IntroductionPhytoplasmas are phloem-limited, insect-transmitted, wall-less, nonculturable plant pathogens from the class Mollicutes. They cause diseases in numerous plant species including fruit, vegetable, cereal, forest, and ornamental crops worldwide (Lee et al., 2000). Molecular methods and interactive online Web software have become the most reliable tools for the detection, identification, and classification of phytoplasma diseases. These methods are most commonly used to amplify either an entire or a specific phytoplasma sequence of 16S rDNA and to generate in silico digestions with a few key enzymes. The latter may help to distinguish the input data from previously recognized patterns (Lee et al., 1998;Khadhair et al., 2008).Following the application of molecular technologies, phytoplasma taxonomy is largely or entirely based on analysis of 16S rRNA gene sequences. "Candidatus Phytoplasma solani" falls within the 16SrXII group containing phytoplasmas such as "Ca. P. japonicum", "Ca. P. fragariae", and "Ca. P. australiense", which infect a wide range of crop plants (Duduk and Bertaccini, 2011; EFSA, 2014).An important ornamental plant, marigold (Tagetes erecta L.) is grown in homes and gardens throughout Turkey. In addition to their ornamental role (Wright, 1979), marigold plants have been used as pharmaceutical plants (Tostle, 1968) and pesticides for the protection of agricultural crops (Morallo and Decena, 1982;Kourany and Arnason, 1988;Rhoades, 1990). Although "Ca. P. solani" has been known on wild marigold, Calendula officinalis (common marigold) (Esmailzadeh-Hosseini et al., 2011), the presence of the disease on T. erecta (marigold) has not been reported. Today, the only phytoplasma disease reported on T. erecta is marigold phyllody, which belongs to the aster yellows group (16SrI), subgroup B (Almeyda- León and Rocha-Peña, 2001;Rojas-Martínez et al., 2003).

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 µg 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.

Van ilinde yetiştiriciliği yapılan yonca (Medicago sativa L.) bitkilerinde 2019 yılında virüslerin oluşturduğu simptomlara benzer belirtiler görülmüştür. Gözlenen belirtiler arasında cüceleşme, yapraklarda mozaik deseni ve sararma, rozetlenme ve küçük yaprak oluşumu yer almaktadır. Simptom gösteren ve göstermeyen bitkilerden toplanan 19 yonca yaprak örneğine, virüs genomunu tespit edebilmek için uygun primer çiftleri ile RT-PCR testi uygulanmıştır. Yonca yaprağı örneklerinden şiddetli belirtiye sahip altısı beklenen büyüklükte 700 bp DNA band oluşturmuştur. Bunlar arasından rastgele seçilen ikisi bir plazmid vektörüne klonlanmıştır. Elde edilen rekombinant plazmidler her iki yönde dizilenmiştir. Dizi analizi sonuçlarına göre infekteli yoncalardaki virüsün Yonca mozaik virüs olduğu açığa çıkarılmıştır. Dizi bilgileri MT210179 ve MT210178 erişim numaraları ile gen bankasına yüklenmiştir ve sırasıyla Alakoy Y9 ve Alakoy Y1 olarak isimlendirilmiştir. Her iki dizi için gen bankasına kayıtlı 16 AMV dizisi ile oluşturulan filogenetik ağaca göre, her iki izolat da nükleotit düzeyinde ABD, Brezilya ve Puglia izolatları ile en yüksek benzerlik ve Güney Kore izolatı ile en düşük benzerlik oranı göstermiştir. Ayrıca her iki dizinin birbirleri arasında, 7 nükleotit değişikliği ile %98.45 oranında nükleotit benzerliği gösterdiği ortaya çıkarılmıştır. Gerçekleştirilen litaratür tarama çalışmalarına göre, bu çalışma Türkiye'nin Van ilinde yetiştirilen yonca bitkilerindeki Yonca mozaik virüsü (AMV)' nün ilk raporu ve moleküler analizidir.

Phytoplasma-type symptoms were noted in tomato (Lycopersicum esculentum L.) in Bingöl province of Turkey. The remarkable symptoms include witch's broom, rosetting, purple curled and crispy leaves, small leaves in the upper branches and excessively elongated calyx. Genomic DNA isolation was performed to identify possible pathogens from the leaves of 11 plants with and without symptom. In the Nested-PCR test performed using universal primer sets, DNA bands of approximately 1200 bp size were obtained in 4 of 11 samples. Randomly chosen two DNA bands primed R16F2n/R16R2 were cloned into an appropriate plasmid vector to further characterizations. The recombinant plasmid DNAs purified were sequenced in both directions. Molecular assays of the 16S rRNA sequence confirmed the existence of the "Canditatus Phytoplasma solani" (16SrXII-A group) (similarity coefficient 1.00) (Accession no: MT279680) and the "Canditatus Phytoplasma trifolii" (16SrVI-A group) (similarity coefficient 1.00) (Accession no: MT279852) in the infected tomato samples. The isolates associated with tomato-phytoplasma were named as 'Bingöl D11' and 'Bingöl D90' isolates, respectively. The phylogenetic dendrogram created also confirmed where both pathogens belong. This current paper is documented in the first record of "Ca. P. solani" (16SrXII-A) and "Ca. P. trifolii" (16SrVI-A) in naturally diseased tomato in Bingöl of Turkey.

Alfalfa mosaic virus (AMV) is one of the most important viral diseases of alfalfa plant among the forage crop, causing significant annual economic losses. The agent is also of potential importance to other cultivars such as tomatoes, potatoes, and peppers in most cases. The identification and phylogenetic relationships of AMV were carried out by reverse-transcription polymerase chain reaction (RT-PCR), following by bacterial cloning. The cDNA of alfalfa samples (12) were subjected to RT-PCR tests using primer pairs, specific for the capsid protein gene (CP) of AMV, resulting in a DNA fragment of approximately 700 bp as expected. The amplicons were directly cloned and then resulting sequences were deposited in GenBank (Acc. No: MW962977, MW962976). The BLASTn analysis of both sequences demonstrated that AMV virus isolates from alfalfa were highly similar to other AMV isolates from various crops in the world, with nucleotide identity ranging from 97 to 99.37%. The results of phylogenetic dendrogram based on CP gene sequences clearly suggested that our isolates are closely related to four AMV isolates from alfalfa in Turkey. To our knowledge, this study is the first report of molecular phylogeny and AMV presence in alfalfa exhibiting yellowing, mottling, and leaves abnormalities in Bingöl province, Turkey.

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

The natural occurrence of 'Candidatus Phytoplasma trifolii' in pear trees (Pyrus communis Linnaeus) is reported here for the first time. In 2017, a total of thirty-five pear trees, two of them exhibiting leaf rolling along the midvein, reddening, bushy appearance, and upright growth symptoms were sampled in different locations in Van province, Turkey. The total deoxyribonucleic acid was extracted from symptomatic and asymptomatic plants. The purified DNA served as a template in nested polymerase chain reaction (nested-PCR) assays, performed to amplify 16S rRNA sequences using universal primer pairs (R16mF2/R16mR1 and R16F2n/R16R2). The resulting PCR products were then cloned into a pGEM T-Easy vector and sequenced bidirectionally. The phytoplasma strain, group, and subgroup identity were determined using the in silico restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal RNA-encoding gene sequences profiling with seventeen distinct restriction enzymes. Of the thirty-five pear samples, only two yielded 1 256 bp and 1 258 bp DNA fragments and were designated as Van-Pr3 (Acc. No. MH709141) and Van-Pr4 (Acc. No. MH730561), respectively. Based on the in silico virtual RFLP pattern analysis of the 16S rRNA sequences, we confirmed the presence of 'Ca. P. trifolii' belonging to the clover proliferation group and both identified phytoplasmas were identical with the similarity coefficient of 1.00 to the reference pattern of 16Sr group VI, subgroup A (Acc. No. AY390261). Here we report that the pear tree is an alternate host of the 'Ca. P. trifolii'.

In the surveys conducted in the province of Bingöl in 2019, melon plants exhibiting foliar deformations, mosaic pattern in different concentrations of green, and vein banding were noticed. Melon specimens were gathered and screened by RT-PCR using capsid protein (CP) gene-specific primer sets to characterize and to ascertain the possible viral agents related to infected plants. About 657 bp and 822 bp DNA fragments were observed in the agarose gel of infected plants, confirming the presence of Cucumber mosaic cucumovirus (CMV) and Watermelon mosaic potyvirus (WMV). Two related DNA fragments from each recovered from agarose gel randomly were cloned in the proper cloning vector and sequenced by nextgeneration sequencing (NGS). Viral CP sequences obtained were deposited in GenBank (NCBI) with accession number MT361015 and MT361016 for CMV and with accession number MT413451 and 437295 for WMV. Sequences analysis revealed that CMV and WMV isolates showed high sequence consensus with their same species, 99.84%, and 99.88%, respectively. Further analysis disclosed that both Bingöl isolates showed the highest sequence similarity with China isolate (DQ399708). The consensus tree created from various CP sequences in different geographies clearly revealed that the two CMV isolates detected in this study are Subgroup IB, in the very close phylogenetic relations with Turkey, Thailand, India, and China's Subgroup IB isolates from diverse plant origins. Moreover, Bingöl WMV isolates exhibited an evolutionary affinity with isolates from melon and zucchini in France and isolate from a watermelon in China. This work is the first scientific evidence showing infection of CMV and WMV of melon plants in Bingöl province of Turkey, supported by high sequence homology and consensus trees.