Kadriye Çağlayan

Genetic Variation and Possible Mechanisms Driving the Evolution of Worldwide Fig mosaic virus Isolates

Fig mosaic virus (FMV) is a multipartite negative-sense RNA virus infecting fig trees worldwide. FMV is transmitted by vegetative propagation and grafting of plant materials, and by the eriophyid mite Aceria ficus. In this work, the genetic variation and evolutionary mechanisms shaping FMV populations were characterized. Nucleotide sequences from four genomic regions (each within the genomic RNAs 1, 2, 3, and 4) from FMV isolates from different countries were determined and analyzed. FMV genetic variation was low, as is seen for many other plant viruses. Phylogenetic analysis showed some geographically distant FMV isolates which clustered together, suggesting long-distance migration. The extent of migration was limited, although varied, between countries, such that FMV populations of different countries were genetically differentiated. Analysis using several recombination algorithms suggests that genomes of some FMV isolates originated by reassortment of genomic RNAs from different genetically similar isolates. Comparison between nonsynonymous and synonymous substitutions showed selection acting on some amino acids; however, most evolved neutrally. This and neutrality tests together with the limited gene flow suggest that genetic drift plays an important role in shaping FMV populations.

Endophytic bacterial community living in roots of healthy and ‘Candidatus Phytoplasma mali’-infected apple (Malus domestica, Borkh.) trees

Abstract‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and ‘Ca. Phytoplasma mali’-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g. Bacillus, Pseudomonas, and Burkholderia) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control ‘Ca. Phytoplasma mali’ in order to develop sustainable approaches for managing AP.

Comparison by Sequence-Based and Electron Microscopic Analyses of Fig mosaic virus Isolates Obtained from Field and Experimentally Inoculated Fig Plants

Fig mosaic disease (FMD) and the fig mite, Aceria ficus, are widespread in different fig growing provinces of Turkey. Fig trees (Ficus carica) cv. Bursa siyahı (D1) and an unknown seedling (D2) that showed typical FMD symptoms and was heavily infested by fig mites were used as donor plants for attempted mite transmissions to healthy fig seedlings. Transmission electron microscopy observations of donor plant samples prior to the transmission tests were performed and showed the presence of double membrane bodies (DMBs) in the palisade mesophyll cells. Electron microscopy of all experimentally inoculated fig seedlings showed the same bodies. This result reinforced the suggestion that an agent that elicits the production of DMBs in infected cells is involved in the etiology of FMD. Double-stranded (ds)RNA analyses were also performed from experimentally inoculated plants, and dsRNAs with sizes approximately 1.30 and 1.96 kb were obtained. Reverse transcription-polymerase chain reaction (RT-PCR) products of 468 and 298 bp specific to Fig mosaic virus (FMV) were amplified from both donor and experimentally inoculated plants. BLAST analyses of nucleotide sequences of these fragments showed 90% identity with FMV for the donor plant and 94 to 96% for experimentally inoculated plants. According to these results, FMV is present in both donor and experimentally inoculated plants in Turkey, and this virus is transmissible by A. ficus from fig plant to fig plant.

DETECTION OF FIG MOSAIC VIRUS IN VIRULIFEROUS ERIOPHYID MITE ACERIA FICUS

Fig leaves showing typical fig mosaic symptoms on cv. Bursa siyahi (donor plant) were cut under a stereo microscope into small pieces each hosting 10 putatively viruliferous eriophyid mites (Aceria ficus Cotte) and placed directly on the top leaves of healthy Cucumis sativus, Chenopodium quinoa. C. amaranticolor, Nicotiana occidentalis, Catharanthus roseus, Fraxinus excelsior plants, and fig seedlings. Donor and test plants were analyzed by electron microscopy, RT-PCR and sequencing, whereas the mites (ErMs) underwent molecular assays using Fig mosaic virus (FMV)-specific primers. Mite-infested leaves of fig seedlings and C. roseus showed small yellowish spots after 10 days and 6 weeks, respectively, whereas no symptoms were observed in other test or control plants for three months. Electron microscopy observations showed the occurrence of double membrane bodies (DMBs) in the palisade cells of donor and mite-inoculated fig plants, but not in C. roseus. However, 302 bp RT-PCR products specific to FMV were amplified from donor and inoculated figs, C. roseus and ErMs. Nucleotide identity with the sequence of the FMV isolate in GenBank (accession No. AM941711.6) was 87%, 89% and 87% for donor plant (JQ708183), inoculated fig seedlings (JQ708184) and C. roseus (JQ408437, JQ408438), respectively. The sequences obtained from ErMs (JQ408432, JQ408436) showed 87% and 88% nucleotide identity with the reference FMV isolate, respectively. When dsRNA extracts were analyzed to confirm virus presence in inoculated periwinkles, a complex dsRNA profile was obtained, suggestive of infection by a multipartite virus or by multiple viruses. Sequence from RT-PCR amplicons of dsRNA (JX040436) showed 88% identity with those the reference FMV isolate (AM941716.1) and the donor plant (JQ708183). According to these results, Madagascar periwinkle (C. roseus) can be retained as a new experimental host for FMV and A. ficus appears to be able to transmit FMV from fig to periwinkle plants.

First report on hop stunt viroid (HSVd) from some Mediterranean countries.

Hop stunt viroid (HSVd) has a very wide host range including most stone fruit trees. Among them, apricot is one of the most important host crops in the Mediterranean basin. In this study non-isotopic molecular hybridisation revealed, for the first time, the presence of HSVd on apricot in four Mediterranean countries (Cyprus, Greece, Morocco and Turkey). The results obtained by this technique were confirmed by northern-blot and RT-PCR analyses. The data presented in this work indicate a wider geographical distribution of this viroid than hitherto known and emphasise the need for this kind of study as part of the control effort.

Potential vectors of Plum pox virus in the Eastern Mediterranean Region of Turkey

Although Plum pox virus (PPV) was first detected in Turkey 44 years ago, the virus is present in a rather limited number of trees. Our recent studies on PPV incidence showed that PPV was introduced rapidly in PPV-free regions and that there are no data available about the role of aphid species and Prunus rootstocks on these new infections. In this study the epide- miological aspect of PPV was studied in Antakya-Hatay, located in the Eastern Mediterranean region of Turkey where PPV was first detected in 2011. The susceptibility of different Prunus rootstocks to PPV was evaluated in an established experimental plot next to a PPV-infected nectarine orchard. Aphid populations were monitored in 2011 and 2012 from the last week of April to the middle of June by the sticky-plant method in both the experimental plot (EP) and the surrounding infected nectarine orchard (SNO). Regularly collected plant samples and aphids were individually tested by DASI-ELISA and squash real-time RT-PCR, respectively. The highest aphid population densities were observed at the end of May in both years. The most abundant aphid species were Aphis gossypii and A. spiraecola both in EP and SNO in both years. The percentage of PPV-viruliferous Myzus persicae, A. fabae, A. gosypii, A. spiraecola, Hyalopterus pruni, Macrosiphon euphorbiae and A. craccivora as estimated by squash real- time RT-PCR were 39.47%, 25.00%, 24.56%, 22.60%, 22.22%, 20.00% and 8.00%, respec- tively. The percentages of viruliferous aphids collected from SNO were 12.5% in A. spiraecola, 12.42% in A. gossypii and 11.11% in H. pruni. At the end of 2012, three Myrobolan 29C and two Adesoto 101 rootstocks were found infected by PPV. Molecular characterization studies showed that PPV-M was the strain present in both the originally infected nectarine plot and the Myrobolan 29C rootstocks.

EVALUATION OF THE SUSCEPTIBILITY OF DIFFERENT PRUNUS ROOTSTOCKS TO NATURAL INFECTION OF PLUM POX VIRUS-T

SUMMARY Plum pox virus (PPV) has been observed in Turkey since 1968, but was not widespread except in apricot and plum trees in home gardens and ornamental parks in restricted areas. Susceptibility of six different Prunus rootstocks to strain PPV-T was assessed under natural inoculum pressure in the Izmir-Aegean region during 2010-2011. Aphid populations were monitored from the first week of April to the middle of June by the stickyplant method one year after the rootstock plantation was established. Aphids collected from different rootstocks were tested individually by squash real-time RT-PCR and all rootstocks were regularly tested by DASI-ELISA. The largest aphid populations were observed at the end of May and the most abundant aphid species as averages over the two years were Myzus persicae (20.15%), Hyalopterus pruni (18.64%), Aphis craccivora (9.04%) and Aphis gossypii (8.36%). In 2011, the highest percentage of viruliferous aphids was found in M. persicae (34.78%), followed by H. pruni (32.50%), Macrosiphum euphorbiae (25.00%), A. gossypii (23.80%), A. spiraecola (12.50%) and A. craccivora (10.00%). Of the six Prunus rootstocks tested, only Nemaguard and Myrobalan 29C were infected by PPV-T, infection rate in 2010 being 6.0% (Nemaguard) and 4.0% (Myrobalan 29C). The infection rate increased to 16.0% for Nemaguard and 14.0% for Myrobalan 29C in 2011. However, the other rootstocks, Prunus marianna GF8.1, Docera6, GF677 and Garnem tested negative for PPV-T throughout 2011. PPV isolates obtained from naturally infected apricot trees (inoculum source) and from infected rootstocks in the experimental plot were characterized as PPV-T and had more than 99.5% nucleotide sequence identity.

FIRST REPORT OF ‘CANDIDATUS PHYTOPLASMA ASTERIS’ (GROUP 16SrI-B) INFECTING SWEET CHERRIES IN TURKEY

Five-year-old sweet cherry (Prunus avium L.) trees, ex- hibiting symptoms typical of phytoplasma diseases were observed in the Turkish province of Usak during 2011. The percentage of symptomatic plants, scattered in the orchards, was nearly 40%. Samples were collected during late spring and early summer from trees showing proliferation of branch- es, off season flowering and decline. In order to establish phytoplasma association with these symptoms, nucleic acid was extracted from leaf midribs of 10 symptomatic and five symptomless plants. Nested PCR assays using universal phy- toplasma primers P1/P7 followed by R16F2n/R2 and by 16R758f/16R1232r (Duduk et al., 2013) provided positive responses for seven of the symptomatic samples. Restriction fragment length polymorphism (RFLP) analysis was per- formed on PCR products using restriction enzymes Tsp509I, Tru1I and AluI. Preliminary RFLP identification was con- firmed by nested PCR assays with primers R16(I)F1/R1 (Lee et al., 1994) followed by RFLP analysis, that allowed phyto- plasma classification in the 16SrI-B subgroup. Since all am- plicons showed identical restriction profile, according to the enzymes and primers employed, one of them was sequenced in both directions using primers R16(I)F1 and R16(I)R1. The 1,006 nucleotide long sequence, deposited in GenBank under the accession No. KF476062, showed 99.0% identity with 16S rDNA from several phytoplasmas related to ‘Candidatus Phytoplasma asteris’, including strains associated with cherry little leaf (GenBank AY034089) and cherry bunchy leaf (Gen- Bank HM067754), that are affiliated to a different 16SrI sub- group (Jomantiene et al., 2011). This is the first report of ‘Ca. P. asteris’ infecting sweet cherries in Turkey.

Phylogenetic analysis of partial sequences from Fig mosaic virus isolates in Turkey

Fig mosaic virus (FMV), which was described recently, is the only characterized causal agent of fig mosaic disease (FMD). It has six RNA segments and belongs to the Bunyaviridae family. In order to determine the genetic diversity of Turkish FMV isolates, the most common fig cultivars showing FMD symptoms were collected from different fig-growing provinces of Turkey. Nucleoprotein (Np) and Glycoprotein (Gp) gene-specific primers of FMV were used for RT-PCR analysis. According to RT-PCR results, 71 of 90 samples from 20 different cultivars and unknown fig seedlings were found to be infected by FMV. Among them, 41 isolates were sequenced and subjected to phylogenetic analyses based on the partial Gp and Np sequences at the amino acid level by the neighbor-joining method. The isolates showed more than 80% identity with reference FMV isolates (Acc. nos. FM991954.1 and FM864225.2). Based on phylogenetic analysis, the sequences clustered into two main groups for Np and Gp regions. Significant relationships between FMV isolates based on geographic origin and cultivars were not observed.

Geographical Distribution of Viroids in Africa and the Middle East

This chapter describes major viroids and viroid diseases reported from at least 22 countries in Africa and the Middle East.