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

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

Plant Disease ◽

10.1094/pdis-01-21-0175-pdn ◽

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.

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

Plant Disease ◽

10.1094/pdis-08-21-1630-pdn ◽

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.

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First Report of Tomato spotted wilt virus in Lettuce Crops in Saudi Arabia

Plant Disease ◽

10.1094/pdis-05-14-0444-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1591-1591 ◽

Cited By ~ 2

Author(s):

M. A. Al-Saleh ◽

I. M. Al-Shahwan ◽

M. A. Amer ◽

M. T. Shakeel ◽

M. H. Ahmad ◽

...

Keyword(s):

Saudi Arabia ◽

Tomato Spotted Wilt Virus ◽

Post Inoculation ◽

Rt Pcr ◽

Specific Primers ◽

Indicator Plants ◽

First Report ◽

Tomato Spotted Wilt ◽

Local Lesions ◽

Wilt Virus

A survey for viruses in open field lettuce crops was carried out in March 2014 in the Al-Uyaynah area, central region of Saudi Arabia. In one plot, more than 50% of the lettuce plants (Lactuca sativa; hybrid: Romaine), with the majority of the affected plants in the edges of the plot, were showing virus-like symptoms such as necrotic lesions, necrosis of the lamina of the younger leaves, and leaf curling, indicating a possible infection by a Tospovirus, possibly Tomato spotted wilt virus (TSWV). Most of them were dead when the field was visited again 3 weeks later. Samples from 10 symptomatic and two asymptomatic plants were collected. Five of the samples from symptomatic and two from asymptomatic plants were mechanically inoculated onto Nicotiana benthamiana and N. glutinosa (three indicator plants of each species were used for each sample) using 0.1 M phosphate buffer (pH 7) containing 0.01M Na2SO3 mM. All the symptomatic lettuce samples were also tested serologically using polyclonal antisera (3) against TSWV, CMV, and by using monoclonal antibodies against potyviruses. Moreover, total RNA was extracted (1) and detection of TSWV was also attempted with reverse transcription (RT)-PCR using species specific primers (4) for a 276-bp fragment of the L RNA segment. In both serological and molecular methods, positive and negative controls were included. All the mechanically inoculated plants with tissue from the symptomatic lettuce plants of N. benthamiana showed chlorotic local lesions followed by systemic top necrosis 2 to 3 weeks post inoculation. Similarly, all inoculated N. glutinosa plants showed necrotic local lesions followed by systemic chlorosis. However, all the indicator plants mechanically inoculated with tissue from asymptomatic lettuce plants gave no reaction. All the symptomatic lettuce samples reacted positively, while asymptomatic samples reacted negatively in ELISA tests with TSWV antiserum and the presence of the virus was further confirmed by RT-PCR by using specific primers (method A) (4). PCR products of two randomly selected positive samples were directly sequenced and BLAST analysis of the obtained sequences (Accession Nos. KJ701035 and KJ701036) revealed 99% nucleotide and 100% amino acid identity with the deposit sequence in NCBI from South Korea (KC261947). Regarding mechanical inoculation, 10 days post-inoculation, both indicator plants showed typical symptoms of TSWV infection, such as necrotic local lesions, systemic necrotic patterns, and leaf deformation. None of the symptomatic plants was found to be infected with either CMV or potyvirus. To our knowledge, this is the first report of TSWV naturally infecting lettuce in Saudi Arabia; therefore, insect vector and weed management are necessary measures to control the virus spread to other crops such as tomato and pepper (2). References: (1) E. Chatzinasiou et al. J. Virol. Meth. 169:305, 2010. (2) E. K. Chatzivassiliou. Plant Dis. 92:1012, 2008. (3) E. K. Chatzivassiliou et al. Phytoparasitica 28:257, 2000. (4) R. A. Mumford et al. J. Virol. Meth. 46:303, 1994.

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First Report of Spinach latent virus in Tomato in New Zealand

Plant Disease ◽

10.1094/pdis-91-2-0228a ◽

2007 ◽

Vol 91 (2) ◽

pp. 228-228 ◽

Cited By ~ 4

Author(s):

B. S. M. Lebas ◽

F. M. Ochoa-Corona ◽

Z. J. Tang ◽

R. Thangavel ◽

D. R. Elliott ◽

...

Keyword(s):

New Zealand ◽

Pcr Amplification ◽

Latent Virus ◽

Replicase Gene ◽

Rt Pcr ◽

Chenopodium Amaranticolor ◽

Systemic Necrosis ◽

First Report ◽

Local Lesions ◽

C 30

A Lycopersicon esculentum (tomato) plant from a commercial property in New Zealand was submitted to the Investigation and Diagnostic Centre for diagnosis in 2003. Fruits had faint yellow ringspots but no obvious symptoms were observed on leaves. No virus particles were observed from tomato and symptomatic herbaceous plants crude sap preparations. Mechanically inoculated Nicotiana clevelandii and N glutinosa developed systemic chlorosis, whereas pinpoint necrotic local lesions were observed on Chenopodium amaranticolor. Chlorotic local lesions were also observed on C. quinoa followed by systemic necrosis. No symptoms were observed on Cucumis sativus, Gomphrena globosa, N. benthamiana, N. sylvestris, or N. tabacum cv. White Burley. Total RNA was extracted from N. glutinosa and C. quinoa leaf samples using the Qiagen (Qiagen Inc., Valencia, CA) Plant RNeasy Kit. Reverse transcription (RT) was carried out by using random hexamer primers and SuperScript II reverse transcriptase (Invitrogen, Frederick, MD) followed with PCR using broad-detection primers targeting the genera Carmovirus, Dianthovirus, Ilarvirus, Tospovirus, (Agdia Inc., Elkhart, IN) and Tombusvirus (2). A positive RT-PCR amplification was obtained only with Ilarvirus primers. The 450-bp product (GenBank Accession No. DQ457000) from the replicase gene had a 97.4% nt and 98.6% aa identity with Spinach latent virus (SpLV; Accession No. NC_003808). An RT-PCR protocol was developed for the specific detection of SpLV. Primers were designed from three SpLV RNA sequences (RNA1: NC_003808; RNA2: NC_003809; RNA3: NC_003810) using the Primer3 software (3). Primers SpLV-RNA1-F (5′-TGTGGATTGGTGGTTGGA-3′) and SpLV-RNA1-R (5′-CTTGCTTGAGGAGAGATGTTG-3′) anneal to the replicase gene from nt 1720 to 2441. Primers SpLV-RNA2-F (5′-GAACCACCGAAACCGAAA-3′) and SpLV-RNA2-R (5′-CCACCTCAACACCAGTCATAG-3′) bind to the polymerase gene from nt 603 to 1038. Primers SpLV-RNA3-F (5′-GCCTTCATCTTTGCCTTTG-3′) and SpLV-RNA3-R (5′-CATTTCATCTGCGGTGGT-3′) amplify the movement protein gene from nt 724 to 936. The predicted amplified product sizes were 722, 436, and 213 bp from RNA1, RNA2, and RNA3, respectively. RT was carried out as described above. PCR was performed in a 20-μl reaction containing 2 μl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM of forward and reverse primers, and 1 U Taq polymerase (Promega, Madison, WI). The PCR amplification cycle was identical for the three primer pairs: denaturation (95°C for 3 min) followed by 37 cycles of 95°C (20 s), 60°C (30 s), and 72°C (30 s) with a final elongation step (72°C for 3 min). The amplified products were analyzed by gel electrophoresis, stained with SYBR Green, and their identities confirmed by sequencing. The tomato sample was grown from seed imported from the Netherlands where SpLV occurs (4). The virus is of potential importance for the tomato industry because of its symptomless infection and high frequency of seed transmission in many plant species (1,4). SpLV has never been detected in other submitted tomato samples. Consequently, SpLV is not considered to be established in New Zealand. To our knowledge, this is the first report of SpLV in tomato. References: (1) L. Bos et al. Neth. J. Plant Pathol. 86:79, 1980. (2) R. Koeing et al. Arch. Virol. 149:1733, 2004. (3) S. Rozen and H. Skaletsky. Page 365 in: Bioinformatics Methods and Protocols. Humana Press, Totowa, NJ, 2000. (4) Z. Stefenac and M. Wrischer. Acta Bot. Croat. 42:1, 1983.

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The occurrence of strawberry virus 1 infecting strawberry in Shandong province, China

Plant Disease ◽

10.1094/pdis-01-21-0038-pdn ◽

2021 ◽

Author(s):

Chengyong He ◽

Dehang Gao ◽

Lingjiao Fan ◽

Tengfei Xu ◽

Fei Xing ◽

...

Keyword(s):

High Throughput Sequencing ◽

De Novo ◽

Shandong Province ◽

Post Inoculation ◽

Rt Pcr ◽

Illumina Hiseq ◽

Total Rna ◽

Sodium Phosphate Buffer ◽

Strawberry Vein Banding Virus ◽

Two Samples

Strawberry (Fragaria × ananassa Duch.) is one of the most important horticultural plants worldwide with high economic and nutritional value. Strawberry associated virus 1 (SaV1) is a putative Cytorhabdovirus isolated from strawberry in Fujian province, China (Ding et al., 2019). Strawberry virus 1 (StrV-1) is another putative Cytorhabdovirus characterized from F. ananassa and F. vesca in Czech Republic (Fránová et al., 2019). The complete genomes of isolates of SaV1 and StrV-1 share 79 to 98% nucleotide (nt) identities. In August 2020, foliar chlorotic spots or streaks were observed in four strawberry cultivars (cv. Honeoye, Mibao, 8128 and All Star) in Yantai, Shandong province, China. To identify the associated viruses, symptomatic leaves from two plants of each cultivar (8 samples) were pooled for high-throughput sequencing (HTS). Total RNA was extracted from the composite sample and used for constructing a cDNA library after ribosomal RNA (rRNA)-depletion. Sequencing was carried out on Illumina Hiseq 4000 (Novogene, China). Raw reads were filtered, trimmed and de novo assembled as described previously (Grabherr et al., 2013; Zhou et al. 2020). The resulting contigs were screened by BLASTn and BLASTx against GenBank database. Subsequent analyses indicated the presence of strawberry vein banding virus, strawberry pallidosis associated virus and strawberry mottle virus in the analyzed sample, which had been reported previously in strawberry (Martin and Tzanetakis, 2013; Shi et al., 2018; Bhagwat et al., 2016). Besides, five contigs ranging from 266 to 6,057 nt were obtained. They shared 87 to 91% nt sequence identity with StrV-1 isolate B (GenBank accession no. MK211271). To confirm StrV-1 infection in the strawberry plants, total RNA was isolated from all eight samples using RNAprep Pure Plant Plus Kit (Tiangen, China). Reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers StrVp1 (Forward: 5ʹ-CATTACTGAAGCATTCCGTG-3′/Reverse: 5ʹ-AGATATCACGCACAGTGAC-3ʹ), and StrVp2 (Forward: 5ʹ-TTGCGCGAAGCGGATGTCCG-3′/Reverse: 5ʹ-GGCTGCCAGAGCGTTGGATG-3ʹ), targeting nt positions 70-1,231 and 7,825-9,348 of StrV-1 isolate B, respectively. Fragments with the expected sizes were amplified from two samples of cv. All Star. The amplicons were cloned, sequenced, and deposited in GenBank under accession no. MW419123-124 and MW645247-248. Both protein encoding sequences shared 91 to 92% and 80 to 84% nt identities with the corresponding sequences of StrV-1 isolate B and SaV1, respectively, indicating that the isolates from this study are genetic variants of StrV-1 and distantly related to SaV1. Crude sap was prepared by homogenizing leaf tissues of StrV-1 infected strawberry in 0.02 mol/L sodium phosphate buffer with 0.45% (w/v) sodium diethyldithiocarbamate thihydrate, then gently rubbed onto five healthy Nicotiana benthamiana plants. Neither the inoculated leaves nor the systemically infected leaves showed obvious symptoms seven days post inoculation. However, StrV-1 was detected by RT-PCR in all five N. benthamiana plants as described above. In addition, a survey of strawberry greenhouses was conducted in August 2020 and approximately 10% of plants in a 667 m2 greenhouse in Yantai had StrV-1-like symptoms. To the best of our knowledge, this is the first report of the occurrence of StrV-1 infecting strawberry in Shandong province, China. Our findings expand the geographic range and genetic diversity of StrV-1 and indicate it could be a potential virus threat to strawberry production in China.

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First Report of Cucumber mosaic virus in Saintpaulia ionantha in Korea

Plant Disease ◽

10.1094/pdis-08-13-0847-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 573-573 ◽

Cited By ~ 1

Author(s):

J. Y. Yoon ◽

G. S. Choi ◽

I. S. Cho ◽

S. K. Choi

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

African Violet ◽

Saintpaulia Ionantha ◽

Local Lesions ◽

Pcr Products ◽

Das Elisa

African violet (Saintpaulia ionantha) is an ornamental species of the family Gesneriaceae and is characterized by fleshy leaves and colorful flowers. This popular, exotic ornamental, originally from Kenya and Tanzania, is vegetatively produced from cutting and tissue culture (1). In May 2013, virus-like foliar symptoms, including a mosaic with dark green islands and chlorosis surrounding the veins, were observed on an African violet plant in a greenhouse located in Icheon, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with a commercial immunostrip kit (Agdia, Elkhart, IN). The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-AV1) caused necrotic local lesions on Chenopodium amaranticolor at 5 days post-inoculation (dpi), while mild to severe mosaic was observed in Nicotiana glutinosa, N. tabacum ‘Samsun NN,’ Cucurbita pepo ‘Super-Top,’ Physalis angulate, and Solanum lycopersicum ‘Unicorn’ 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ∼28 nm in diameter, respectively. To verify whether CMV-AV1 is the cause of disease symptoms observed in African violet, virus-free African violet (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-AV1. At 8 weeks after inoculation, all plants produced systemic mosaic and chlorosis surrounding veins, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated African violet plants remained symptomless and virus-free. The presence of CMV-AV1 in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CPTALL3 and CPTALL5 (2), amplifying the entire CP gene and part of an intergenic region and 3′-noncoding region of CMV RNA3. RT-PCR products (960 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-AV1 CP sequence (Accession No. AB842275) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-AV1 infection. CMV may pose a major threat for production of African violet since the farming of African violet plants is performed using the vegetative propagation of the African violet leaves in Korea. In particular, mosaic and chlorosis symptoms in African violet cause damage to ornamental quality of African violet. To our knowledge, this is the first report of CMV infection of African violet in the world. References: (1) S. T. Baatvik. Fragm. Flor. Geobot. Suppl. 2:97, 1993. (2) S. K. Choi et al. J. Virol. Methods 83:67, 1999.

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First Report of Cucumber mosaic virus in Catharanthus roseus in Korea

Plant Disease ◽

10.1094/pdis-03-14-0230-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1283-1283

Author(s):

S.-K. Choi ◽

I.-S. Cho ◽

G.-S. Choi ◽

J.-Y. Yoon

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Catharanthus Roseus ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

Bedding Plant ◽

Local Lesions ◽

Pcr Products ◽

Das Elisa

Catharanthus roseus, commonly known as Madagascar rosy periwinkle (also called vinca), is a tropical perennial herb of the family Apocyanaceae. Periwinkle is a bedding plant widely used in Korea because of its drought tolerance, low maintenance, and varied flower colors. In May 2013, virus-like foliar symptoms, including a mosaic with malformation of leaves, were observed on a periwinkle plant in a greenhouse located in Chonbuk Province, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with an immune-strip kit developed by our laboratory. The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia, Elkhart, IN). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-Vin) caused necrotic local lesions on Chenopodium amaranticolor at 5 days-post-inoculation (dpi), while mild to severe mosaic was observed in Capsicum annuum, Cucumis sativus, Cucurbita pepo ‘Cheonggobong,’ Nicotiana glutinosa, N. tabacum‘Samsun NN,’ Physalis angulate, and Solanum lycopersicum ‘Pink-Top’ 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ~28 nm in diameter, respectively. To verify whether CMV was the causal agent for the disease symptoms observed in naturally infected periwinkle, virus-free periwinkle (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-Vin. At 6 weeks after inoculation, all plants produced systemic mosaic and distortion of leaves, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated periwinkle plants remained symptomless and virus-free. The presence of CMV-Vin in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with a RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CMVCPFor and CMVCPRev (1), which amplified the entire CP gene. RT-PCR products (657 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from a positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-Vin CP sequence (Accession No. AB910598) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-Vin infection. Being perennial, periwinkle plants could serve as a reservoir for CMV to infect other ornamentals and cultivated crops (2). To our knowledge, this is the first report of CMV infection on periwinkle in Korea. References: (1) S. K. Choi et al. Virus Res. 158:271, 2011. (2) P. Palukaitis et al. Adv. Virus. Res. 41:281, 1992.

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First report of Cherry virus F infecting Japanese plum in Korea

Plant Disease ◽

10.1094/pdis-08-20-1725-pdn ◽

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.

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Updating the Quarantine Status of Prunus Infecting Viruses in Australia

Viruses ◽

10.3390/v12020246 ◽

2020 ◽

Vol 12 (2) ◽

pp. 246 ◽

Cited By ~ 1

Author(s):

Wycliff M. Kinoti ◽

Narelle Nancarrow ◽

Alison Dann ◽

Brendan C. Rodoni ◽

Fiona E. Constable

Keyword(s):

Polymerase Chain Reaction ◽

High Throughput Sequencing ◽

Mottle Virus ◽

Rt Pcr ◽

Chain Reaction ◽

Virus Family ◽

First Report ◽

Hop Stunt Viroid ◽

Polymerase Chain ◽

Stem Pitting

One hundred Prunus trees, including almond (P. dulcis), apricot (P. armeniaca), nectarine (P. persica var. nucipersica), peach (P. persica), plum (P. domestica), purple leaf plum (P. cerasifera) and sweet cherry (P. avium), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus.

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First Report of Grapevine Pinot gris virus (GPGV) in grapevine in France

Plant Disease ◽

10.1094/pdis-10-14-1008-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 293-293 ◽

Cited By ~ 25

Author(s):

M. Beuve ◽

T. Candresse ◽

M. Tannières ◽

O. Lemaire

Keyword(s):

Large Scale ◽

De Novo ◽

Pinot Noir ◽

Rt Pcr ◽

Wine Quality ◽

First Report ◽

Italian Isolate ◽

Pcr Products ◽

Grapevine Pinot Gris Virus ◽

Plant Pathol

Grapevine Pinot gris virus (GPGV), belonging to the genus Trichovirus of the family Betaflexiviridae, was first identified by siRNA sequencing in northern Italy in 2012, in the grapevine varieties Pinot gris, Traminer, and Pinot Noir, which exhibited mottling and leaf deformation (1), and in asymptomatic vines, with a lower frequency. Since 2012, this virus has also been reported in South Korea, Slovenia, Greece (3), Czech Republic (2), Slovakia (2), and southern Italy (4). In 2014, GPGV was identified by Illumina sequencing of total RNAs extracted from leaves of the Merlot variety (Vitis vinifera) grafted onto Gravesac rootstock originated from a vineyard in the Bordeaux region of France. This Merlot plant exhibited fanleaf-like degeneration symptoms associated with Tomato black ring virus (TBRV) infection. Cuttings were collected in 2010 and maintained thereafter in a greenhouse. The full-length genome was assembled either de novo or by mapping of the Illumina reads on a reference GPGV genome (GenBank FR877530) using the CLC Genomics workbench software (CLC Bio, Qiagen, USA). The French GPGV isolate “Mer” (7,223 nucleotides, GenBank KM491305) is closely related to other European GPGV sequences; it exhibits 95.4% nucleotide identity with the reference Italian isolate (NC_015782) and 98 to 98.3% identity with Slovak isolates (KF134123 to KF134125). The higher divergence between French and Italian GPGV isolates was mainly due to differences in the 5′ extremity of the genome, as already shown with the Slovak GPGV isolates. RNA extracted from phloem scrapings of 19 cv. Merlot vines from the same plot collected in 2014 were analyzed by RT-PCR using the specific primer pair Pg-Mer-F1 (5′-GGAGTTGCCTTCGTTTACGA-3′) and Pg-Mer-R1 (5′-GTACTTGATTCGCCTC GCTCA-3′), designed on the basis of alignments of all available GPGV sequences from GenBank. The resulting amplicon of 770 bp corresponded to a fragment of the putative movement protein (MP) gene. Seven (35%) of the tested plants gave a strong positive amplification. Three RT-PCR products were directly sequenced and showed 99.3 to 99.5% identity within the MP gene of the GPGV-Mer isolate. Given the mixed viral infection status of the vines found infected by GPGV, it was not possible to associate a specific symptomatology with the presence of GPGV. Furthermore, similar RT-PCR tests were also performed on RNA extracts prepared from two plants of cv. Carignan that originated from a French grapevine collection, exhibiting fanleaf-like symptoms without any nepovirus detection. These samples similarly gave a strong positive amplification. The sequences obtained from the two Carignan vines showed 98.4 and 97.8% identity with the GPGV-Mer isolate. To our knowledge, this is the first report of GPGV in France. GPGV has been discovered in white and red berry cultivars, suggesting that its prevalence could be important in European vineyards (2). Further large-scale studies will be essential to determine the world prevalence of GPGV and to evaluate its potential effects on yield and on wine quality, as well as to shed light on GPGV epidemiology. Of particular concern is whether, like the other grapevine-infecting Trichovirus, Grapevine berry inner necrosis virus (GPGV) can be transmitted by the eryophid mite Colomerus vitis. References: (1) A. Giampetruzzi et al. Virus Res. 163: 262, 2012. (2) M. Glasa et al. Arch. Virol. 159: 2103, 2014. (3) G. P. Martelli, J. Plant Pathol. 96: S105, 2014. (4) M. Morelli et al. J. Plant Pathol. 96:431, 2014.

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