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    Detection and Molecular Characterization of two 'Candidatus Phytoplasma Trifolii' Isolates Infecting Peppers at the Same Ecological Niche
    (Friends Science Publ, 2017) Oksal, Hatice Digdem; Apak, Fulya Kaya; Oksal, Ercin; Tursun, Nihat; Sipahioglu, Hikmet Murat
    Pepper (Capsicum annuum L.) cultivars exhibiting phytoplasma like symptoms including yellowing, flower sterility, necrosis, stunting and small leaves of lateral shoots were collected in Spring 2016 from Malatya province (Turkey). Leaf samples of the most common annual weeds and leafhoppers nearby symptomatic peppers were also sampled. Nested-PCR and virtual computer-simulated restriction fragment length polymorphism (virtual RFLP) methods have been implemented to ascertain and characterize the phytoplasma-associated disease. Using universal primer pairs in nested-PCR DNA fragments of approximately 1.2 kb were amplified from 3 pepper samples. None of the weed and leafhopper samples were reacted positive in PCR reactions. Next-generation sequencing (NGS) was used to sequence the amplified PCR fragments of two samples. The presence of 'Candidatus Phytoplasma trifolii' infections were confirmed by the analysis of 16S rDNA sequence and virtual-RFLP. Molecularly characterized isolates were designated as TR1 and TR2 (Acces. no: KY321932 and KY568694). Both isolates were identified as members of the clover proliferation phytoplasma group (subgroup 16SrVI-A) in pepper plants as strains of 'Ca. Phytoplasma trifolii'. Sequence alignment of the two 'Ca. Phytoplasma trifolii' isolates revealed a low level of genetic diversity. However, the restriction enzyme patterns of both isolates particularly in MseI profiles were differed from reference patterns of all previously established 'Ca. Phytoplasma trifolii' isolates in the world. Particularly, the TR2 isolate showed a point mutation comparing TR1 isolate and with reference strain (AY390261) of 'Ca. Phytoplasma trifolii'. This is the first report of 'Ca. Phytoplasma trifolii' isolates naturally infecting pepper plants from Turkey. (C) 2017 Friends Science Publishers
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    First report of Candidatus Phytoplasma solani on a new host marigold (Tagetes erecta L.)
    (Tubitak Scientific & Technological Research Council Turkey, 2016) Alp, Sevket; Usta, Mustafa; Sipahioglu, Hikmet Murat; Guller, Abdullah
    Marigold (Tagetes erecta L.) plants, also called Mexican or Aztec marigold, with symptoms of shoot proliferation, dwarfing, and reddening were observed in ornamental gardens of Van Province (Turkey). Five plants, two of them showing reddening and three symptomless plants, were sampled at the end of September 2014. Genomic DNA isolated from symptomatic and nonsymptomatic plant leaves was used to amplify 16S rDNA fragments by nested polymerase chain reaction (PCR). Of the 5 marigold samples tested by PCR, only the two showing reddening symptoms yielded the expected 1.2-kb DNA fragments. Amplified PCR fragments were cloned into a plasmid vector and transformed into competent Escherichia coli strain JM 109. Recombinant plasmid DNA was isolated and sequenced bidirectionally. The provided sequences were 1244 bp and 1245 bp in length and were designated as isolate 1 and isolate 2, respectively. BLAST analysis of the 16S rDNA sequence and virtual restriction fragment length polymorphism (RFLP) analysis confirmed the presence of the phytoplasma Candidatus Phytoplasma solani. The in silico virtual RFLP pattern of isolate 1, based on the 16S rDNA F2n/R2 fragment, was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959). Isolate 1 was identified as a member of 16SrXII-A. Based on the same analyses, isolate 2 showed molecular characteristics different from reference patterns of all previously established 16Sr groups and subgroups. The most similar was the reference pattern of 16Sr group XII, subgroup A (GenBank accession no.: AF248959), with a similarity coefficient of 0.97. This is the first report of naturally occurring Ca. P. solani affecting T. erecta, which shows that this plant species is an alternate host of the agent.
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    Molecular Analysis of 'Candidatus Phytoplasma trifolii' and 'Candidatus Phytoplasma solani' Associated with Phytoplasma Diseases of Tomato (PDT) in Turkey
    (Friends Science Publ, 2018) Usta, Mustafa; Guller, Abdullah; Sipahioglu, Hikmet Murat
    Tomato plants displaying severe fruit deformation, flower sterility, aerial rooting, purplish leaves and leaf rolling were observed in tomato fields at Van province (Turkey). Samples were collected, and total DNA was extracted from symptomatic and asymptomatic plants. Nested polymerase chain reaction (nested-PCR) assays were performed to amplify 16S rDNA sequences for molecular detection using universal primer pairs. Out of 100 tested tomato samples, 11% of tomato samples yielded a DNA fragment of 1.25 kb. Amplified PCR products were then cloned into pGEM T-Easy vector and sequenced using new generation DNA sequencing (NGS) system. The virtual restriction fragment length polymorphism (RFLP) analysis of 16S rDNA sequences and molecular detections were allowed to characterize possible phytoplasmas associated with diseased plants. Our results revealed the presence of two Phytoplasma species belonging to two different ribosomal groups; 'Candidatus Phytoplasma trifolii' (16Sr VI-A group) (Acces no. MF564268, MG732925) and 'Candidatus Phytoplasma solani' (16SrXII-A group) (Acces no. KY579358, MF576263). Despite a high variation in their similarity coefficient of'Ca. P. solani' VTS2 (0.91) and 'Ca. P. trifolii' VTT1 (0.88) isolates, the infected tomato plants generally displayed similar disease symptoms during field observations. Due to its commercial interest, co-existing of these phytoplasmas in tomato fields is of great phytosanitary significance not only for tomato plants but also for other crops such as vegetables, ornamentals and field crops. With this study, 'Ca. P. trifolii' associated with phytoplasma diseases of tomato (PDT) has been reported for the first time in tomato in Turkey. (C) 2018 Friends Science Publishers
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    Molecular Cloning and Sequence Analysis of the its Region of Nuclear Ribosomal DNA for Species Identification in Dodders (Cuscuta; Convolvulaceae)
    (Friends Science Publ, 2017) Keskin, Fatma; Kaya, Ilhan; Usta, Mustafa; Demir, Ibrahim; Sipahioglu, Hikmet Murat; Nemli, Yildiz
    Dodder (Cuscuta sp.) is an obligate parasitic plant that is very difficult to control. In plants the internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal DNA (nrDNA) has been considered one of the most important sequences for phylogenetic analysis. Here we report the analysis of nrDNA's ITS sequences as an efficient tool to study the phylogeny of dodders collected from various provinces of Eastern Anatolia (Turkey). Genomic DNA of six dodder samples belonging to 4 distinct species was extracted from body tissue samples. The sequences of 18S rRNA, ITS-1, 5.8S rRNA, ITS-2 and 26S rRNA regions of 4 Cuscuta species were determined by molecular cloning and sequencing. The identity of cloned fragments was compared to determine sequence identity using NCBI database. Bootstrap analysis of nrDNA of C. approximate, C. lupuliformis, C. campestris and C. babylonica indicated high sequence identity with similar sequences belonging to different geographical origins of the world retrieved from NCBI database. Our results clearly showed that the most stable secondary structure derived from the sequences obtained by universal ITS4 and ITS5 primers is very efficient tool for identification of Cuscuta species when used in combination with phylogenetic analysis. (C) 2017 Friends Science Publishers
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    Pokeweed (Phytolacca americana L.) antiviral protein inhibits Zucchini yellow mosaic virus infection in a dose-dependent manner in squash plants
    (Tubitak Scientific & Technological Research Council Turkey, 2017) Sipahioglu, Hikmet Murat; Kaya, Ilhan; Usta, Mustafa; Unal, Murat; Ozcan, Dilek; Ozer, Meryem; Guller, Abdullah
    Pokeweed antiviral protein (PAP) of Phytolacca americana L. (pokeweed) is a single-chain ribosome-inactivating protein (RIP) characterized by its ability to depurinate plant ribosomes. Here, we isolated, cloned, and expressed the ribosome inactivating protein (RIP) gene, designated as pokeweed antiviral protein type 1 (PAP I), from the summer leaves of pokeweed collected from the Black Sea region (Turkey). Our findings presented here provide direct evidence that exogenous application of PAP I causes concentration-dependent inhibition of Zucchini yellow mosaic virus (ZYMV) infection on squash plants. Squash plants were exposed to PAP I protein with and without DMSO for four consecutive days. Regular spraying of approximately 30 kDa recombinant PAP I at 2 mu g mL(-1) concentration prevented treated plants from mechanical virus infection. PAP I showed antiviral activity in 9 plants out of 15 inoculated plants. Remarkably, simultaneous application of PAP, DMSO, and ZYMV did not prevent virus infection, suggesting that PAP did not have any effect on viral RNA. In the absence of ZYMV the purified peptide was not cytotoxic for squash plants, although a reduction of plant size, possibly caused by host ribosome depurination, was observed.