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

Genetic diversity and population structure of tomato brown rugose fruit virus (ToBRFV) variants from Antalya province, Turkey

In this study, bioinformatic analyses were carried out according to the fully coded CP and MP gene regions of the agent, using six novel tomato brown rugose fruit virus (ToBRFV) variants obtained from the production greenhouses in Antalya, where the infection was first detected in Turkey and global variants. Molecular evolutionary analyses using both CP and MP gene regions showed that all variants were distributed in three major clades. Population dynamics studies for both gene regions have shown that there was very low nucleotide diversity and haplotype diversity. The low haplotype diversity for the CP and MP genes indicated almost no recombination status. A strong negative selection was determined for CP and MP gene regions, dN/dS= 0.0877 and dN/dS=0.2104, respectively. Neutrality test results revealed that ToBRFV populations are in an expansion phase. Pairwise comparisons were performed between populations separated in the geographic hierarchy as American, European, and Asian variants, and the findings showed intense gene flow and high genetic similarity (FST<0,33 and migration rate >1). The results of this study reveal the recent population structure of the virus and suggest that necessary precautions should be taken in the international seed trade against contaminated seeds

Antiviral and Antifungal Activity of Biologically Active Recombinant Bouganin Protein from Bougainvillea spectabilis Willd

Bouganin antiviral protein (BAP) gene, one of the ribosome inactivating proteins, isolated from Bougainvillea spectabilis Willd. was cloned, expressed and the antiviral and antifungal activities were investigated. The full-length bouganin antiviral protein gene was amplified by reverse transcription-PCR using mRNA as template extracted from mature leaves. The coding region of bouganin gene was cloned into prokaryotic expression vector pETDuet-1 after amplification with end to end gene specific primers. The recombinant plasmid was transformed into Escherichia coli cells BL21(DE3)pLysS and the expression of BAP gene was induced by isopropyl β-D thiogalactopyranoside (IPTG). Bouganin antiviral protein having a molecular mass of 28 kDa has been isolated from transformed bacterial colonies. Antiviral activity of bouganin was assayed against Zucchini yellow mosaic virus (ZYMV) by a mechanical inoculation test. The antifungal activity of purified recombinant protein was tested against pathogenic and non-pathogenic Rhizoctonia solani, Trichoderma harzianum, and Fusarium oxysporum fungi using disc diffusion method. The increased amount of antiviral protein reduced the disease severity caused by ZYMV. The bouganin antiviral protein was inhibited the growth of R. solani by 30.7% and of T. harzianum by 20% after 72 h compared to control. No growth inhibition was observed for F. oxyporum. All plants including controls treated with in vitro expressed BAP protein exhibited severe growth reduction compared with negative control (not treated) plants.&nbsp

Coat protein of alfalfa mosaic alfamovirus (AMV) from Türkiye: genetic inference and in silico docking analysis for potential antiphytoviral purposes

In 2021, a study was conducted in the Denizli region of Türkiye to investigate the phylogenetic relationship and presence of alfalfa mosaic alfamovirus (AMV) infecting pepper plants exhibiting viral disease symptoms. A total of 57 samples were collected, of which twenty-four tested positive for AMV with reverse transcriptase polymerase chain reaction (RT-PCR) assays. Samples from pepper plants displaying virus symptoms gave positive bands on the agarose gel, while healthy plants yielded negative results. One of the positive samples was randomly selected, cloned and sequenced. This sequence of the Denizli AMV isolate (753 bp) was recorded in the GenBank database with accession number OQ845956. The nucleotide sequence showed a high nucleotide consensus of 97%-99% compared with the nucleotide sequences of the same variants from different origins in GenBank. According to the phylogenetic tree generated through the Neighbour Joining (NJ) method, this AMV isolate belongs to the same group as Iranian isolates from various of hosts. Furthermore, in silico docking analysis of the coat protein (CP) of the AMV isolate and promising 12 essential oil compounds was performed to enable potential antiviral drug development. Docking study showed that eucalyptol, eugenol and carvacrol can make important contributions to the advancement of drug-based strategies for the managing of plant viruses by interacting with the virus coat protein of high binding energies, -5.3, -5.2 and -5.0 kcal mol-1, respectively. Although the presence of AMV in Denizli province has been reported previously, this study reports the phylogenetic relationships and docking analysis of the new AMV isolate in pepper crops

Current status and molecular phylogeny of some economically important viruses infecting wheat crops of Şırnak province, Turkey

A survey of yellow dwarf viruses (YDVs) -associated diseases of wheat plants was performed for the first time in the Şırnak Province, Turkey. In plants wheat cultivated areas, symptoms such as diminished leaf size, dwarfing, yellowing, and reddening of leaves wheat plants were observed in cultivated areas. A total of 441 specimens were collected regardless of showing symptoms and assayed for Barley/Cereal Yellow Dwarf Viruses [B/CYDV] association by PCR tests using virus coat protein gene (CPG)-specific primer pairs. These assays demonstrated that 13.38% (55 samples) were positive to the occurrence of BYDV-PAV (9.73%) and CYDV-RPV (0.73%) for wheat viruses, but no BYDV-MAV, -SGV, and -RMV infections of BYDVs were found in PCR assays of collected samples. In addition, double infection of BYDV-PAV and CYDV-RPV was detected in 12 samples with a 2.91% infection rate. The CPG sequences of randomly selected two isolates were revealed using bacterial cloning and sequencing. Sequences obtained were deposited in GenBank under Accession numbers OL685734 and OL685736 for BYDV-PAV; and OL685735 and OL685737 for      CYDV-RPV. BLASTn analysis of CPG sequences of four isolates shared high nucleotide homology with their intraspecies isolates, with 97.35-98.67% for two BYDV-PAV and 97.72-97.89 for two CYDV-RPV. The consensus tree constructed classified B/CYDV-associated wheat viruses with their phylogenetically closest individuals from Turkey and the world. This survey is the first report of BYDV-PAV and CYDV-RPV associated with wheat plants in Şırnak Province of Turkey.Keywords: BYDVs; Coat protein gene; Incidence; Sequencing; Şırna

Current status and molecular phylogeny of some economically important viruses infecting wheat crops of Şırnak province, Turkey

A survey of yellow dwarf viruses (YDVs) -associated diseases of wheat plants was performed for the first time in the Şırnak Province, Turkey. In plants wheat cultivated areas, symptoms such as diminished leaf size, dwarfing, yellowing, and reddening of leaves wheat plants were observed in cultivated areas. A total of 441 specimens were collected regardless of showing symptoms and assayed for Barley/Cereal Yellow Dwarf Viruses [B/CYDV] association by PCR tests using virus coat protein gene (CPG)-specific primer pairs. These assays demonstrated that 13.38% (55 samples) were positive to the occurrence of BYDV-PAV (9.73%) and CYDV-RPV (0.73%) for wheat viruses, but no BYDV-MAV, -SGV, and -RMV infections of BYDVs were found in PCR assays of collected samples. In addition, double infection of BYDV-PAV and CYDV-RPV was detected in 12 samples with a 2.91% infection rate. The CPG sequences of randomly selected two isolates were revealed using bacterial cloning and sequencing. Sequences obtained were deposited in GenBank under Accession numbers OL685734 and OL685736 for BYDV-PAV; and OL685735 and OL685737 for      CYDV-RPV. BLASTn analysis of CPG sequences of four isolates shared high nucleotide homology with their intraspecies isolates, with 97.35-98.67% for two BYDV-PAV and 97.72-97.89 for two CYDV-RPV. The consensus tree constructed classified B/CYDV-associated wheat viruses with their phylogenetically closest individuals from Turkey and the world. This survey is the first report of BYDV-PAV and CYDV-RPV associated with wheat plants in Şırnak Province of Turkey.Keywords: BYDVs; Coat protein gene; Incidence; Sequencing; Şırna

First Report of ‘Candidatus Phytoplasma australasia’ Strain Related to Witches’-Broom of Tomato in Türkiye

Phytoplasmas are dangerous bacteria severely infecting agricultural production worldwide. In the present study, the identification of phytoplasmas infecting tomato plants showing symptoms such as small leaves, flower abnormalities, stunting, witches' broom, and reddening was performed. Five plants, two symptomatic and three asymptomatic, were tested to verify phytoplasma infection. Total DNA isolated from 5 leaf samples was used as a template for PCR reactions. The phytoplasma agents were confirmed in the two symptomatic samples. BLASTn search of 16S rRNA of two sequences shared identity similarity of 99.84% with ‘Candidatus Phytoplasma australasia’. Computer-simulated virtual RFLP profiles show that the 16S rRNA sequences is identical to the reference pattern of the 16SrII-D subgroup, with a similarity coefficient of 1.00. Based on BLAST, virtual RFLP, and phylogenetic dendrogram, the identified phytoplasma strains are enclosed in the 16SrII-D subgroup. This is the first report of tomato witches' broom disease related to 16SrII-D subgroup phytoplasma strains in the Antalya province of Türkiye