Coat Protein Sequences Research Articles

Molecular characterization of chickpea chlorotic dwarf virus and peanut witches\u2019 broom phytoplasma associated with chickpea stunt disease and identification of new host crops and leafhopper vectors in India

An investigation was carried out to identify and characterize the phytoplasma and viruses associated with the chickpea varieties showing severe stunting, leaf reddening, yellowing and phyllody symptoms during the summer season of 2018–2019 and 2019–2020 in eight states of India. The average disease incidence was recorded from 3 to 32% in different states. The presence of chickpea chlorotic dwarf virus (CpCDV) was confirmed in thirty-seven chickpea samples by amplification of CpCDV coat protein gene and sequence comparison analysis. No record of association of luteovirus, polerovirus and cucumovirus could be detected in any of the symptomatic chickpea samples by RT-PCR assay. Brassica nigra, B. juncea, Lens culinaris, two weeds (Heteropogan contartus, Aeschynomene virginica) and one leafhopper (Amarasca biguttula) were identified as new putative hosts for CpCDV. Association of peanut witches’ broom phytoplasma was confirmed in twenty-eight chickpea samples, Sesamum indicum, five weeds hosts and two leafhopper species (Exitianus indicus, Empoasca motti) using nested PCR assays with primer pairs P1/P7 and R16F2n/R16Rn. The results of phytoplasma association in plants and leafhopper samples were further validated by using five multilocus genes (secA, rp, imp, tuf and secY) specific primers. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene and five multilocus genes confirmed the identity of association of 16SrII-C and 16SrII-D subgroups of phytoplasmas strain with chickpea samples collected from Andhra Pradesh (AP), Telangana, Karnataka, Madhya Pradesh, Uttar Pradesh and New Delhi. Mixed infection of phytoplasma (16SrII-D) and CpCDV was also detected in symptomatic chickpea samples from AP and Telangana. The reports of association of 16SrII-C subgroup phytoplasma in chickpea and 16SrII-D subgroup phytoplasma in C. sparsiflora and C. roseus are the new host records in world and from India, respectively.

Full genome sequence of a new three-segment gammapartitivirus from the phytopathogenic fungus Alternaria tenuissima on cotton in China

In this study, a new double-stranded RNA (dsRNA) virus, Alternaria tenuissima partitivirus 1 (AttPV1), was isolated from Alternaria tenuissima strain XJ-BZ-2-6, a phytopathogenic fungus infecting cotton in China. The genome of AttPV1 comprised three dsRNAs of 1,785 nt (dsRNA1), 1,545 nt (dsRNA2), and 1,537 nt (dsRNA3) in length, the nucleotide sequence of which was determined using reverse transcription polymerase chain reaction, random-primed clones, and RNA-ligase-mediated rapid amplification of cDNA ends. dsRNA1 had a single open reading frame encoding a putative 61.54-kDa RNA-dependent RNA polymerase (RdRp). dsRNA2 and dsRNA3 were predicted to encode putative coat proteins (CPs) of 47.90 kDa and 46.25 kDa, respectively. The RdRp domain shared 63.54-73.17% amino acid sequence identity with members of the genus Gammapartitivirus. Phylogenetic trees based on RdRp or CP sequences showed that AttPV1 clustered with members of the genus Gammapartitivirus. Hence, these results indicate that AttPV1 is a new gammapartitivirus from A. tenuissima.

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.

A novel tepovirus, Agave virus T, identified by the analysis of the transcriptome data of blue agave (Agave tequilana).

The genome sequence of a novel RNA virus was identified by analyzing transcriptome data obtained from the stem sample of a blue agave (Agave tequilana) plant. Sequence comparison and phylogenetic analysis showed that the RNA virus, Agave virus T (AgVT), was a new member of the genus Tepovirus in the family Betaflexiviridae. AgVT genome had three open reading frames: a 1605-amino acid (aa) replicase (REP), 355-aa movement protein (MP), and 220-aa coat protein (CP). Phylogenetic analyses based on the REP, MP, and CP sequences of AgVT, previously reported tepoviruses, and other Betaflexiviridae viruses revealed that tepoviruses could be classified into two subclades: "potato virus T (PVT)-clade" and "Prunus virus T (PrVT)-clade." PVT, the type species and founding member of the genus Tepovirus, belong to "PVT-clade" along with AgVT, while the other five tepoviruses belong to "PrVT-clade." The genome sequence of AgVT may be useful for studying the phylogenetic relationships between tepoviruses and other closely related viruses. Keywords: Agave virus T; Tepovirus; Betaflexiviridae; blue agave; Agave tequilana.

A Mixed Infection of Helenium Virus S With Two Distinct Isolates of Butterbur Mosaic Virus, One of Which Has a Major Deletion in an Essential Gene.

Multiple carlaviruses infect various ornamental plants, often having limited host ranges and causing minor symptoms, yet often reducing yield or quality. In this study we have identified a mixed infection of butterbur mosaic virus (ButMV) and helenium virus S (HelVS) from a plant of veronica (Veronica sp.) showing foliar mosaic and distortion. Carlavirus-like particles were observed by transmission electron microscopy (TEM), and RNA from partially purified virions was amplified by random RT-PCR, yielding clones of 439–1,385 bp. Two partially overlapping clones including coat protein (CP) sequence, and two of four partial replicase clones, were closely related to ButMV-J (AB517596), previously reported only from butterbur (Petasites japonicus) in Japan. Two other partial replicase clones showed lower identity to multiple carlaviruses. Generic primers which amplify the 3′-terminal region of multiple carlaviruses yielded clones of three distinct sequences: (1) with 98% nt identity to HelVS; (2) ButMV-A, showing 82% nt identity to ButMV-J; and (3) ButMV-B, with 78% nt identity to each of ButMV-J and ButMV-A. Further amplification of upstream fragments revealed that ButMV-B had an internal deletion in TGB1, confirmed using isolate-specific primers. Near-complete genomes of both ButMV-A and ButMV-B were obtained from next-generation sequencing (NGS), confirming the deletion within ButMV-B, which is presumably maintained through complementation by ButMV-A. HelVS was previously reported only from Helenium hybrids and Impatiens holstii. A near-complete HelVS genome was obtained for the first time by NGS from the same sample. Additional Veronica hybrids infected with HelVS were identified by TEM and RT-PCR, including cv. ‘Sunny Border Blue’ which was also subjected to NGS. This resulted in assembly of an 8,615 nt near-complete HelVS genome, with high identity to that from the mixed infection. The predicted CP sequence has 96% amino acid (aa) identity to HelVS from helenium (Q00556). Other ORFs show a maximum of 54% (TGB3) to 68% (NABP) aa identity to the equivalent ORFs of other carlaviruses. These results demonstrate for the first time maintenance by complementation of a carlavirus isolate with a major deletion in an essential gene, and confirm that HelVS is a distinct species in the genus Carlavirus.

Population genetic analysis of lettuce big-vein disease viruses and their vector fungi Olpidium virulentus in Ankara province, Turkey

Lettuce big-vein disease (LBVD) is caused by either Mirafiori lettuce big-vein virus (MiLBVV) alone, or co-infected with Lettuce big-vein associated virus (LBVaV). Both viruses are transmitted by the fungal vector Olpidium spp. In the present study, population genetics of these two viruses and their fungal vector, O. virulentus, were analyzed, using the nucleotide sequences of the virus coat protein (CP) partial genes and the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of the vector fungi. The analysis was conducted along with all previously reported LBVD viruses and Olpidium spp. populations. We used conventional PCR techniques to identify the viruses in lettuce samples and their fungal vector in soil samples collected from the root zone of field-grown lettuce plants in the Ankara province of Turkey. Phylogenetic analyses showed that LBVD viruses had at least two evolutionary lineages, and Ankara LBVD isolates were more closely related to the European populations. Also, we found that there is a strong negative selection pressure in the partial CP genes of LBVD viruses isolated from Ankara, due to the effective terrestrial climate. The negative selection pressure indicates balancing selection within Turkish isolates and other populations. The population of O. virulentus isolates from Ankara and other geographic regions were branched into five sub-clades, and there was no geographical separation in these isolates. Our analysis also showed Olpidium spp. to be monophyletic, and showed speciation among the isolates.

Identification of Divergent Isolates of Banana Mild Mosaic Virus and Development of a New Diagnostic Primer to Improve Detection.

Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the Musa germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol.

Bioinformatics analysis of putative causes for сross-reactive antibodies interacting with antigens derived from various pathogenic human papillomaviruses

Human papillomaviruses (HPVs) belong to highly abundant resulting in sexually transmitted virus infections, and cause cervical cancer holding place 4 among most common cancer types in women. In 2012, there were registered 266,000 death cases and 528,000 new cases. At present, three HPV prophylactic vaccines were generated worldwide: bivalent Cervarix, quadrivalent Gardasil and nonavalent Gardasil-9. Examining such vaccines uncovered that they are able to induce anti-HPV antibody production against viral antigens lacked in vaccine formula. The mechanism of such crossneutralizing antibodies recognizing antigens derived from various HPV pathogenic types remains unknown. In our study we attempted to uncover putative basis underlying cross-reactive interaction between vaccine-induced antibodies and non-vaccine antigens by bioinformatical approaches, that might allow optimize generation of future candidate vaccines and obtain more effective polyvalent immunobiological preparations against HPV. We used amino acid sequences of L1 coat protein of four top high-risk oncogenic HPV types (16, 18, 31 and 45) in the study. Work sequences were retrieved from the International Data Base of NCBI (National Center for Biotechnology Information) and aligned by using Clustal Omega’ and BioEdit software. A search and analysis of distinct antigenic determinant (epitopes) were performed by using software suite BepiPred-2.0: Sequential B-Cell Epitope Predictor, DiscoTope 2.0 Server, and SYFPEITHI. Bioinformatics data revealed pronounced potential of cross-neutralizing vaccine-induced antibodies and non-vaccines antigens derived from high-risk pathogenic types HPV 16, 18, 31 and 45 owing to the similarity in antigenic determinants (epitopes). Common linear determinants for T- and B-cells were found in all four types of L1 protein counterparts. In addition, similar three-dimensional B-cell determinants were discovered in HPV16 L1 and HPV18 L1. Antigenic determinants derived from HPV16 L1 and HPV31 L1 exhibited most close similarity. Hence, while immunizing with HPV16 L1, a more pronounced and moderate cross-reactive antibodies interacting with HPV31 L1 as well as HPV18 L1 and HPV45 L1 antigens, respectively, should be expected. Inversely, immunization with HPV18 L1might elicit active and less efficient crossneutralizing response with HPV45 L1 as well as HPV16 L1 and HPV31 L1, respectively.

Characterization and complete genome sequences of two novel variants of the family Closteroviridae from Chinese kiwifruit.

Two distinct closterovirus-like genome sequences (termed AdV-1 v1 and v2) were identified in Actinidia chinensis var. deliciosa ‘Miliang-1’ that had no disease symptoms using high-throughput sequencing. Using overlapping reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, the genomic sequences of AdV-1 v1 and v2 were confirmed as 17,646 and 18,578 nucleotides in length, respectively. The two complete genomes contained 9 and 15 open reading frames, respectively, coding for proteins having domains typical of Closteroviridae, such as RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h) and coat protein (CP). Sequence analysis showed that the amino acid sequences of RdRp, HSP70h, and CP of the two variants exhibited high similarity (> 80%), while their genomic organization was somewhat different. This suggested that the two viral genomes identified here are variants of the family Closteroviridae in a single kiwifruit host. Furthermore, phylogenetic relationship analysis revealed that the two variants had a closer relationship with the unclassified virus Persimmon virus B (PeVB) and Actinidia virus 1 (AcV-1) than with other members of the family Closteroviridae, as did their genomic organization. It is speculated that the two variants, together with PeVB and AcV-1 belong to a new subfamily of Closteroviridae.

Detection and characterization of a second carlavirus in Rosa sp.

A new virus resembling members in the genus Carlavirus was identified in an Out of Yesteryear rose (Rosa sp.) by high-throughput sequencing. The virus was discovered during the screening of a rose virus collection belonging to Foundation Plant Services (UC-Davis). The full genome of the virus is 8825 nt long, excluding a poly(A) tail, and includes six predicted genes coding for replicase, triple gene block, coat protein (CP), and nucleic acid binding protein. The closest relative of the putative virus is rose virus A (RVA; genus Carlavirus), with 75% and 78% aa sequence identity in the replicase and CP, respectively. The relationship with RVA and other carlaviruses was supported by phylogenetic analyses using replicase and CP sequences. Based on genome organization, sequence identity, and phylogenetic analysis, the virus found in the Out of Yesteryear plant represents a new member of the genus Carlavirus and is provisionally named "rose virus B" (RVB). Further testing by reverse transcription PCR confirmed the presence of RVB in the original source and seven additional rose selections from the same collection.

Chimeric Tobamoviruses With Coat Protein Exchanges Modulate Symptom Expression and Defence Responses in Nicotiana tabacum.

In the pathogen infection and host defence equilibrium, plant viruses have evolved to efficiently replicate their genomes, to resist the attack from host defence responses and to avoid causing severe negative effect on growth and metabolism of the hosts. In this study, we generated chimeric tobacco mosaic virus (TMV) variants, in which the coat protein (CP) sequences were substituted with that of cucumber green mottle mosaic virus (CGMMV) or pepper mild mottle virus (PMMoV) to address the role of these in virus infection and host symptomology. The results showed that the chimeric viruses (TMV-CGCP or TMV-PMCP) induce stunting and necrotic symptoms in tobacco plants. We analyzed the transcriptomic changes in tobacco plants after infection of TMV and its chimeras using a high-throughput RNA sequencing approach and found that infection of the chimeric TMV induced significant up-regulation of host defence responsive genes together with salicylic (SA) or abscisic acid (ABA) responsive genes, but down-regulation of auxin (Aux) responsive genes. We further confirmed the increase in the levels of SA and ABA, together with the reduced levels of Aux after infection of chimeric TMV in tobacco plants. These data suggest novel roles of tobamovirus CP in induction of host symptoms and defence responses.

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.

Molecular Detection of Potato Viruses in Bangladesh and Their Phylogenetic Analysis.

Potato (Solanum tuberosum) is a major food source in the whole world including Bangladesh. Viral diseases are the key constraint for sustainable potato production by reducing both quality and quantity. To determine the present status of eight important potato viruses in Bangladesh, tuber samples were collected from three major potato growing regions (Munshiganj, Jessore and Bogra districts) in January–February 2017 and February 2018. Reverse transcription polymerase chain reaction (RT-PCR) with coat protein (CP)-specific primers were used to amplify CP sequences of the respective viruses, and confirmed by sequencing, which were deposited in the GenBank. Results indicated that the tuber samples were subjected to Potato leafroll virus (PLRV), Potato virus X (PVX), Potato virus Y (PVY), Potato virus S (PVS), Potato virus H (PVH), Potato aucuba mosaic virus (PAMV) and Potato virus M (PVM) infection, whereas mixed infections were very common. Phylogenetic analysis revealed that the PLRV from this study was closely related to a Canadian and a Chinese isolate, respectively; PVX was closely related to a Canadian and a Chinese isolate, respectively; PVY was closely related to a Chinese isolate; PVS was closely related to a Chinese and an Iranian isolate, respectively; PAMV was closely related to a Canadian isolate; PVH was closely related to a Huhhot isolate of China; and PVM was closely related to an Indian and an Iranian isolate, respectively. As far as we know, PAMV in this study is the first report in Bangladesh. These findings will provide a great scope for appropriate virus control strategies to virus free potato production in Bangladesh.

Molecular diversity of barley yellow dwarf virus-PAV from China and the Czech Republic

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.

Molecular variability of the coat protein gene of prunus necrotic ringspot virus on sweet and sour cherry in Bulgaria

Prunus necrotic ringspot virus (PNRSV) is a stone fruit tree pathogen. Field surveys were conducted in the spring of 2018 and 2019 in 58 commercial and four collection sweet and sour cherry orchards in six regions of Bulgaria. Leaf samples from 3214 trees showing and not showing virus-like symptoms were tested by enzyme linked immunosorbent assay (ELISA) for PNRSV infection. The average infection level of 5.1% was higher in sour cherry (15.5%), than in sweet cherry trees (1.8%). The coat protein (CP) sequences of 25 isolates were determined. The obtained sequences were 596 or 675–681 nucleotides (nts) in length, translated to 198 or 225–227 amino acids (aa), respectively. Sequence comparisons showed 94–100% nucleotide and 90–100% amino acid identities among the 25 isolates. Phylogenetic analysis using nucleotide and amino acid sequences of Bulgarian isolates and cherry isolates retrieved from the GenBank database confirmed clustering into the previously reported PV96-II, PV32-I, PE5-III and CH30 phylogroups. Twenty Bulgarian isolates belonged to PV96-II and five isolates to PV32-I phylogroup. No correlation was noted between PV96-II phylogrouping and the sampling region/and or symptoms of infected trees of studied isolates, while a connection to a certain degree with geographical origin of the isolates in PV32-I phylogroup, but not with the symptomatology was observed.

Occurrence and genetic diversity of groundnut rosette assistor virus in western Kenya

Groundnut (Arachis hypogaea) is an important legume in western Kenya, but yields are low and declining. Pests and diseases are ranked high among the yield reducing factors. Groundnut rosette disease (GRD) is the main disease and can cause up to 100% yield loss. Groundnut rosette disease is transmitted by the groundnut aphid, Aphis craccivora Koch and caused by two synergistic viruses; groundnut rosette assistor virus (GRAV) and groundnut rosette virus (GRV) associated with a satellite-ribonucleic acid (sat-RNA). Inadequate information was available on the occurrence and molecular diversity of GRAV in western Kenya, which is a limiting factor on proper diagnosis and management. This study determined the occurrence and molecular diversity of GRAV on groundnuts in western Kenya. A survey of GRD was conducted in 144 farms in Bungoma and Kakamega counties during the short and long rains of 2016–2017. A total of 301 symptomatic leaf samples were collected and preserved until use. Incidence and severity data was collected and subjected to pairwise comparison of means using the standardized disease diagnostic score sheet at P ≤ 0.05. Screening for resistance/tolerance to GRD was done on five popular legume varieties (Arachis hypogaea, Phaseolus vulgaris, Vigna unquiculata, Glycine max and Vigna radiata) and one solanaceous Physalis peruviana. The plants were monitored for GRD symptoms development in the screenhouse for 8 weeks. Total RNA was extracted from the leaf samples using RNeasy Mini Kit (Qiagen) according to the manufacturers’ protocol. DNA libraries were prepared and sequenced on the MiSeq platform (Illumina). Phylogenetic analyses and comparisons were performed using the MEGA X software. From the sequences obtained, primers were designed for use in RT-PCR. Groundnut rosette disease was prevalent in all the surveyed areas with chlorotic rosette symptoms being dominant followed by green rosette and mosaic rosette. Groundnut rosette disease incidence ranged between 31 and 67%, but was higher in Bungoma (67%) than Kakamega (61%) county, while the short rains had higher incidence than the long rains season. All screened indicator plants developed typical GRD viral symptoms and tested positive for GRAV. Nucleotide sequences of GRAV coat protein (CP) gene revealed 97–99% identity among the western Kenya isolates and those from Ghana, Malawi and Nigeria respectively. The fact that GRD occurs wherever groundnuts are grown in western Kenya, is of great concern and may be the reason for the observed low yields. Incorporation of GRD resistance genes in the local cultivars/varieties may be the only practical solution.

Molecular characterization of viruses causing maize lethal necrosis disease in South-Rift region, Kenya

The first report of Maize lethal necrosis (MLN) disease in Africa was in Bomet, Kenya, nine years ago. It has since spread in East and Central African (ECA) countries, causing massive yield losses. Currently, 90% of the preferred commercial maize germplasm grown by farmers in Kenya is susceptible to MLN disease. As such, the disease has continued to pose a serious challenge to food security in the ECA region. This study sought to characterize the MLN, causing viruses present in the maize leaf samples collected from the South-Rift region. Using total RNA extracted from 60 leaf samples collected from Bomet, Kericho, and Kisumu Counties, reverse transcription-polymerase chain reaction (RT-PCR) was carried out. The PCR products with the strongest bands were purified and sequenced using the Sanger sequencing technique. The results showed that samples from the three counties were positive for maize chlorotic mottle virus (MCMV) (MH645622 and MH645621) and sugarcane mosaic virus (SCMV) (MH645623, MH645624 and MH645625) and negative to wheat streak mosaic virus (WSMV). The coat protein (CP) sequences of MCMV isolates were closely related to the sequences of MCMV isolates, which had been previously reported from Eastern and Sub-Saharan Africa. For the CP sequences of SCMV isolates, only one sequence of the isolate KCO59 was similar to the sequence of a Kenyan isolate (JX286708). Sequences of isolates KCO5 and KCO24 were not identical to those of the Kenyan isolate (JX286708). Based on these results, in the surveyed counties, isolates of SCMV are genetically diverse, while those of MCMV are not. There exists a new variant of SCMV, which appears to be the main potyvirus in synergism with MCMV in causing MLN disease in Kenya. Key words: Maize lethal necrosis (MLN), maize chlorotic mottle virus (MCMV), sugarcane mosaic virus (SCMV), wheat streak mosaic virus (WSMV).

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).

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically.

A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of Nicotiana benthamiana with the binary vector pCambia-crTMV-CPPLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.