scholarly journals First report of peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and mume virus A (MuVA) from Mei (Prunus mume) in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Yuhong Zhang ◽  
Jun Zhou ◽  
Binhui Zhan ◽  
shifang li ◽  
Zhixiang Zhang
Keyword(s):  
Prunus Mume ◽  
Sequencing Data ◽  
Rt Pcr ◽  
Illumina Hiseq ◽  
New Host ◽  
First Report ◽  
Interveinal Chlorosis ◽  
Sequence Identity ◽  
Two Samples ◽  
Stem Pitting

Mei (Prunus mume Sieb. et Zucc.), widely distributed in East Asian countries for both fruiting- and flowering-purposes, is susceptible to viral infections (Marais et al. 2018). Infection by plum bark necrosis stem pitting-associated virus (PBNSPaV) or little cherry virus 2 (LChV-2) possibly caused overall yield loss in mei in Japan due to incomplete flower development, low fruit bearing rate, and interveinal chlorosis (Numaguchi et al. 2019). Virus-like disease showing mosaic, interveinal chlorosis, vein clearing, or necrotic spot on leaf was observed in mei trees in Beijing, Wuhan, Wuxi, and Nanjing in spring and early summer from 2017 to 2018. Symptomatic leaves collected from the four regions were pooled as two samples for RNA-sequencing (RNA-seq) analysis. After ribosomal RNA (rRNA)-depletion, total RNA extracted by TRNzol reagent (TIANGEN, China) was subjected to library construction using NEBNext Ultra RNA Library Prep Kit (NEB, MA, USA) and sequenced on an Illumina Hiseq 4000 (Novogene, China). Sequencing data was filtered, screened, and assembled as described previously (Zhou et al. 2020) to generate contigs, following by BLAST-x/n search in viral genomes in GenBank. We identified >300 contigs (208-10756 nt) homologous to Asian prunus virus 1 and Asian prunus virus 2 (APV1 and 2), mume virus A (MuVA), PBNSPaV, and peach leaf pitting-associated virus (PLPaV), with 71-100% of nucleotide sequence identity values. APV1 and 2 have been reported in mei in China (Wang et al. 2018), here, we focused on the other three viruses. Contigs homologous to these three viruses were further assembled into three scaffolds of 14,224 nt, 1107 nt, and 753 nt for PBNSPaV, MuVA, and PLPaV, respectively. The scaffold of PBNSPaV (MW217574) nearly covered the whole genome of the isolate VIC3 from Australia (LC523039.1) (Kinoti et al. 2020) with 92.30% of sequence identity; the scalffold of MuVA (MW217572) covered 14.50% of the genome of the isolate pm14 from Japan (NC 040568.1) (Marais et al.2018) with 98.47% sequence identity; the scaffold of PLPaV (MW217573) covered 15.26% of the genome of the isolate XJ-6 from peach (KY867750.1) (He et al. 2017) with 85.23% sequence identity. Presence of the three viruses were verified by RT-PCR detection using designed specific primers for PBNSPaV (Forward: 5′-CAACAAAACTCCCACAGCGG-3 [positions 4014-4033, NC_009992.1] / Reverse: 5′-GCCAAAAGAAGTGCTGGTGG-3′ [positions 4659-4640, NC_009992.1]), MuVA (Forward: 5′-AAGAGAATTACTTCAATGCCCTC-3′ [positions 171-194, NC_040568.1] / Reverse: 5′-GATATCCAAGATACGATTAGCCAG-3′ [positions 533-510, NC_040568.1]), and PLPaV (Forward: 5′-GCTATATCTCAACAACTGCAAGAA-3 [positions 5798-5821, KY867750.1] / Reverse: 5′- GAGTGATACATAGTCCACAGAGAT-3′[ positions 6045-6022, KY867750.1]). The amplified 626, 350 and 251 bp fragments of PBNSPaV, MuVA and PLPaV had 91.47%, 98.07% and 81.89% sequence identity to their respective reference sequences. This is the first report of PBNSPaV and MuVA infecting mei in China, and more importantly, the first report of a new host for PLPaV. In addition, 30 collected leaf samples from Nanjing and Wuhan were analyzed by RT-PCR and 15, 6, and 5 samples tested positive to PLPaV, PBNSPaV, and MuVA, respectively. Although it is difficult to link a particular virus with the observed symptoms due to mixed infections, the symptoms were significantly associated with viral infection because almost all symptomatic leaf samples were virus(es)-positive. Further studies would be required to determine the distribution and impact of these viruses on mei trees and other stone fruits species and to understand the possibility that mei trees may play a role in PLPaV epidemiology.

scholarly journals First Report of Pepper vein yellows virus Infecting Hot Pepper in Sudan

Plant Disease ◽  
2014 ◽  
Vol 98 (10) ◽  
pp. 1446-1446 ◽  
Author(s):  
A. Alfaro-Fernández ◽  
E. E. ElShafie ◽  
M. A. Ali ◽  
O. O. A. El Bashir ◽  
M. C. Córdoba-Sellés ◽  
...  
Keyword(s):  
Leaf Size ◽  
The Philippines ◽  
Hot Pepper ◽  
Virus Species ◽  
Rt Pcr ◽  
New Host ◽  
First Report ◽  
Central Sudan ◽  
Two Samples ◽  
Pepper Vein Yellows Virus

In two successive winters (2009 and 2010), 14 hot pepper (Capsicum annuum) samples showing unusual symptoms were surveyed in permanently irrigated seasonal vegetable gardens along the Blue Nile in central Sudan (specifically in Gezira State). Symptoms included leaf curling, leaf deformation, reduced leaf size, leaf puckering, interveinal yellowing, vein clearing, or yellow patches. Total RNA was extracted from symptomatic leaves and analyzed by reverse transcription (RT)-PCR with degenerate primer pairs that amplify different viral species within the family Luteoviridae (1). Amplification of a 340-bp fragment of the coat protein gene (CP) was obtained in all the collected samples analyzed. The amplified fragments were purified and sequenced (Accession Nos. KC685313 to 26), showing 99, 97, and 95 to 99% nucleotide identities to Pepper yellows virus (PYV, accession no. FN600344 from Turkey), Pepper vein yellows virus (PeVYV, AB594828 from Japan) and Pepper yellow leaf curl virus (PYLCV, HM439608 from Israel), respectively. These three viruses belong to the genus Polerovirus and are considered synonyms of the same virus species PeVYV described with those names in different countries (3). Two samples were also tested by RT-PCR with the general Polerovirus primer pair Pol-G-F and Pol-G-R, which amplified a 1.1-kb product spanning the 3′ half of the RNA-dependent RNA polymerase (RdRp) to the 5′ half of CP and movement protein (2). The amplified fragments (KC692834 and KC692833) showed 97, 96, and 95% nt identity with PYV (FN600344), PeVYV (JX427533), and PYLCV (HM439608), respectively. The presence of the recently described Polerovirus PeVYV is the first report of detection in pepper in Sudan. PeVYV has recently been identified in seven other countries (India, Indonesia, Mali, the Philippines, Spain, Taiwan, and Thailand) and on one new host, Solanum nigrum, which suggests this new Polerovirus species poses a potentially wide geographical distribution and a global threat for pepper crops (3,4). References: (1) A. D. Abraham et al. Afr. J. Biotechnol. 7:414, 2008. (2) D. Knierim et al. Plant Pathol. 59:991, 2010. (3) D. Knierim et al. Arch. Virol. 158:1337, 2013. (4) F. Villanueva et al. Plant Dis. 97:1261, 2013.

scholarly journals First Report of Cucumber green mottle mosaic virus Infecting Greenhouse Cucumber in Canada

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 701-701 ◽  
Author(s):  
K.-S. Ling ◽  
R. Li ◽  
W. Zhang
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Cucumis Sativus ◽  
Disease Incidence ◽  
The United States ◽  
Rt Pcr ◽  
First Report ◽  
Sequence Identity ◽  
Two Samples ◽  
Greenhouse Cucumber

In early 2013, greenhouse cucumber growers in Alberta, Canada, observed virus-like disease symptoms on mini-cucumber (Cucumis sativus) crops (e.g., ‘Picowell’). Two types of symptoms were commonly observed, green mottle mosaic and necrotic spots. In the early infection, young leaves of infected cucumber plants displayed light green mottle and blisters. The infected plants were stunted in growth, with darker green blisters and green mottle mosaic symptoms on mature leaves. Disease incidence varied from one greenhouse to another. In some severe cases, diseased plants were widely distributed inside the greenhouse, resulting in 10 to 15% yield losses based on grower's estimation. Nine symptomatic samples were collected and subjected to total RNA isolation using the TRIzol reagent (Invitrogen, Carlsbad, CA). Laboratory analyses were conducted using real-time RT-PCR systems for Cucumber green mottle mosaic virus (CGMMV) (1), Melon necrotic spot virus (MNSV, Ling, unpublished), and Squash mosaic virus (SqMV) (3). All nine samples were positive for CGMMV and seven of them were in mixed infections with MNSV. Two samples were selected for validation for the presence of CGMMV using conventional RT-PCR (2) with a new primer set (CGMMVMP F1: 5′-ATGTCTCTAAGTAAGGTGTC-3′ and CGMMV3′UTR R1: 5′-TGGGCCCCTACCCGGGG-3′) and two previous online published primer sets, one for CGMMV MP (5′ TAAGTTTGCTAGGTGTGATC-3′, GenBank Accession No. AJ250104 and 5′ ACATAGATGTCTCTAAGTAAG-3′, AJ250105), and another for CGMMV CP (5′ ACCCTCGAAACTAAGCTTTC-3′, AJ243351 and 5′ GAAGAGTCCAGTTCTGTTTC-3′, AJ243352). The expected sizes of RT-PCR products were obtained and sequenced directly. Sequences from these three products overlapped and generated a 1,282-bp contig (KF683202). BLASTn analysis to the NCBI database showed 99% sequence identity to CGMMV isolates identified in Asia, including China (GQ277655, KC852074), India (DQ767631), Korea (AF417243), Myanmar (AB510355), and Taiwan (HQ692886), but only 92% sequence identity to other CGMMV isolates identified in Europe, including Spain (GQ411361) and Russia (GQ495274), and 95% to CGMMV isolate from Israel (KF155231). The strong sequence identity to the CGMMV Asian isolates suggests that the Canadian CGMMV isolate identified in Alberta was likely of Asian origin. In two bioassay experiments using one sample prepared in 0.01 M phosphate buffer, the similar green mottle mosaic symptoms were observed on systemic leaves in the mechanically inoculated plants and the presence of CGMMV, but not MNSV, was confirmed through real-time RT-PCR on four different cucurbits, including three Cucumis sativus cultivars (six plants in ‘Marketer,’ five plants in ‘Poinsett 76,’ six plants in ‘Straight 8’), seven plants of C. melo ‘Athena,’ six plants of C. metulifer (PI201681), and two plants of Citrullus lanatus ‘Charleston Gray.’ To our knowledge, CGMMV has only been reported in Asia, Europe, and the Middle East, and this is the first report of CGMMV in the American continents. CGMMV is highly contagious and is seed borne on cucurbits. With the increasing trend in growing grafted watermelon and other cucurbits in the United States and elsewhere, it is even more important now that a vigilant seed health test program for CGMMV should be implemented. References: (1) H. Chen et al. J. Virol. Methods 149:326, 2008. (2) K.-S. Ling et al. Plant Dis. 92:1683, 2008. (3) K.-S. Ling et al. J. Phytopathol. 159:649, 2011.

scholarly journals First Report of Tomato apical stunt viroid in Tomato in Tunisia

Plant Disease ◽  
2006 ◽  
Vol 90 (4) ◽  
pp. 528-528 ◽  
Author(s):  
J. Th. J. Verhoeven ◽  
C. C. C. Jansen ◽  
J. W. Roenhorst
Keyword(s):  
Crop Production ◽  
Plant Protection ◽  
Chenopodium Quinoa ◽  
Test Plant ◽  
Production Facility ◽  
Rt Pcr ◽  
First Report ◽  
Stunt Viroid ◽  
Sequence Identity ◽  
Two Samples

In May 2005, the Plant Protection Service in the Netherlands received two tomato (Lycopersicon esculentum) plant specimens for diagnosis from a protected crop production facility of 2.5 ha near Kebili in Tunisia. Growth of the plants was reduced and leaves were chlorotic and brittle. Ripening of the fruits was delayed and their storage life was reduced from 3 weeks to 1 week. The grower reported that initially only 5% of plants showed symptoms; however, the number of symptomatic plants increased quickly to 100% as a result of increasing temperatures in the production facility. Test plant species Chenopodium quinoa, Datura stramonium, Nicotiana glutinosa, N. hesperis-67A, N. occidentalis-P1, and L. esculentum ‘Money-maker’ were mechanically inoculated with sap from the affected plants. Symptoms including chlorosis and stunting were observed only on L. esculentum. Reverse transcriptase-polymerase chain reaction (RT-PCR) with universal pospiviroid primers Pospi1-RE/FW (2) yielded amplicons of the expected size (196 bp) for each of the two samples. One of these amplicons was sequenced and showed the highest identity to the four isolates of Tomato apical stunt viroid (TASVd) in the NCBI Gen-Bank. Subsequently, the complete sequence of the Tunisian isolate (Gen-Bank Accession No. DQ144506) was determined by sequencing the am-plicon obtained after RT-PCR using primers developed for the detection of Citrus exocortis viroid (CEVd) (1). The isolate consisted of 363 nucleotides and showed the highest sequence identity (96.7%) to tomato isolates of TASVd from Indonesia and Israel (GenBank Accession Nos. X06390 and AY062121, respectively), 92.6% to a tomato isolate from the Ivory Coast (GenBank Accession No. K00818), and 87.7% to an isolate from Solanum pseudocapsicum (GenBank Accession No. X95293). The next highest sequence identity was 81.5% to an isolate of CEVd (GenBank Accession No. X53716). On the basis of these results, the viroid was identified as TASVd. To our knowledge, this is the first report of TASVd in Tunisia. Reference: (1) N. Önelge. Turkish J. Agric. For. 21:419, 1997. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

scholarly journals Cucurbit chlorotic yellows virus infecting Rehmannia glutinosa was detected in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Kun Zhang ◽  
Xinjian Zhuang ◽  
Xiao Guo ◽  
Hongmei Xu ◽  
Zhen He ◽  
...  
Keyword(s):  
De Novo ◽  
Viral Disease ◽  
Henan Province ◽  
Plant Sample ◽  
Herbaceous Plant ◽  
Rehmannia Glutinosa ◽  
Rt Pcr ◽  
Illumina Hiseq ◽  
Two Samples ◽  
Cucurbit Chlorotic Yellows Virus

Rehmannia glutinosa Libosch. is a perennial herbaceous plant of the family Scrophulariaceae. Its roots can be used as traditional Chinese medicine. The asexual reproduction by vegetative organ of R. glutinosa lead to an increased viral disease that seriously affects its yield and quality (Kwak et al. 2020; Kwak et al. 2018; Ling and Liu 2009). Leaves of R. glutinosa in Wenxian County, Henan Province, China showed symptoms of chlorosis, mosaic and irregular yellow in August 2019. In general, the older leaves at the base or middle of the plant (sample 2# and 5#) first became irregular yellowing, followed by a gradual extend to the leaves at the top (Supplementary Fig. S1A). Six plants (2#, 3#, 5#, 7#, 8#, and 9#) with these symptoms were collected. The total RNA was extracted and its siRNAs were obtained. High-throughput siRNA sequencing (Sangon, Shanghai, China) was performed on Illumina Hiseq 2000 platform with paired-end method after siRNA library construction (NEBNext Ultra II RNA Library Prep Kit, NEB, UK). Sequencing files were treated with Illumina’s CASAVA pipeline (version 1.8). The length of the resulting reads with adaptor removed were mostly distributed ranging from 21-24 nt (Supplementary Fig. S1B). The Velvet Software 0.7.31 (k=17) was taken to do de novo assembling, and the contigs (∼13,000, Contigs > 300 bp) were used to perform BLASTN against GenBank database. Two viruses, Rehmannia mosaic virus (ReMV) and cucurbit chlorotic yellows virus (CCYV), were frequently appeared in analyzed six symptomatic samples. To further identify the infection of CCYV to R. glutinosa, ten samples with virus-infected symptoms were randomly collected. Total protein and RNAs were extracted for RT-PCR and ELISA (HALING. Shanghai, China). A specific pair of primers (Supplementary Table S1) were designed to amplify the 753-bp length coat protein (CP) gene of CCYV. The result showed that two samples appeared a specific band of expected size on the agarose gel, which indicated that they were infected by CCYV (Supplementary Fig. S1C, Upper panel). The same result was obtained by ELISA assay (Supplementary Fig. S1D). The amplified CP fragment of CCYV was recycled and purified by TIANgel Midi Purification Kit (Tiangen, Beijing, China), followed by cloned into pMD19-T (TaKaRa, Dalian, China) and transformed into E. coli DH5a.Ten separate clones were selected and sequenced (Sangon, Shanghai, China) after PCR verification. The obtained sequences (GenBank accession No. MW521380 & MW521381) were analyzed by BLASTN and bioEdit software (version 7.2.3). The results showed 100% identity with the CCYV CP sequences that mainly derived from infected cucurbit. To confirm the occurrence and distribution of CCYV and ReMV in planting area, the other twenty-four samples (20 with chlorosis and stunt symptoms and 4 with invisible symptoms) were randomly collected for RT-PCR in different regions of Henan Province (Supplementary Table S1). The results showed that the CCYV and ReMV infection rate were 20.5% and 61.7%, respectively. Co-infection of the CCYV and ReMV was 5.8% in fields (Supplementary Table S2). In sum, these results indicated the CCYV can naturally infect R. glutinosa in China. CCYV is transmitted by white-fly in a semi-persistent manner and mainly damages cucurbits (Orfanidou et al. 2017). CCYV has been discovered in many places (Huang et al. 2010). To date, there is no report about CCYV infecting R. glutinosa in nature. This is the first report of CCYV naturally infect R. glutinosa in China.

scholarly journals Updating the Quarantine Status of Prunus Infecting Viruses in Australia

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 246 ◽  
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.

scholarly journals First Report of Tomato chlorosis virus Infecting Tomato in Georgia

Plant Disease ◽  
2011 ◽  
Vol 95 (7) ◽  
pp. 881-881 ◽  
Author(s):  
S. Sundaraj ◽  
R. Srinivasan ◽  
C. G. Webster ◽  
S. Adkins ◽  
K. Perry ◽  
...  
Keyword(s):  
Nucleic Acid Hybridization ◽  
N Gene ◽  
Natural Occurrence ◽  
Rt Pcr ◽  
Specific Primers ◽  
Tomato Production ◽  
First Report ◽  
Interveinal Chlorosis ◽  
Cp Gene ◽  
Tomato Chlorosis Virus

Tomato yellow leaf curl virus (TYLCV) and Tomato spotted wilt virus (TSWV) are prevalent in field-grown tomato (Solanum lycopersicum) production in Georgia. Typical TYLCV symptoms were observed during varietal trials in fall 2009 and 2010 to screen genotypes against TYLCV at the Coastal Plain Experiment Station, Tifton, GA. However, foliar symptoms atypical of TYLCV including interveinal chlorosis, purpling, brittleness, and mottling on upper and middle leaves and bronzing and intense interveinal chlorosis on lower leaves were also observed. Heavy whitefly (Bemisia tabaci (Gennadius), B biotype) infestation was also observed on all tomato genotypes. Preliminary tests (PCR and nucleic acid hybridization) in fall 2009 indicated the presence of TYLCV, TSWV, Cucumber mosaic virus, and Tomato chlorosis virus (ToCV); all with the exception of ToCV have been reported in Georgia. Sixteen additional symptomatic leaf samples were randomly collected in fall 2010 and the preliminary results from 2009 were used to guide testing. DNA and RNA were individually extracted using commercially available kits and used for PCR testing for ToCV, TYLCV, and TSWV. Reverse transcription (RT)-PCR with ToCV CP gene specific primers (4) produced approximately 750-bp amplicons from nine of the 16 leaf samples. Four of the nine CP gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 100.0% identical with each other (GenBank Accession Nos. HQ879840 to HQ879843). They were 99.3 to 99.5%, 97.2 to 97.5%, and 98.6 to 98.9% identical to ToCV CP sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. The presence of ToCV was confirmed by amplifying a portion of the HSP70h gene using the primers HSP-1F and HSP-1R (1). RT-PCR produced approximately 900-bp amplicons in the same nine samples. Four HSP70h gene amplicons were purified and directly sequenced in both directions. The sequences were 99.4 to 99.7% identical to each other (Accession Nos. HQ879844 to HQ879847). They were 99.2 to 99.5%, 98.0 to 98.4%, and 98.9 to 99.3% identical to HSP70h sequences from Florida (Accession No. AY903448), Spain (Accession No. DQ136146), and Greece (Accession No. EU284744), respectively. TYLCV was also detected in all 16 samples by PCR using degenerate begomovirus primers PAL1v 1978 and PARIc 496 (3) followed by sequencing. TSWV was also detected in two of the ToCVinfected samples by RT-PCR with TSWV N gene specific primers (2) followed by sequencing. To our knowledge, this is the first report of the natural occurrence of ToCV in Georgia. Further studies are required to quantify the yield losses from ToCV alone and synergistic interactions between ToCV in combination with TSWV and/or TYLCV in tomato production in Georgia. References: (1) T. Hirota et al. J. Gen. Plant Pathol. 76:168, 2010. (2) R. K. Jain et al. Plant Dis. 82:900, 1998. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) L. Segev et al. Plant Dis. 88:1160, 2004.

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 Chilli veinal mottle virus Infecting Tomato (Solanum lycopersicum) in China

Plant Disease ◽  
2014 ◽  
Vol 98 (11) ◽  
pp. 1589-1589 ◽  
Author(s):  
F.-F. Zhao ◽  
D.-H. Xi ◽  
J. Liu ◽  
X.-G. Deng ◽  
H.-H. Lin
Keyword(s):  
Mosaic Virus ◽  
Southwest China ◽  
Sichuan Province ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Tomato Plants ◽  
First Report ◽  
Two Samples ◽  
Leaf Distortion ◽  
Chilli Veinal Mottle Virus

Chilli veinal mottle virus (ChiVMV), a potyvirus, is widespread over the world. In China, it was first reported in chili pepper (Capsicum annuum) in Hainan Province (south China) in 2006 (2). Subsequently, it was reported in tobacco (Nicotiana tabacum) in Yunnan Province (southwest China) in 2011 (1). Sichuan Province is one of the largest vegetable producing areas of China. In May 2012, tomatoes with leaves displaying virus-infected symptoms like mottling, mosaic, narrowing, or curling were observed in several fields of Chengdu, eastern Sichuan Province, southwest China. Of the 20 fields we investigated, four fields with 90% tomato plants were infected. During 2012 and 2013, six samples were collected from symptomatic tomato leaves based on different symptoms and locations. All six samples were assayed by western blotting using polyclonal antisera (Cucumber mosaic virus [CMV], Tobacco mosaic virus [TMV]) obtained from Agdia (Elkhart) and one antiserum to ChiVMV obtained from Yunnan Academy of Agricultural Science (China). Two samples from Pengzhou and one sample from Shuangliu exhibiting mosaic leaves were positive for TMV, one sample from Pixian exhibiting narrowing leaves was positive for CMV, and the other two samples from Shuangliu exhibiting mottle and leaf distortion were positive for ChiVMV. Total RNAs was extracted from all six samples and healthy tomato leaves using Trizol reagent (Invitrogen), First-strand cDNA synthesis primed with oligo(dT) by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed using primer pairs ChiVMV-CP F (5′-GCAGGAGAGAGTGTTGATGCTG-3′) and ChiVMV–CP R (5′-(T)16AACGCCAACTATTG-3′), which were designed to direct the amplification of the entire capsid protein (CP) gene and 3′ untranslated region (3′-UTR) of ChiVMV (GenBank Accession No. KC711055). The expected 1,166-bp DNA fragment was amplified from the two tomato samples from Shuangliu that were positive for ChiVMV in the western blot tests, but not from the others. The obtained fragments were purified and cloned into the PMD18-T vector (TaKaRa) and sequenced. The sequencing results showed that the two ChiVMV isolates from tomato in Shuangliu were identical (KF738253). Nucleotide BLAST analysis revealed that this ChiVMV isolate shared ~84 to 99% nucleotide identities with other ChiVMV isolates available in GenBank (KC711055 to KF220408). To fulfill Koch's postulates, we isolated this virus by three cycle single lesion isolation in N. tabacum, and mechanically inoculated it onto tomato leaves. The same mottle and leaf distortion symptoms in systemic leaves were observed. Subsequent RT-PCR, fragment clone, and sequence determination tests were repeated and the results were the same. All the evidence from these tests revealed that the two tomato plants were infected by ChiVMV. To our knowledge, this is the first report of ChiVMV naturally infecting tomato in China. It shows that ChiVMV is spreading in China and is naturally infecting a new solanaceous crop in the southwest area, and the spread of the virus may affect tomato crop yields in China. Thus, it is very important to seek an effective way to control this virus. References: (1) M. Ding et al. Plant Dis. 95:357, 2011. (2) J. Wang et al. Plant Dis. 90:377, 2006.

scholarly journals First Report of Tomato chlorotic dwarf viroid in Greenhouse Tomatoes in Arizona

Plant Disease ◽  
2009 ◽  
Vol 93 (10) ◽  
pp. 1075-1075 ◽  
Author(s):  
K.-S. Ling ◽  
J. Th. J. Verhoeven ◽  
R. P. Singh ◽  
J. K. Brown
Keyword(s):  
Direct Sequencing ◽  
Leaf Tissue ◽  
Natural Occurrence ◽  
Rt Pcr ◽  
Nucleotide Substitutions ◽  
Tomato Production ◽  
First Report ◽  
Sequence Identity ◽  
Tomato Chlorotic Dwarf Viroid ◽  
Plant Pathol

Tomato chlorotic dwarf viroid (TCDVd), a member of the genus Pospivroid, family Pospiviroidae, was first identified on greenhouse tomato (Solanum lycopersicum) in Canada (2). Since then, it has also been reported elsewhere, e.g., on tomato in Colorado (4). During 2006 in Arizona, tomato plants in a large greenhouse facility with continuous tomato production exhibited viroid-like symptoms of plant stunting and chlorosis of the young leaves. Symptomatic plants were often located along the edge of the row, indicating the presence of a mechanical transmissible agent. Approximately 4% of the plants in this greenhouse were symptomatic in 2008. Symptoms were distinctly different from those caused by Pepino mosaic virus (PepMV), a virus that was generally present in this greenhouse and also in our test samples. Other commonly occurring tomato viruses were ruled out by serological, PCR, or reverse transcription (RT)-PCR tests in multiple laboratories. RT-PCR with two sets of universal pospiviroid primers, PospiI-FW/RE and Vid-FW/RE (4), yielded amplicons of the expected sizes of 196 and 360 bp in three samples collected from symptomatic plants. Direct sequencing of the amplicons revealed that the genome was 360 nt and 100% identical to the type TCDVd from Canada (GenBank Accession No. AF162131) (2). Mechanical inoculation with leaf tissue extract from four samples to plants of the tomato ‘Money-Maker’ resulted in the same viroid-like symptoms and TCDVd was confirmed in these plants by RT-PCR and sequencing. In both 2007 and 2008, 18 samples were tested using primers PSTVd-F and PSTVd-R (1), which are capable of amplifying the full TCDVd genome. Analysis of the sequences from the amplicons revealed two genotypes of TCDVd. The first genotype (GenBank Accession No. FJ822877) was identical to the type TCDVd and found in 11 samples from 2007 and one from 2008. The second genotype (GenBank Accession No. FJ822878) was 361 nt, differing from the first by nine nucleotide substitutions, 2 insertions, and 1 deletion. This second genotype was found in 7 and 17 samples from 2007 and 2008, respectively, and showed the highest sequence identity (97%) to a Japanese tomato isolate (AB329668) and a much lower sequence identity (92%) to a U.S. isolate previously identified in Colorado (AY372399) (4). The origin of TCDVd in this outbreak is not clear. The genotype identified first could have been introduced from a neighboring greenhouse where the disease was observed before 2006 and where this genotype also was identified in 2007. The second genotype may have been introduced from infected seed since TCDVd has recently been shown to be seed transmitted in tomato (3). To our knowledge, this is the first report of natural occurrence of TCDVd in Arizona. References: (1) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (2) R. P. Singh et al. J. Gen. Virol. 80:2823, 1999. (3) R. P. Singh and A. D. Dilworth. Eur. J. Plant Pathol. 123:111, 2009. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

scholarly journals First Report of Grapevine Kizil Sapak Virus in Grapevine in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Fang Ren ◽  
Zunping Zhang ◽  
Xudong Fan ◽  
Guojun Hu ◽  
Yafeng Dong
Keyword(s):  
Complete Genome ◽  
High Throughput Sequencing ◽  
Pairwise Comparison ◽  
Bioinformatic Analysis ◽  
Rt Pcr ◽  
Illumina Hiseq ◽  
First Report ◽  
Hop Stunt Viroid ◽  
Pcr Products ◽  
Rapid Amplification

Grapevine Kizil Sapak virus (GKSV) is a novel member of the family Betaflexiviridae classified into the proposed genus Fivivirus within the subfamily Trivirinae. It was first discovered in USA from a grapevine originating from Turkmenistan (Al Rwahnih et al. 2019) and later in France from a grapevine accession from Iran (Marais et al. 2020). In October 2019, an asymptomatic grapevine cv. ‘Crimson Seedless’ (native to USA) was collected from Xinjiang province in China and analyzed by high-throughput sequencing (HTS). Ribosome-depleted RNA preparations were used for library synthesis followed by HTS on an Illumina HiSeq X-ten platform. A total of 29,141,024 cleaned reads were obtained, and 7,878 contigs were generated using CLC Genomics Workbench 9.5 (QIAGEN). One long contig (7,328 bp) showed 88.2% nucleotide (nt) identity with the sequence of GKSV-127 (MN172165) via Blastx, with an average coverage of 284-X. Bioinformatic analysis of the remaining contigs showed the presence of Grapevine leafroll-associated virus 4, Grapevine rupestris vein feathering virus, Grapevine fabavirus, grapevine yellow speckle viroid-1 (GYSVd-1), GYSVd-2 and Hop stunt viroid in the sample. The presence of GKSV was checked by RT-PCR using the primer GKSV-F/R (Al Rwahnih et al. 2019); the 1,240 bp PCR product was cloned using a pTOPO-T vector (Aidlab, China) and sequenced. In pairwise comparison, the obtained nt sequences shared 92.6 to 95.2% identity to the corresponding HTS sequence, confirming the presence of GKSV in the sample. The complete GKSV genome sequence was obtained as two pieces of overlapping DNA sequence using primers GKSV-20A/20B (5’-TAGTCTGGATTTCCCTACCT/5’-CTCCCTAAACTGATTTGATG) and GKSV-25A/25B (5’-GCCACTGGTGAATGAAAAGA/5’-CTAAATGAATGGGCAGGTAT) designed based on the HTS-generated sequence. The 5’ and 3’ termini were determined by rapid amplification of cDNA ends using SMARTer RACE 5’/3’ Kit (Takara, Dalian, China). The complete genome of GKSV isolate CS (MW582898) comprised 7,604 nt (without the polyA tail) and shared 77.8 to 89.2% identities with the other nine reported GKSV isolates, among which it shared the highest nt identity (89.2%) with GKSV-127. In phylogenetic analysis based on complete or nearly complete genome sequences of representative members of Betaflexiviridae, GKSV-CS clustered with the nine known GKSV isolates, forming a subclade with GKSV-127 (Supplementary Fig. 1). To determine the incidence and distribution of GKSV in China, 476 grapevine samples of 75 cultivars were collected from 20 provinces and tested by RT-PCR using primers GKSV-F/R (Al Rwahnih et al. 2019) and Vini-F1/R1 (Marais et al. 2020). The results showed that 0.42% (2 of 476) of the samples tested positive with both primers, including samples GKSV-CS and a ‘Black Monukka’ grape (native to India) also sampled from Xinjiang. Both PCR products of ‘Black Monukka’ were cloned and sequenced (MZ311588 to MZ311602) and they showed 85.1 to 88.9% nt identities to the GKSV-CS sequence. This is the first report of GKSV infecting grapevine in China. Although the pathogenicity of GKSV is yet to be determined, it has been found in several countries such as USA (Al Rwahnih et al. 2019), France (Marais et al. 2020) and China (this study). Both positive samples in this study were collected from Nanjiang region in Xinjiang province, indicating the sporadic occurrence of GKSV in this area.

Sign in / Sign up

Export Citation Format

Share Document