Quantitative real-time PCR and high-resolution melting (HRM) analysis for strain-specific monitoring of fluorescent pseudomonads used as biocontrol agents against soil-borne pathogens of food crops

Fluorescent pseudomonads colonizing roots of crop plants and producing antifungal metabolites are regarded as a reliable alternative to chemical fungicides against soil-borne phytopathogens. Key factors in successful pathogen control are presence and activity at the appropriate concentration, time, and place of biocontrol agents. Thus, quantification methods to monitor population dynamics are pivotal to the development of reliable application protocols. Real-time PCR is nowadays the most widespread cultureindependent technique for the detection and enumeration of different target sequences. Here, its implementation with high resolution melting analysis as a powerful tool to accurately discriminate microbial inoculants is discussed

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease. \ua9 2017 The Author(s

Agroinoculation of Grapevine Pinot Gris Virus in tobacco and grapevine provides insights on viral pathogenesis

The Grapevine Pinot Gris disease (GPG-d) is a novel disease characterized by symptoms such as leaf mottling and deformation, which has been recently reported in grapevines, and mostly in Pinot gris. Plants show obvious symptoms at the beginning of the growing season, while during summer symptom recovery frequently occurs, manifesting as symptomless leaves. A new Trichovirus, named Grapevine Pinot gris virus (GPGV), which belongs to the family Betaflexiviridae was found in association with infected plants. The detection of the virus in asymptomatic grapevines raised doubts about disease aetiology. Therefore, the primary target of this work was to set up a reliable system for the study of the disease in controlled conditions, avoiding interfering factor(s) that could affect symptom development. To this end, two clones of the virus, pRI::GPGV-vir and pRI::GPGV-lat, were generated from total RNA collected from one symptomatic and one asymptomatic Pinot gris grapevine, respectively. The clones, which encompassed the entire genome of the virus, were used in Agrobacterium-mediated inoculation of Vitis vinifera and Nicotiana benthamiana plants. All inoculated plants developed symptoms regardless of their inoculum source, demonstrating a correlation between the presence of GPGV and symptomatic manifestations. Four months post inoculum, the grapevines inoculated with the pRI::GPGV-lat clone developed asymptomatic leaves that were still positive to GPGV detection. Three to four weeks later (i.e. ca. 5 months post inoculum), the same phenomenon was observed in the grapevines inoculated with pRI::GPGV-vir. This observation perfectly matches symptom progression in infected field-grown grapevines, suggesting a possible role for plant antiviral mechanisms, such as RNA silencing, in the recovery process.</div

Dodder transmission of two different MLOs from plum trees affected by “leptonecrosis”

5nonenoneLoi N.; Carraro L.; Musetti R; Pertot I.; Osler R.Loi, Nazia; Carraro, L.; Musetti, Rita; Pertot, I.; Osler, R

COMPARISON BETWEEN ELISA AND NORTHERN DOT BLOT HYBRIDIZATION TECHNIQUES FOR BYDV DETECTION IN TRITICINAE SPECIES

5nonenoneLOI N; DE PACE C; OSLER R; CASA R; PORCEDDU ELoi, Nazia; DE PACE, C; Osler, R; Casa, R; Porceddu, E