Identification of Ilarviruses in almond and cherry fruit trees using nested PCR assays

In this study nested PCR assays have been developed for the detection of Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV) modifying a previously reported assay for the generic detection of ilarviruses. In all cases one generic upstream primer was used along with a virus-specific downstream primer in respective nested PCR assays. The application of the same thermocycling profile allowed all amplifications to run in parallel. Ilarvirus isolates from different hosts were used for the evaluation of the detection range of the assays, which were afterwards applied for screening almond and cherry plant material. In almond trees the incidence of PNRSV and PDV was 41% and 21.5%, respectively. In cherry orchards the opposite was observed with PDV (56.6%) being the prevalent virus followed by PNRSV (19.4%). Mixed infections with both viruses were also encountered in approximately 10 and 17% of cherry and almond trees, respectively. ApMV was not detected in any of the samples tested. This is the first extensive survey conducted in Greece in order to monitor the distribution of these viruses using molecular assays. Keywords: Prune dwarf virus, Prunus necrotic ringspot virus, Apple mosaic virus, cherry, almond, nested PC

Molecular characterization of the coat protein gene of prunus necrotic ringspot virus infecting peach in Montenegro

In 2011 and 2012, two major peach-growing areas in Montenegro were surveyed for the occurrence of eleven peach viruses. Samples collected from 105 peach and nectarine trees were tested by Reverse transcription-polymerase chain reaction (RT-PCR) using sets of primers specific for the detection of Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Cherry mottle leaf virus (CMLV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Peach mosaic virus (PMV), Peach rosette mosaic virus (PRMV), Tobacco ringspot virus (TRSV) and Tomato ringspot virus (ToRSV). The occurrence of Strawberry latent ringspot virus (SLRSV) was examined using nested RT-PCR, while a Real-time quantitative RT-PCR method was applied for detection of Plum pox virus (PPV). PPV, PNRSV and PDV were detected in 42.9, 24.8 and 0.9 % of samples. Mixed viral infections were recorded in 6.7 % of samples, while results showed the absence of ACLSV, ApMV, PMV, CMLV, SLRSV, PRMV, TRSV and ToRSV from all assayed samples. In particular, the PCR products of complete coat protein (CP) gene was cloned and sequenced from fourteen PNRSV isolates. A high identity (87.5–100 %) was found between CP nucleotide sequences of Montenegrin isolates and other PNRSV isolates from different hosts and geographic regions retrieved from GenBank. CP nucleotide sequence comparison, as well as phylogenetic reconstruction confirmed the clustering of the isolates into three molecular groups (PV-96, PV-32 and PE-5), whereby Montenegrin isolates were separated into two phylogroups (PV-96 and PE-5). Comparative sequence and phylogenetic analysis showed that clustering of various isolates was not associated with geographic and host origin

Genome Modification Approaches to Improve Performance, Quality, and Stress Tolerance of Important Mediterranean Fruit Species (Olea europaea L., Vitis vinifera L., and Quercus suber L.).

In the last decades the interest on traditional Mediterranean fruits highly increased, not only due to the constant demand of consumers for new crop alternatives, but also due to the identification in such species of molecules with important properties for human health (e.g. resveratrol from grapes and oleuropein from olives). Efforts to improve the production capacity and fruit quality in such fruit species, as well as the resistance to biotic and abiotic stress, was achieved by plant breeders using mainly classical breeding approaches (e.g. selection, hybridization and mutagenesis), nevertheless, breeding support by plant tissue culture techniques, marker assisted selection, as well as by genome modification, was also used. Here we will present the state of the art related with the production of transgenic plants in three Mediterranean fruit species with important impact on the economy, olive, grapevine and cork. The achievements, problems and future perspectives will be discussed.This work was financially supported by national funds through FCT (Foundation for Science and Technology) under the Project UID/AGR/00115/2013, PTDC/BIA-BQM/28539/2017, by the Project OLEAVALOR (ALT20-03-0145-FEDER000014) funded by FEDER funds through the Program Alentejo 2020; Hélia Cardoso and Susana Serrazina were supported by FCT through post-doc fellowship SFRH/BPD/109849/2015 and SFRH/BPD/108653/2015, respectively; Andreia Figueiredo was also supported by the investigator FCT program IF/00819/2015