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    Antiviral and Antifungal Activity of Biologically Active Recombinant Bouganin Protein from Bougainvillea spectabilis Willd
    (Ankara Univ, Fac Agr, 2018) Guller, Abdullah; Siphioglu, Hikmet Murat; Usta, Mustafa; Demirer Durak, Emre
    Bouganin antiviral protein (BAP) gene. one of the ribosome inactivating proteins, isolated from Bougainvillea speclabilis 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 beta-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 Rhizocionia solani. Trichoderma harzianum, and Fusarium arysporurn 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. solcati by 30.7% and of T harzianum by 20% after 72 h compared to control. No growth inhibition was observed for F oayporurn. All plants including controls treated with in vitro expressed BAP protein exhibited severe growth reduction compared with negative control (not treated) plants.
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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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    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.