Recovery phenomena in apricot trees cv. Bergeron infected by European Stone Fruit Yellows in the province of Trento (Italy)

Recovery phenomena, have been intensively studied for apple proliferation and grapevine yellows. Several recent findings in apricot orchards in Friuli-Venezia Giulia indicate the importance of this strategy also for the control of European Stone Fruit Yellows (ESFY) infections, caused by \u201cCandidatus Phytoplasma prunorum\u201d. The aim of this work was to investigate the occurrence of recovery in 2 experimental orchards of apricot cv. Bergeron on \u201cWavit\u201d, located in the province of Trento, where the disease has been constantly spreading since 2000. This included visual inspections for typical symptoms and a diagnostic method based on Spot Real-Time Reverse Transcription PCR assay. The results obtained showed that three different groups of plants infected by ESFY were present: many plants with typical symptoms, some recovered and some symptomless infected plants. To date no interesting plant genotypes showing resistance to ESFY are available, moreover most of the insecticide applications have no efficacy in controlling the vector than the disease. Both use of pathogen-free propagation material and resort to natural recovery could be adopted as an integrated approach for the control of this phytoplasma disease. For those reasons the research will continue with the aim to quantify the concentration of the pathogen in the recovered plants

A multiplex Real Time Taqman RT-PCR assay for the detection of grapevine flavescence doree and bois noir phytoplasmas using crude sap extracts.

Flavescence dor\ue9e (FD) and Bois noir (BN) are the most important and spread grapevine yellows diseases in Europe and are caused by phytoplasmas of the 16SrV and 16SrXII-A groups, respectively. Detection of BN and FD phytoplasmas is particularly difficult, as they are generally present in low concentrations and unevenly distributed in the grapevine phloem. Many efforts recently focused on developing of a rapid and sensitive method for phytoplasma detection. In this study a triplex real time TaqMan RT-PCR methodology has been set up to simultaneously, but specifically, amplify a 16SrDNA gene fragment of FD and BN phytoplasmas and a 18SrDNA gene region of the host plant using a crude sap extract as template. The use of RNA as template, instead of DNA, takes advantage from the high ribosomal-RNA copy number present in pathogen cells and provides an evidence of viability and metabolic activity of phytoplasma, which is not provided by DNA-based detection. During our study an efficient and rapid method for GY phytoplasmas detection in different host plants has been obtained. Absence of nucleic acid extraction step allows to greatly decrease both costs and time necessary to perform the assay. Therefore the method presented is a rapid, specific and sensitive alternative to conventional nested-PCR. We successfully applied this technique to the detection of BN and FD phytoplasmas on more than three hundred samples of grapevine, strawberry, tomato, wild host plants and vector insects

Evaluation of Plum Pox Virus sensibility on different stone fruit varieties in Emilia-Romagna region (Italy)

Between 2003 and 2011, about 200 stone fruit varieties were inoculated by chip budding with PPV M strain, grown in a screen house and then surveyed for symptom expression on leaves, flowers and fruits. During the following season in particular 126 peach and nectarine, 45 apricot and 28 plum varieties and seedlings (from breeding programs) under evaluation were assessed in the present work. A large number of varieties showed discoloration and mottling on the young leaves in spring; furthermore all peach and nectarine selections with rosaceous flowers appeared infected by PPV with strong symptoms on petals. Many peach and apricot cultivars and seedlings, but only one plum variety, presented fruit deformations with typical rings and mottling. Only a few accessions (2 peaches and 10 apricots) belonging to \u201cancient\u201d germplasm or to seedlings did not appear affected by typical symptoms on leaves and fruits, nor were positive to PPV by ELISA and Real Time RT-PCR assays. These experimental data revealed high sensibility of stone fruit germplasm to PPV-M as confirmed by the information gathered in the Emilia-Romagna region, by inspectors of the local Plant Protection Service. In particular during 15 years of orchards surveys 36 varieties of apricot, 37 of plum, 102 of nectarine and 83 of peach (both traditional and promising varieties) were infected, showing typical symptoms, as a result of the natural PPV spread in the field

A multiplex RT-PCR assay capable of distinguishing beet necrotic yellow vein virus type A and B

Primers were developed which could distinguish the A and B types of Beet necrotic yellow vein virus (BNYVV) using a multiplex reverse-transcription polymerase chain reaction (mRT-PCR). RNA was extracted from 72 BNYVV isolates from Asia, Europe and North America, and the type of each isolate determined using an established single strand conformation polymorphisms (SSCP) detection method. An area of the ‘triple gene block’ region on RNA 2 was amplified and sequenced from 16 isolates of the A and B types. These sequences were aligned and two sets of PCR primers were designed to amplify unique areas common to each type. One assay produced a single 324 base-pair RT-PCR fragment from samples containing the A type and the other produced a 178 base-pair product from samples containing the B type. Fragment length differed sufficiently to allow both assays to be run in a single PCR tube. Results obtained using the new multiplex RT-PCR assay were consistent with those from the established SSCP method for all 72 reference samples

New insights into kiwifruits-infecting viruses in Italy

In Italy, for protecting kiwifruit (Actinidia spp.) production by restraining the spread of new and harmful diseases on both nurseries and orchards, a specific Decree has been issued by the Italian Ministry for Agriculture, Food and Forest Policies (Decree 7th February 2011 \u2013 published in the Official Journal of the Italian Republic n. 69, 25th March 2011). According to this Decree, primary source material must be free, at least, from 6 different virus species: Actinidia virus A (AVA), Alfa alfa mosaic virus (AMV), Apple stem grooving virus (ASGV), Citrus leaf blotch virus (CLBV), Cucumber mosaic virus (CMV), Ribgrass mosaic virus (RMV), and also from the Stolbur phytoplasma (Candidatus Phytoplasma solanii). Deeper information on biological and molecular proprieties of kiwifruit viruses is therefore necessary to ensure the development of diagnostic tools for a rapid, sensitive and reliable analysis of kiwifruit vegetative propagation material. A CMV isolate (K35) has been recently identified in kiwifruit plants collected in the Emilia Romagna (northern Italy). A deep molecular characterization of this isolate has been developed in order to establish the virus origin. Moreover agroinfectious K35 clones have been produced with the purpose to infect new kiwifruit plants on which to perform studies on natural transmission of the virus and evaluate performances of developed detection methodologies. New viral isolates have been identified during survey carried out in 2011. Molecular characterization is in progress, but these new reports suggest that viral infections occur more extensively than expected on Italian kiwifruit nurseries and orchards

Virus infecting kiwifruit in Italy

Kiwifruit (Actinidia spp.) is an important horticultural crop grown in temperate region. In Italy and in other EU countries, kiwifruit has been considered as a relatively disease-free ornamental crop and consequently neither passaport regulation nor certification system for this species are been developed: no additional declaration for imported material must be provided and any specific rules for plant propagation into the EU need to be observed. In recent years a number of fungal and bacterial diseases have been recorded, including Botrytis cinerea and Pseudomonas syringae pv. actinidiae. Moreover a new strain of apple stem grooving virus has been identified during post entry quarantine in New Zealand in A. chinensis from China. More recent work has demonstrated that Actinidia spp. can be infected by a range of viruses belonging to the genera Tobamovirus, Potexvirus, Vitivirus and also by Alfa alfa mosaic virus, Cucumber mosaic virus and Citrus leaf blotch-like virus. During spring 2010 kiwifruit plants showing a range of symptoms, including vein clearing, interveinal mottling, mosaics and chlorotic ring spots were collected from both nurseries and orchards in Italy. Transmission electron microscopy of sap or partially purified extracts from kiwifruit plants detected both rod-shaped and flexuous particles. Viruses were tentatively transmitted by mechanical inoculation to several herbaceous indicator plants, including Nicothiana benthamiana, N. occidentalis and Chenopodium quinoa. Molecular charcterization is currently in progress to identify the viruses