We explored the idea of whether electropotential waves (EPWs) primarily act as vehicles for systemic spread of Ca²⁺ signals. EPW-associated Ca²⁺ influx may trigger generation and amplification of countless long-distance signals along the phloem pathway given the fact that gating of Ca²⁺-permeable channels is a universal response to biotic and abiotic challenges. Despite fundamental differences, both action and variation potentials are associated with a sudden Ca²⁺ influx. Both EPWs probably disperse in the lateral direction, which could be of essential functional significance. A vast set of Ca²⁺-permeable channels, some of which have been localized, is required for Ca²⁺-modulated events in sieve elements. There, Ca²⁺-permeable channels are clustered and create so-called Ca²⁺ hotspots, which play a pivotal role in sieve element occlusion. Occlusion mechanisms play a central part in the interaction between plants and phytopathogens (e.g. aphids or phytoplasmas) and in transient re-organization of the vascular symplasm. It is argued that Ca²⁺-triggered systemic signalling occurs in partly overlapping waves. The forefront of EPWs may be accompanied by a burst of free Ca²⁺ ions and Ca²⁺-binding proteins in the sieve tube sap, with a far-reaching impact on target cells. Lateral dispersion of EPWs may induce diverse Ca²⁺ influx and handling patterns (Ca²⁺ signatures) in various cell types lining the sieve tubes. As a result, a variety of cascades may trigger the fabrication of signals such as phytohormones, proteins, or RNA species released into the sap stream after product-related lag times. Moreover, transient reorganization of the vascular symplasm could modify cascades in disjunct vascular cells.

Engineered nanoscale materials (ENMs) are considered emerging contaminants since they are perceived as a potential threat to the environment and the human health. The reactions of living organisms when exposed to metal nanoparticles (NPs) or NPs of different size are not well known. Very few studies on NPs-plant interactions have been published, so far. For this reason there is also great concern regarding the potential NPs impact to food safety. Early genotoxic and phytotoxic effects of cerium oxide nanoparticles (nCeO2) and titanium dioxide nanoparticles (nTiO2) were investigated in seedlings of Hordeum vulgare L. Caryopses were exposed to an aqueous dispersion of nCeO2 and nTiO2 at respectively 0, 500, 1000 and 2000 mg l-1 for 7 days. Genotoxicity was studied by Randomly Amplified Polymorphism DNA (RAPDs) and mitotic index on root tip cells. Differences between treated and control plants were observed in RAPD banding patterns as well as at the chromosomal level with a reduction of cell divisions. At cellular level we monitored the oxidative stress of treated plants in terms of reactive oxygen species (ROS) generation and ATP content. Again nCeO2 influenced clearly these two physiological parameters, while nTiO2 were ineffective. In particular, the dose 500 mg l-1 showed the highest increase regarding both ROS generation and ATP content; the phenomenon were detectable, at different extent, both at root and shoot level. Total Ce and Ti concentration in seedlings was detected by ICP-OES. TEM EDSX microanalysis demonstrated the presence of aggregates of nCeO2 and nTiO2 within root cells of barley. nCeO2 induced modifications in the chromatin aggregation mode in the nuclei of both root and shoot cells.

Metallic nanoparticles (MeNPs) can be formed in living plants by reduction of the metal ions absorbed as soluble salts. It is very likely that plant metabolism has an important role in MeNP biosynthesis. The in vivo formation of silver nanoparticles (AgNPs) was observed in Brassica juncea, Festuca rubra and Medicago sativa. Plants were grown in Hoagland's solution for 30 days and then exposed for 24 h to a solution of 1,000 ppm AgNO3. In the leaf extracts of control plants, the concentrations of glucose, fructose, ascorbic acid, citric acid and total polyphenols were determined. Total Ag content in plant fractions was determined by inductively coupled plasma atomic emission spectroscopy. Despite the short exposure time, the Ag uptake and translocation to plant leaves was very high, reaching 6,156 and 2,459 mg kg−1 in B. juncea and F. rubra, respectively. Ultrastructural analysis was performed by transmission electron microscopy (TEM), and AgNPs were detected by TEM X-ray microanalysis. TEM images of plant fractions showed the in vivo formation of AgNPs in the roots, stems and leaves of the plants. In the roots, AgNPs were present in the cortical parenchymal cells, on the cell wall of the xylem vessels and in regions corresponding to the pits. In leaf tissues, AgNPs of different sizes and shapes were located close to the cell wall, as well as in the cytoplasm and within chloroplasts. AgNPs were not observed in the phloem of the three plant species. This is the first report of AgNP synthesis in living plants of F. rubra. The contents of reducing sugars and antioxidant compounds, proposed as being involved in the biosynthesis of AgNPs, were quite different between the species, thus suggesting that it is unlikely that a single substance is responsible for this process.

One hundred twenty-six endophytic microorganisms isolated from grapevine leaves showing anomalous symptoms of downy mildew were tested on grapevine leaf disks as biocontrol agents against Plasmopara viticola. Among the 126 microorganisms, only five fungal isolates completely inhibited the sporulation of P. viticola; all of them were identified as Alternaria alternata. Ultrastructural analyses were carried out by transmission electron microscopy to observe cellular interactions between P. viticola and A. alternata in the grapevine leaf tissue. Cytological observations indicated that, even without close contact with A. alternata, the P. viticola mycelium showed severe ultrastructural alterations, such as the presence of enlarged vacuoles or vacuoles containing electron-dense precipitates. Haustoria appeared necrotic and irregularly shaped or were enclosed in callose-like substances. Therefore, a toxic action of A. alternata against P. viticola was hypothesized. To examine the production of toxic low-molecular-weight metabolites by A. alternata, we analyzed the fungal liquid culture by thin layer chromatography and proton magnetic resonance spectroscopy. The main low-molecular-weight metabolites produced by the endophyte were three diketopiperazines: cyclo(l-phenylalanine-trans-4-hydroxy-l-proline), cyclo(l-leucine-trans-4-hydroxy-l-proline), and cyclo(l-alanine-trans-4-hydroxy-l-proline). When applied at different concentrations to both grapevine leaf disks and greenhouse plants, a mixture of the three diketopiperazines was very efficacious in limiting P. viticola sporulation.

Aureobasidium pullulans and Epicoccum nigrum are frequently reported as endophytes of various crops, including grapevine (Vitis vinifera). Because of their potential role as biological control agents against grapevine pathogens, we examined the occurrence of A. pullulans and E. nigrum in two grapevine varieties (Merlot and Prosecco) in Italian vineyards where spontaneous recovery from phytoplasma disease is recurrent. Species-specific primers for A. pullulans and two genetically distinct strains of E. nigrum were designed in variable regions of ITS1 and ITS2. Primer specificity was confirmed by polymerase chain reaction using purified DNA from other fungal endophytes that are usually encountered during isolation attempts from grapevine tissues and from several other strains of A. pullulans and E. nigrum isolated from other sources. In order to determine the occurrence of the two endophytes in grapevine plants, DNA was extracted from shoots of 44 grapevines collected in six vineyards from different localities of northeast Italy. Both endophytes were detected and their identity was confirmed by restriction fragment length polymorphism (RFLP) patterns obtained from reference strains. RFLP analyses confirmed the presence of two E. nigrum strains belonging to different RFLP groups in grapevine. The molecular methods described allowed a sensitive, specific, and reliable identification of the two endophytes in grapevine.