Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), Shallot yellow stripe virus (SYSV), and Plantago asiatica mosaic virus (PlAMV) are five of the economically important viruses infecting lilies (Lilium spp.) worldwide. In order to prevent the occurrence and spread of these viruses, it is necessary to develop a rapid, effective, and sensitive detection method for the simultaneous detection and specific quantification of these viruses. In this study, specific primers and probes for multiplex TaqMan real-time PCR assays designed from conserved regions of the coat protein sequence of each virus were used for the simultaneous detection of these viruses in lilies (Lilium spp.). The optimal concentration of primers and probes and reaction annealing temperature were 20 µM and 55.9 °C, respectively. The detection limits of the assay were 1.33 × 102, 1.27 × 101, 1.28 × 101, 2.33 × 102, and 2.01 × 102 copies·μL−1 for LSV, LMoV, CMV, SYSV, and PlAMV, respectively. Specificity was determined using seven viral pathogens of lilies. Variability tests of intra- and inter-assays showed high reproducibility with coefficients of variation <2%. The multiplex TaqMan real-time PCR assay was used to detect these viruses from lily samples in China. In brief, our developed assay showed high specificity, sensitivity, and reproducibility for the simultaneous detection and differentiation of five lily-infecting viruses and can be used for certification and quarantine programs.

Characterization of Chickpea Chlorotic Dwarf Virus (CpCDV) Associated with Chickpea Stunt Disease

Background: Stunt disease is becoming the major yield limiting factors for the chickpea production and its occurrence has been reported form different states of India. The symptoms of stunt disease caused by chickpea chlorotic dwarf virus are difficult to distinguish Mastrevirus-infected plant from other disease-causing pathogens. Therefore, it’s an imperative for precise detection of causal agent of the disease for development of management strategy against chickpea stunt.Methods: Survey for the incidence of stunt disease with most characteristic symptoms of leaf reddening and yellow orange typical to Mastrevirus infection was conducted in chickpea fields. The causal agent of the stunt was characterized and described through conventional and virus-specific PCR-based diagnostic technique.Result: The study revealed that maximum of 60% of the chickpea stunt was observed in three districts of Uttar Pradesh with an average incidence of 12.90%. The PCR amplification using CpCDV-specific primers encoding coat protein resulted in an expected amplicon size of 350bp. The comparison of the partial coat protein sequence of virus revealed that maximum homology of 98.70% with previously identified chickpea chlorotic dwarf virus (CpCDV) strains, indicating that CpCDV associated with the chickpea stunt. Based on molecular characterization, chickpea stunt disease caused by Chickpea chlorotic dwarf virus (ssDNA), belongs to the genus Mastrevirus which is also responsible for the lentil stunt disease.

Identification and molecular characterization of onion yellow dwarf virus (OYDV) in Allium cepa crops in Poland

Onion yellow dwarf virus is distributed worldwide significantly reducing yield of crops from the Allium genus. The aim of the study was the detection and molecular characterization of newly identified OYDV isolates infecting onions in Poland. The virus was detected by transmission electron microscopy and RT-PCR techniques using two pairs of diagnostic primers: OYDV-NibCPF1/R1 and OYDV-CPF2/R2. The specificity of obtained RT-PCR products was confirmed by Sanger sequencing and received viral coat protein sequence was used for phylogenetic analysis. The phylogenetic analysis was carried out using CP sequences of the new Polish onion isolate obtained in this study and 37 other sequences of OYDV retrieved from GenBank. The analysis revealed that the Polish OYDV isolate is the most similar to the OYDV isolates derived from onions from Argentina and Germany, which may indicate their common origin. Moreover, it was observed that the Polish onion and garlic isolates are very diverse and belong to different phylogroups.

Requirements for the Packaging of Geminivirus Circular Single-Stranded DNA: Effect of DNA Length and Coat Protein Sequence

Geminivirus particles, consisting of a pair of twinned isometric structures, have one of the most distinctive capsids in the virological world. Until recently, there was little information as to how these structures are generated. To address this, we developed a system to produce capsid structures following the delivery of geminivirus coat protein and replicating circular single-stranded DNA (cssDNA) by the infiltration of gene constructs into plant leaves. The transencapsidation of cssDNA of the Begomovirus genus by coat protein of different geminivirus genera was shown to occur with full-length but not half-length molecules. Double capsid structures, distinct from geminate capsid structures, were also generated in this expression system. By increasing the length of the encapsidated cssDNA, triple geminate capsid structures, consisting of straight, bent and condensed forms were generated. The straight geminate triple structures generated were similar in morphology to those recorded for a potato-infecting virus from Peru. These finding demonstrate that the length of encapsidated DNA controls both the size and stability of geminivirus particles.

The molecular characterization of the coat protein sequence and differentiation of CMV- subgroup I on tobacco from native flora in Turkey

Cucumber mosaic virus (CMV) has a broad plant-host range and a wide ecological zone distribution. Virus-like symptoms were observed on tobacco fields of Adiyaman province (Turkey) showing conspicuous mottling, greenish mosaic patterns and severe malformations of leaves. A total of forty tobacco samples tested positive against CMV by reverse transcription polymerase chain reaction (RT-PCR) using coat protein gene specific primers. Five randomly chosen CMV isolates were cloned into pGEM T-Easy vector and transformed into Escherichia coli JM109 strain. The recombinant bacterial clones containing insert-DNA were further purified and sequenced bidirectionally. In multiplex-RT-PCR studies carried out, it was found that all 40 CMV isolates belong to Subgroup I by resulting a 593 bp long DNA fragments. CMV subgroup IA was found to predominate in 4 out of 5 tobacco samples and CMV subgroup IB was found in 1 out of 5 CMV-positive samples by comparing the isolates with CMV reference isolates in phylogenetic tree. However, no Subgroup II sequences were found by multiplex RT-PCR using discriminating primers. The nucleic acid sequences were analyzed for the investigation of diversity of coat protein (CP) sequences of 5 CMV isolates. The sequence similarity ranged from 94.2-100% with the CMV subgroup I isolates infecting diverse plants in other regions of the world. The evolutionary tree revealed that the CMV IA Adiyaman isolates exhibited a genetic affinity with Australian and Spanish isolates. However, the CMV IB Adiyaman isolate showed a close genetic relationship with only the Australian isolates. To our knowledge, this study shows for the first time the occurrence of CMV IA and IB isolates infecting cultured tobacco plants in Adiyaman province.

Introduction pages

Notulae Botanicae Horti Agrobotanici Cluj-Napoca (NBHA), Issue 2, Volume 48, 2020: The papers published in this issue Vol 48 No 2 (2020) represent new exciting researches in different topics of life science, respectively in plant science, horticulture, agronomy and crop science. Among the interesting articles we invite you to find news about Development of SCAR markers related to heat tolerance in Kentucky bluegrass; The molecular characterization of the coat protein sequence and differentiation of CMV- subgroup I on tobacco from native flora in Turkey; Genetic diversity of Polish cultivars of common wheat (Triticum aestivum L.) based on molecular and protein markers; Somatic embryo induction and Agrobacterium-mediated transformation of embryonic callus tissue in Phoebe bournei, an endangered woody species in Lauraceae; Phenolic composition and antioxidant activity of red, rosé and white wines originating from Romanian grape cultivars etc. Notulae Botanicae Horti Agrobotanici Cluj-Napoca journal has moved to online-only publication at the start of 2017; beginning in 2019, the journal appears quarterly. The new Impact Factor communicated by ISI Clarivate, June 29, 2020, is IF 2019 = 1.168 (position 149 of 234 journals, Q3 in Plant Sciences). New metrics in Scopus – Elsevier (June 22, 2020): CiteScore 1.40 (#43/84 in Horticulture); SJR 0.35 - Q2, #41/90 in Horticulture (SJR Scimago Journal).

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.

Preliminary molecular characterization of some Citrus tristeza Closterovirus isolates infecting Croatian citrus

Citrus tristeza Closterovirus (CTV) is widespread in major citrus-growing regions of the world often causing destructive diseases. Citrus samples were taken from orchards in the Croatian coastal region. CTV was detected in two symptomless field trees of Satsuma mandarins and one diseased lemon tree. Double-stranded RNA was isolated from the field trees and the dsRNA patterns were compared in polyacrylamide gels. The same dsRNA extracts were used as templates in RT-PCR experiments amplifying the CTV coat protein sequence. Amplicons were subjected to SSCP and RFLP analyses. The results indicate greater similarity between CTV isolates from Satsuma mandarins than between these two and the lemon isolate.

In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

Bacteriophage GA coat protein formed self-assembly competent virus-like particles (VLPs) have been expressed previously in bacterial and yeast cells. On the basis of our previous experiments on the yeast vector pESC-URA / S. cerevisiae system containing two oppositely oriented promoters, new constructions were created with point-mutations in coat protein to mimic phage MS2-like RNA binding characteristics. Simultaneously, the MS2 operator sequence was added to mRNA desired for packaging. After the introduction of single-point mutations (S87N, K55N, R43K) and double-point mutations (S87N + K55N and S87N + R43K), the coat protein’s ability to form VLPs was retained, but yield from cells was decreased. Exchange of the 87th Ser to Asn in coat protein sequence in combination with bacteriophage MS2 translational operator provided specific packaging of the gene of interest (GFP). Although non-specific nucleic acid sequences were packaged, the remarkable specificity for packaging of the gene of interest can be achieved using the described approach.