scholarly journals First Report of Ageratum enation virus, Betasatellite and Alphasatellite Causing Leaf Curl and Enation Disease of Amaranthus hypochondriacus in India

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1285-1285 ◽  
Author(s):  
A. Srivastava ◽  
S. Kumar ◽  
S. K. Raj
Keyword(s):  
Sequence Data ◽  
Disease Incidence ◽  
Rolling Circle Amplification ◽  
Nucleotide Identity ◽  
Post Inoculation ◽  
Specific Primers ◽  
Rolling Circle ◽  
Amaranthus Hypochondriacus ◽  
Close Phylogenetic Relationship ◽  
Leaf Curl

During a survey in February 2011, severe symptoms of upward leaf curling, vein enation on lower side of the leaves, and shortening of internodes were observed on 20 out of 117 Amaranthus hypochondriacus plants (17% disease incidence) examined at breeding plots of CSIR-NBRI, Lucknow. These symptoms are typical of begomovirus infection. PCR with begomovirus-specific primers (3) produced the expected ~1.1-kb product from DNA extracts of 20 symptomatic plants but not from a non-symptomatic plant, suggesting the association of a begomovirus. The full-length begomoviral genome from a representative sample was amplified by rolling circle amplification using Ø-29 DNA polymerase and digested by BamHI, which resulted in a ~2.7 kb product when electrophoresed in 1.0% agarose gel. The product obtained was cloned, sequenced, and sequence data of 2,753 nucleotides was deposited in GenBank (Accession No. JF682242). BLASTn analysis revealed 97 to 98% nucleotide identity and forms a distinct clade with Ageratum enation virus (AEV) isolates. This shows the virus in A. hypochondriacus to be an isolate of AEV. The separate PCRs were also performed with betasatellite and alphasatellite specific primers (1,2) that resulted in ~1.3-kb amplicons from all samples, suggesting their association. The amplification products were cloned and sequenced. An analysis of betasatellite (JX512904) revealed highest 98% nucleotide identity and close phylogenetic relationship with Ageratum leaf curl betasatellite (ALCB, JQ710745). The alphasatellite (JX512905) showed highest 95% identity and close relationship with Hibiscus leaf curl alphasatellite (HLCA, FN794199). This shows the betasatellite and alphasatellite in A. hypochondriacus to be isolates of ALCB and HLCA, respectively. The partial direct repeat clones of the begomovirus (pCAM-AEV), betasatellite (pCAM-ALCB), alphasatellite (pCAM-HLCA) were generated and mobilized into Agrobacterium tumefaciens strain GV3101 and infiltrated in A. hypochondriacus seedlings. The plants inoculated with pCAM-AEV, pCAM-ALCB, and pCAM-HLCA; pCAM-AEV and pCAM-ALCB developed severe leaf curl and enation symptoms on 5/5 plant at 35 days post inoculation, which were similar to those of naturally infected plants, satisfying Koch's postulates. On the other hand, plants inoculated with pCAM-AEV alone or in combination with pCAM-HLCA developed mild symptoms. Plants inoculated with pCAM-ALCB and pCAM-HLCA did not develop symptoms. The results here show that leaf curl and enation disease of A. hypochondriacus in India is caused by AEV and ALCB and that an alphasatellite may be associated with symptomatic plants. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. E. Bull et al. Mol. Biotechnol. 23:83, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

scholarly journals Identification of Chili leaf curl virus Causing Leaf Curl Disease of Petunia in Oman

Plant Disease ◽  
2014 ◽  
Vol 98 (4) ◽  
pp. 572-572 ◽  
Author(s):  
A. A. Al-Shihi ◽  
S. Akhtar ◽  
A. J. Khan
Keyword(s):  
Indian Subcontinent ◽  
Rolling Circle Amplification ◽  
Rapid Evolution ◽  
Post Inoculation ◽  
Multiple Sequence ◽  
Rolling Circle ◽  
Begomovirus Genome ◽  
Leaf Curl Virus ◽  
Leaf Curl Disease ◽  
Leaf Curl

Petunias (Petunia × hybrida) are the most important ornamental plants in Oman. In 2012, petunias were observed in public parks and airport landscape in Dhofar region with symptoms of upward leaf curling, yellowing and vein clearing, and size reduction in leaves. Almost all plants in the surveyed landscape showed high infestation of Bemisia tabaci and symptoms that suggested infection with a begomovirus. Six symptomatic samples were collected from three different sites. All symptomatic samples were found PCR-positive with diagnostic primers for begomovirus (3) when DNA extracted from infected leaves was used as template. Nucleic acids extracted from the symptomatic leaves were used to amplify circular DNA molecules by rolling circle amplification method. The amplified concatameric products were digested with restriction enzyme PstI, which yielded a product ∼2.8 kb in size. The putative begomovirus fragment was cloned and sequenced in both orientations. Partial sequences of six clones were 99 to 100% similar and thus only two clones, PT-2 and PT-3, were fully sequenced. The whole genomes of both clones were 2,761 bp, and both were deposited in GenBank under accession numbers HF968755 and HF968756 for the isolates PT-2 and PT-3, respectively. Both sequences had six open reading frames; Rep, TrAP, REn, and C4 genes in complementary sense; and CP and V2 genes in virion-sense, typical of the begomovirus genome organization. Upon alignment, the two sequences showed 99.4% nucleotide identity with each other, thus representing isolates of a single begomovirus species. BlastN comparison showed PT-2 and PT-3 from petunia were 94 to 95% identical to the sequences of ChCLV from Oman (JN604490 to JN604500), which were obtained from other hosts. ClustalV multiple sequence alignment showed that isolates PT-2 and PT-3 shared maximum sequence identity of 93.3 and 92.8%, respectively, with an isolate of ChLCV-OM (JN604495). According to ICTV rules for begomoviruses, PT-3 should be considered to be a new strain of ChLCV-OM and PT-2 a variant of the already existing ChLCV-OM strain. We propose the name for this new strain as the “Petunia strain” of Chili leaf curl virus (ChLCV-Pet). Two infectious clones were constructed from the PT-2 and PT-3 sequences, clones as 1.75-genome sequences in a binary vector, suitable for agroinfection to confirm their infectivity. Both clones, PT-2 and PT-3, produced typical leaf curl disease symptoms upon inoculation on petunia 18 days post inoculation. The presence of the same virus in symptomatic field infected and inoculated petunia was confirmed by Southern blot using 650 bp DIG labeled probe prepared from CP region of PT-3 isolate. ChLCV-OM, a monopartite begomovirus, is widely associated with leaf curl disease of tomato and pepper in Oman, with its origin traced to the Indian subcontinent (2). Identification of a new strain of ChLCV from petunia provides evidence of an ongoing rapid evolution of begomoviruses in this region. Although petunia has been tested as an experimental host for some begomoviruses (1,4), this is the first report of petunia as natural host for ChLCV, a begomovirus previously reported in tomato and pepper in Oman. References: (1) Cui et al. J. Virol. 78:13966, 2004. (2) Khan et al. Virus Res. 177:87, 2013. (3) Khan et al. Plant Dis. 97:1396, 2013. (4) Urbino et al. Arch. Virol. 149:417, 2003.

scholarly journals Frequent occurrence of Mungbean yellow mosaic India virus in tomato leaf curl disease affected tomato in Oman

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
M. S. Shahid ◽  
M. Shafiq ◽  
M. Ilyas ◽  
A. Raza ◽  
M. N. Al-Sadrani ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Monopartite Begomoviruses ◽  
Tomato Leaf ◽  
Bipartite Begomovirus ◽  
Rolling Circle ◽  
Tomato Leaf Curl Disease ◽  
Planting Material ◽  
Next Generation Sequencing Ngs ◽  
Tomato Leaf Curl ◽  
Leaf Curl

Abstract Next generation sequencing (NGS) of DNAs amplified by rolling circle amplification from 6 tomato (Solanum lycopersicum) plants with leaf curl symptoms identified a number of monopartite begomoviruses, including Tomato yellow leaf curl virus (TYLCV), and a betasatellite (Tomato leaf curl betasatellite [ToLCB]). Both TYLCV and ToLCB have previously been identified infecting tomato in Oman. Surprisingly the NGS results also suggested the presence of the bipartite, legume-adapted begomovirus Mungbean yellow mosaic Indian virus (MYMIV). The presence of MYMIV was confirmed by cloning and Sanger sequencing from four of the six plants. A wider analysis by PCR showed MYMIV infection of tomato in Oman to be widespread. Inoculation of plants with full-length clones showed the host range of MYMIV not to extend to Nicotiana benthamiana or tomato. Inoculation to N. benthamiana showed TYLCV to be capable of maintaining MYMIV in both the presence and absence of the betasatellite. In tomato MYMIV was only maintained by TYLCV in the presence of the betasatellite and then only at low titre and efficiency. This is the first identification of TYLCV with ToLCB and the legume adapted bipartite begomovirus MYMIV co-infecting tomato. This finding has far reaching implications. TYLCV has spread around the World from its origins in the Mediterranean/Middle East, in some instances, in live tomato planting material. The results here may suggest that begomoviruses which do not commonly infect tomato, such as MYMIV, could be spread as a passenger of TYLCV in tomato.

scholarly journals Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 3010-3018 ◽  
Author(s):  
Yuanjian Qiu ◽  
Song Zhang ◽  
Haodong Yu ◽  
Zhiyou Xuan ◽  
Liu Yang ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Close Association ◽  
Phylogenetic Analyses ◽  
Rolling Circle Amplification ◽  
Field Investigation ◽  
Broussonetia Papyrifera ◽  
New Members ◽  
Rolling Circle ◽  
Mulberry Leaf ◽  
Leaf Curl

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Use of RCA-PCR assay for detection of Begomovirus infection in Bhut Jolokia (Capsicum assamicum) in Tezpur region of Assam, India

2016 ◽  
Vol 6 (2) ◽  
pp. 64-69
Author(s):  
Ajitabh Bora ◽  
Hemant Kumar Gogoi ◽  
Mohan C. Kalita
Keyword(s):  
Rolling Circle Amplification ◽  
Viral Disease ◽  
Viral Pathogen ◽  
Rolling Circle ◽  
North East India ◽  
North East ◽  
Detection Assay ◽  
A Genome ◽  
Bhut Jolokia ◽  
Leaf Curl

Bhut Jolokia (Capsicum assamicum), one of the hottest chilli in the world is a chilli cultivar, endemic to North East India and is extensively cultivated in the states of Assam, Nagaland and Manipur. The demand of this chilli is very high in domestic as well as in the international market due to its extreme hotness and pleasant aroma. This plant is severely affected by leaf curl disease caused by Begomovirus, leading to total crop loss. Accurate diagnosis of this viral disease at seedling stage, prior to transplanting, is essential to prevent further spread of the disease. With an aim to device a rapid and accurate detection assay, Rolling Circle Amplification (RCA)-Polymerase Chain Reac-tion (PCR) technique was used for detection of the viral pathogen. RCA tech-nique was employed to increase the viral template and PCR was conducted using a degenerate primer pair. With the help of this assay, 1.4 kbp segment of DNA-A genome of the Begomovirus was amplified. Phylogeny revealed close proximity of the isolate with Tomato leaf curl Bangladesh virus. This is the first report on characterization of Begomovirus infecting Bhut Jolokia in Tezpur region of Assam.

scholarly journals Multiply-primed rolling circle amplification of human papillomavirus using sequence-specific primers

Virology ◽  
2012 ◽  
Vol 432 (1) ◽  
pp. 57-62 ◽  
Author(s):  
Yanara Marincevic-Zuniga ◽  
Inger Gustavsson ◽  
Ulf Gyllensten
Keyword(s):  
Human Papillomavirus ◽  
Rolling Circle Amplification ◽  
Specific Primers ◽  
Rolling Circle

scholarly journals New Record of Catharanthus yellow mosaic virus and a Betasatellite Associated with Lethal Leaf Yellowing of Kalmegh (Andrographis paniculata) in Northern India

Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 292-292 ◽  
Author(s):  
A. Khan ◽  
S. T. Saeed ◽  
A. Samad
Keyword(s):  
Sequence Analysis ◽  
Mosaic Virus ◽  
Rolling Circle Amplification ◽  
Andrographis Paniculata ◽  
Yellow Vein ◽  
Yellow Mosaic Virus ◽  
Mechanical Transmission ◽  
Specific Primers ◽  
Leaf Yellowing ◽  
Leaf Curl

Andrographis paniculata (Family Acanthaceae), also called Kalmegh, is a medicinal herb in India well-known for its various pharmaceutical properties (1). In August 2012, during a survey in the northern parts of India, several Kalmegh plants in Barabanki District of Uttar Pradesh Province showed typical virus-like symptoms along with prominent lethal leaf yellowing. The infected plants initially showed some chlorotic streaks, which later turned completely yellow, ultimately leading to premature death. Mechanical/sap inoculation failed to transmit the pathogen. Based on the symptomology, a heavy infestation of whiteflies (Bemisia tabaci) in the infected fields, and lack of mechanical transmission, the association of a begomovirus was suspected. The disease incidence was calculated to be about 15 to 20% on the basis of plant population. Twenty samples from naturally infected plants of A. paniculata were collected from various field locations. Total genomic DNA from the symptomatic and non-symptomatic samples was isolated by the modified CTAB method (4). The initial PCR-based detection was performed using begomovirus coat protein gene specific primers (forward 5′-ATGGCGAAGCGACCAG-3′ and reverse 5′-TTAATTTGTGACCGAATCAT-3′), which generated an amplicon of 771 bp in most of the (17/20) symptomatic samples. No amplification was obtained in healthy or non-symptomatic plant samples. The full-length genome was amplified via rolling-circle amplification (RCA) according to the manufacturer's instructions using random hexamer primers and φ29 DNA polymerase. A portion of the RCA product (1 μl) was subjected to digestion with different restriction enzymes, out of which BamHI yielded DNA fragments of approximately 2.7 and 1.3 kb, corresponding to DNA-A and β satellite molecules, respectively. These fragments were eluted from the gel and cloned into the suitable restriction site of pGreen0029 vector. The positive clones were checked by restriction digestion. Twelve out of 20 clones were found to be positive and sequenced. The complete genome sequences of DNA A (2,754 bp) and β (1,366 bp) satellites were deposited in the GenBank database with the accession numbers KM359406 and KM359407, respectively. The absence of DNA-B molecule was ascertained, as no PCR amplification was detected with DNA-B-specific primers. Sequence analysis showed highest nucleotide identity (90%) with Catharanthus yellow mosaic virus (CYMV) (HE580234) and ≤85% identity with other begomoviruses of the database. Sequence analysis of the associated betasatellite showed 96% identity with Andrographis yellow vein leaf curl betasatellite (KC967282). CYMV was first reported on Catharanthus roseus with no associated betasatellite from Pakistan (2). However, this is the first report of CYMV along with a betasatellite infecting A. paniculata in India. Recently a begomovirus (Eclipta yellow vein virus) infection was reported on A. paniculata in association with Andrographis yellow vein leaf curl betasatellite from India for the first time (3); now the crop has also become a host of CYMV. Thus, this study highlights the spread of CYMV from its preliminary host to a new host plant (A. paniculata), across the South Asian countries. Therefore, it is important to take measures for the management of its transmitting vector so as to curtail the spread of the virus to new economically and commercially important crops. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) M. Ilyas et al. Arch. Virol. 158:505, 2013. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) S. P. S. Khanuja et al. Plant Mol. Biol. Rep. 17:1, 1999.

scholarly journals Cucurbit leaf crumple virus (CuLCrV) associated to watermelon disease in Campeche state, Mexico

Plant Disease ◽  
2020 ◽  
Author(s):  
Edgar Antonio Rodríguez-Negrete ◽  
Rafael Jordan-Ramírez ◽  
Norma Elena Leyva-López ◽  
Jesus Mendez-Lozano
Keyword(s):  
Citrullus Lanatus ◽  
Infectious Clone ◽  
Rolling Circle Amplification ◽  
Yield Reduction ◽  
Specific Primers ◽  
Genebank Accession ◽  
Squash Leaf Curl Virus ◽  
Leaf Curl Virus ◽  
Cucurbit Leaf Crumple Virus ◽  
Leaf Curl

An annual recurrent disease causing yield reduction in cultivated watermelon (Citrullus lanatus) was documented by the growers in different farms of Campeche state, Mexico. In April 2019 and March 2020 open field grown watermelon plants showed symptoms such as leaf curling, crumpling, and leaf basal or apical necrosis (Figure S1), with an incidence ranging from 30 up to 80%. These plants also presented high populations of whitefly, especially in the most affected fields. In order to identify the causal agent of the disease, a total of 22 symptomatic watermelon plants were collected in four locations from Campeche state. Total nucleic acids (DNA and RNA) were extracted from these leaf samples. Initially, RT-PCR analysis was performed with specific primers (Table S1) for cucurbit-infecting Crinivirus transmitted by whitefly but the expected size PCR product for those viruses was not amplified in any of these samples. To investigate the presence of cucurbit-infecting begomoviruses, PCR was performed by using specific primers for those begomoviruses reported in Mexico and north/central America including Squash leaf curl virus (SLCV), Watermelon chlorotic stunt virus (WmCSV), Melon chlorotic leaf curl virus (MCLCuV), and Cucurbit leaf crumple virus (CuLCrV) (Table S1). Only the expected amplicon size of ~1089 bp for CuLCrV was amplified from DNA extracts from all 22 watermelon samples, suggesting a single cucurbit-associated virus. The putative complete genome of the CuLCrV Campeche isolate was amplified by circular DNA enrichment using a Rolling Circle Amplification (RCA) procedure from two representative samples, followed by enzymatic digestion using BamHI, EcoRI, KpnI, and SacI enzymes (Inoue-Nagata et al., 2004). Expected linearized full-length viral components (~2.7 kb) were obtained with EcoRI and SacI, and both products, from one selected sample, were cloned in to pGreen0029 vector and were fully sequenced. Sequence analysis of the EcoRI clone, designated as LV2019Camp_A (deposited in GenBank accession no. MW273384) revealed the highest identity of 97.52% to CuLCrV DNA-A isolate Baja California Sur isolate (GeneBank accession no. MN625831.1), whereas the KpnI clone, designated as LV2019Camp_B (deposited in GenBank accession no. MW273385), shared 94.87% identity with DNA B of CuLCrV isolate Arizona (GeneBank accession no. AF327559.1). Subsequently, CuLCrV isolate Campeche-derived agroinfectious clone, was obtained by constructing a partial dimeric tandem repeat of both DNA-A and DNA-B components (Bang et al., 2014). Twelve watermelon plants were agroinfiltrated with the infectious clone at the fourth true leaf stage, resulting in symptomatic plants (11/12) exhibiting leaf yellowing, curling, and crumpling 15 days after agroinfiltrated (Figure S1), and CuLCrV infection was confirmed by PCR specific detection using DNA extract from non-inoculated leaves. Previously CuLCrV has been detected in the USA (Arizona, Texas, California, Florida, South Carolina, and Georgia), and north Mexico (Coahuila) infecting cucurbits including squash, cucumber, cantaloupe, pumpkin, and watermelon (Brown et al., 2000., Keinath et al., 2018), in both single and mixed infection with other whitefly transmitted RNA viruses (CYSDV, genera Crinivirus), and DNA viruses (SLCV, genera Begomovirus) (Kuo et al., 2007). To our knowledge, this is the first report of CuLCrV infecting a cucurbit crop in the Campeche state from the Yucatán peninsula, in Mexico.

scholarly journals First Report of Chilli leaf curl India virus Infecting Mentha spicata (Neera) in India

Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 164-164 ◽  
Author(s):  
S. T. Saeed ◽  
A. Khan ◽  
B. Kumar ◽  
P. V. Ajayakumar ◽  
A. Samad
Keyword(s):  
Sequence Analysis ◽  
Indian Subcontinent ◽  
Disease Incidence ◽  
Rolling Circle Amplification ◽  
Full Length ◽  
Yellow Vein ◽  
Mechanical Transmission ◽  
Mentha Spicata ◽  
First Report ◽  
Leaf Curl

Mint (Mentha spp.; family Lamiaceae) is an important essential oil-bearing crop cultivated on the Indian subcontinent as a cash crop for the international market and industrial purposes. Since May 2010, typical symptoms such as yellow vein, leaf yellowing, mosaic, crinkling, and cupping were observed, which led to significant yield loss in spearmint (M. spicata var. Neera) at CIMAP experimental fields and farmers' fields of Badaun, Rampur, and Moradabad regions of Uttar Pradesh province, India. Disease incidence was recorded in the range of 40 to 50%. Mentha spp. has been reported to be affected by many viral diseases (3). Due to the absence of fungal/bacterial infection, lack of mechanical transmission of the pathogen, and presence of whiteflies in the fields, the causal pathogen was suspected to be a begomovirus. Total genomic DNA was extracted from the leaves of naturally infected and healthy samples of Mentha by the CTAB protocol. Eighteen symptomatic samples were collected from different location of fields and screened for the presence of begomovirus. DNA from these samples was used as PCR template to amplify a 771-bp fragment using begomovirus coat protein (CP) gene specific primers. Eleven of 18 (61.1%) samples were found positive. PCR products were cloned into the pGEM-T Easy (Promega) and sequenced using the universal M13F/M13R primers showed sequence similarity with Chilli leaf curl India virus. To amplify the full-length DNA-A/B and a possible β-satellite, a second detection method was used: rolling circle amplification (RCA) using the TempliPhi 100 Amplification System (GE Healthcare). RCA products were digested independently with various restriction enzymes: BamHI, EcoRI, EcoRV, HincII, HindIII, SacI, and KpnI. Digested products were resolved on 1% agarose gel and the bands corresponding to ~2.7 and ~1.3 kb were purified using Nucleospin Gel and PCR Clean-up Kit and cloned into the respective sites of pGreen0029 vector. The sequence of full-length DNA-A (2,749 bp) and β-satellite component (1,347-bp) were obtained and deposited in NCBI GenBank with accession nos. KF312364 and KF364485, respectively. The sequence analysis showed maximum nucleotide identity (99%) with Chilli leaf curl India virus (FM877858) and distant affinities (≤88%) with other begomoviruses. The sequence analysis of isolated β-satellite showed 93% identity with Ageratum yellow vein virus satellite (AJ252072.1). No presence of DNA-B was detected using the universal primer PBL1v2040/PCRc1 (2), thus confirming it to be a monopartite begomovirus (1). Viruliferous whiteflies (Bemisia tabaci) proved Koch's postulation by inducing similar symptoms on healthy plants while aphids (Myzus persicae) failed to transmit the virus. To our knowledge, this is the first report of Chilli leaf curl India virus infecting M. spicata var. Neera in India. Mint is widely grown together with other reported hosts of begomoviruses, and thus could pose a serious threat as future expansion of begomovirus to new crops. Hence, the development of resistant varieties coupled with the implementation of adapted integrated pest management strategies would be essential for successful production of mint crops. References: (1) Y. Kumar et al. Plant Pathol. 60:1040, 2011. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) I. E. Tzanetakis et al. Plant Dis. 94:4, 2010.

scholarly journals A workflow for accurate metabarcoding using nanopore MinION sequencing

2020 ◽  
Author(s):  
Bilgenur Baloğlu ◽  
Zhewei Chen ◽  
Vasco Elbrecht ◽  
Thomas Braukmann ◽  
Shanna MacDonald ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Rolling Circle Amplification ◽  
Error Rates ◽  
Read Length ◽  
Taxonomic Assignment ◽  
Major Drawback ◽  
Rolling Circle ◽  
Sequencing Platform ◽  
Sequencing Platforms

AbstractMetabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short-read high-throughput sequencing platforms. The Oxford Nanopore MinION™, a portable sequencing platform, represents a low-cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%.To test if the MinION™ represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full-length consensus DNA barcodes (658bp of cytochrome oxidase I - COI) for a bulk mock sample of 50 aquatic invertebrate species. By applying two different laboratory protocols, we generated two MinION™ runs that were used to build consensus sequences. We also developed a novel Python pipeline, ASHURE, for processing, consensus building, clustering, and taxonomic assignment of the resulting reads.We were able to show that it is possible to reduce error rates to a median accuracy of up to 99.3% for long RCA fragments (>45 barcodes). Our pipeline successfully identified all 50 species in the mock community and exhibited comparable sensitivity and accuracy to MiSeq. The use of RCA was integral for increasing consensus accuracy, but it was also the most time-consuming step during the laboratory workflow and most RCA reads were skewed towards a shorter read length range with a median RCA fragment length of up to 1262bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding but we recommend the exploration of other isothermal amplification procedures to improve consensus length.

scholarly journals First Report of Sweet potato leaf curl virus on Blue Morning Glory in Greece

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 700-700 ◽  
Author(s):  
E. Fiallo-Olivé ◽  
N. I. Katis ◽  
J. Navas-Castillo
Keyword(s):  
Sweet Potato ◽  
Rolling Circle Amplification ◽  
Morning Glory ◽  
Yellow Vein ◽  
Nucleotide Identity ◽  
Potato Leaf ◽  
First Report ◽  
Crete Island ◽  
Leaf Curl Virus ◽  
Leaf Curl

Blue morning glory (Ipomoea indica, Convolvulaceae) plants are widespread along the Greek coast, where they grow as weeds in addition to being cultivated as ornamentals. Yellow vein symptoms are frequently observed on these plants. These symptoms are similar to those reported for isolates of Sweet potato leaf curl virus (SPLCV) infecting I. indica in Italy and Spain (1,3). SPLCV belongs to the sweepoviruses, a unique group within the genus Begomovirus in the family Geniminiviridae that infects sweet potato (I. batatas) crops around the world. In May 2013, three leaf samples of I. indica showing yellow vein symptoms were collected in Kolymbari (Crete Island), where ~50% of the observed plants were symptomatic, and five asymptomatic leaf samples were collected in Kremasti and Mandriko (Rhodes Island). Total DNA, isolated from all samples, was used as a template in rolling-circle amplification (RCA) using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK) and the product was digested with a set of restriction endonucleases. The samples from Kolymbari and one sample from Kremasti yielded amplification products that were shown to contain a single BamHI site. The DNA fragments of ~2.8 kbp obtained from one sample from each island were cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA). Inserts of two clones from the Kolymbari sample and one clone from the Kremasti sample were completely sequenced (Macrogen, Seoul, South Korea). Sequences were aligned with available sequences of sweepoviruses using MUSCLE and pairwise identity scores were calculated with SDT as described (4). The sequences obtained from Kolymbari (2,830 nt, GenBank Accession Nos. KF697069 and KF697070) were 98.8% similar between them and showed the highest nucleotide identity (97.7%) with a SPLCV isolate obtained from an I. indica plant in Sicily Island (Italy) (AJ586885) (1). The sequence obtained from Kremasti (2,804 nt, KF697071) showed the highest nucleotide identity (92.4%) with a SPLCV isolate (previously named as Ipomoea yellow vein virus, which is currently a synonym of SPLCV [2]) obtained from an I. indica plant from southern Spain (EU839578) (3). Nucleotide sequence identities were above the 91% threshold for begomovirus species demarcation (2), thus confirming that the begomoviruses found infecting I. indica in Greece are isolates of SPLCV. It is worth to note that the infected I. indica plant from Kremasti did not show any conspicuous symptoms, thus highlighting the importance of this species as an alternative host for SPLCV, which could thus affect the sweet potato crop that is grown in Greece in familiar plots. To our knowledge, this is the first report of SPLCV in Greece. References: (1) R. W. Briddon et al. Plant Pathol. 55:286, 2006. (2) ICTV Geminiviridae Study Group. New species and revised taxonomy proposal for the genus Begomovirus (Geminiviridae). ICTV. Retrieved from http://talk.ictvonline.org/files/proposals/taxonomy_proposals_plant1/ m/plant04/4720.aspx , 20 November 2013. (3) G. Lozano et al. J. Gen. Virol. 90:2550, 2009. (4) B. Muhire et al. Arch. Virol. 158:1411, 2013.

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