Transient silencing of the grapevine gene VvPGIP1 by agroinfiltration with a construct for RNA interference.

Grapevine is an economically important crop, and the recent completion of its genome makes it possible to study the function of specific genes through reverse genetics. However, the analysis of gene function by RNA interference (RNAi) in grapevine is difficult, because the generation of stable transgenic plants has low efficiency and is time consuming. Recently, transient expression of genes in grapevine leaves has been obtained by Agrobacterium tumefaciens infiltration (agroinfiltration). We therefore tested the possibility to silence grapevine genes by agroinfiltration of RNAi constructs. A construct to express a double strand RNA (dsRNA) corresponding to the defense-related gene VvPGIP1, encoding a polygalacturonase-inhibiting protein (PGIP), was obtained and transiently expressed by agroinfiltration in leaves of grapevine plants grown in vitro. Expression of VvPGIP1 and accumulation of PGIP activity were strongly induced by infiltration with control bacteria, but not with bacteria carrying the dsRNA construct, indicating that the gene was efficiently silenced. In contrast, expression of another defense-related gene, VST1, encoding a stilbene synthase, was unaffected by the dsRNA construct. We have therefore demonstrated the possibility of transient down-regulation of grapevine genes by agroinfiltration of constructs for the expression of dsRNA. This system can be employed to evaluate the effectiveness of constructs that can be subsequently used to generate stable RNAi transgenic plants.

The effects of fungicides on non-target mites can be mediated by plant pathogens.

Field tests are useful for the evaluation of the pesticides' impact in realistic use situations. However, the distinction between the direct and indirect effects of a pesticide is not always possible in field, with consequences on the conclusions about pesticides toxicity. Generalist predatory mites belonging to the Phytoseiidae family are widely considered as non-target organisms in pesticide side-effect evaluations. Plant pathogens of several cultivated plants can be of importance as food resources for various phytoseiids. Pesticides with fungicidal activity may have a direct impact on phytoseiids, but they can also have an indirect effect reducing food availability for predatory mites. Here, we present the results of field experiments performed on grapevine, where we investigate whether the availability of the plant pathogen grape downy mildew has an effect on fungicides impact on the predatory mites Typhlodromus pyri Scheuten and Amblyseius andersoni (Chant). In these experiments we used fungicides characterized by differential selectivity to predatory mites in laboratory: copper compounds, folpet, and mancozeb. Results indicated that the abundance of predatory mites was associated with the plant pathogen foliar symptoms presence. The presence of predatory mites was different among treatments in response of the toxicological traits of a pesticide (direct effects), but also as consequences of differential plant pathogen availability induced by fungicide applications (indirect effects). During the investigation, the variable plant pathogen spread on untreated control determined contrasting results on pesticides effect. We segregated the direct effects from the indirect food resource-mediated effects including a non-toxic reference in the experimental protocols.

Occurrence of black aspergilli and ochratoxin A on grapes in Italy.

Ochratoxin A (OTA) in wine is linked to contamination by several Aspergillus species. In 2003-2007, grape samples collected in Italy were surveyed for the presence of OTA and OTA-producing fungi. A. niger aggregate was the prevalent species. A. carbonarius, which is considered the main source of OTA in grapes, was mostly found in Southern Italy. The year and the environment had an important influence on the development of the black Aspergillus populations. Testing with ELISA showed OTA to be present in about 30% of the samples. Samples from Southern Italy showed the highest occurrence (45%) and also the highest OTA concentration, sometimes higher than 2 µg/L. The values decreased progressively the further North the samples were taken.

A new TaqMan method for the identification of phytoplasmas associated with grapevine yellows by real-time PCR assay.

Three real-time PCR systems for direct detection of phytoplasmas associated to Flavescence dorée (FD), Bois noir (BN) and aster yellows (AY) diseases were developed. TaqMan probes and primers were designed on the 16S ribosomal RNA sequences of phytoplasma genome. A further TaqMan assay, targeting a grapevine gene encoding for the chloroplast chaperonin 21, was developed in order to check the DNA quality and to verify the absence of PCR inhibition. A comparison between real-time PCR and conventional nested-PCR methods for phytoplasma detection was carried out on several reference samples from grapevine, periwinkle, other host plants and insect species. Detection of FD, BN and AY phytoplasma DNA on infected specimens was rapid, specific and reproducible. Sensitivity was as high as nested-PCR assay. The two procedures were then used on about 450 samples collected from grapevines showing yellows symptoms. The results showed that real-time PCR approach for phytodiagnostic purposes was more advantageous than nested-PCR method with regard to rapidity of the assay and reduced risk of sample cross contamination. These new protocols represent an improvement of existing analytical methods and could be used as a reliable diagnostic procedure in certification and control programs.

On the role of H2O2 in the recovery of grapevine (Vitis vinifera cv. Prosecco) from Flavescence dorée disease.

In the present work, we compared hydrogen peroxide (H2O2) localisation and the activities/contents of antioxidant enzymes and metabolites in the leaf tissues of grapevine (Vitis vinifera L. cv. Prosecco) plants showing different sanitary status, namely diseased by Flavescence dorée, healthy or recovered. Polymerase chain reaction analysis revealed that the pathogen associated with Flavescence dorée (proposed as 'Candidatus Phytoplasma vitis') was detected in the leaf tissues of symptomatic plants, but was not observed in either the healthy or recovered plants. Hydrogen peroxide accumulated in the phloem plasmalemma of recovered grapevine leaves, but was not detected in either healthy or diseased material. When compared to diseased or healthy plants, recovered plants had distinctly lower extractable levels of catalase and ascorbate peroxidase, two enzymes primarily involved in the scavenging of excess H2O2 generated in different cell compartments. Among healthy, diseased and recovered leaves there was no significant difference in the amount of 2-thiobarbituric acid-reactive substances, which are assumed to reflect the extent of peroxidative breakdown of membrane lipids. Therefore, it is suggested that recovery from Flavescence dorée disease in grapevine might be associated with a long-term, sustained and tissue-specific accumulation of H2O2 in leaves, which reduces numbers or prevents further infection by Flavescence dorée phytoplasma. Recovered grapevine plants might be able to achieve such H2O2 accumulation through a selective and presumably stable downregulation of enzymatic H2O2 scavengers, without altering the levels of other antioxidant systems and without incurring an increased oxidative risk.

From field to health: a simple way to increase the nutraceutical content of grape as shown by NO-dependent vascular relaxation.

Polyphenolic grapevine components involved in plant resistance against pathogens possess various pharmacological properties that include nitric oxide (NO)-dependent vasodilation and anti-inflammatory and free radical scavenging activities, which may explain the protective effect of moderate red wine consumption against cardiovascular disease. The aim of this work was (a) to verify the possibility that preharvest treatments of grapevine with a plant activator, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), could lead to an enriched nutraceutical potential of wine and (b) to characterize the profile of metabolites responsible for pharmacological activity. Plant spraying at the end of veraison, with a water suspension of BTH (0.3 mM), led to increased whole anthocyanin content as confirmed by HPLC comparative analysis. Extracts from berry skins of BTH-treated grapevines caused NO-dependent vasorelaxation, with a concentration-response curve that was significantly shifted to the left of the control non-BTH-treated curve. Moreover, 1:1000 dilutions of berry extracts from BTH-treated plants significantly increased basal production of guanosine 3',5'-cyclic monophosphate (cGMP) in human vascular endothelial cells when compared to the corresponding extracts of untreated plants. These results show that BTH treatment increases anthocyanin content of grape extracts, as well as their ability to induce NO-mediated vasoprotection. No increase of anthocyanin content was observed in the wine extracts from BTH-treated vines. It is concluded that BTH treatment could be exploited to increase the nutraceutical potential of grapes.

Induction of resistance to gray mold with benzothiadiazole modifies amino acid profile and increases proanthocyanidins in grape: primary versus secondary metabolism.

Field treatments of grapevine (cv. Merlot) with the plant activator benzothiadiazole (BTH, 0.3 mM) induced resistance against gray mold caused by Botrytis cinerea. Both incidence and severity of the disease were reduced. The resistance was associated with an increase of total polyphenols in berry skins, in particular, the proanthocyanidin fraction, that increased up to 36%. The amino acid profile of leaves was also modified by treatments, particularly lysine, that augmented 4-fold. Other amino acids involved in resistance mechanisms to either biotic or abiotic stress increased as well. These results indicate that BTH treatments can be used to control gray mold, thereby limiting an excessive use of fungicides, and could be exploited to increase the content of micronutrients of high nutritional value, arising from both primary and secondary metabolisms.

Benzothiadiazole enhances resveratrol and anthocyanin biosynthesis in grapevine, meanwhile improving resistance to Botrytis cinerea.

Pre-harvest multiple treatments of grapevine (cv. Merlot) with the plant activator benzothiadiazole (BTH, 0.3 mM) enhanced trans-resveratrol content in berries by about 40%. An even more striking effect was observed on anthocyanin synthesis, particularly on malvidine 3-glucoside, malvidine 3-(6-O-acetyl)glucoside and malvidine 3-(6-O-p-coumaroyl)glucoside, whose amounts were more than doubled. These data were obtained with a novel and time-saving HPLC method, set up for the simultaneous detection of stilbenes and anthocyanins, using an RF-10Axl fluorimetric detector instrument, with excitation at 330 nm and emission at 374 nm, and a SPD-Avp UV detector with absorption at 520 nm. Furthermore, BTH treatments induced systemic acquired resistance in grapevine, as assessed by inoculating clusters from treated and untreated plants with Botrytis cinerea. Disease severity, estimated according to the percentage of infected berries per cluster, was significantly reduced in grapes from BTH-treated plants. These results indicate that BTH treatments, besides improving the content of two important classes of nutraceuticals, with their well-known antioxidant, antitumoral, and phytoestrogenic activities, could be exploited in vineyard to protect grape against gray mould infection, thereby limiting an excessive use of fungicides