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.

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.

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.

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.

Hyperspectral Reflectance of Light-Adapted Leaves Can Predict Both Dark- and Light-Adapted Chl Fluorescence Parameters, and the Effects of Chronic Ozone Exposure on Date Palm (Phoenix dactylifera).

High-throughput and large-scale measurements of chlorophyll a fluorescence (ChlF) are of great interest to investigate the photosynthetic performance of plants in the field. Here, we tested the capability to rapidly, precisely, and simultaneously estimate the number of pulse-amplitude-modulation ChlF parameters commonly calculated from both dark- and light-adapted leaves (an operation which usually takes tens of minutes) from the reflectance of hyperspectral data collected on light-adapted leaves of date palm seedlings chronically exposed in a FACE facility to three ozone (O3) concentrations (ambient air, AA; target 1.5 × AA O3, named as moderate O3, MO; target 2 × AA O3, named as elevated O3, EO) for 75 consecutive days. Leaf spectral measurements were paired with reference measurements of ChlF, and predictive spectral models were constructed using partial least squares regression. Most of the ChlF parameters were well predicted by spectroscopic models (average model goodness-of-fit for validation, R2: 0.53-0.82). Furthermore, comparing the full-range spectral profiles (i.e., 400-2400 nm), it was possible to distinguish with high accuracy (81% of success) plants exposed to the different O3 concentrations, especially those exposed to EO from those exposed to MO and AA. This was possible even in the absence of visible foliar injury and using a moderately O3-susceptible species like the date palm. The latter view is confirmed by the few variations of the ChlF parameters, that occurred only under EO. The results of the current study could be applied in several scientific fields, such as precision agriculture and plant phenotyping.

Drought stress modulates secondary metabolites in Brassica oleracea L. convar. acephala (DC) Alef, var. sabellica L.

BACKGROUND: Consumer preference today is for the consumption of functional food and the reduction of chemical preservatives. Moreover, the antimicrobial properties and health-promoting qualities of plant secondary metabolites are well known. Due to forecasted climate changes and increasing human population, agricultural practices for saving water have become a concern. In the present study, the physiological responses of curly kale Brassica oleracea L. convar. Acephala (DC) var. sabellica to drought stress and the impact of water limitation on the concentration of selected secondary metabolites were investigated under laboratory-controlled conditions. RESULTS: Results indicated that drought stress increased the content of trans-2-hexenal, phytol and δ-tocopherol, and decreased chlorophyll content. Moreover, drought stress increased antioxidant capacity and the expression of AOP2, a gene associated with the biosynthesis of aliphatic alkenyl glucosinolates, and of three genes - TGG1, TGGE and PEN2 - encoding for myrosinases, the enzymes involved in glucosinolate breakdown. CONCLUSION: The present study shows that water limitation during the growing phase might be exploited as a sustainable practice for producing curly kale with a high concentration of nutritionally important health-promoting bioactive metabolites. © 2019 Society of Chemical Industry.

How Quercus ilex L. saplings face combined salt and ozone stress: a transcriptome analysis.

BACKGROUND: Similar to other urban trees, holm oaks (Quercus ilex L.) provide a physiological, ecological and social service in the urban environment, since they remove atmospheric pollution. However, the urban environment has several abiotic factors that negatively influence plant life, which are further exacerbated due to climate change, especially in the Mediterranean area. Among these abiotic factors, increased uptake of Na + and Cl - usually occurs in trees in the urban ecosystem; moreover, an excess of the tropospheric ozone concentration in Mediterranean cities further affects plant growth and survival. Here, we produced and annotated a de novo leaf transcriptome of Q. ilex as well as transcripts over- or under-expressed after a single episode of O3 (80 nl l-1, 5 h), a salt treatment (150 mM for 15 days) or a combination of these treatments, mimicking a situation that plants commonly face, especially in urban environments. RESULTS: Salinity dramatically changed the profile of expressed transcripts, while the short O3 pulse had less effect on the transcript profile. However, the short O3 pulse had a very strong effect in inducing over- or under-expression of some genes in plants coping with soil salinity. Many differentially regulated genes were related to stress sensing and signalling, cell wall remodelling, ROS sensing and scavenging, photosynthesis and to sugar and lipid metabolism. Most differentially expressed transcripts revealed here are in accordance with a previous report on Q. ilex at the physiological and biochemical levels, even though the expression profiles were overall more striking than those found at the biochemical and physiological levels. CONCLUSIONS: We produced for the first time a reference transcriptome for Q. ilex, and performed gene expression analysis for this species when subjected to salt, ozone and a combination of the two. The comparison of gene expression between the combined salt + ozone treatment and salt or ozone alone showed that even though many differentially expressed genes overlap all treatments, combined stress triggered a unique response in terms of gene expression modification. The obtained results represent a useful tool for studies aiming to investigate the effects of environmental stresses in urban-adapted tree species.

Variations in physiological and biochemical traits of oak seedlings grown under drought and ozone stress.

Despite the huge biodiversity characterizing the Mediterranean environment, environmental constraints, such as high sunlight and high temperatures alongside with dry periods, make plant survival hard. In addition, high irradiance leads to increasing ozone (O3 ) concentrations in ambient air. In this era of global warming, it is necessary to understand the mechanisms that allow native species to tolerate these environmental constraints and how such mechanisms interact. Three Mediterranean oak species (Quercus ilex, Quercus pubescens and Quercus cerris) with different features (drought tolerant, evergreen or deciduous species) were selected to assess their biometrical, physiological and biochemical responses under drought and/or O3 stress (80-100 nl l(-1) of O3 for 5 h day(-1) for 77 consecutive days). Leaf visible injury appeared only under drought stress (alone or combined with O3 ) in all three species. Drought × O3 induced strong reductions in leaf dry weight in Q. pubescens and Q. cerris (-70 and -75%, respectively). Alterations in physiological (i.e. decrease in maximum carboxylation rate) and biochemical parameters (i.e. increase in proline content and build-up of malondialdehyde by-products) occurred in all the three species, although drought represented the major determinant. Quercus ilex and Q. pubescens, which co-occur in dry environments, were more tolerant to drought and drought × O3 . Quercus ilex was the species in which oxidative stress occurred only when drought was applied with O3 . High plasticity at a biochemical level (i.e. proline content) and evergreen habitus are likely on the basis of the higher tolerance of Q. ilex.

Impact of mechanical mowing and chemical treatment on phytosociological, pedochemical and biological parameters in roadside soils and vegetation.

Many chemical and non-chemical strategies have been applied to control weeds in agricultural and industrial areas. Knowledge regarding the effects of these methods on roadside vegetation is still poor. A 2-year field experiment was performed along a road located near Livorno (Tuscany, central Italy). Eight plots/strips were identified, of which four were subjected to periodical mechanical mowing and the remaining four were treated with a chemical herbicide based on glyphosate (the producer's recommended rates were used for the selective control of broad-leaved weeds). Our results clearly showed that roadside soil and vegetation are a significant reservoir of anthropogenic activities which have a strong negative effect on several phytosociological, pedochemical and biological parameters. Compared with conventional mechanical mowing, chemical treatment induced (i) a significant increase in organic matter in the upper plot layers (+18%), and (ii) a marked reduction in weed height throughout the entire period of the experiment. Irrespectively of the kind of treatment, no significance differences were detected in terms of (i) biological quality of soil (the abundance and diversity of arthropod communities did not change), and (ii) plant elemental content (bulk concentrations of analysed trace elements had a good fit within ranges of occurrence in the "reference plant"). The glyphosate partially controlled broad-leaved weeds and this moderate efficacy is dependent upon the season/time of application. In conclusion, the rational and sustainable use of chemical herbicides may be a useful tool for the management of roadside vegetation.

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.

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.

Leaf and Flower Extracts from the Dwarf Elder (Sambucus ebulus): Toxicity and Repellence against Cosmopolitan Mosquito-Borne Diseases Vectors.

As there has been no scientific evidence of the bioactivity of Sambucus ebulus (Adoxaceae) extracts against insects, we chemically characterized S. ebulus leaves and flowers extracted in methanol and water. The crude extracts, phenolic compounds, and amino acids isolated were tested as larvicides against the fourth-instar larvae of Aedes albopictus and Culex pipiens (Diptera: Culicidae). To understand their mode of action, we evaluated the in vitro acetylcholinesterase (AChE) inhibitor effect of the crude extracts on the two mosquito larvae through a colorimetric method. Furthermore, the deterrent effect of the crude extracts against ovipositing Ae. albopictus females was assessed in the open field. Twelve phenylpropanoids and fourteen amino acids were detected in the extracts, with a prevalence of hydroxycinnamic acids and nonaromatic amino acids. The most toxic compound to Ae. albopictus larvae after 24 h was gallic acid, followed by the crude S. ebulus leaf extract; on Cx. pipiens, it was the crude flower extract. The AChE test showed higher inhibition on both mosquito species exerted by the leaf extract if compared to the flower extract, and it also deterred oviposition by Ae. albopictus females starting from the third day. The results indicated that vegetal extracts could effectively help in the integrated vector management of mosquitoes.

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.

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.

Signaling molecules and cell death in Melissa officinalis plants exposed to ozone.

KEY MESSAGE: The study focuses on the interaction between reactive oxygen species and hormones that regulate the programmed cell death in plants of Melissa officinalis exposed to ozone. Interaction between hormone and redox signaling pathways has been investigated in ozone-stressed (200 ppb, 5 h) lemon balm to verify if the response resembles the biotic defense reactions. In comparison to controls, plants exhibited foliar injury and the cell death was induced by (1) biphasic production of hydrogen peroxide and superoxide radical; (2) hormonal regulation of ozone-induced lesion formation with a significant production of ethylene, salicylic, jasmonic and abscisic acid; (3) ozone degradation to reactive oxygen species and their detoxification by some enzymatic (such as superoxide dismutase) and non-enzymatic antioxidant systems (such as ascorbic acid, glutathione and carotenoids), that worked in cooperation without providing a defense against free radicals (such as confirmed by the modification of the antioxidant properties of leaf tissue). This integrated view showed that reactive oxygen species interact with hormonal signaling pathway regulating cell death and the sensitivity of lemon balm to ozone.

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.

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.

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.

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.

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.