Ozone Treatment as an Approach to Induce Specialized Compounds in Melissa officinalis Plants.

Plants are constantly subjected to environmental changes that deeply affect their metabolism, leading to the inhibition or synthesis of "specialized" compounds, small organic molecules that play a fundamental role in adaptative responses. In this work, Melissa officinalis L. (an aromatic plant broadly cultivated due to the large amounts of secondary metabolites) plants were exposed to realistic ozone (O3) dosages (80 ppb, 5 h day-1) for 35 consecutive days with the aim to evaluate its potential use as elicitor of specialized metabolite production. Ozone induced stomatal dysfunction throughout the whole experiment, associated with a low photosynthetic performance, a decrease in the potential energy conversion activity of PSII, and an alteration in the total chlorophyll content (-35, -36, -10, and -17% as average compared to the controls, respectively). The production of hydrogen peroxide at 7 days from the beginning of exposure (+47%) resulted in lipid peroxidation and visible injuries. This result suggests metabolic disturbance within the cell and a concomitant alteration in cell homeostasis, probably due to a limited activation of antioxidative mechanisms. Moderate accumulated doses of O3 triggered the accumulation of hydroxycinnamic acids and the up-regulation of the genes encoding enzymes involved in rosmarinic acid, phenylpropanoid, and flavonoid biosynthesis. While high accumulated doses of O3 significantly enhanced the content of hydroxybenzoic acid and flavanone glycosides. Our study shows that the application of O3 at the investigated concentration for a limited period (such as two/three weeks) may become a useful tool to stimulate bioactive compounds production in M. officinalis.

Signalling responses in the bark and foliage of canker-susceptible and -resistant cypress clones inoculated with Seiridium cardinale.

The necrotrophic fungus Seiridium cardinale is the main responsible for Cypress Canker Disease (CCD), a pandemic affecting many Cupressaceae worldwide. The present study aims to elucidate the signalling of the early responses in the bark and foliage of CCD-susceptible and -resistant C. sempervirens clones to S. cardinale inoculation (SI and RI, respectively). In the bark of SI, a peaking production of ethylene (Et) and jasmonic acid (JA) occurred at 3 and 4 days post inoculation (dpi), respectively, suggesting an attempted plant response to the pathogen. A response that, however, was ineffective, as confirmed by the severe accumulation of malondialdehyde by-products at 13 dpi (i.e., lipid peroxidation). Differently, Et emission peaked in RI bark at 3 and 13 dpi, whereas abscisic acid (ABA) accumulated at 1, 4 and 13 dpi, resulting in a lower MDA accumulation (and unchanged levels of antioxidant capacity). In the foliage of SI, Et was produced at 1 and 9 dpi, whereas JA and salicylic acid (SA) accumulated at 1 and 3 dpi. Conversely, an increase of ABA and SA occurred at 1 dpi in the RI foliage. This outcome indicates that some of the observed metabolic alterations, mainly occurring as local defence mechanisms, might be able to gradually shift to a systemic resistance, although an accumulation of MDA was observed in both SI and RI foliage (but with an increased antioxidant capacity reported only in the resistant clone). We believe that the results reported here will be useful for the selection of clones able to limit the spread and damage of CCD.

Untangling the role of leaf age specific osmoprotectant and antioxidant responses of two poplar clones under increasing ozone concentrations.

Plants possess different degrees of tolerance to abiotic stress, which can mitigate the detrimental effect of environmental inputs affecting carbon balance. Less is known about the functions of osmoprotectants in scavenging of reactive oxygen species (ROS), generated at different sites depending on leaf age. This study aimed to clarify the osmotic adjustments adopted by old and young leaves of Oxford and I-214 poplar clones [differing in ozone (O3) sensitivity] to cope with three levels of O3 [ambient (AA), and two elevated O3 levels]. In both clones, the impact of intermediate O3 concentrations (1.5 × AA) on ROS production appeared to be leaf age-specific, given the accumulation of hydrogen peroxide (H2O2) observed only in old leaves of the Oxford plants and in young leaves of the I-214 ones (2- fold higher than AA and +79%, respectively). The induction of an oxidative burst was associated with membrane injury, indicating an inadequate response of the antioxidative systems [decrease of lutein and ß-carotene (-37 and -85% in the old leaves of the Oxford plants), accumulation of proline and tocopherols (+60 and +12% in the young leaves of the I-214 ones)]. Intermediate O3 concentrations reacted with unsaturated lipids of the plasma membrane in old and young leaves of the Oxford plants, leading to an increase of malondialdehyde by-products (more than 2- fold higher than AA), while no effect was recorded for I-214. The impact of the highest O3 concentrations (2.0 × AA) on ROS production did not appear clone-specific, which may react with cell wall components by leading to oxidative pressure. Outcomes demonstrated the ability of young leaves of I-214 plants in contain O3 phytotoxic effects.

Biocontrol Agents of Fusarium Head Blight in Wheat: A Meta-Analytic Approach to Elucidate Their Strengths and Weaknesses.

The use of biocontrol agents (BCAs) coping with fungal pathogens causing Fusarium head blight (FHB) is a compelling strategy for disease management, but a better elucidation of their effectiveness is crucial. Meta-analysis is the analysis of the results of multiple studies, which is typically performed to synthesize evidence from many possible sources in a formal probabilistic manner. This meta-analytic study, including 30 pathometric, biometric, physiochemical, genetic, and mycotoxin response variables reported in 56 studies, evidences the BCA effects on FHB in wheat. The effectiveness of BCAs of FHB in wheat in terms of pathogen abundance and disease reductions, biomass and yield conservation, and mycotoxin prevention/control was confirmed. BCAs showed higher efficacy (i) in studies published more recently; (ii) under controlled conditions; (iii) in high susceptible wheat cultivars; (iv) when Fusarium inoculation and BCA treatment did not occur directly on the plant (i.e., at the seed and kernel levels) in terms of disease development and mycotoxin control, and vice versa in terms of biomass conservation; (v) if Fusarium inoculation and BCA treatment occurred by spraying spikes in terms of yield; (vi) at 15 to 21 days post Fusarium inoculation or BCA treatment; and (vii) if they were filamentous fungi. However, BCAs overall were less efficacious than conventional agrochemicals, especially in terms of pathogen abundance and FHB reductions, as well as of mycotoxin prevention/control, although inconsistencies were reported among the investigated moderator variables. This study also highlights the complexity of reaching a good balance among BCA effects, and the need for further research.

An assessment of ozone risk for date palm suggests that phytotoxic ozone dose nonlinearly affects carbon gain.

Tropospheric ozone (O3) is a significant phytotoxic air pollutant that has a negative impact on plant carbon gain. Although date palm (Phoenix dactylifera L.) is a globally important crop in arid or semi-arid regions, so far O3 risk assessment for this species has not been reported. This study estimated leaf- and plant-level photosynthetic CO2 uptake for understanding how elevated levels of O3 affects date palm biomass growth. Ozone risks to date palm plants were assessed based on exposure- (AOT40) or flux-based indices (Phytotoxic Ozone Dose, PODy, where y is a threshold of uptake). For this purpose, plants were exposed to three levels of O3 [ambient air, AA (45 ppb as daily average); 1.5 × AA; 2.0 × AA] for 92 days in an O3 Free-Air Controlled Exposure facility. According to the model simulations, the negative effects of O3 on plant-level net photosynthetic CO2 uptake were attributed to reduced gross photosynthetic carbon gain and increased respiratory carbon loss. Season-long O3 exposure and elevated temperatures promoted the negative O3 effect because of a further increase of respiratory carbon loss, which was caused by increased leaf temperature due to stomatal closure. POD1 nonlinearly affected the photosynthetic CO2 uptake, which was closely related to the variation of dry mass increment during the experiment. Although the dose-response relationship suggested that a low O3 dose (POD1 < 5.2 mmol m-2) may even positively affect photosynthetic CO2 uptake in date palms, stomatal O3 uptake at the current ambient O3 levels has potentially a negative impact on date palm growth. The results indicate 5.8 mmol m-2 POD1 or 21.1 ppm h AOT40 as critical levels corresponding to a 4% reduction of net CO2 uptake for date palm, suggesting that this species can be identified as a species moderately sensitive to O3.

Exogenous application of melatonin protects bean and tobacco plants against ozone damage by improving antioxidant enzyme activities, enhancing photosynthetic performance, and preventing membrane damage.

Ozone (O3) pollution is harmful to plants and ecosystems. Several chemicals have been evaluated to protect plants against O3 deleterious effects. However, they are not adequately efficient and/or the environmental safety of their application is questioned. Hence, new chemicals that provide sufficient protection while being safer for environmental application are needed. This study investigates the response of two O3-sensitive plant species (Phaseolus vulgaris L. cv. Pinto and Nicotiana tabacum L. cv. Bel-W3) leaf-sprayed with deionized water (W, control), ethylenediurea (EDU, 1 mM) or melatonin at lower (1 mM) or higher (3 mM) concentrations (Mel_L and Mel_H, respectively), and then exposed to a square wave of 200 ppb O3, lasting 1 day (5 h day-1) for bean and 2 days (8 h day-1) for tobacco. In both species, the photosynthetic activity of O3-exposed plants was about halved. O3-induced membrane damage was also confirmed by increased malondialdehyde (MDA) byproducts compared to control (W). In EDU- and Mel-treated bean plants, the photosynthetic performance was not influenced by O3, leading to reduction of the incidence and severity of O3 visible injury. In bean plants, Mel_L mitigated the detrimental effect of O3 by boosting antioxidant enzyme activities or osmoprotectants (e.g. abscisic acid, proline, and glutathione transferase). In Mel_L-sprayed tobacco plants, O3 negatively influenced the photosynthetic activity. Conversely, Mel_H ameliorated the O3-induced oxidative stress by preserving the photosynthetic performance, preventing membrane damage, and reducing the visible injuries extent. Although EDU performed better, melatonin protected plants against O3 phytotoxicity, suggesting its potential application as a bio-safer and eco-friendlier phytoprotectant against O3. It is worth noting that the content of melatonin in EDU-treated plants remained unchanged, indicating that the protectant mode of action of EDU is not Mel-related.

Isolation and characterization of Fusarium spp. From unhatched eggs of Caretta caretta in Tuscany (Italy).

Sea Turtle Egg Fusariosis (STEF) is a worldwide emergent fungal disease affecting eggs and causing embryos mortality in turtle's nests such as those of Caretta caretta. It is caused by a complex of species belonging to Fusarium genus, particularly those included in the Fusarium Solani Species Complex (FSSC). During the samplings carried out in summer 2020 along the Tuscany coastlines (Italy), C. caretta eggs showed clinical signs resembling those caused by STEF. A total of 32 fungal isolates were obtained from lesioned eggs whose molecular characterization allowing identifying as belonging to FSSC / Neocosmospora spp., Fusarium oxysporum Species Complex (FOSC) / F. oxysporum and Fusarium nodosum, i.e., fungal genera and speciesincluding also well-known plant pathogens. Isolates inoculated on several plant hosts did not result in any pathogenic activity but F. nodosum causing, on wheat spikes, disease symptoms.This is the first time F. nodosum has been isolated from portions of eggs showing evident signs of fungal infection. This work represents the first report of Fusarium spp. isolated from C. caretta eggs showing lesions resembling those caused by STEF on Tuscan coast thus posing a significant concern to loggerhead sea turtle conservation also in this region.

Physiological and Biochemical Responses Induced by Plum Pox Virus and Plum Bark Necrosis Steam Pitting Associated Virus in Tuscany Autochthonous Plum cv. Coscia di Monaca.

The present study focused on trees of Tuscany autochthonous plum cv. Coscia di Monaca in order to evaluate the presence of viruses and elucidate the physiological and biochemical responses to virus infections under real field conditions. Among the several investigated viruses, plums tested positive only to plum pox virus (PPV) and plum bark necrosis steam pitting associated virus (PBNSPaV), occurring as both singular and co-infections. This is the first report of PBNSPaV in a Tuscany orchard. Furthermore, the present study not only confirmed the detrimental effects of PPV on the carbon dioxide assimilation rate due to both stomatal limitations and mesophyll impairments, but also showed that although PBNSPaV did not induce such photosynthetic impairments when occurring as singular infection, it enhanced this damaging effect when present as a co-infection with PPV, as confirmed by a severe decrease in the chlorophyll content. Infection-specific responses in terms of accessory pigments (i.e., carotenoids and xanthophylls), as well as sugars and organic acids, were also reported, these being likely related to photoprotective mechanisms and osmotic regulations under virus-induced oxidative stress. Overall, the results here presented represent an important step to fill knowledge gaps about the interaction of plant viruses and autochthonous Prunus cultivars.

Functional responses of two Mediterranean pine species in an ozone Free-Air Controlled Exposure (FACE) experiment.

Effects of the phytotoxic and widespread ozone (O3) pollution may be species specific, but knowledge on Mediterranean conifer responses to long-term realistic exposure is still limited. We examined responses regarding to photosynthesis, needle biochemical stress markers and carbon and nitrogen (N) isotopes of two Mediterranean pine species (Pinus halepensis Mill. and Pinus pinea L.). Seedlings were grown in a Free-Air Controlled Exposure experiment with three levels of O3 (ambient air, AA [38.7 p.p.b. as daily average]; 1.5 × AA and 2.0 × AA) during the growing season (May-October 2019). In P. halepensis, O3 caused a significant decrease in the photosynthetic rate, which was mainly due to a reduction of both stomatal and mesophyll diffusion conductance to CO2. Isotopic analyses indicated a cumulative or memory effect of O3 exposure on this species, as the negative effects were highlighted only in the late growing season in association with a reduced biochemical defense capacity. On the other hand, there was no clear effect of O3 on photosynthesis in P. pinea. However, this species showed enhanced N allocation to leaves to compensate for reduced photosynthetic N- use efficiency. We conclude that functional responses to O3 are different between the two species determining that P. halepensis with thin needles was relatively sensitive to O3, while P. pinea with thicker needles was more resistant due to a potentially low O3 load per unit mass of mesophyll cells, which may affect species-specific resilience in O3-polluted Mediterranean pine forests.

Mushroom-Based Supplements in Italy: Let's Open Pandora's Box.

Mushrooms and derivates are well known to the scientific community for having different health benefits and exhibit a wide range of pharmacological activities, including lipid-lowering, antihypertensive, antidiabetic, antimicrobic, antiallergic, anti-inflammatory, anticancer, immunomodulating, neuroprotective and osteoprotective actions. In Europe, medical mushrooms are mainly marketed in the form of food supplements as single components or combined with other nutraceuticals. In this context, the first peculiarity that distinguishes it is the safety established through the "history of consumption" that characterizes that mushroom. However, the cultivation of medicinal mushrooms on a large scale is performed mainly in China, where most of the production facilities do not have internationally recognized good manufacturing practices, despite that many European companies that sell myotherapies are supplied by Chinese manufacturers. This is particularly evident in Italy, where an arsenal of mushroom products is marketed in the form of powders and extracts not always of ascertained origin and sometimes of doubtful taxonomic identification, and thus not meeting the quality criteria required. The growing interest in mycotherapy involves a strong commitment from the scientific community to propose supplements of safe origin and genetic purity as well as to promote clinical trials to evaluate its real effects on humans. The purpose of this research is to analyze different mushroom-based dietary supplements used in medicine as monotherapy on the Italian market and to evaluate their composition and quality. The molecular identification of the sequences with those deposited in GenBank allowed for identifying 6 out of 19 samples, matching with those deposited belonging to the species indicated in the label, i.e., Lentinula edodes (samples 1, 4, 12 and 18) and Ganoderma lucidum (samples 5 and 10). Samples containing Ganoderma, labeled in the commercial product as G. lucidum, showed sequences that showed homology of 100% and 99% with G. resinaceum and G. sichuanense. An additional investigation was carried out in order to determine the active fungal ingredients, such as ergosterol, aflatoxins, heavy metals, nicotine and total glucan. The results obtained and shown in the manuscript highlight how the data were not only in line with what is expected with respect to what is indicated in the labels.

Ozone-Induced Biochemical and Molecular Changes in Vitis vinifera Leaves and Responses to Botrytis cinerea Infections.

To investigate how plants cope with multi-stress conditions, we analyzed the biochemical and molecular changes of Vitis vinifera leaves subjected to single or sequential double stresses (infection by Botrytis cinerea (Bc) and ozone (O3, 100 ppb for 3 h) treatment). In Bc+/O3- leaves, the hydrogen peroxide (H2O2) induction (observed at 12 and 24 h from the end of treatment (FET)) triggered a production of ethylene (Et; +35% compared with Bc-/O3- leaves), which was preceded by an increase of salicylic acid (SA; +45%). This result confirms a crosstalk between SA- and Et-related signaling pathways in lesion spread. The ozone induced an early synthesis of Et followed by jasmonic acid (JA) and SA production (about 2-fold higher), where Et and SA signaling triggered reactive oxygen species production by establishing a feedback loop, and JA attenuated this cycle by reducing Et biosynthesis. In Bc+ + O3+ leaves, Et peaked at 6 and 12 h FET, before SA confirmed a crosstalk between Et- and SA-related signaling pathways in lesion propagation. In O3+ + Bc+ leaves, the H2O2 induction triggered an accumulation of JA and Et, demonstrating a synergistic action in the regulation of defence reactions. The divergence in these profiles suggests a rather complex network of events in the transcriptional regulation of genes involved in the systemic acquired resistance.

Sustainable Strategies to Counteract Mycotoxins Contamination and Cowpea Weevil in Chickpea Seeds during Post-Harvest.

Mycotoxins contamination and pest infestation of foods and feeds represent a pivotal threat for food safety and security worldwide, with crucial implications for human and animal health. Controlled atmosphere could be a sustainable strategy to reduce mycotoxins content and counteract the vitality of deleterious organisms in foodstuff. Ozone treatment (O3, 500 ppb for 30, 60 or 90 min) and high nitrogen concentration (N2, 99% for 21 consecutive days) were tested in the post-harvest management of four batches of Cicer arietinum grains to control the presence of mycotoxigenic fungi and their secondary metabolites, as well as pest (i.e., Callosobruchus maculatus) infestation. At the end of the treatment, O3 significantly decreased the incidence of Penicillium spp. (by an average of -50%, independently to the time of exposure) and reduced the patulin and aflatoxins content after 30 min (-85 and -100%, respectively). High N2 concentrations remarkably reduced mycotoxins contamination (by an average of -94%) and induced pest mortality (at 100% after 5 days of exposure). These results confirm the promising potential of O3 and N2 in post-harvest conservation strategies, leading to further investigations to evaluate the effects on the qualitative characteristics of grains.

Horticultural Therapy May Reduce Psychological and Physiological Stress in Adolescents with Anorexia Nervosa: A Pilot Study.

Studies in psychiatric populations have found a positive effect of Horticultural therapy (HCT) on reductions in stress levels. The main objective of the present pilot study was to evaluate the impact of the addition of HCT to conventional clinical treatment (Treatment as Usual, TaU) in a sample of six female adolescents with anorexia nervosa restricting type (AN-R), as compared to six AN-R patients, matched for sex and age, under TaU only. This is a prospective, non-profit, pilot study on patients with a previous diagnosis of AN-R and BMI < 16, recruited in 2020 in clinical settings. At enrolment (T0) and after treatment completion (TF), psychiatric assessment was performed. At T0, all the patients underwent: baseline electrocardiogram acquisition with a wearable chest strap for recording heart rate and its variability; skin conductance registration and thermal mapping of the individual's face. An olfactory identification test was administered both to evaluate the olfactory sensoriality and to assess the induced stress. One-way analyses of variance (ANOVAs) were performed to analyze modifications in clinical and physiological variables, considering time (T0, TF) as a within-subjects factor and group (experimental vs. control) as between-subjects factors. When the ANOVA was significant, post hoc analysis was performed by Paired Sample T-tests. Only in the HCT group, stress response levels, as measured by the biological parameters, improved over time. The body uneasiness level and the affective problem measures displayed a significant improvement in the HCT subjects. HCT seems to have a positive influence on stress levels in AN-R.

The Effects of Elevated Tropospheric Ozone on Carbon Fixation and Stable Isotopic Signatures of Durum Wheat Cultivars with Different Biomass and Yield Stability.

Tropospheric ozone (O3) enrichment caused by human activities can reduce important crop yields with huge economic loss and affect the global carbon cycle and climate change in the coming decades. In this study, two Italian cultivars of durum wheat (Claudio and Mongibello) were exposed to O3 (80 ppb, 5 h day-1 for 70 consecutive days), with the aim to investigate the changes in yield and biomass, ecophysiological traits, and stable carbon and nitrogen isotope values in plants, and to compare the stable isotope responses under environmental stressors. Both cultivars showed a relative O3 tolerance in terms of photosynthetic performance, but in cultivar Mongibello, O3 was detrimental to the grain yield and plant biomass. The δ13C values in the leaves of plants identified that the impact of O3 on CO2 fixation by RuBisCO was dominant. The δ15N value showed significant differences between treatments in both cultivars at seven days from the beginning of the exposure, which could be considered an early indicator of ozone pollution. Under increasingly frequent extreme climates globally, the relationships among stable isotope data, ecophysiological traits, and agronomic parameters could help breed future cultivars.

Antioxidants and Phytohormones Act in Coordination to Regulate Sage Response to Long Term Ozone Exposure.

Antioxidants and phytohormones are hallmarks of abiotic stress responses in plants. Although it is known that they can offer cell protection or accelerate programmed cell death (PCD) depending on the level of stress, the involvement of these metabolites in stress acclimation is still not fully elucidated. Here, we showed the role of antioxidants and phytohormones in Salvia officinalis tolerance to long-term ozone (O3) exposure (120 ppb for 36 days, 5 h day-1). Salicylic acid (SA) content was increased under O3 throughout the whole experiment (+150%, as average compared with control), being required to maintain the cellular redox state and potentiate defense responses. This accumulation was induced before the production of ethylene (ET), suggesting that ET was controlled by SA during O3 exposure to modulate the magnitude of chlorosis formation and the cell redox balance (by regulating ascorbate and glutathione levels). The synthesis and/or regeneration of these antioxidants did not protect membranes from lipid peroxidation, as demonstrated by the accumulation of malondialdehyde (+23% as average). However, these processes of lipid oxidation did not include the synthesis of the membrane breakdown products, as confirmed by the unchanged values of jasmonic acid, thus indicating that this compound was not involved in the regulation of PCD strategies.

Season-long exposure of bilberry plants to realistic and future ozone pollution improves the nutraceutical quality of fruits.

Ozone (O3) is a phytotoxic air pollutant capable of limiting plant yield and growth, and altering the quality of edible plant products. This study aimed to investigate the effects of long-term O3 exposure at realistic and future concentrations (applied during fruit development) not only on morphological, physiological, and biochemical plant/leaf traits of Vaccinium myrtillus but also on its fruit yield and quality. Three-year-old saplings were grown from May to July under three levels of O3 concentration [1.0, 1.5 and 2.0 times the ambient air concentrations, denoted as AA, 1.5_AA and 2.0_AA], using a new-generation O3 Free Air Controlled Exposure system. Ozone induced oxidative pressure and membrane denaturation as confirmed by the accumulation of anion superoxide, hydrogen peroxide (•O2-: +39 and + 29%; H2O2: +55 and + 59% in 1.5_AA and 2.0_AA, respectively, compared with AA), and malondialdehyde by-product (1.4- and 2.5-fold higher than AA, in 1.5_AA and 2.0_AA, respectively). The observed oxidative burst likely affected several cellular structures interested by photosynthetic processes (e.g., decrease of the maximum rate of carboxylation: -30%). This constraint likely induced a decline in plant vitality and a different partitioning of biomass allocation between above and below organs. An accelerated maturation of bilberries due to O3 was reported, suggesting that plants grown under harsher environmental conditions suffered from metabolic changes associated with early ripening. Increasing O3 concentrations might be responsible for an alteration of the ratio between oxidation and reduction processes mechanisms that was followed by a loss of integrity of membranes, so limiting the availability of energy/resources, triggering enzymatic oxidation of phenols to red/purple pigments, and promoting fruit maturation. To the best of our knowledge, this is the first research showing that long-term O3 exposure during bilberry fruit development influenced not only several plant/leaf traits, but also fruit nutraceutical quality at the time of harvest.

Transient Waterlogging Events Impair Shoot and Root Physiology and Reduce Grain Yield of Durum Wheat Cultivars.

Durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn) is a staple crop of the Mediterranean countries, where more frequent waterlogging events are predicted due to climate change. However, few investigations have been conducted on the physiological and agronomic responses of this crop to waterlogging. The present study provides a comprehensive evaluation of the effects of two waterlogging durations (i.e., 14 and 35 days) on two durum wheat cultivars (i.e., Svevo and Emilio Lepido). An integrated analysis of an array of physiological, biochemical, biometric, and yield parameters was performed at the end of the waterlogging events, during recovery, and at physiological maturity. Results established that effects on durum wheat varied depending on waterlogging duration. This stress imposed at tillering impaired photosynthetic activity of leaves and determined oxidative injury of the roots. The physiological damages could not be fully recovered, subsequently slowing down tiller formation and crop growth, and depressing the final grain yield. Furthermore, differences in waterlogging tolerance between cultivars were discovered. Our results demonstrate that in durum wheat, the energy maintenance, the cytosolic ion homeostasis, and the ROS control and detoxification can be useful physiological and biochemical parameters to consider for the waterlogging tolerance of genotypes, with regard to sustaining biomass production and grain yield.

Stress markers and physiochemical responses of the Mediterranean shrub Phillyrea angustifolia under current and future drought and ozone scenarios.

Mediterranean plants are particularly threatened by the exacerbation of prolonged periods of summer drought and increasing concentrations of ground-level ozone (O3). The aims of the present study were to (i) test if selected markers (i.e., reactive oxygen species, ROS; malondialdehyde, MDA; photosynthetic pigments) are able to discriminate the oxidative pressure due to single and combined stress conditions, and (ii) elucidate the physiochemical adjustments adopted by Phillyrea angustifolia (evergreen woody species representative of the maquis, also known as narrow-leaved mock privet) to perceive and counter to drought and/or O3. Plants were grown from May to October under the combination of two levels of water irrigation [i.e., well-watered (WW) and water-stressed (WS)] and three levels of O3 [i.e., 1.0, 1.5 and 2.0 times the ambient air concentrations, i.e. AA (current O3 scenario), 1.5 × AA and 2.0 × AA (future O3 scenarios), respectively], using a new-generation O3 Free Air Controlled Exposure (FACE) system. Overall, this species appeared relatively sensitive to drought (e.g., net CO2 assimilation rate and stomatal conductance significantly decreased, as well as total chlorophyll and carotenoid contents), and tolerant to O3 (e.g., as confirmed by the absence of visible foliar injury, the unchanged values of total carotenoids, and the detrimental effects on stomatal conductance, total chlorophylls and terpene emission only under elevated O3 concentrations). The combination of both stressors led to harsher oxidative stress. Only when evaluated together (i.e., combining the information provided by the analysis of each stress marker), ROS, MDA and photosynthetic pigments, were suitable stress markers to discriminate the differential oxidative stress induced by drought and increasing O3 concentrations applied singly or in combination: (i) all these stress markers were affected under drought per se; (ii) hydrogen peroxide (H2O2) and MDA increased under O3per se, following the gradient of O3 concentrations (H2O2: about 2- and 4-fold higher; MDA: +22 and + 91%; in 1.5 × AA_WW and 2.0 × AA_WW, respectively); (iii) joining together the ROS it was possible to report harsher effects under 2.0 × AA_WS and 1.5 × AA_WS (both anion superoxide and H2O2 increased) than under 2.0 × AA_WW (only H2O2 increased); and (iv) MDA showed harsher effects under 2.0 × AA_WS than under 1.5 × AA_WS (increased by 49 and 18%, respectively). Plants activated physiological and biochemical adjustments in order to partially avoid (e.g., stomatal closure) and tolerate (e.g., increased terpene emission) the effects of drought when combined with increasing O3 concentrations, suggesting that the water use strategy (isohydric) and the sclerophyllous habit can further increase the plant tolerance to environmental constraints in the Mediterranean area.

In the tripartite combination ozone-poplar-Chrysomela populi, the pollutant alters the plant-insect interaction via primary metabolites of foliage.

Ozone (O3)-induced metabolic changes in leaves are relevant and may have several ecological significances. Here, variations in foliar chemistry of two poplar clones (Populus deltoides × maximowiczii, Eridano, and P. × euramericana, I-214) under a chronic O3 treatment (80 ppb, 5 h d-1 for 10 consecutive days) were investigated. The aim was to elucidate if leaf age and/or O3-sensitivity (considering Eridano and I-214 as O3-sensitive and O3-resistant, respectively) can affect suitability of poplar foliage for Chrysomela populi L. (Coleoptera Chrysomelidae), in terms of palatability. Comparing controls, only low amino acid (AA) contents were reported in Eridano [about 3- and 4-fold in mature and young leaves (ML and YL, respectively)], and all the investigated primary metabolites [i.e. water soluble carbohydrates (WSC), proteins (Prot) and AA] were higher in YL than in ML of I-214 (+23, +54 and + 20%, respectively). Ozone increased WSC only in YL of Eridano (+24%, i.e. highest values among samples; O3 effects are always reported comparing O3-treated plants with the related controls). A concomitant decrease of Prot was observed in both ML and YL of Eridano, while only in YL of I-214 (-41, -45 and -51%, respectively). In addition, O3 decreased AA in YL of Eridano and in ML of I-214 (-40 and -14%, respectively). Comparing plants maintained under charcoal-filtered air, total ascorbate (Asc) was lower in Eridano in both ML and YL (around -22%), and abscisic acid (ABA) was similar between clones; furthermore, higher levels of Asc were reported in YL than in ML of Eridano (+19%). Ozone increased Asc and ABA (about 2- and 3-fold, respectively) in both ML and YL of Eridano, as well as ABA in YL of I-214 (about 2-fold). Comparing leaves maintained under charcoal-filtered air, the choice feeding test showed that the 2nd instar larvae preferred YL, and the quantity of YL consumed was 9 and 4-fold higher than ML in Eridano and I-214, respectively. Comparing leaves exposed to O3-treatment, a significant feeding preference for YL disks was also observed, regardless of the clone. The no-choice feeding test showed that larval growth was slightly higher on untreated YL than on untreated ML (+19 and + 10% in Eridano and I-214, respectively). The body mass of larvae fed with O3-treated YL was also significantly higher than that of larvae fed with untreated YL (3- and 2-fold in Eridano and I-214). This study highlights that realistic O3 concentrations can significantly impact the host/insect interactions, a phenomenon dependent on leaf age and O3-sensitivity of the host.

The Biosynthesis of Phenolic Compounds is an Integrated Defence Mechanism to Prevent Ozone Injury in Salvia officinalis.

Specialized metabolites constitute a major antioxidant system involved in plant defence against environmental constraints, such as tropospheric ozone (O3). The objective of this experiment was to give a thorough description of the effects of an O3 pulse (120 ppb, 5 h) on the phenylpropanoid metabolism of sage, at both biochemical and molecular levels. Variable O3-induced changes were observed over time among the detected phenylpropanoid compounds (mostly identified as phenolic acids and flavonoids), likely because of their extraordinary functional diversity. Furthermore, decreases in the phenylalanine ammonia-lyase (PAL), phenol oxidase (PPO), and rosmarinic acid synthase (RAS) activities were reported during the first hours of treatment, probably due to an O3-induced oxidative damage to proteins. Both PAL and PPO activities were also suppressed at 24 h from the beginning of exposure, whereas enhanced RAS activity occurred at the end of treatment and at the recovery time, suggesting that specific branches of the phenolic pathways were activated. The increased RAS activity was accompanied by the up-regulation of the transcript levels of genes like RAS, tyrosine aminotransferase, and cinnamic acid 4-hydroxylase. In conclusion, sage faced the O3 pulse by regulating the activation of the phenolic biosynthetic route as an integrated defence mechanism.