Next-generation sequencing identification and multiplex RT-PCR detection for viruses infecting cigar and flue-cured tobacco.

BACKGROUND: Early, precise and simultaneous identification of plant viruses is of great significance for preventing virus spread and reducing losses in agricultural yields. METHODS AND RESULTS: In this study, the identification of plant viruses from symptomatic samples collected from a cigar tobacco planting area in Deyang and a flue-cured tobacco planting area in Luzhou city, Sichuan Province, China, was conducted by deep sequencing of small RNAs (sRNAs) through an Illumina sequencing platform, and plant virus-specific contigs were generated based on virus-derived siRNA sequences. Additionally, sequence alignment and phylogenetic analysis were performed to determine the species or strains of these viruses. A total of 27930450, 21537662 and 28194021 clean reads were generated from three pooled samples, with a total of 105 contigs mapped to the closest plant viruses with lengths ranging from 34 ~ 1720 nt. The results indicated that the major viruses were potato virus Y, Chilli veinal mottle virus, tobacco vein banding mosaic virus, tobacco mosaic virus and cucumber mosaic virus. Subsequently, a fast and sensitive multiplex reverse transcription polymerase chain reaction assay was developed for the simultaneous detection of the most frequent RNA viruses infecting cigar and flue-cured tobacco in Sichuan. CONCLUSIONS: These results provide a theoretical basis and convenient methods for the rapid detection and control of viruses in cigar- and flue-cured tobacco.

Molecular analysis of Greek isolates of cucumber mosaic virus from vegetables shows a low prevalence of satellite RNAs and suggests the presence of host-associated virus strains.

Cucumber mosaic virus (CMV) is a generalist pathogen that infects many economically important crops in Greece. The present study was designed to evaluate the genetic variability of Greek CMV isolates in combination with their satellite RNAs (satRNAs). To achieve this goal, 77 CMV isolates were collected from symptomatic Greek vegetables, mainly tomatoes and cucurbits, alongside their neighboring crops, during a four-year period from 2015 to 2018. Phylogenetic analysis of a partial coat protein (CP) gene segment revealed that all of the isolates belong to CMV subgroups IA and IB and that they are closely related to previously reported Greek isolates. It should be noted, however, that the latter mainly included tomato isolates. Network analysis of the evolutionary relationships among the CP sequences of the Greek isolates in comparison to the corresponding sequences obtained from the GenBank database indicated two predominant common ancestors and at least three differentiated peripherals, and possibly host-associated (tomatoes, legumes, cucurbits) haplogroups (strain groups). More specifically, host-adaptive evolution can be postulated regarding the tomato isolates in subgroup IB. Necrogenic or non-necrogenic satRNAs were detected in four samples from tomato and melon, and this is the first report of non-necrogenic satRNAs in CMV in Greece.

Co-existence of chlorosis inducing strain of Cucumber mosaic virus with tospoviruses on hot pepper (Capsicum annuum) in India.

Cucumo- and tospoviruses are the most destructive viruses infecting hot pepper (chilli). A diagnostic survey was conducted to assess the prevalence of cucumo and tospoviruses in chilli growing tracts of Tamil Nadu. Infected plants showing mosaic with chlorotic and necrotic rings, veinal necrosis, mosaic mottling, leaf filiformity and malformation were collected. Molecular indexing carried out through reverse transcription polymerase chain reaction (RT-PCR) with coat protein gene specific primer of Cucumber mosaic virus (CMV) and tospovirus degenerate primer corresponding to the L segment (RdRp). Ostensibly, amplifications were observed for both CMV and tospoviruses as sole as well for mixed infections. The sequence analysis indicated that the Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) to be involved with CMV in causing combined infections. The co-infection of CMV with CaCV was detected in 10.41% of the symptomatic plant samples and combined infection of CMV with GBNV was recorded in around 6.25% of the symptomatic plants surveyed. The amino acid substitution of Ser129 over conserved Pro129 in coat protein of CMV implies that CMV strain involved in mixed infection as chlorosis inducing strain. Further, the electron microscopy of symptomatic plant samples explicated the presence of isometric particles of CMV and quasi spherical particles of tospoviruses. This is the first molecular evidence for the natural co-existence of chlorosis inducing CMV strain with CaCV and GBNV on hot pepper in India.

"Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay".

Cucumber mosaic virus (CMV) is a widespread plant virus infecting important vegetables, plantation and flower crops. Currently, CMV is detected by enzyme-linked immunosorbent assays (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. ELISA requires polyclonal antibodies and is time-consuming. PCR requires skilled manpower and complex procedures of RNA isolation as well as a thermal cycler. To overcome these difficulties, a portable rapid, simple and visual fluorescence-based reverse transcription-recombinase polymerase amplification (portable RT-exo-RPA) assay for the detection of CMV was developed. A specific primer pair of 30-33 bp targeting a conserved region of the coat protein (CP) gene of CMV and a probe to function in the RT-exo-RPA assays were designed and synthesized. A total of 62 symptomatic as well as 58 asymptomatic banana plant samples, collected from banana orchards located in Jalgaon, Maharashtra, India, were evaluated for CMV infections using crude leaf extracts as templates by a reverse transcription-recombinase polymerase amplification (RT-RPA) assay as well as a real-time RT-exo-RPA assay and the results were compared with those of a reverse transcription-polymerase chain reaction (RT-PCR) assay using purified total plant RNAs as templates. CMV was as efficiently detected using the crude leaf extract template in the RT-RPA and real-time RT-exo-RPA assays as using the purified RNA template in the RT-PCR assay. To dispense with the use of real-time PCR, a portable RT-exo-RPA assay was developed and the alternative methods for the visualization of CMV detection using either a fluorometer or direct viewing with a UV transilluminator were evaluated. To our knowledge, this is the first report of the rapid and reliable diagnosis of CMV infections by a real-time RT-exo-RPA assay using a crude leaf extract as template.

Use of Fluorescent Microsphere-Based Assay for Detection of Three Cucurbit-Infecting Viruses.

In this study, we describe multiplex polymerase chain reaction (PCR) coupled with the LiquiChip assay for the identification of Zucchini yellow mosaic virus, Cucumber green mottle mosaic virus, and Cucumber mosaic virus by coamplification with plant mRNA as an internal control. Multiplex reverse-transcription (RT)-PCR products were subjected to allele-specific primer extension, then hybridized to carboxylated microspheres with unique fluorescent identifiers followed by detection using the LiquiChip 200 workstation. This assay is highly specific for distinguishing individual viruses from a mixed viral population and is 10 times more sensitive than multiplex RT-PCR. In addition, the establishment of this method enabled the detection of cucurbit viruses in field samples.

Cucumber mosaic virus Isolates from Greek Legumes are Associated with Satellite RNAs that are Necrogenic for Tomato.

Worldwide, Cucumber mosaic virus (CMV) is the causal agent of many economically important diseases. Based on immunological or molecular analysis, three distinct subgroups of CMV isolates can be identified (IA, IB, and II). In addition, some CMV isolates are associated with satellite RNAs (satRNAs), a type of noncoding transcript that may alter the symptoms of CMV infections. This study presents an analysis of CMV isolates occurring in legumes in Greece in respect to their genetic diversity, and the presence and diversity of their satRNA. Phylogenetic analysis of the CMV coat protein sequence of 18 legume and 5 tomato CMV isolates collected throughout Greece classified them within subgroups IA and IB, with a limited genetic diversity. The CMV satRNAs found in nine field legumes exhibiting mild symptoms and in one tomato with a necrotic syndrome contained a functional necrogenic motif; therefore, they were grouped within the necrogenic group of CMV-satRNAs. The necrotic phenotype was expressed in all legume CMV isolates containing necrogenic satRNAs when mechanically inoculated onto tomato plants. To our knowledge, this is the first observation that legumes host necrogenic CMV-satRNAs. The possible role of legumes in the epidemiology of CMV and necrogenic satRNA complex is discussed.

Proteomics analysis of Xiangcaoliusuobingmi-treated Capsicum annuum L. infected with Cucumber mosaic virus.

Among different viruses, Cucumber mosaic virus (CMV) has the most extensive host range, being capable of infecting over 1200 species, and causes severe damage worldwide. Xiangcaoliusuobingmi (B1), a candidate plant immune activator drug, exhibited significant protective effects against CMV. However, its potential mechanism is still unknown. In this study, we found the defensive enzyme activities of peroxidase (POD), phenylalanine ammonia lyase (PAL), superoxide dismutase (SOD) and catalase (CAT) can be enhanced by B1. Meanwhile, we found RT-qPCR assay results of the defensive gene expression can be improved by B1 in capsicum. Moreover, we analyze the result of label-free proteomics, B1 could trigger abscisic acid (ABA) pathway. All data provide a more understanding about the response to infect CMV capsicum activeted by B1 in the level of the plant physiology and biochemistry, gene and protein.

[Molecular identification of cucumber mosaic virus in woad (Isatis tinctoria) with mosaic disease in Beijing].

To detect possible pathogenic virus(es) in woad (Isatis tinctoria) cultivated at Institute of Medicinal Plant Development in Beijing, reverse transcription(RT)-PCR was performed using total RNA of symptomatic woad leaves with primers for poty-, polero-, tobamovirus, broad bean wilt virus 2(BBWV2) and cucumber mosaic virus (CMV). A 657 bp fragment was amplified from symptomatic woad using CMV primers. Sequencing and BLAST analysis indicated that this fragment shared 99% nucleotide identity and 100% amino acid identity with CMV-Vi isolate. The isolate was named CMV-Isatis tinctorial (CMV-It). Phylogenetic analysis based on nucleotide sequences of CP genes showed that CMV-It clustered with CMV-K and belonged to subgroup I. To our knowledge, this is first identification of CMV in woad by RT-PCR and the CP gene was analyzed. This work provided data for research and control of woad mosaic disease.

Virome analysis of lily plants reveals a new potyvirus.

Lily plants exhibiting virus-like symptoms of leaf yellowing, twisting and brownish necrotic spots were collected, and next-generation sequencing of small RNAs was conducted to identify the associated viruses. Cucumber mosaic virus, lily symptomless virus and a hitherto unrecorded potyvirus, tentatively named "lily yellow mosaic virus" (LYMV), were detected. The genomic RNA of LYMV was 9811 nt in length, encoding a large polyprotein of 3,124 amino acids with a predicted Mr of 353.3 kDa. BLAST analysis showed that LYMV shared a high degree of amino acid sequence identity with Thunberg fritillary mosaic virus (55%), bean yellow mosaic virus (52%), clover yellow vein virus (51%), leek yellow stripe virus (51%), and lily mottle virus (52%), and these viruses clustered together in a phylogenetic tree.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.

Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily.

Characterization of siRNAs derived from cucumber mosaic virus in infected tobacco plants.

This study characterized the viral small interfering RNAs (vsiRNAs) from Nicotiana tabacum cv. Samsun infected with a cucumber mosaic virus (CMV) 2b-deficient mutant. Most vsiRNAs were 21 -22 nucleotides in length and the 5'-terminal ends were dominated by A and U, respectively. The observed vsiRNAs were heterogeneously distributed throughout the CMV genome; however, most of the vsiRNAs were derived from sense strands, as opposed to antisense strands. These results demonstrate the conserved and specific function of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins in tobacco. Finally, it was revealed that vsiRNAs target abundant host genes, indicating complex roles for CMV vsiRNAs during the development of symptoms.

Genetic diversity, distant phylogenetic relationships and the occurrence of recombination events among cucumber mosaic virus isolates from zucchini in Poland.

In recent years, the occurrence of cucumber mosaic virus (CMV) has been noted in zucchini crops in Poland. Beside characteristic isolates, which displayed mosaics and chlorosis on infected plants, new necrotic isolates have also been identified. Here, we analysed the molecular variability of 27 isolates of CMV collected from zucchini in various regions of the country. Sequence and phylogenetic analysis based on the genes encoding the coat (CP) and movement (MP) proteins revealed that the Polish isolates belong to two subgroups: IA and II, with the prevalence of subgroup II. New recombinant variants with an IA-MP/II-CP pattern for RNA3 were also detected.

Development of a molecular assay for the detection of Cucumber mosaic virus and the discrimination of its subgroups I and II.

A nucleic acid based test for the detection of the economically important plant virus Cucumber mosaic virus (CMV) based on the Luminex xTAG technology was developed. This technology has the advantage of allowing the simultaneous detection of various targets. Applying this method, we prove the presence of CMV in general and differentiate between its two subgroups I and II for which significant differences concerning severity of symptoms and virulence have been reported. For the development of the test procedure the coat protein gene sequences of 29 CMV isolates were cloned, sequenced and classified into subgroups. Sequences from GenBank were used to design primers. Additionally, a subgroup specific ELISA was conducted for comparison. This work is part of a project which aims to develop a test for the simultaneous detection of various plant pathogens (viral, bacterial and fungal) in plant material.

A genetically novel, narrow-host-range isolate of cucumber mosaic virus (CMV) from rosemary.

An isolate of cucumber mosaic virus (CMV), designated CMV-Rom, was isolated from rosemary (Rosmarinus officinalis) plants in several locations near Avignon, France. Laboratory studies showed that, unlike typical CMV isolates, CMV-Rom has a particularly narrow host range. It could be transmitted by aphids Aphis gossypii and Myzus persicae, but with low efficacy compared to a typical CMV isolate. Phylogenetic analysis of the nucleotide sequences of the CMV-Rom genomic RNAs shows that this isolate does not belong to any of the previously described CMV subgroups, IA, IB, II or III.

Aphid performance changes with plant defense mediated by Cucumber mosaic virus titer.

BACKGROUND: Cucumber mosaic virus (CMV) causes appreciable losses in vegetables, ornamentals and agricultural crops. The green peach aphid, Myzus persicae Sulzer (Aphididae) is one of the most efficient vectors for CMV. The transmission ecology of aphid-vectored CMV has been well investigated. However, the detailed description of the dynamic change in the plant-CMV-aphid interaction associated with plant defense and virus epidemics is not well known. RESULTS: In this report, we investigated the relationship of virus titer with plant defense of salicylic acid (SA) and jasmonic acid (JA) during the different infection time and their interaction with aphids in CMV-infected tobacco plants. Our results showed that aphid performance changed with virus titer and plant defense on CMV-inoculated plants. At first, plant defense was low and aphid number increased gradually. The plant defense of SA signaling pathway was induced when virus titer was at a high level, and aphid performance was correspondingly reduced. Additionally, the winged aphids were increased. CONCLUSION: Our results showed that aphid performance was reduced due to the induced plant defense mediated by Cucumber mosaic virus titer. Additionally, some wingless aphids became to winged aphids. In this way CMV could be transmitted with the migration of winged aphids. We should take measures to prevent aphids in the early stage of their occurrence in the field to prevent virus outbreak.

Temporal analysis of reassortment and molecular evolution of Cucumber mosaic virus: Extra clues from its segmented genome.

Cucumber mosaic virus (CMV) is a damaging pathogen of over 200 mono- and dicotyledonous crop species worldwide. It has the broadest known host range of any virus, but the timescale of its evolution is unknown. To investigate the evolutionary history of this virus, we obtained the genomic sequences of 40 CMV isolates from brassicas sampled in Iran, Turkey and Japan, and combined them with published sequences. Our synonymous ('silent') site analyses revealed that the present CMV population is the progeny of a single ancestor existing 1550-2600 years ago, but that the population mostly radiated 295-545 years ago. We found that the major CMV lineages are not phylogeographically confined, but that recombination and reassortment is restricted to local populations and that no reassortant lineage is more than 251 years old. Our results highlight the different evolutionary patterns seen among viral pathogens of brassica crops across the world.

Sequence analysis and genetic diversity of five new Indian isolates of cucumber mosaic virus.

Cucumber mosaic virus (CMV) is an important virus since it causes severe losses to many economically important crops worldwide. Five new isolates of CMV were isolated from naturally infected Hippeastrum hybridum, Dahlia pinnata, Hemerocallis fulva, Acorus calamus and Typhonium trilobatum plants, all exhibiting severe leaf mosaic symptoms. For molecular identification and sequence analyses, the complete coat protein (CP) gene of these isolates was amplified by RT-PCR. The resulting amplicons were cloned and sequenced and isolates were designated as HH (KP698590), DP (JF682239), HF (KP698589), AC (KP698588) and TT (JX570732). For study of genetic diversity among these isolates, the sequence data were analysed by BLASTn, multiple alignment and generating phylogenetic trees along with the respective sequences of other CMV isolates available in GenBank Database were done. The isolates under study showed 82-99% sequence diversity among them at nucleotide and amino acid levels; however they showed close relationships with CMV isolates of subgroup IB. In alignment analysis of amino acid sequences of HH and AC isolates, we have found fifteen and twelve unique substitutions, compared to HF, DP and TT isolates, suggesting the cause of high genetic diversity.

Genotyping of Cucumber mosaic virus isolates in western New York State during epidemic years: Characterization of an emergent plant virus population.

In the early 2000s an epidemic of cucumber mosaic virus (CMV) spread within the Midwestern and Eastern US affecting snap and dry bean (Phaseolus vulgaris L.) cultivation. Fifty one CMV isolates from this period were partially characterized from varied hosts by sequencing a section from each of the three genomic RNAs. Aside from one subgroup II strain from pepper, all isolates, including those from snap bean, fell within the IA subgroup. The nucleotide sequence diversity of virus populations sampled at multiple sites and at different years was significantly higher than that of a population from single site in a single year, although in general the number of polymorphisms was low (<11%). Complementary DNA (cDNA) clones of Bn57, a representative isolate from snap bean, were engineered for the production of infectious in vitro RNA transcripts initiated from a T7 promoter. Infections from these cDNAs resulted in symptoms consistent with those of the original field isolate, indicating that a satellite RNA is not involved in symptom expression in snap bean. These infectious clones were used to assess symptom determinants and the effects of virus infection on plant growth. Inoculations with pseudorecombinants derived from Bn57 and the non-bean infecting strain Fny confirmed RNA2 as a specific determinant for snap bean infection. Bn57, along with almost all isolates identified in this study contained the Y631 locus in the 2a protein, a determinant for systemic infection in bean. The presence of this locus extended to all non-bean hosts except two pepper infecting isolates. Infection by Bn57 in snap bean had a significant effect on pod number and mass with a 55 and 41 percent reduction in greenhouse assays, respectively. To our knowledge Bn57 is the first CMV strain isolated from P. vulgaris to be fully sequenced and cloned, providing a useful tool for analyses of CMV-host interactions.

Molecular characterization of cucumber mosaic virus isolates infecting tomato in Hamedan and Tehran provinces of Iran.

Here we identified four isolates, MS, 3H, 50A, and 2K of cucumber mosaic virus (CMV) infecting tomato, on the basis of their non-coding intergenic region and a part of the coat protein (CP) sequence in the CMV genomic RNA3. The sequences from the four isolates were compared with other previously characterized isolates of CMV isolated from different plant species across the globe. Sequence comparisons revealed that the two CMV isolates from Hamedan province (MS and 3H) had the highest sequence identity with CMV-G10 (98%), which was previously reported as a severe Hellenic tomato isolate of CMV, while the CMV isolates from Tehran province, including CMV-2K (isolated from Karaj region) and CMV-50A (isolated from Varamin region), had the highest sequence identity with that of CMV-ALF (99%). Phylogenetic analysis of the nucleotide sequences showed that CMV-MS and CMV-3H belong to group IB, while CMV-2K and CMV-50A belong to group IA. This is the first report on the molecular characterization of novel isolates of CMV infecting tomato plants in Iran.