scholarly journals First Report of Apricot vein clearing-associated virus Infecting flowering apricot (Prunus mume) in the United States

Plant Disease ◽  
2021 ◽  
Author(s):  
Maher Al Rwahnih ◽  
Nourolah Soltani ◽  
Reid Soltero Brisbane ◽  
Tongyan Tian ◽  
Deborah Anne Golino
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
The United States ◽  
Cdna Libraries ◽  
Mechanical Transmission ◽  
Viral Agent ◽  
Sequencing Analysis ◽  
First Report ◽  
Vein Clearing ◽  
Biological Studies

Apricot vein clearing-associated virus is the type species of genus Prunevirus, family Betaflexiviridae. The virus was first discovered from an Italian apricot tree (Prunus armeniaca) showing leaf vein clearing and mottling symptoms (Elbeaino et al. 2014). Since then, apricot vein clearing-associated virus (AVCaV) has been reported in symptomatic and asymptomatic plants from other countries (Marais et al. 2015; Kinoti et al. 2017; Kubaa et al. 2014). In 2018, a domestic selection of a flowering apricot (P. mume cv. Peggy Clarke) (PC01) with no discernible foliar virus-like symptoms was received for inclusion in the Foundation Plant Services (UC-Davis) collection. The plant originated from a private Prunus collection located in California. Total nucleic acids (TNA) were isolated from PC01 leaves using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific). The TNA were analyzed for a panel of 15 Prunus-infecting viruses by reverse-transcription quantitative PCR (RT-qPCR) (Diaz-Lara et al. 2020). In addition, to screen for sap-transmissible viruses, young leaves of PC01 were homogenized in inoculation buffer and were rubbed onto leaves of herbaceous indicator plants, Chenopodium amaranticolor, C. quinoa, Cucumis sativus, and Nicotiana clevelandii (Rowhani et al. 2005). The source PC01 tested negative for the 15 screened viruses. Interestingly, vein clearing symptoms were observed on leaves of C. quinoa and C. amaranticolor plants (Figure S1). These results suggested the presence of a mechanically transmissible virus in PC01. To determine the identity of mechanically transmissible viral agent, symptomatic C. quinoa and PC01 plant were advanced for high throughput sequencing analysis. Aliquots of TNA from PC01 and C. quinoa were rRNA-depleted and used for cDNA library preparation with TruSeq Stranded Total RNA kit (Illumina). The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). A total of 47,261,138 and 8,812,296 single-end reads were obtained from cDNA libraries of PC01 and C. quinoa, respectively. The de novo assembly generated near-complete contigs resembling AVCaV genome ) from both PC01 and C. quinoa, which were 99.8% identical at the nucleotide level. The longest contig (8,342 nucleotides, 73.5x coverage depth) obtained from PC01 was further completed using SMARTer RACE 5’/3’ kit (Takara Bio). The complete genome sequence of AVCaV-PC01 is 8,364 nucleotides long (GenBank: MK170158). The full-length virus genome was compared with GenBank database using BLASTn, which the best hit corresponded to KY132099 with 98% identity. Additionally, AVCaV infection was confirmed in both PC01 selection and the symptomatic C. quinoa by RT-PCR as previously described (Marais et al. 2015). Lastly, symptomatic leaves of C. quinoa were used in leaf dip method to visualize virus particles by transmission electron microscope. As a result, flexuous rod-shaped virions were observed from leaf dips of symptomatic C. quinoa plants (Figure S2). Therefore, our results represent the first report of AVCaV in California, USA. Furthermore, mechanical transmission of an AVCaV isolate infecting flowering apricot to herbaceous hosts was confirmed. Field surveys and biological studies are underway to determine the prevalence of AVCaV in commercial orchards and assess its effect on tree performance.

scholarly journals First report of Cherry virus F infecting Japanese plum in Korea

Plant Disease ◽  
2020 ◽  
Author(s):  
Yeonhwa Jo ◽  
Hoseong Choi ◽  
Jin Kyong Cho ◽  
Won Kyong Cho
Keyword(s):  
Czech Republic ◽  
High Throughput Sequencing ◽  
Prunus Avium ◽  
De Novo ◽  
Protein Database ◽  
Specific Primers ◽  
The Czech Republic ◽  
First Report ◽  
Japanese Plum ◽  
Leaf Spots

Cherry virus F (CVF) is a tentative member of the genus Fabavirus in the family Secoviridae, consisting of two RNA segments (Koloniuk et al. 2018). To date, CVF has been documented in only sweet cherry (Prunus avium) in the Czech Republic (Koloniuk et al. 2018), Canada, and Greece. In May 2014, we collected leaf samples from four symptomatic (leaf spots and dapple fruits) and two asymptomatic Japanese plum cultivars (Sun and Gadam) grown in an orchard in Hoengseong, South Korea, to identify viruses and viroids infecting plum trees. Total RNA from individual plum trees was extracted using two commercial kits: Fruit-mate for RNA Purification Kit (Takara, Shiga, Japan) and RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). We generated six mRNA libraries from the six different plum cultivars for RNA-sequencing using the TruSeq RNA Library Preparation Kit v2 (Illumina, CA, U.S.A.) as described previously (Jo et al. 2017). The mRNA libraries were paired-end (2 X 100 bp) sequenced with a HiSeq 2000 system (Macrogen, Seoul, Korea). The raw sequence reads were de novo assembled by Trinity program v. 2.8.6, with default parameters (Haas et al. 2013). The assembled contigs were subjected to BLASTX search against the non-redundant protein database in NCBI. Of the two asymptomatic cultivars, the transcriptome of asymptomatic plum cv. Gadam contained five contigs specific to CVF. Two and three contigs were specific to CVF RNA1 (2,571 reads, coverage 42.15%) and RNA2 (2,025 reads, coverage 53.04%), respectively. The size of these five contigs ranged from 241 to 5,986 bp. Contigs of 5,986 and 3,867 bp in length, referred to as CVF isolate Gadam RNA1 (GenBank MN896996) and RNA2 (GenBank MN896995), respectively, were subjected to BLASTP search against NCBI’s non-redundant protein database. The results showed that the polyprotein sequences of RNA1 and RNA2 shared 95.3% and 93.11% amino acid identities with isolates SwC-H_1a from the Czech Republic (GenBank acc. no. AWB36326) and Stac-3B_c8 from Canada (AZZ10055), respectively. To confirm the infection of CVF in cv. Gadam, RT-PCR was conducted using CVF RNA1-specific primers designed based on the CVF reference genome sequences (MH998210 and MH998216), including 5’-CCACCAAATAGGCAAGAGGTCAC-3’ (position 3190–3212) and 5’-CACAATCACCATCAATGGTCTCTGC-3’ (position 3742–3766), and CVF RNA2-specific primers, including 5’-CTGCTTTATGATGCTAGACATCAAGATG-3’ (position 1015–1042) and 5’-ACAATAGGCATGCTCATCTCAACCTC-3’ (position 1594–1619). We amplified 577-bp RNA1-specific and 605-bp RNA2-specific amplicons that were cloned and then performed Sanger sequencing. Sequencing of the cloned amplicons for isolate Gadam RNA1 (GenBank MN896993) and RNA2 (GenBank MN896994) revealed values of 99.48% and 99.17% nucleotide identity to that of RNA1 and RNA2 determined by high-throughput sequencing, respectively. Additionally, we tested five plants for each of the six plum cultivars grown in the same orchard. The detection of CVF was carried out through PCR using the primers and protocol described above. Of the 30 trees, CVF was detected in three trees of cv. Gadam by both primer pairs. To our knowledge, this is the first report of CVF infecting Japanese plum and the first report of the virus in Korea. However, its prevalence in other Prunus species, including apricot, European plum, and peach, should be further elucidated.

scholarly journals Association of a Novel DNA Virus with the Grapevine Vein-Clearing and Vine Decline Syndrome

2011 ◽  
Vol 101 (9) ◽  
pp. 1081-1090 ◽  
Author(s):  
Yu Zhang ◽  
Kashmir Singh ◽  
Ravneet Kaur ◽  
Wenping Qiu
Keyword(s):  
United States ◽  
Small Rnas ◽  
The United States ◽  
Wide Distribution ◽  
Whole Genome Sequence ◽  
Cdna Libraries ◽  
Dna Virus ◽  
Vein Clearing ◽  
Vine Decline ◽  
Midwest Region

A severe vein-clearing and vine decline syndrome has emerged on grapevines (Vitis vinifera) and hybrid grape cultivars in the Midwest region of the United States. The typical symptoms are translucent vein-clearing on young leaves, short internodes and decline of vine vigor. Known viral pathogens of grapevines were not closely associated with the syndrome. To obtain a comprehensive profile of viruses in a diseased grapevine, small RNAs were enriched and two cDNA libraries were constructed from a symptomatic grapevine and a symptomless grapevine, respectively. Deep sequencing of the two cDNA libraries showed that the most abundant viral small RNAs align with the genomes of viruses in the genus Badnavirus, the family Caulimoviridae. Amplification of the viral DNA by polymerase chain reaction allowed the assembly of the whole genome sequence of a grapevine DNA virus, which shared the highest homology with the Badnavirus sequences. This is the first report of a DNA virus in grapevines. The new DNA virus is closely associated with the vein-clearing symptom, and thus has been given a provisional name Grapevine vein clearing virus (GVCV). GVCV was detected in six grapevine cultivars showing vein-clearing and vine decline syndrome in Missouri, Illinois, and Indiana, suggesting its wide distribution in the Midwest region of the United States. Discovery of DNA viruses in grapevines merits further studies on their epidemics and economic impact on grape production worldwide.

scholarly journals First Report of Beet necrotic yellow vein virus Infecting Spinach in California

Plant Disease ◽  
2010 ◽  
Vol 94 (5) ◽  
pp. 640-640 ◽  
Author(s):  
H.-Y. Liu ◽  
B. Mou ◽  
K. Richardson ◽  
S. T. Koike
Keyword(s):  
Spinacia Oleracea ◽  
Sandwich Elisa ◽  
Chenopodium Quinoa ◽  
Yellow Vein ◽  
Mechanical Transmission ◽  
Polymyxa Betae ◽  
Rt Pcr ◽  
First Report ◽  
Vein Clearing ◽  
Rigid Rod

In 2009, plants from two spinach (Spinacia oleracea) experimental fields in Monterey County and one commercial spinach field in Ventura County of California exhibited vein-clearing, mottling, interveinal yellowing, and stunting symptoms. For experimental fields, up to 44% of spinach plants have symptoms. With a transmission electron microscope, rigid rod-shaped particles with central canals were observed from plant sap of the symptomatic spinach. Analysis with a double-antibody sandwich-ELISA assay for Beet necrotic yellow vein virus (BNYVV) showed that all 10 symptomatic plants we tested were positive and 5 asymptomatic plants were negative. Symptomatic spinach from both counties was used for mechanical transmission experiments. Chenopodium quinoa, Tetragonia expansa, and Beta vulgaris (sugar beet) showed chlorotic local lesions and B. macrocarpa and spinach showed vein-clearing, mottling, and systemic infections. To further confirm the presence of BNYVV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from field- and mechanically inoculated symptomatic spinach plants using an RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR. Forward and reverse primers specific to the BNYVV RNA-3 P25 protein gene from the beet isolate were used (2). Amplicons of the expected size (approximately 860 bp) were obtained. Four RT-PCR products were sequenced and the sequences were identical (GenBank Accession No. GU135626). Sequences from the spinach plants had 97 to 99% nucleotide and 94 to 100% amino acid identity with BNYVV RNA-3 P25 protein sequences available in the GenBank. On the basis of the data from electron microscopy, indicator plants, serology, and cDNA sequencing, the virus was identified as BNYVV. BNYVV has been reported from spinach fields in Italy (1). To our knowledge, this is the first report of BNYVV occurring naturally on spinach in California. Since BNYVV is transmitted by the zoospores of the soil-inhabiting plasmodiophorid Polymyxa betae, it could be a new threat to spinach production in the state. References: (1) C. R. Autonell et al. Inf. Fitopatol. 45:43, 1995. (2) H.-Y. Liu and R. T. Lewellen, Plant Dis. 91:847, 2007.

scholarly journals First report of apple rubbery wood virus 1 in apple in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Guojun Hu ◽  
Yafeng Dong ◽  
Zunping Zhang ◽  
Xudong Fan ◽  
Fang Ren ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Rna Virus ◽  
Rt Pcr ◽  
Apple Rootstocks ◽  
Apple Trees ◽  
Illumina Hiseq ◽  
First Report ◽  
Sequence Contigs ◽  
Apple Stem

More than 30 viral and subviral pathogens infect apple (Malus domestica, an important fruit crop in China) trees and rootstocks, posing a threat to its production. With advances in diagnostic technologies, new viruses including apple rubbery wood virus 1 (ARWV-1), apple rubbery wood virus 2 (ARWV-2), apple luteovirus 1 (ALV), and citrus virus A (CiVA) have been detected (Beatriz et al. 2018; Rott et al. 2018; Hu et al. 2021). ARWV-1 (family Phenuiviridae) is a negative-sense single-stranded RNA virus with three RNA segments (large [L], medium [M], and small [S]). It causes apple rubbery wood disease (Rott et al. 2018) and is found in apple rootstocks, causing leaf yellowing and mottle symptoms in Korea (Lim et al. 2018). To determine virus prevalence in apple trees in China, 200 apple leaf and shoot samples were collected from orchards in Hebei (n = 26), Liaoning (40), Shandong (100), Yunnan (25), and Shanxi (4), and Inner Mongolia (5) in 2020. Total RNA was extracted from the shoot phloem or leaf (Hu et al., 2015) and subjected to reverse transcription (RT)-PCR to detect apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple necrotic mosaic virus (ApNMV), apple scar skin viroid (ASSVd), ARWV-2, ARWV-1, ALV, and CiVA, using primers specific to respective viruses (Supplementary Table 1). The prevalence of ACLSV, ASPV, ASGV, ApNMV, ASSVd, ARWV-2, ARWV-1, ALV and CiVA was found to be 75.5%, 85.5%, 86.0%, 43.0%, 4.0%, 48.5%, 10.5%, 0% and 0%, respectively (Supplementary Table 2). Among the 21 positive samples for ARWV-1, three, five and 13 samples were from Hebei, Liaoning, and Shandong, respectively. Five ARWV-1-positive samples (cultivars Xinhongjiangjun, Xiangfu-1, Xiangfu-2 and Tianhong) showed leaf mosaic symptoms. To confirm ARWV-1 by RT-PCR, amplicons from Xiangfu-1 and Tianhong were cloned into the pMD18-T vector (Takara, Dalian, China), and three clones of each sample were sequenced. BLASTn analyses demonstrated that the sequences (accession nos. MW507810–MW507811) shared 96.9%–98.9% identity with ARWV-1 sequences (MH714536, MF062127, and MF062138) in GenBank. An lncRNA library was prepared for high-throughput sequencing (HTS) with the Illumina HiSeq platform using Xiangfu-1 RNA. A total of 71,613,294 reads were obtained. De novo assembly of the reads revealed 135 viral sequence contigs of ACLSV, ASGV, ASPV, ApNMV, ARWV-1, and ARWV-2. The sequences of contig-100_88981 (302 nt) and contig-100_25701 (834 nt) (accession nos. MW507821 and MW507820) matched those of segment S from ARWV-1, whereas the sequences of contig-100_6542 (1,660 nt) and contig-100_27 (7,364 nt) (accession nos. MW507819 and MW507818) matched those of segments M and L, respectively. To confirm the HTS results, fragments of segments L (744 bp), M (747 bp), and S (554 bp) from Xiangfu-1 and Tianhong were amplified (Supplementary Table 1) and sequenced. The sequences (accession nos. MW507812–MW507817) showed 94.8%–99.9% nucleotide identity with the corresponding segments of ARWV-1. Co-infection of ARWV-1 with ApNMV and/or ARWV-2 was confirmed in 17/21 ARWV-1-positive samples. The prevalence of ARWV-1/ApNMV, ARWV-1/ARWV-2, and ARWV-1/ApNMV/ARWV-2 infections was 61.9%, 71.4%, and 52.4%, respectively. To our knowledge, this is the first report of ARWV-1 infecting apple trees in China. Further research is needed to determine whether and how ARWV-1 affects apple yield and quality.

scholarly journals First report of grapevine virus L infecting grapevine in southeast France

Plant Disease ◽  
2022 ◽  
Author(s):  
Laurence Svanella ◽  
Armelle Marais ◽  
Thierry Candresse ◽  
Marie Lefebvre ◽  
Jerome Lluch ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Protein Gene ◽  
The United States ◽  
Read Length ◽  
Nucleic Acid Binding ◽  
Grapevine Virus ◽  
Total Rna ◽  
First Report ◽  
Grapevine Virus A ◽  
Hop Stunt Viroid

Grapevine virus L (GVL) is a recently described vitivirus (family Betaflexiviridae) with a positive-sense single-stranded RNA genome. It has so far been reported from China, Croatia, New-Zealand, the United States and Tunisia (Debat et al. 2019; Diaz-Lara et al. 2019; Alabi et al. 2020; Ben Amar et al. 2020). It has significant genetic variability (up to 26% of nucleotide divergence between isolates) and the existence of four phylogroups has been proposed (Alabi et al. 2020). In the frame of a project investigating the possible links between grapevine trunk diseases and grapevine virome, viral high throughput sequencing (HTS)-based testing was performed on symptomatic and asymptomatic grapevines collected in July 2019 in vineyards of four areas in France (Bourgogne, Charentes, Gard, Gironde) corresponding to five cultivars of Vitis vinifera (Cabernet franc, Cabernet Sauvignon, Chardonnay, Sauvignon, Ugni blanc). Total RNAs were purified from powder of 105 trunk wood samples using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and RNA-seq libraries were prepared using Zymo-Seq RiboFree Total RNA Library Prep Kit (Ozyme, Saint Cyr l’Ecole, France). HTS was performed on a S4 lane of Illumina NovaSeq 6000 using a paired-end read length of 2x150 bp. The trimmed sequence reads obtained from Chardonnay plants CH30-75M (99.9 M) and CH37-19S (114 M) from a vineyard in Gard were analyzed using CLC Genomics Workbench v21 (Qiagen, Courtaboeuf, France) and revealed complex mixed infections. Besides contigs representing a complete GVL genome (average scaffold coverage: 6,197x and 2,970x, respectively), contigs from grapevine rupestris stem pitting virus (1,697x ; 1,124x), grapevine virus A (82x ; 95x), grapevine pinot gris virus (1,475x ; 866x), grapevine leafroll-associated virus 3 (5,122x ; 1,042x), hop stunt viroid (13,783x ; 29,514x) and grapevine yellow speckle viroid 1 (690x ; 1158x) were also identified. Plant CH37-19S was also co-infected by grapevine rupestris vein feathering virus (164x). The GVL contigs integrated respectively 320,000 and 152,000 reads (corresponding to 0.32% and 0.11% of filtered/trimmed reads, respectively). The GVL genomic sequences from each sample (7,616 nt) have been deposited in GenBank (Accession nos. OK042110 and OK042111, respectively). The two contigs are nearly identical (99.9% nt identity) and share respectively 97.5% and 95.9% with GVL-KA from the USA (MH643739) and GVL-RS from China (MH248020), the closest isolates present in GenBank. To confirm the presence of GVL, the original grapevines were resampled in the field and total RNAs were extracted as described above from cambial scrappings and leaves. Total RNAs were used for RT-PCR tests using primers targeting a 279-bp fragment corresponding to the 3’ end of the coat protein gene and part of the nucleic acid binding protein gene (Debat et al. 2019). The Sanger-derived sequences from the amplicons shared 100% nt identities with the corresponding sequences of the HTS assembled genomes, confirming the presence of GVL in both tissues of both grapevine samples. To our knowledge, this represents the first report of the occurrence of GVL in vineyards in France. Given the complex mixed infection present in the two analyzed grapevines, no conclusions can be drawn on the pathogenicity of GVL. Further efforts are needed to better understand GVL distribution and its potential pathogenicity to grapevine. References Alabi, O J., et al. 2020. Arch. of Virol. 165:1905-1909. Ben Amar, A., et al. 2020. Plant disease 104:3274. Debat, H., et al. 2019. Eur J Plant Pathol. 155:319. Diaz-Lara, A., et al. 2019. Arch. of Virol. 164:2573. Acknowledgments The authors are grateful to the “Plan National Dépérissement du Vignoble” (Mycovir project) for the financial support

scholarly journals First report of Rose leaf rosette-associated virus infecting rose (Rosa spp.) in California, USA

Plant Disease ◽  
2021 ◽  
Author(s):  
Nourolah Soltani ◽  
Deborah Anne Golino ◽  
Maher Al Rwahnih
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Rna Isolation ◽  
Complete Sequence ◽  
Nucleotide Sequences ◽  
Ringspot Virus ◽  
Reference Sequence ◽  
Rosa Multiflora ◽  
First Report ◽  
Putative Coat Protein

Rose leaf rosette-associated virus (RLRaV) is a member of genus Closterovirus, family Closteroviridae. The virus was first discovered in China in 2015 from a mixed infected wild rose (Rosa multiflora Thunb.) showing small leaf rosettes on branches, dieback and severe decline symptoms (He et al. 2015). In 2013, a rose plant (cv. Roses Are Red) was introduced to Foundation Plant Services (FPS, UC-Davis) rose collection. The plant was originated from a private rose breeder collection located in California. In 2019, total nucleic acids (TNA) were isolated from leaf tissues of one asymptomatic plant (Roses Are Red plant) using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific, USA). Extracted TNA were screened by reverse-transcription quantitative PCR (RT-qPCR) for six common viruses infecting roses, including prunus necrotic ringspot virus (PNRSV), apple mosaic virus (ApMV), rose spring dwarf associated virus (RSDaV), rose yellow vein virus (RYVV), rose rosette virus (RRV), and blackberry chlorotic ringspot virus (BCRV); however, the results were negative. Therefore, the sample was subjected to high throughput sequencing (HTS). Briefly, TNA was depleted of rRNA and advanced for cDNA library preparation using TruSeq Stranded Total RNA kit (Illumina, USA). HTS was performed on Illumina NextSeq 500 platform. The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). HTS generated 23.6 million 75 nucleotide (nt) single-end raw data reads. De novo assembly generated a contig (16,528 nts) resembling RLRaV reference sequence (KJ748003) with 74% identity at the nucleotide level. Putative coat protein and heat shock protein 70-like protein were identified based on >90% identity with RLRaV genes. To confirm HTS results, RT-PCR was performed using two primer sets, 1) Clo-F4916 (5’-GGTGTTCCAACGCTATCGTG-3’) and Clo-R5215 (5’- TGTCCTCAAACCGCCTACAT-3’) targeting nucleotide sequences of putative polyprotein 1a, and 2) Clo-F10006 (5’-GATTCCGCGGACGAATTAAT-3’) and Clo-R10311 (5’-GGTAACCGAAAGGTAAAGTATTC-3’) targeting nucleotide sequences of putative protein p25. The RLRaV amplicons with expected size of 300 nt were confirmed using bidirectional Sanger sequencing. The near-complete sequence of the new RLRaV isolate was deposited in GenBank under accession number MW056181. In addition, HTS analysis showed that RLRaV was in mixed infection with two mycoviruses (rose cryptic virus with 8,267 mapped reads and rose partitivirus with 7,283 mapped readss). To our knowledge, this is the first report of RLRaV affecting roses in California. Further research is needed to determine the prevalence of RLRaV in California as well as evaluation of RLRaV effect on rose performance.

scholarly journals First Report of Cowpea Aphid-Borne Mosaic Potyvirus from Cowpeas Grown Commercially in the U.S.

Plant Disease ◽  
1997 ◽  
Vol 81 (8) ◽  
pp. 959-959 ◽  
Author(s):  
A. S. Kline ◽  
E. J. Anderson
Keyword(s):  
Chenopodium Quinoa ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mechanical Transmission ◽  
First Report ◽  
Chlorotic Mottle ◽  
Cowpea Aphid ◽  
Healthy Control ◽  
Systemic Infections ◽  
The U.S

Cowpea aphid-borne mosaic potyvirus (CABMV) is one of several seed-borne viruses known to limit cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata) production in Africa, Europe, and Asia, but CABMV has not been reported on commercially grown cowpeas in the United States (1). However, a sesame (Sesamum indicum L.)-infecting isolate of CABMV was recently characterized from plants growing near cowpea introduction plots in Georgia (2). In February 1997, we received samples of three seed lots of cowpea cv. Chinese Red that had been harvested in southern Texas during 1996. Approximately 28% of the plants grown from these seed lots expressed strong mosaic symptoms on primary and trifoliate leaves. Viruslike symptoms were reproduced following mechanical transmission to plants of Chinese Red cowpea, Nicotiana benthamiana, and soybean (Glycine max L.) cv. Lee. When Coronet and Pinkeye Purple Hull-BVR cowpeas were inoculated with sap extracts from symptomatic Chinese Red plants, chlorotic lesions developed on inoculated leaves, but only Coronet plants supported symptomless systemic infections. Similarly inoculated plants of Chenopodium quinoa (L.) and common bean (Phaseolus vulgaris L.) cvs. Pinto and Black Valentine developed localized chlorotic lesions. In Ouchterlony gel diffusion assays, extracts from symptomatic cowpea plants did not react with antisera to blackeye cowpea mosaic potyvirus (BlCMV), cucumber mosaic cucu-movirus (CMV), southern bean mosaic sobemovirus, cowpea mosaic comovirus, cowpea severe mosaic comovirus, or cowpea chlorotic mottle bromovirus. In the indirect enzyme-linked immunosorbent assay, sap extracts from symptomatic plants reacted with antiserum to CABMV, giving OD values at A405 of 0.10 to 0.25, and reacted weakly with antiserum to BlCMV, with OD values at A405 less than 0.035. Extracts from healthy control plants gave OD values at A405 less than 0.010. No positive reactions were obtained with antisera to bean yellow mosaic potyvirus, peanut mottle potyvirus, soybean mosaic potyvirus, or CMV. To our knowledge, this is the first report of CABMV in commercially grown cowpea from the U.S. References: (1) A. G. Gillaspie et al. Plant Dis. 79:388, 1995. (2) H. R. Pappu et al. Arch. Virol. 142:1, 1997.

scholarly journals First report of grapevine virus H (GVH) in grapevine in Greece

Plant Disease ◽  
2021 ◽  
Author(s):  
Polina Panailidou ◽  
Leonidas Lotos ◽  
Chrysoula-Lyto Sassalou ◽  
E. Gagiano ◽  
Gerhard Pietersen ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Virus Genome ◽  
Composite Sample ◽  
Grapevine Virus ◽  
Northern Greece ◽  
Total Rna ◽  
First Report ◽  
The Usa

Grapevine virus H (GVH) is a member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae, order Tymovirales) that infects grapevine (Candresse et al., 2018). GVH was first identified in a symptomless grapevine of an unknown cultivar from Portugal in 2018 (Candresse et al. 2018), and since then the virus has been reported only from California (Diaz‑Lara et al. 2019). Several vitiviruses have been detected in Greek vineyards (Avgelis and Roubos 2000; Dovas and Katis 2003a; 2003b; Panailidou et al. 2019; Lotos et al. 2020), but no information was available on the presence of GVH. In the fall of 2020, in order to investigate the virome of a commercial vineyard of the cultivar Assyrtiko in northern Greece, a composite sample was made of leaves and petioles from nine vines exhibiting leafroll disease symptoms. Total RNA was extracted from the composite sample according to the protocol of White et al. (2008) and subjected to rRNA depletion, library construction (TruSeq Stranded Total RNA kit), and high-throughput sequencing (HTS) in a NovaSeq6000 platform (Illumina Inc.) at Macrogen (Korea). The resulting ~42 million 101-nt paired-end reads were analyzed in Geneious Prime 2020, and the assembled de novo contigs were subjected to a local BLASTn search, which revealed the presence of 18 grapevine infecting viruses and viroids, among which also a GVH-like contig (GeA-9). GeA-9 was 7,404 nucleotides (nt) long, covering 99.4% of the full virus genome and shared 98.2 % nt identity with a GVH isolate from the USA (MN716768). To confirm the presence of GVH, the nine samples of the cultivar Assyrtiko, used initially to produce the composite sample for HTS analysis, were tested individually by RT-PCR, using the primers GVH_F_2504 (5’-CTGCTTCGCTGAACATATGC-3’) and GVH_R_2835 (5’-ATCATTRTGATCGAGAGAGTAGTG-3’) that amplify a 331-nt segment of ORF1. GVH was detected in five out of the nine tested samples and one of these was reamplified and subjected to Sanger sequencing. The fragment of ORF1 obtained by Sanger sequencing (MW460005) was 97.5% identical to the nucleotide sequence of the corresponding GVH-like de novo contig (GeA-9) from HTS analysis and it shared 97.2% nt identity with GVH sequences reported from Portugal and USA, respectively (NC_040545 and MN716768), confirming the presence of GVH in the tested samples. This is the first report of GVH in grapevine in Greece, thus further increasing the number of vitiviruses known to infect Greek vineyards and also expanding the number of geographic locations in which GVH is recorded so far.

scholarly journals First report of Coguvirus eburi infecting pear (Pyrus communis) in South Africa

Plant Disease ◽  
2021 ◽  
Author(s):  
Kayleigh Bougard ◽  
Hans Jacob Maree ◽  
Gerhard Pietersen ◽  
Julia Christine Meitz-Hopkins ◽  
Rachelle Bester
Keyword(s):  
South Africa ◽  
High Throughput Sequencing ◽  
De Novo ◽  
Rna Extraction ◽  
Pyrus Communis ◽  
Rt Pcr ◽  
Disease Symptoms ◽  
Total Rna ◽  
First Report ◽  
Pcr Assays

Coguvirus eburi is a member of the genus Coguvirus in the family Phenuviridae (Khun et al., 2020). The species Coguvirus eburi was established to include citrus virus A (CiVA), which is a negative-sense, single-stranded RNA virus that was first found infecting sweet orange in southern Italy via high-throughput sequencing (HTS) (Navarro et al., 2018). This virus was also found to infect pome fruits in France, such as pear (Svanella-Dumas et al., 2019). More recently CiVA infections have been associated with impietratura disease in citrus (Beris et al. 2021). In the summer of 2021, leaf samples were collected from a pear tree (Pyrus communis cv. Bosc, B175) in the Koue Bokkeveld, South Africa as part of a virus survey. Sample B175 displayed no visual disease symptoms. One gram of leaf petioles was used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). Ribo-depleted RNA was prepared (Ribo-Zero Plant kit) and a sequencing library constructed (Illumina TruSeq Stranded Total RNA). The RNA library was paired-end (2 × 100 bp) sequenced on an Illumina HiSeqX instrument (Macrogen, South Korea). A total of 47,750,152 reads were obtained. Raw data was trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly performed with CLC Genomics Workbench 11.0.1 (Qiagen) (Default parameters) using high quality reads yielded 75250 contigs. BLASTn analysis identified two viral contigs with high nucleotide (nt) identity to apple stem pitting virus (ASPV) and CiVA. The CiVA contig was 9400 nts and on closer examination, a concatemer of CiVA RNA1 and RNA2. The concatenation occurred due to the characteristic near-identical nucleotides shared at the 5’ and 3’ ends of RNA1 and RNA2 of these negative-stranded RNA viruses (Navarro et al., 2018). After splitting and curation, the RNA1 contig was 6664 nts and the RNA2 contig 2686 nts. A total of 51397 and 34820 reads were used to construct these contigs resulting in an average depth of coverage of 761 and 1281 for RNA1 and RNA2, respectively. The contigs had the highest nt identity to the complete CiVA GenBank accessions MT720885.1 (95.53%) and MW148460.1 (96.03%), spanning 99.6% and 98.1 % of the genomes of RNA1 and RNA2, respectively. These contigs were submitted as partial genomes to GenBank as accessions MZ463039 and MZ463040. Reverse transcription polymerase chain reaction (RT-PCR) was used to validate the presence of CiVA in sample B175. Two RT-PCR assays, directed at RNA1 and RNA2 respectively (Bester et al. (2021)) were used to generate amplicons. Amplicon sequences were confirmed with bi-directional Sanger sequencing. Twenty-one additional samples from the same orchard as B175 as well as other samples from the Koue Bokkeveld and Elgin areas, including cultivars Abate (10 samples), Forelle (10 samples), Early Bon Chretien (3 samples), Packham’s Triumph (12 samples) and Rosemarie (3 samples), were all surveyed for CiVA using the same RT-PCR assays as mentioned above. Thirty-six of the 59 samples tested were positive for CiVA, which further confirms the presence and wide-spread distribution of this virus in the limited survey conducted in pears in South Africa. However, no association with any disease symptoms or specific cultivar were identified. This is the first report of CiVA infecting pear in South Africa. This study therefore contributed to investigating the distribution of this virus and will assist the South African plant material certification scheme to assess the incidence of CiVA in South Africa.

scholarly journals First report of eggplant mottled crinkle virus infecting eggplant in Greece.

Plant Disease ◽  
2021 ◽  
Author(s):  
Despoina Beris ◽  
Ioanna Malandraki ◽  
Oxana Kektsidou ◽  
Christina Varveri
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Solanum Melongena ◽  
Post Inoculation ◽  
The European Union ◽  
Rt Pcr ◽  
Systemic Necrosis ◽  
Capsid Protein Gene ◽  
First Report ◽  
Local Lesions

During winter 2020-2021, a severe virus-like disease outbreak was observed in eggplant (Solanum melongena L.) hybrids ‘Monarca’ (F1) and ‘Angela’ (F1) growing under protected conditions in Heraklion, Crete, Greece. In three greenhouses, the percentage of infected plants reached 100% leading to crop abandonment. Symptoms included leaf mottling and yellowing accompanied with plant stunting and apical necrosis. Extensive fruit damage was due to severe malformation and necrotic lesions on the calyx, peduncle and the endocarp (Sup. Fig. 1). To identify the causal agent, total RNA was extracted from a symptomatic eggplant fruit with PureLink™ RNA Mini Kit (ThermoFisher Scientific, USA), which was subjected to high throughput sequencing (HTS) analysis (Illumina Inc., USA). The de novo assembly of the obtained 25 million, 75 bp, single-end reads with Geneious Prime (Biomatters, New Zealand) and the annotation of the resulting contigs with BLASTn revealed the presence of only eggplant mottled crinkle virus (EMCV, genus Tombusvirus) in the sample. The assembled sequence of EMCV isolate from Greece (EMCV-Gr, GenBank Acc. No. MW716271) was 4764 bp in length, covering the full genome of the virus and showing 96.3 % nucleotide (nt) identity with an isolate identified from calla lilies (Zantedeschia sp.) in Taiwan (AM711119). Five symptomatic and seven asymptomatic ‘Monarca’ (F1) eggplants, as well as two symptomatic ‘Angela’ (F1) eggplants were tested by RT-PCR that targeted the capsid protein gene of the virus (Dombrovsky et al., 2009). PCR products of 1184 bp were obtained from the seven symptomatic samples and their Sanger sequencing revealed 100 % nt identity with the respective HTS-derived EMCV sequence. No product was obtained from the analysis of the asymptomatic samples. Mechanical sap transmission of the HTS analysed eggplant sample resulted in necrotic local lesions on Nicotiana rustica and Chenopodium quinoa, necrotic local lesions plus systemic necrosis on N. tabacum cv. Xanthi-nc, cv. Samsun and N. glutinosa, systemic collapse of N. benthamiana, and leaf mottling plus stunting of pepper cv. Yolo Wonder plants (Sup. Fig. 1I). Although no symptoms were observed on tomato plants cv. Ace 55, systemic EMCV infection was detected by RT-PCR. To establish the relationship between the disease and EMCV, infected tissue from N. benthamiana plants was used for the mechanical inoculation of virus-tested negative eggplant seedlings cv. Black beauty. Necrotic spots, shoot necrosis, leaf mottling and mosaic, symptoms were observed (Sup. Fig. J) on the test plants ten days post inoculation and the presence of the virus was confirmed by RT-PCR as described. To the best of our knowledge this is the first report of EMCV infecting eggplant in Greece. The virus was originally described in eggplant in Lebanon (Makkouk et al., 1981) and it is mainly present outside the European Union (EU) territory, including India, Japan, Taiwan, Iran and Israel (Dombrovsky et al., 2009 and references therein). A latent EMCV infection was detected in pear in Italy (Russo et al., 2002) and the virus is considered by the European Food Safety Authority as an exotic virus of the genera Cydonia, Malus, and Pyrus that meets all the criteria to qualify as an EU quarantine pest (Bragard et al., 2019). Τhe severity of the disease observed in Crete leading to the destruction of eggplant greenhouse cultivations, constitutes EMCV as an emerging threat to eggplant and other solanaceous crops for Greece and Europe.

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