for at least 6 months and causes a significant delay for the distribution of the germplasm. We evaluated the sensitivity of different protocols for BanMMV detection from in vitro plants to accelerate the testing process. We first used corm tissues from 137 in vitro plants and obtained a diagnostic sensitivity (DSE) of only 61% when testing four plants per accession. After thermotherapy was carried out to eliminate BanMMV infection, the meristem was recovered and further grown in vitro. The same protocol was evaluated in parallel on the corm tissue surrounding the meristem, as a rapid screening to evaluate virus therapy success, and was compared to the results obtained following the standard protocol. The obtained results showed 28% false negatives when conducting testing from corm tissues, making this protocol unsuitable in routine processes. Furthermore, RT-PCR and high-throughput sequencing (HTS) tests were applied on tissues from the base (n = 39) and the leaves (n = 36). For RT-PCR, the average DSE per sample reached 65% from either the base or leaves. HTS was applied on 36 samples and yielded 100% diagnostic specificity (DSP) and 100% DSE, whatever the sampled tissue, allowing the identification of a new Betaflexiviridae species infecting Musa. These results suggest that a reliable diagnostic of BanMMV from in vitro plants using RT-PCR or HTS technologies might represent an efficient alternative for testing after greenhouse cultivation.

Detection of Banana mild mosaic virus and Banana virus X by polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR)

2007 ◽  
Vol 142 (1-2) ◽  
pp. 41-49 ◽  
Author(s):  
Pierre-Yves Teycheney ◽  
Isabelle Acina ◽  
Benham E.L. Lockhart ◽  
Thierry Candresse
Keyword(s):  
Mosaic Virus ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Degenerate Oligonucleotide ◽  
Mild Mosaic Virus

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

Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1320-1320 ◽  
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.

scholarly journals Identification of Divergent Isolates of Banana Mild Mosaic Virus and Development of a New Diagnostic Primer to Improve Detection

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1045
Author(s):  
Marwa Hanafi ◽  
Rachid Tahzima ◽  
Sofiene Ben Kaab ◽  
Lucie Tamisier ◽  
Nicolas Roux ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
High Throughput Sequencing ◽  
Plant Viruses ◽  
Mild Mosaic ◽  
Genome Variability ◽  
Molecular Tests ◽  
Pcr Products ◽  
Reverse Primer ◽  
Mild Mosaic Virus ◽  
Germplasm Accessions

Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the Musa germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol.

scholarly journals VIRUS NILAM: IDENTIFIKASI, KARAKTER BIOLOGI DAN FISIK, SERTA UPAYA PENGENDALIANNYA

2015 ◽  
Vol 34 (1) ◽  
pp. 1 ◽  
Author(s):  
Miftakhurohmah Miftakhurohmah ◽  
Rita Noveriza
Keyword(s):  
Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Tobacco Necrosis Virus ◽  
Rt Pcr ◽  
Cymbidium Mosaic Virus ◽  
Peanut Stripe Virus ◽  
Mild Mosaic Virus ◽  
Wilt Virus ◽  
Broad Bean Wilt Virus

Infeksi virus pada tanaman nilam dapat menyebabkan penurunan produksi dan kualitas minyak. Sembilan jenis virus diidentifikasi menginfeksi tanaman nilam, yaitu Patchouli mosaic virus (PatMoV), Patchouli mild mosaic virus (PatMMV), Telosma mosaic virus (TeMV), Peanut stripe virus (PStV), Patchouli yellow mosaic virus (PatYMV), Tobacco necrosis virus (TNV), Broad bean wilt virus 2 (BBWV2), Cucumber mosaic virus (CMV), dan Cymbidium mosaic virus (CymMV). Kesembilan virus tersebut memiliki genom RNA, tetapi panjang dan bentuk partikelnya berbeda. Deteksi dan identifikasi berdasarkan bagian partikel virus dapat dilakukan secara serologi dengan teknik ELISA dan secara molekuler dengan RT-PCR. Gejala awal tanaman nilam terserang virus yaitu mosaik atau belang pada daun pucuk dan pada gejala berat tanaman menjadi kerdil. Infeksi virus dapat bersifat tunggal, tetapi ada pula infeksi oleh beberapa virus. Virus menular secara mekanis dan sebagian melalui penyambungan dan vektor. TNV, BBWV2, dan CMV memiliki kisaran inang yang luas, sedangkan virus yang lain inangnya terbatas. Virus nilam umumnya memiliki titik panas inaktivasi dan titik batas pengenceran yang tinggi, sedangkan ketahanan in vitro tidak stabil. Pendekatan terbaik pengendalian virus ialah menggunakan bahan tanaman bebas virus atau tahan virus dan pengendalian vektor. Tanaman bebas virus dapat diperoleh melalui kultur meristem, sedangkan pengendalian vektor dapat menggunakan pestisida nabati atau kimia.

scholarly journals First Report of Ranunculus mild mosaic virus on Ranunculus asiaticus in Yunnan Province, China

Plant Disease ◽  
2008 ◽  
Vol 92 (11) ◽  
pp. 1585-1585 ◽  
Author(s):  
J. H. Wang ◽  
S. Zhao ◽  
X. M. Yang
Keyword(s):  
Mosaic Virus ◽  
Yunnan Province ◽  
Disease Incidence ◽  
Viral Disease ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Potential Threat ◽  
First Report ◽  
Ranunculus Asiaticus ◽  
Mild Mosaic Virus

In June 2007, a new viral disease occurred in commercial fields of Ranunculus asiaticus in the Yunnan Province of China. Infected plants exhibited mosaic symptoms and growth abnormalities. Viral disease incidence for this ornamental crop host in the Yunnan Province was estimated to range from 10 to 20%. Electron microscopic examination of negatively stained leaf-dip preparations from symptomatic plants identified long, flexuous linear particles (approximately 800 nm). The samples were tested using indirect antigen-coated plate (ACP)-ELISA. ACP-ELISA results showed that the leaf samples from symptomatic plants reacted positively to the potyvirus group antibody (Agdia Inc., Eklhart, IN). Total nucleic acid extracted from symptomatic plants was tested using reverse transcription (RT)-PCR with primers (S 5′-GGNAAAAYAGYGGNCARCC-3′; M4: 5′-GTTTTCCCAGTCACGAC-3′ [N = A, G, C, or T; Y = C or T; and R = A or G]) designed to amplify the 3′ terminal region of genomic RNA of the genus Potyvirus (1). RT-PCR produced a 1,650-bp amplification product that was cloned and sequenced (GenBank Accession No. EU684747). The sequenced portion showed 90 and 99% identity with the Ranunculus mild mosaic virus (RMMV) isolates (GenBank Accession Nos. DQ152191 and EF445546) from Italy and Israel, respectively (2). To our knowledge, this is the first report of RMMV in China. Infection from this virus may cause losses for cut-flower production of Ranunculus asiaticu and it is also a potential threat for international trade of Ranunculus germplasm. References: (1) J. Chen and J. P. Chen. Chin. J. Virol. 18:371, 2002. (2) M. Turina et al. Phytopathology 96:560, 2006.

scholarly journals Evaluation of Four Species of Wild Yams, as Potential Natural Reservoirs of Potyviruses Infecting Yams Cultivated in Togo

2019 ◽  
pp. 1-9
Author(s):  
Kwasi Dzola Ayisah ◽  
Mawuli Kossivi Aziadekey ◽  
Yawovi Mawuena Dieudonné Gumedzoe
Keyword(s):  
Mosaic Virus ◽  
Control Method ◽  
Elisa Test ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Efficient Control ◽  
Mild Mosaic Virus ◽  
Wild Yams ◽  
Pcr Test

Yams cultivation in Togo is hampered by diseases caused by Potyviruses, mainly Yam mosaic virus (YMV) and Yam mild mosaic virus (YMMV). To understand the Potyviruses dissemination mechanism and to develop an efficient control method, the present study aims to establish the role of wild yams species as potential natural reservoirs of these pathogens. As such, Potyvirus susceptibility assessment was performed on four wild yams, D. dumetorum, D. bulbifera, D. togoensis and, D. smilacifolia, which grow spontaneously in yam fields in Togo. For this, phytosanitary surveys were carried out on yam fields and forests near yam plots, in July 2018 at the long rainy season, covering 27 localities in Maritime, Central and Plateaux regions of Togo, during which wild yam leaves were sampled for viruses identification. The leaves samples were analyzed first by ACP-ELISA test to detect Potyviruses using universal anti-potyvirus monoclonal antibodies, and then by RT-PCR test to identify YMV and YMMV, using respectively pairs of primers YMV1&YMV2 (196 pb) and YMV-CP-2F & YMV-UTR-1R (249 pb). Then 140 seedlings obtained from seeds of the four wild yams, were inoculated with YMV isolate 20-601/06. ACP-ELISA test revealed that only the leaves samples of D. dumetorum and D. togoensis, collected in Plateaux region, were infected by Potyviruses, with respectively 24.24% and 6.25% of incidence rate. But these samples were positive for neither YMV nor YMMV at RT-PCR test. However, after the inoculations, respectively 20% of seedlings of D. dumetorum, 52.5% of D. bulbifera, 64% of D. togoensis, and 3.33% of D. smilacifolia, were infected by YMV. This suggests a high potential of these yams, mostly D. bulbifera and D. togoensis, to become natural reservoirs for YMV, under high pressure of the viruses and their vectors. These wild yams control in and around yam fields can help limit Potyviruses infections.

scholarly journals First Report of Olive mild mosaic virus and Sowbane mosaic virus in Spinach in Greece

Plant Disease ◽  
2012 ◽  
Vol 96 (8) ◽  
pp. 1230-1230 ◽  
Author(s):  
M. E. Gratsia ◽  
P. E. Kyriakopoulou ◽  
A. E. Voloudakis ◽  
C. Fasseas ◽  
I. E. Tzanetakis
Keyword(s):  
Mosaic Virus ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
First Report ◽  
Type Isolate ◽  
Sequence Identity ◽  
Leaf Deformation ◽  
Mild Mosaic Virus ◽  
Necrotic Spots ◽  
Plant Pathol

Uncommon, viruslike symptoms (yellowing, line patterns, leaf deformation, and necrosis), were observed in spinach fields in the Marathon area, Greece in 2004. Seedlings from the same seed lot, grown in the greenhouse, also developed the same viruslike symptoms, indicating that the causal agent(s) of the disorder is seed-transmissible. Spinach seedlings of the same variety but a different lot and herbaceous indicators (Chenopodium quinoa, C. amaranticolor, Sonchus oleraceus, and Nicotiana benthamiana) were mechanically inoculated with infected material. Spinach developed yellowing or necrotic spots whereas indicators showed variety of symptoms including mosaic, vein banding, and necrotic lesions. Virus purifications, double-stranded RNA extractions, cloning, and sequencing (2,3) followed by a combination of molecular (reverse transcription [RT]-PCR and immunocapture RT-PCR) and serological (ELISA) techniques with antisera provided by Dr. Avgelis were performed as described (4), verifying the presence of two viruses in the diseased seedlings: Sowbane mosaic virus (SoMV), a sobemovirus, was present in spinach and indicators with mottling and leaf deformation, whereas Olive mild mosaic virus (OMMV), a necrovirus, was present in plants with necrotic spots. All RT-PCR products amplified with primers SoMV-F (5′-CAAATGGTCTTGGTCAGCAGTC)/SoMV-R (5′-GCATACGCTCGACGATCTG) and OMMV-F (5′-CAAACCCAGCCTGTGTTCGATG)/OMMV-R (5′-CATCAGTTTGGTAATCCATTGA) were sequenced and found to confirm the other results. The SoMV-spinach isolate polyprotein gene sequence (GenBank Accession No. DQ450973) has 95% sequence identity with the type isolate from C. quinoa (GenBank Accession No. GQ845002), whereas the OMMV-spinach isolate (GenBank Accession No. JQ288895) has 92% sequence identity with the OMMV type isolate from olive (GenBank Accession No. AY616760). SoMV has been found to naturally infect spinach in the Netherlands (1) and, to our knowledge, this is the first report on spinach in Greece. The presence of OMMV in spinach is, to our knowledge, the first report worldwide. Its natural host range is limited to olive, tulip, and now spinach. OMMV might be transmitted by Olpidium spp. and may, according to data of its close relatives, persist in the soil for several decades. Pollen- and seedborne viruses (PSVs) like sobemoviruses and necroviruses are of particular importance for a crop like spinach where crop increase takes place in small, seed production-designated areas. If a PSV spreads in such an area it has the potential to become a major problem for the industry, especially when it remains undetected. Infected seed can be shipped worldwide with PSVs, causing diseases and becoming endemic in areas where they were absent. For this reason and the fact that field losses can exceed 50%, rigorous monitoring for the presence of SoMV and OMMV in seed fields is essential to minimize the possibility of the viruses moving to new areas. References: (1) L. Bos and N. Huijberts. Eur. J. Plant Pathol. 102:707, 1996. (2) S. M. Girgis et al., Eur. J. Plant Pathol. 125:203, 2009, (3) I. E. Tzanetakis et al. J. Virol. Methods 124:73, 2005. (4) I. E. Tzanetakis et al. Virus Res. 121:199, 2006.

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