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    植物病毒合辑Plant Virus

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    Strawberry vein banding virus P6 protein intracellular transport and an important domain identification
    PAN Yuan, ZHOU Xiu-hong, LI Shuai, FENG Ming-feng, SHI Man-ling, ZUO Deng-pan, JIANG Xi-zi, CHEN Jing, HU Ya-hui, ZHANG Xiang-xiang, JIANG Tong
    2018, 17 (09): 2031-2041.   DOI: 10.1016/S2095-3119(18)61978-1
    Abstract344)      PDF (13957KB)(228)      
    Strawberry vein banding virus (SVBV)-infected strawberry cells contain cytoplasmic inclusions with isometric particles.  To identify the components of the inclusions, green fluorescent protein (GFP) was fused to the carboxy-terminus (C-terminus) of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves.  Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies (IBs).  To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4´,6-diamidino-2-phenylindole (DAPI).  We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes.  To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum (ER), the microfilament marker protein (GFP-ABD2-GFP), microtubules marker protein (mCherry-MAP65-1) and ER marker protein (mCherry-HDEL) were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum.  Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells.  The P6 protein contains one C-terminal nuclear localization signal (NLS) region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression.  These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions.  In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD. 
     
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    Two mutations in the truncated Rep gene RBR domain delayed the Wheat dwarf virus infection in transgenic barley plants
    Pavel Cejnar, Ludmila Ohnoutková, Jan Ripl, Tomá? Vl?ko, Jiban Kumar Kundu
    2018, 17 (11): 2492-2500.   DOI: 10.1016/S2095-3119(18)62000-3
    Abstract249)      PDF (20567KB)(190)      
    Wheat dwarf virus (WDV), an important cereal pathogen, is closely related to Maize streak virus (MSV), a model virus of the Mastrevirus genus.  Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication-associated (RepA) gene (WDVRepA215) and the WDV RepA gene with a mutated retinoblastoma-related protein (RBR) interaction domain (WDVRepA215RBRmut) were cloned into the pIPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation.  A detailed study of T1-generation plants infected by leafhoppers (Psammotettix alienus) fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection.  A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBRmut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred.  Both WDVRepA215 and WDVRepA215RBRmut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants. 
     
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    Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle (Catharanthus roseus)
    LI Ya, XU Mei-rong, DAI Ze-han, DENG Xiao-ling
    2018, 17 (11): 2501-2508.   DOI: 10.1016/S2095-3119(18)61918-5
    Abstract276)      PDF in ScienceDirect      
    Candidatus Liberibacter asiaticus (CaLas), an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing (HLB) in citrus.  CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues.  Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder (Cuscuta spp.).  To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting.  The movement of the inoculum and associated titer changes were observed over time in various tissues.  CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe.  The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively.  The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus.  The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.
     
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    Limited infection by ‘Candidatus Liberibacter asiaticus’ in ‘Valencia’ sweet orange trees in the presence of Citrus tristeza virus
    FU Shi-min, Cristina Gouin, ZHOU Chang-yong, John S. Hartung
    2019, 18 (10): 2284-2293.   DOI: 10.1016/S2095-3119(19)62605-5
    Abstract77)      PDF in ScienceDirect      
    Huanglongbing (HLB) is the most destructive disease of citrus and is associated with ‘Candidatus Liberibacter asiaticus’ (CLas), a member of the α-proteobacteria. Citrus tristeza virus (CTV) is another pathogen of citrus with very great historic as well as current importance. Both CLas and CTV are phloem-restricted pathogens. A severe CTV isolate, CTV-B6, and CLas-B232 induce a group of symptoms of phloem dysfunction that overlap, but the mild isolate CTV-B2 does not cause any loss to commercial trees. Prior inoculation and establishment of CLas-B232 did not affect subsequent establishment of either CTV-B2 or CTV-B6, while super infection by CLas-B232 was reduced by prior establishment of CTV-B2 and to a lesser extent by prior infection with CTV-B6. Trees co-infected with CTV-B6 and CLas-B232 developed more severe symptoms, typical of CTV-B6, than either of the two pathogens co-infected with CTV-B2. In this study, we confirmed that CLas established in the rootlets earlier and with higher concentration than in leaves. The distribution of CLas in the plant infected by CLas-B438 alone and with CTV-B2 fits a previously proposed model but CLas was more sporadically distributed in a plant co-infected by CLas and CTV-B2 than in a plant infected by CLas alone. These biological phenomena are aligned with previously analyzed transcriptome data and the study provides a novel idea that mild CTV strains may provide some protection against CLas by limiting its multiplication and spread. The protective effect may be due to opposite regulation of key host defense pathways in response to CTV-B2 and CLas-B438.
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    Sugarcane mosaic virus infection of model plants Brachypodium distachyon and Nicotiana benthamiana
    XU Jing-sheng, DENG Yu-qing, CHENG Guang-yuan, ZHAI Yu-shan, PENG Lei, DONG Meng, XU Qian, YANG Yong-qing
    2019, 18 (10): 2294-2301.   DOI: 10.1016/S2095-3119(19)62572-4
    Abstract68)      PDF in ScienceDirect      
    Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is a causal pathogen of sugarcane mosaic disease, and it is widespread in regions where sugarcane (Saccharum spp. hybrids) is grown.  It is difficult to investigate the molecular mechanism of pathogen infection in sugarcane because of limited genomic information.  Here, we demonstrated that SCMV strain FZ1 can systemically infect Brachypodium distachyon inbred line Bd21 and Nicotiana benthamiana through inoculation, double antibody sandwich enzyme-linked immunosorbent, transmission electron microscopy, and reverse transcription PCR assays.  The leaves of Bd21 developed mosaic symptoms, while the leaves of N. benthamiana showed no obvious symptoms under the challenge of SCMV-FZ1.  We concluded that B. distachyon inbred line Bd21 is a promising experimental model plant compared with N. benthamiana for study on the infectivity of SCMV.  This is the first report on the SCMV infection of model plants B. distachyon inbred line Bd21 and N. benthamiana, which will shed light on the mechanism of SCMV infection of sugarcane and benefit sugarcane breeding against sugarcane mosaic disease.
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    Quantitative analysis of the interaction of heterologous viruses with Plum pox virus in C5 HoneySweet transgenic plums
    Khushwant Singh, Tereza Neubauerová, Jiban Kumar Kundu
    2019, 18 (10): 2302-2310.   DOI: 10.1016/S2095-3119(18)62136-7
    Abstract75)      PDF in ScienceDirect      
    Stone fruits are an important crop in most parts of the world and are heavily challenged by several viruses including Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Apple chlorotic leaf spot virus (ACLSV).  We validated the PPV resistance in C5 plum plants (commercially known as HoneySweet) grown in the Czech Republic for more than 16 years in a field trial experiment under natural environmental conditions.  We quantified single (PPV-Rec) and mixed viruses (PPV-Rec+ACLSV, PPV-Rec+PDV and PPV-Rec+ACLSV+PDV) in C5 transgenic plums inoculated for the period 2016 to 2018.  The accumulation of PPV-Rec was high (~5.43E+05 copies) compared with that of ACLSV (~8.70E+04 copies) in the inoculated graft of C5 transgenic plants.  Leaves close to the inoculum sources showed a differential level of virus titre in single and mixed infections (~10 to ~5×102 copies).  C5 plants with permanent virus pressure showed 103- to 105-fold fewer copies of viruses than those of the inoculated graft.  We observed high accumulation of conserved miRNAs such as miR167, miR69 and miR396 in C5 plants co-infected with PPV, ACLSV and PDV that are associated with its resistance against viruses.  Overall, i) C5 transgenic plums showed high resistance to PPV infection, and a low level (~32 copies) of PPV only accumulated in some grafted plants, ii) high accumulation of PPV was found in inoculated grafts in single PPV infection and mixed infections, iii) heterologous virus infection sustained by ACLSV or PDV did not suppress PPV resistance, and iv) high and low conserved microRNAs accumulated in C5 plants.
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    Molecular, serological and biological characterization of a novel Apple stem pitting virus strain from a local pear variety grown in China
    LI Liu, ZHENG Meng-meng, MA Xiao-fang, LI Yuan-jun, LI Qing-yu, WANG Guo-ping, HONG Ni
    2019, 18 (11): 2549-2560.   DOI: 10.1016/S2095-3119(19)62636-5
    Abstract82)      PDF in ScienceDirect      
    Apple stem pitting virus (ASPV) is an important causal agent of pear diseases.  Nowadays, the infection status and molecular characteristics of the virus in old pear trees have never been investigated.  In this study, we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar ‘Chili’ specifically grown at Laiyang area in China.  ASPV-LYC possesses a chimeric genome consisting of 9 273 nucleotides excluding a poly(A) tail at its 3´ end and harboring a recombination region in its open reading frame (ORF1) with Aurora-1 and KL9 identified as the major and minor parents.  Western blot analysis with antisera against recombinant coat proteins (CPs) of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates, but with differential activities.  Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates.  The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.
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    Evaluation of the biocontrol potential of Aspergillus welwitschiae against the root-knot nematode Meloidogyne graminicola in rice (Oryza sativa L.)
    LIU Ying, DING Zhong, PENG De-liang, LIU Shi-ming, KONG Ling-an, PENG Huan, XIANG Chao, LI Zhong-cai, HUANG Wen-kun
    2019, 18 (11): 2561-2570.   DOI: 10.1016/S2095-3119(19)62610-9
    Abstract70)      PDF in ScienceDirect      
    The root-knot nematode Meloidogyne graminicola is considered one of the most devastating pests in rice-producing areas, and nematicides are neither ecofriendly nor cost effective.  More acceptable biological agents are required for controlling this destructive pathogen.  In this study, the biocontrol potential of Aspergillus welwitschiae AW2017 was investigated in laboratory and greenhouse experiments.  The in vitro ovicidal and larvicidal activities of A. welwitschiae metabolites were tested on M. graminicola in laboratory experiments.  The effect of A. welwitschiae on the attraction of M. graminicola to rice and the infection of rice by M. graminicola was evaluated in a greenhouse.  The bioagent AW2017 displayed good nematicidal potential via its ovicidal and larvicidal action.  The best larvicidal activity was observed at a concentration of 5×AW2017, which caused an 86.2% mortality rate at 48 h post inoculation.  The highest ovicidal activity was recorded at a concentration of 5×AW2017, which resulted in an approximately 47.3% reduction in egg hatching after 8 d compared to the control.  Under greenhouse conditions, the application of A. welwitschiae significantly reduced the root galls and nematodes in rice roots compared to the control.  At a concentration of 5×AW2017, juveniles and root galls in rice roots at 14 d post inoculation (dpi) were reduced by 24.5 and 40.5%, respectively.  In addition, the attraction of M. graminicola to rice roots was significantly decreased in the AW2017 treatment, and the development of nematodes in the AW2017-treated plants was slightly delayed compared with that in the PDB-treated control plants.  The results indicate that A. welwitschiae is a potential biological control agent against M. graminicola in rice.
     
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    Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus
    LIU Mei, LIU Li-ming, WU Hui-jie, KANG Bao-shan, GU Qin-sheng
    2020, 19 (1): 153-163.   DOI: 10.1016/S2095-3119(19)62647-X
    Abstract90)      PDF in ScienceDirect      
    Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter (SGP) is extremely important for constructing viral vectors.  Although Cucumber green mottle mosaic virus (CGMMV)-based virus vectors have been constructed, SGP of the coat protein (CP) has not yet mapped.  To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site (TSS) as previous study identified by random amplification of cDNA ends (RACE).  Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein (MP) and CP.  To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction.  The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS.  For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein (EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS.  To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis.  Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced.  To our best knowledge, this is the first mapping of CGMMV SGP.
     
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    Molecular and biological characterization of melon-infecting squash leaf curl China virus in China 
    WU Hui-jie, LI Meng, HONG Ni, PENG Bin, GU Qin-sheng
    2020, 19 (2): 570-577.   DOI: 10.1016/S2095-3119(19)62642-0
    Abstract107)      PDF in ScienceDirect      
    It has been reported that squash leaf curl China virus (SLCCNV) infects some Cucurbitaceae crops except for melon (Cucumis melo L.).  A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China.  In this study, the pathogen was identified as SLCCNV through biological and molecular characterization.  The isolate (SLCCNV-HN) possess a bipartite genome, DNA-A (HM566112.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062251.1) pumpkin and SLCCNV-Hn61 (AM260205.1) squash isolates from China, whereas DNA-B (HM566113.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062252.1).  Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and -B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin, SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China, forming an independent cluster.  Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%, the systemic symptoms in melon showed identical to those of melon plants infected in fields.  Additionally, melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95% (19/20) showed leaf curl and dwarf symptoms 15 days post transmission, thereby fulfilling Koch’s postulates.  Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus.  To the best of our knowledge, this is the first characterization of melon-infecting SLCCNV through its genome, infectious clone and transmission.
     
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    Prevalence and genetic diversity of grapevine fabavirus isolates from different grapevine cultivars and regions in China
    FAN Xu-dong, ZHANG meng-yan, ZHANG Zun-ping, REN Fang, HU Guo-jun, DONG Ya-feng
    2020, 19 (3): 768-774.   DOI: 10.1016/S2095-3119(19)62677-8
    Abstract89)      PDF in ScienceDirect      
    A total of 288 grapevine samples of 61 different grapevine cultivars, collected from 22 provinces and regions, were analyzed by reverse transcription-PCR (RT-PCR) for the presence of grapevine fabavirus (GFabV).  PCR detection results showed the incidences of GFabV were 12.8% (30/235) and 48.1% (25/52) in the asymptomatic and symptomatic vines, respectively.  The genetic diversity of GFabV isolates was analyzed based on partial nucleotide and encoded amino acid sequences of the RNA1 and RNA2 polyprotein genes.  Phylogenetic analyses of the RNA1 and RNA2 gene sequences divided the GFabV isolates into five well-defined groups.  Groups 1, 2, and 4 comprised only Chinese isolates.  This article represents the first report for the prevalence and genetic diversity of GFabV in grapevines grown in China.
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    Monoclonal antibody-based serological detection of potato virus M in potato plants and tubers
    ZHANG Yu, GAO Yan-ling, HE Wan-qin, WANG Ya-qin, QIAN Ya-juan, ZHOU Xue-ping, WU Jian-xiang
    2020, 19 (5): 1283-1291.   DOI: 10.1016/S2095-3119(19)62755-3
    Abstract74)      PDF in ScienceDirect      
    Potato virus M (PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide.  To investigate and control this viral disease, efficient and specific detection techniques are needed.  In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies (MAbs).  Four highly specific and sensitive murine MAbs, i.e., 1E1, 2A5, 8A1 and 17G8 were prepared through a conventional hybridoma technology.  Using these four MAbs, we have developed an antigen-coated plate (ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers.  PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240 (w/v, g mL–1) by the dot-ELISA or at 1:163 840 (w/v, g mL–1) by the ACP-ELISA.  The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection.  Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China.  The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing.  We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM.  These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.
     
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    Virome identification in wheat in the Czech Republic using small RNA deep sequencing
    Khushwant SINGH, Jana JAROŠOVÁ, Jan FOUSEK, CHEN Huan, Jiban Kumar KUNDU
    2020, 19 (7): 1825-1833.   DOI: 10.1016/S2095-3119(19)62805-4
    Abstract103)      PDF in ScienceDirect      
    High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA (sRNA) population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample.  In the present sRNA deep-sequencing analysis of virus-infected wheat samples in the Czech Republic, samples were firstly tested for barley yellow dwarf viruses (BYDVs), wheat streak mosaic virus (WSMV) and wheat dwarf virus (WDV) using ELISA, RT-PCR and PCR.  Subsequent sRNA sequencing of these samples yielded more than ~60 million single-end 50-bp reads with high confidence for nine field samples of wheat.  Overall, 16.5% of reads were virus-specific and 83.5% were mapped to the host.  More 21-nt reads (~7.7E+06 reads) were found than 24-nt (~6.20E+06 reads) or 22-nt (~4.30E+06 reads) reads.  De novo assembly of the high-quality contigs revealed the presence of three earlier reported viruses in the Czech Republic: BYDVs (31.48%), WSMV (24.23%) and WDV (26.66%).  We also showed the presence of cereal yellow dwarf virus (14.33%; two species CYDV-RPS and CYDV-RPV (family Luteoviridae/Polerovirus) and wheat yellow dwarf virus (WYDV, 3.30%; Luteoviridae).  Phylogenetic analysis showed CYDV and WYDV grouped separately from BYDVs.  Furthermore, several recombination breakpoints were found among the groups of yellow dwarf viruses (BYDVs, CYDV, and WYDV).  Using RNA deep sequencing, we confirmed the presence of the three known viruses (BYDVs, WSMV, and WDV) and the first record of two species of CYDV and WYDV in wheat in the Czech Republic.
     
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    A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types
    REN Fang, ZHANG Zun-ping, FAN Xu-dong, HU Guo-jun, ZHANG Meng-yan, DONG Ya-feng
    2020, 19 (7): 1834-1841.   DOI: 10.1016/S2095-3119(19)62784-X
    Abstract76)      PDF in ScienceDirect      
    To develop a rapid and high-sensitivity method for detection of grapevine virus E (GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method (RT-qPCR) was established.  This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventional RT-PCR.  An excellent linear correlation (R2=0.997) and a high amplification efficiency (E=97.5%) were obtained from the standard curve of this method.  Reproducibility tests revealed that the coefficients of variation in the intra- and inter-assay results were 0.31–1.03% and 0.82–2.62%, respectively, indicating a good reproducibility.  The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types.  The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR.  The detection rates in spring, summer, autumn and winter increased gradually.  Samples in autumn and winter were best for detection, and the detection rates of most samples were 80–100%, which were 10 to 40% higher than conventional RT-PCR.  In general, old petioles and branches were the best tissues for GVE detection.  The detection rates of these samples in each season were all 100%, which were 20 to 40% higher than conventional RT-PCR.  The second highest rates were in the old leaf, with detection rates for RT-qPCR of 80–100% in all seasons, which were 20 to 40% higher than conventional RT-PCR.  GVE could be difficultly detected in young leaves by conventional RT-PCR, and the detection rates were only 0–50%, while by RT-qPCR the rates could increase to 0–80%.  A total of 33 out of 363 samples (belonging to 68 cultivars) from 20 regions in China were detected to be positive by RT-qPCR (9.1%), which was more than twice the rate of the conventional RT-PCR (3.9%). 
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    Identification of a novel emaravirus infecting lilac through next-generation sequencing
    WANG Ya-qin, SONG Yu, CAO Meng-ji, CHENG Qi, WU Jian-xiang, HU Tao
    2020, 19 (8): 2064-2071.   DOI: 10.1016/S2095-3119(19)62806-6
    Abstract85)      PDF in ScienceDirect      
    Emaraviruses are multipartite negative-sense single-stranded viruses that usually infect flowering plants.  Using unbiased next-generation sequencing (NGS) approach, a novel virus from lilac leaves with yellow mottle symptom was identified.  Five viral genomic RNA segments were detected and completely sequenced.  The molecular characteristics of the viral RNAs and proteins resemble those of members of the genus Emaravirus.  The virus was found to be mechanically transmissible to Nicotiana benthamiana. Sequence alignment and phylogenetic analyses of the virus support the proposal of creating a new species in the genus Emaravirus for which the name lilac chlorotic ringspot-associated virus (LiCRaV) is proposed. 
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    Decreasing detection frequency of MITE (MCLas-A) in the population of ‘Candidatus Liberibacter asiaticus’ recently collected in southern China
    CUI Xue-jin, ZENG Chun-hua, LIU Ke-hong, TENG Cai-ling, ZHOU Chang-yong, WANG Xue-feng
    2020, 19 (10): 2597-2601.   DOI: 10.1016/S2095-3119(20)63217-8
    Abstract72)      PDF in ScienceDirect      
    An active miniature inverted-repeat transposable element (MITE), MCLas-A, was previously identified from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease).  To explore the recent transposition status of MCLas-A, 389 ‘Ca. L. asiaticus’ strains collected from nine regions in China were amplified using a specific primer set and three representative ‘Ca. L. asiaticus’ strains were analyzed by next-generation sequencing (NGS) approach.  PCR and genomic analysis showed that the entire MCLas-A was only present in 1.80% (7/389) and the jumping-out type of the MITE was predominant (81.23%) in samples tested, suggesting high frequency transposition occurred in ‘Ca. L. asiaticus’ strains recently collected from China.  Biological roles of transposition of the active MITE remain to be determined.
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    Molecular diversity of barley yellow dwarf virus-PAV from China and the Czech Republic
    May Oo kHINE, brozenká MICHAELA, LIU Yan, Jiban kumar kUNDU, WANG Xi-feng
    2020, 19 (11): 2736-2745.   DOI: 10.1016/S2095-3119(20)63241-5
    Abstract79)      PDF in ScienceDirect      
    Wheat yellow dwarf disease (BYD), caused by different species of barley/cereal yellow dwarf viruses (B/CYDVs), is one of the most serious cereal diseases in China and the Czech Republic.  Because genetic diversity of the virus directly influences disease epidemiology, the molecular diversity and population structure of 24 Chinese isolates and 16 the Czech Republic isolates of BYDV-PAV from different regions in two countries were analyzed by sequencing their coat protein (CP) and readthrough protein (RTP) domain (RTD) genes and comparing the sequences with six CP and 16 RTP sequences of BYDV-PAV isolates from the NCBI database based on nucleotide identity position, phylogenetic analysis and nucleotide diversity.  Nucleotide identities between the Chinese and the Czech Republic isolates for the CP were 76.6–99.4%, 73.9–89.1% for RTD (ORF5), respectively.  The Chinese and the other country isolates showed 74.7–99.2% nucleotide identity for RTP (ORF3+ORF5).  Phylogenetic analysis of CP sequences showed that 20 Chinese isolates clustered in the same clade, but the other four Chinese isolates clustered in another clade with the isolates from the Czech Republic and other counties.  The population of BYDV-PAV in China had greater nucleotide variability and was more divergent than that in the Czech Republic.  Geographical and ecological factors but not hosts might contribute to the population differences in the two countries.
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    Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection
    YAN Zhi-yong, ZHAO Mei-sheng, MA Hua-yu, LIU Ling-zhi, YANG Guang-ling, GENG Chao, TIAN Yan-ping, LI Xiang-dong
    2021, 20 (7): 1871-1879.   DOI: 10.1016/S2095-3119(20)63275-0
    Abstract101)      PDF in ScienceDirect      
    Tomato brown rugose fruit virus (ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry.  So far, it has spread to 10 countries in America, Asia, and Europe.  In 2019, ToBRFV was identified in Shandong Province (ToBRFV-SD), China.  In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits.  ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102.  All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD.  The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel.  In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus (TMV).  Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus.  The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.
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    Robust molecular detection of the new Tomato brown rugose fruit virus in infected tomato and pepper plants from Turkey
    Hakan FIDAN, Pelin SARIKAYA, Kubra YILDIZ, Bengi TOPKAYA, Gozde ERKIS, Ozer CALIS
    2021, 20 (8): 2170-2179.   DOI: 10.1016/S2095-3119(20)63335-4
    Abstract89)      PDF in ScienceDirect      
    Tomato brown rugose fruit virus (ToBRFV) causes severe fruit loss in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants.  It is an emerging Tobamovirus that is spreading globally.  The major challenge is to develop a reliable method for the detection of the virus, and to better characterize the symptoms it causes.  The aims of this study, therefore, were to characterize the symptom development on tomato and pepper plants, and to establish a reliable detection method for the virus.  Following infection of the tomato and pepper plants with ToBRFV, the leaves turned chlorotic, mosaic or mottled, while the fruit became rugose, necrotic and marbled, and showed discoloration with yellow or brown spots.  Transmission electron microscopy (TEM) revealed single rod-like virus particles characteristic of the Tobamoviruses.  Classical reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) with specific primers and probes confirmed that the virus is ToBRFV.  We found that the resistance genes from tomato, Tm-22, and pepper, L1, L2, L3 and L4, did not confer resistance to ToBRFV.  Here, we present a PCR-based method as a diagnostic test for detecting ToBRFV in infected seeds.  This method will help to prevent further spread of the virus in commercial seeds.
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    Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy
    HU Guo-jun, DONG Ya-feng, ZHANG Zun-ping, FAN Xu-dong, REN Fan
    2021, 20 (9): 2463-2470.   DOI: 10.1016/S2095-3119(20)63336-6
    Abstract85)      PDF in ScienceDirect      
    Vitis vinifera 87-1 plants infected by grapevine fleck virus (GFkV) and grapevine rupestris stem pitting-associated virus (GRSPaV) were used as the plant materials for virus elimination treatment.  This study evaluated the effects of ribavirin at different concentrations (15 and 25 μg mL–1; R15 and R25, respectively), thermotherapy (37°C; T), and the combination of ribavirin and thermotherapy (R15+T and R25+T) on eliminating viruses from grapevine plants in vitro.  Both R15 and R25 had phytotoxic effects and weakened plant growth.  Thermotherapy positively affected the growth of grapevine plants.  Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25.  The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively.  The survival rates of regenerated plants after all treatments were >68.0%.  Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses.  The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively.  The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively.  The elimination rate of GFkV was 23.9% higher in R25 (35/44) than in R15 (25/45), and that of GRSPaV was 7.0% higher in R25 than in R15.  The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.  
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    Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica
    CHEN Wei-yu, YU Jia, XU Heng, LU Xin-yu, DAI Ting-ting, TIAN Yue-e, SHEN Dan-yu, DOU Dao-long
    2021, 20 (10): 2696-2705.   DOI: 10.1016/S2095-3119(20)63459-1
    Abstract75)      PDF in ScienceDirect      
    Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation, affecting both yield and quality.  Thus, rapid and accurate detection of P. parasitica plays an important role in disease management.  The aim of this study was to develop a simple diagnostic method to detect P. parasitica infection by combining recombinase polymerase amplification and lateral flow strips (LF-RPA).  To establish the LF-RPA assay of P. parasitica, the primers and probe designed based on the Ypt1 gene were tested for specificity to P. parasitica, which showed no cross-reactivity with DNAs of other related oomycete species.  The LF-RPA assay detected the amount of genomic DNA of P. parasitica which was as low as 1 pg.  To make the LF-RPA assay useful in low-resource settings, four simplified DNA extraction methods were compared, after which the LF-RPA assay was applied, with no specialized equipment, to analyze a diverse range of citrus tissues by using a simplified PEG-NaOH method for DNA extraction.  This method was successful in detecting P. parasitica in infected plant samples within 30 min.  Combining the LF-RPA assay and a simplified DNA extraction method could be a potential  detection test for P. parasitica, especially in areas with limited resources.
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    Three Sclerotinia species as the cause of white mold on pea in Chongqing and Sichuan of China
    DENG Dong, SUN Su-li, DU Chen-zhang, XIANG Chao, LONG Jue-chen, CHEN Wei-dong, ZHU Zhen-dong
    2021, 20 (11): 2957-2965.   DOI: 10.1016/S2095-3119(21)63629-8
    Abstract84)      PDF in ScienceDirect      
     
    White mold of pea caused by Sclerotinia sclerotiorum is a common disease in China.  However, we discovered that the diverse Sclerotinia species could cause white mold on pea plants in Chongqing and Sichuan of China during recent disease surveys.  Thus, the objective of this study was to confirm the causal agents from diseased pea plants.  The obtained isolates of white mold from Chongqing and Sichuan were identified by morphological characters and molecular characterization to determine the pathogen species, and their pathogenicity was confirmed on pea through completing Koch’s postulates.  Fungal isolates of Sclerotinia-like were obtained from diseased plants or sclerotia.  Based on morphological characteristics and molecular characterization, 30 isolates were identified to three species, six isolates as S. minor, seven as S. sclerotiorum, and 17 as S. trifoliorum.  In pathogenicity tests on pea cultivars Zhongwan 4 and Longwan 1, all 30 isolates caused typical symptoms of white mold on the inoculated plants, and the inoculated pathogens were re-isolated from the diseased plants.  This study confirmed that white mold of pea was caused by three Sclerotinia species, S. sclerotiorum, S. minor and S. trifoliorum in Chongqing and Sichuan. It is the first report that S. minor and S. trifoliorum cause white mold of pea in Southwest China.
     
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    Three sensitive and reliable serological assays for detection of potato virus A in potato plants
    WU Jia-yu, ZHANG Yu, ZHOU Xue-ping, QIAN Ya-juan
    2021, 20 (11): 2966-2975.   DOI: 10.1016/S2095-3119(20)63492-X
    Abstract99)      PDF in ScienceDirect      
    Vegetative propagation of seed potato often allows passaging of viruses to seed tubers, resulting in significant yield losses and reduction of potato tuber quality.  Thus, virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.  Among the reported potato-infecting viruses, potato virus A (PVA) is considered as one of the most important viruses in potato-growing regions worldwide.  This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies (MAbs) (2D4, 8E11, 14A6 and 16H10) using purified PVA virions as an immunogen.  Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.  Using these four MAbs, this study developed antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.  The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327 680 (w/v, g mL–1) by ACP-ELISA or up to 1:10 240 by Dot-ELISA.  The Tissue print-ELISA is the quickest and easiest approach among the three serological assays, and is more suitable for onsite large-scale potato screening programs.  Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.  Hence, the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys. 
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