Aphicidal Activity and Phytotoxicity of Citrus sinensis Essential-Oil-Based Nano-Insecticide.

Due to its high polyphagy, Aphis gossypii is considered a key pest of many crops, and it can feed on hundreds of plant species belonging to the families Cucurbitaceae, Malvaceae, Solanaceae, Rutaceae, and Asteraceae. The control of this pest mainly relies on synthetic insecticides whose adverse effects on the environment and human health are encouraging researchers to explore innovative, alternative solutions. In this scenario, essential oils (EOs) could play a key role in the development of ecofriendly pesticides. In this study, the development of a citrus peel EO-based nano-formulation and its biological activity against A. gossypii both in the laboratory and field were described and evaluated. The phytotoxicity towards citrus plants was also assessed. The developed nano-insecticide highlighted good aphicidal activity both in the laboratory and field trials, even at moderate EO concentrations. However, the highest tested concentrations (4 and 6% of active ingredient) revealed phytotoxic effects on the photosynthetic apparatus; the side effects need to be carefully accounted for to successfully apply this control tool in field conditions.

Abiotic and Herbivory Combined Stress in Tomato: Additive, Synergic and Antagonistic Effects and Within-Plant Phenotypic Plasticity.

BACKGROUND: Drought, N deficiency and herbivory are considered the most important stressors caused by climate change in the agro- and eco-systems and varied in space and time shaping highly dynamic and heterogeneous stressful environments. This study aims to evaluate the tomato morpho-physiological and metabolic responses to combined abiotic and herbivory at different within-plant spatial levels and temporal scales. METHODS: Leaf-level morphological, gas exchange traits and volatile organic compounds (VOCs) profiles were measured in tomato plants exposed to N deficiency and drought, Tuta absoluta larvae and their combination. Additive, synergistic or antagonistic effects of the single stress when combined were also evaluated. Morpho-physiological traits and VOCs profile were also measured on leaves located at three different positions along the shoot axes. RESULTS: The combination of the abiotic and biotic stress has been more harmful than single stress with antagonistic and synergistic but non-additive effects for the morpho-physiological and VOCs tomato responses, respectively. Combined stress also determined a high within-plant phenotypic plasticity of the morpho-physiological responses. CONCLUSIONS: These results suggested that the combined stress in tomato determined a "new stress state" and a higher within-plant phenotypic plasticity which could permit an efficient use of the growth and defense resources in the heterogeneous and multiple stressful environmental conditions.

Monoterpene Synthase Genes and Monoterpene Profiles in Pinus nigra subsp. laricio.

In the present study, we carried out a quantitative analysis of the monoterpenes composition in different tissues of the non-model conifer Pinus nigra J.F. Arnold subsp. laricio Palib. ex Maire (P. laricio, in short). All the P. laricio tissues examined showed the presence of the same fourteen monoterpenes, among which the most abundant were ß-phellandrene, α-pinene, and ß-pinene, whose distribution was markedly tissue-specific. In parallel, from the same plant tissues, we isolated seven full-length cDNA transcripts coding for as many monoterpene synthases, each of which was found to be attributable to one of the seven phylogenetic groups in which the d1-clade of the canonical classification of plants' terpene synthases can be subdivided. The amino acid sequences deduced from the above cDNA transcripts allowed to predict their putative involvement in the biosynthesis of five of the monoterpenes identified. Transcripts profiling revealed a differential gene expression across the different tissues examined, and was found to be consistent with the corresponding metabolites profiles. The genomic organization of the seven isolated monoterpene synthase genes was also determined.

Diterpene Resin Acids and Olefins in Calabrian Pine (Pinus nigra subsp. laricio (Poiret) Maire) Oleoresin: GC-MS Profiling of Major Diterpenoids in Different Plant Organs, Molecular Identification and Expression Analysis of Diterpene Synthase Genes.

A quali-quantitative analysis of diterpenoid composition in tissues obtained from different organs of Pinus nigra subsp. laricio (Poiret) Maire (Calabrian pine) was carried out. Diterpene resin acids were the most abundant diterpenoids across all the examined tissues. The same nine diterpene resin acids were always found, with the abietane type prevailing on the pimarane type, although their quantitative distribution was found to be remarkably tissue-specific. The scrutiny of the available literature revealed species specificity as well. A phylogeny-based approach allowed us to isolate four cDNAs coding for diterpene synthases in Calabrian pine, each of which belonging to one of the four groups into which the d3 clade of the plants' terpene synthases family can be divided. The deduced amino acid sequences allowed predicting that both monofunctional and bifunctional diterpene synthases are involved in the biosynthesis of diterpene resin acids in Calabrian pine. Transcript profiling revealed differential expression across the different tissues and was found to be consistent with the corresponding diterpenoid profiles. The isolation of the complete genomic sequences and the determination of their exon/intron structures allowed us to place the diterpene synthase genes from Calabrian pine on the background of current ideas on the functional evolution of diterpene synthases in Gymnosperms.

Plasticity, exudation and microbiome-association of the root system of Pellitory-of-the-wall plants grown in environments impaired in iron availability.

The investigation of the adaptive strategies of wild plant species to extreme environments is a challenging issue, which favors the identification of new traits for plant resilience. We investigated different traits which characterize the root-soil interaction of Parietaria judaica, a wild plant species commonly known as "Pellitory-of-the-wall". P. judaica adopts the acidification-reduction strategy (Strategy I) for iron (Fe) acquisition from soil, and it can complete its life cycle in highly calcareous environments without any symptoms of chlorosis. In a field-to-lab approach, the microbiome associated with P. judaica roots was analyzed in spontaneous plants harvested from an urban environment consisting in an extremely calcareous habitat. Also, the phenolics and carboxylates content and root plasticity and exudation were analyzed in P. judaica plants grown under three different controlled conditions mimicking the effect of calcareous environments on Fe availability: results show that P. judaica differentially modulates root plasticity under different Fe availability-impaired conditions, and that it induces, to a high extent, the exudation of caffeoylquinic acid derivatives under calcareous conditions, positively impacting Fe solubility.

The Assessment and the Within-Plant Variation of the Morpho-Physiological Traits and VOCs Profile in Endemic and Rare Salvia ceratophylloides Ard. (Lamiaceae).

Salvia ceratophylloides (Ard.) is an endemic and rare plant species recently rediscovered as very few individuals at two different Southern Italy sites. The study of within-plant variation is fundamental to understand the plant adaptation to the local conditions, especially in rare species, and consequently to preserve plant biodiversity. Here, we reported the variation of the morpho-ecophysiological and metabolic traits between the sessile and petiolate leaf of S. ceratophylloides plants at two different sites for understanding the adaptation strategies for surviving in these habitats. The S. ceratophylloides individuals exhibited different net photosynthetic rate, maximum quantum yield, light intensity for the saturation of the photosynthetic machinery, stomatal conductance, transpiration rate, leaf area, fractal dimension, and some volatile organic compounds (VOCs) between the different leaf types. This within-plant morpho-physiological and metabolic variation was dependent on the site. These results provide empirical evidence of sharply within-plant variation of the morpho-physiological traits and VOCs profiles in S. ceratophylloides, explaining the adaptation to the local conditions.

Single and Combined Abiotic Stress in Maize Root Morphology.

Plants are continually exposed to multiple stresses, which co-occur in nature, and the net effects are frequently more nonadditive (i.e., synergistic or antagonistic), suggesting "unique" responses with respect to that of the individual stress. Further, plant stress responses are not uniform, showing a high spatial and temporal variability among and along the different organs. In this respect, the present work investigated the morphological responses of different root types (seminal, seminal lateral, primary and primary lateral) of maize plants exposed to single (drought and heat) and combined stress (drought + heat). Data were evaluated by a specific root image analysis system (WinRHIZO) and analyzed by uni- and multivariate statistical analyses. The results indicated that primary roots and their laterals were the types more sensitive to the single and combined stresses, while the seminal laterals specifically responded to the combined only. Further, antagonistic and synergistic effects were observed for the specific traits in the primary and their laterals and in the seminal lateral roots in response to the combined stress. These results suggested that the maize root system modified specific root types and traits to deal with different stressful environmental conditions, highlighting that the adaptation strategy to the combined stress may be different from that of the individual ones. The knowledge of "unique or shared" responses of plants to multiple stress can be utilized to develop varieties with broad-spectrum stress tolerance.

Profiling Volatile Terpenoids from Calabrian Pine Stands Infested by the Pine Processionary Moth.

Terpenoids make up the biggest and most diversified class of chemical substances discovered in plants, encompassing over 40,000 individual compounds. In conifers, the production of terpenoids, either as oleoresin or emitted as volatile compounds, play an important role in the physical and chemical defence responses against pathogens and herbivores. In the present work, we examined, for the first time to the best of our knowledge, the terpenic defensive relations of Calabrian pine (Pinus nigra subsp. laricio (Poiret) Maire), facing the attack of the pine processionary moth (Thaumetopoea pityocampa (Denis and Schiffermüller, 1775)), brought about in the open on adult plant individuals growing at two distinct forest sites. Among the volatile terpenoids emitted from pine needles, bornyl acetate [(4,7,7-trimethyl-3-bicyclo[2.2.1]heptanyl) acetate] was the most frequently and selectively associated with the infestation, increasing during the period of most intense trophic activity of the caterpillars (defoliation), and decreasing thereafter. Although further work is needed to clarify whether the observed response reflects defence reactions and/or they are involved in communication among the infested plants and their biotic environment, the present results boost the currently growing interest in the isolation and characterization of plant secondary metabolites that can be used to control pests, pathogens, and weeds.

A sonic root detector for revealing tree coarse root distribution.

This study assesses the reliability of a non-destructive method for determining the in situ distribution of tree coarse roots within a scaled distance 6-fold the DBH by comparing the results with the actual 3D root architecture revealed by invasive methods. The root architecture of 22-year-old olive trees was determined non-destructively with a Root Detector device (Fakopp Enterprise Bt) using sonic speed and directly by a 3D digitizer (Fastrak, Polhemus) after soil removal. The radial and vertical distributions of the coarse root biomass and diameter in the soil as determined by the 3D digitizer were correlated with the root map detected by sonic speed. A highly significant correlation was observed between the coarse root biomass distribution and the sonic speed within 30 cm of soil depth, but this correlation decreased with increasing distance from the trunk, up to 120 cm. No correlations were observed between sonic speed and root diameter. The Root Detector was able to map the coarse roots of the olive tree in the soil environment, but only under certain conditions. First, root detection was more efficient within 30 cm of soil depth, provided that more than 35% of the total biomass of lateral roots occurs within this depth range. Second, the distance of 120 cm from the trunk, scaled as 6-fold the DBH, may be considered the threshold over which the sonic speed and the detection of roots markedly decreased. Third, Root Detector technology is unable to detect root size in terms of geometric parameters such as root diameter.

On the Evolution and Functional Diversity of Terpene Synthases in the Pinus Species: A Review.

In the biosynthesis of terpenoids, the ample catalytic versatility of terpene synthases (TPS) allows the formation of thousands of different molecules. A steadily increasing number of sequenced plant genomes invariably show that the TPS gene family is medium to large in size, comprising from 30 to 100 functional members. In conifers, TPSs belonging to the gymnosperm-specific TPS-d subfamily produce a complex mixture of mono-, sesqui-, and diterpenoid specialized metabolites, which are found in volatile emissions and oleoresin secretions. Such substances are involved in the defence against pathogens and herbivores and can help to protect against abiotic stress. Oleoresin terpenoids can be also profitably used in a number of different fields, from traditional and modern medicine to fine chemicals, fragrances, and flavours, and, in the last years, in biorefinery too. In the present work, after summarizing the current views on the biosynthesis and biological functions of terpenoids, recent advances on the evolution and functional diversification of plant TPSs are reviewed, with a focus on gymnosperms. In such context, an extensive characterization and phylogeny of all the known TPSs from different Pinus species is reported, which, for such genus, can be seen as the first effort to explore the evolutionary history of the large family of TPS genes involved in specialized metabolism. Finally, an approach is described in which the phylogeny of TPSs in Pinus spp. has been exploited to isolate for the first time mono-TPS sequences from Pinus nigra subsp. laricio, an ecologically important endemic pine in the Mediterranean area.

Allelopathic potential of Artemisia arborescens: isolation, identification and quantification of phytotoxic compounds through fractionation-guided bioassays.

The aerial part of Artemisia arborescens L. (Asteraceae) was extracted with water and methanol, and both extracts were fractionated using n-hexane, chloroform, ethyl acetate and n-butanol. The potential phytotoxicity of both crude extracts and their fractions were assayed in vitro on seed germination and root growth of lettuce (Lactuca sativa L.), a sensitive species largely employed in the allelopathy studies. The inhibitory activities were analysed by dose-response curves and the ED 50 were estimated. Crude extracts strongly inhibited both germination and root growth processes. The fraction-bioassay indicated the following hierarchy of phytotoxicity for both physiological processes: ethyl acetate ≥ n-hexane > chloroform ≥ n-butanol. On the n-hexane fraction, GC-MS analyses were carried out to characterise and quantify some of the potential allelochemicals. Twenty-one compounds were identified and three of them, camphor, trans-caryophyllene and pulegone were quantified.

Phytotoxic activity of foliar volatiles and essential oils of Calamintha nepeta (L.) Savi.

Foliar volatiles and essential oils of Calamintha nepeta (L.) Savi, a Mediterranean plant species belonging to the Labiatae family, were investigated for their phytotoxic activities on seed germination and root growth of crops (Lactuca sativa L. and Raphanus sativus L.) and weed species (Lolium perenne L. and Amaranthus retroflexus L.). Foliar volatiles of C. nepeta (L.) Savi strongly inhibited both germination and root growth of lettuce, and its essential oils, especially at 125, 250 and 500 µL/L, inhibited both processes in lettuce, radish and A. retroflexus L. species, while displaying a little effect on L. perenne L. By GC-MS, 28 chemicals were identified: 17 monoterpenes, 8 sesquiterpenes, 1 diterpene and 2 miscellaneous. Pulegone was the main constituent of the C. nepeta (L.) Savi essential oils. The terpenic components of essentials oils were probably responsible for the phytotoxic activities.

Nitrate uptake along the maize primary root: an integrated physiological and molecular approach.

The spatial variability of the nitrate (NO3-) uptake along the maize primary root axis was investigated at physiological and molecular levels. Net NO3- uptake rate (NNUR) and its kinetic parameters, together with the gene expression of a high-affinity NO3- transporter (NRT2.1), were evaluated. The activity and the expression of plasma membrane H⁺ -ATPase (PM H⁺ -ATPase), key enzyme in plant nutrition, were also analysed. The NNUR showed a heterogeneous spatial pattern along the root, where the regions closer to the root tip early exhibited higher capacity to absorb NO3- than the basal regions, because of a higher maximum NNUR and faster induction of the inducible high-affinity transport system (iHATS), the presence of the high-affinity transport system (HATS) also at external NO3- concentrations >100 µm and an improved NO3- transport because of lower K(m) values. ZmNRT2.1 transcript abundances were not spatially correlated with NNUR, suggesting that post-translational effects or NAR2 protein co-expression could be involved. By contrast, PM H⁺ -ATPase displayed a similar spatial-temporal pattern as that of nitrate uptake, resulting in higher activity in the root tip than in the basal regions. Increased activities of the enzyme after nitrate supply resulted in enhanced expression of MAH3 and MAH4, PM H⁺ -ATPase subfamily II genes, while MAH1 was not expressed.

Short-term effects of coumarin along the maize primary root axis.

The short-term effects of coumarin on three different maize primary root zones, transition zone (TZ, 3 mm), and two non-growing zones (NGZ1 and NGZ2 at 20 and 50 mm, respectively), were studied in order to investigate the effect of the allelochemical on maize root elongation rate (RER). The RER, plasma membrane (pm) H (+) -ATPase activity, quantitative pH changes and cell membrane potentials were evaluated. The results showed that coumarin caused at the TZ 1) an increased RER; 2) an enhancement of pm H (+) -ATPase activity and proton extrusion; and 3) a transient depolarization followed by a hyperpolarization of cell membrane potential. These observations were not evident in the NGZ1 and NGZ2 of the maize root. Coumarin-treatment in the NGZ1 did not change RER, but caused a membrane depolarization, while the NGZ2 was mostly insensitive to the allelochemical. These data suggested that the primary maize root was sensitive to coumarin within a 20 mm section from the root tip, but the more distal NGZ2 was not involved in coumarin-elicited physiological responses.

Cadmium adsorption on vermiculite, zeolite and pumice: batch experimental studies.

Batch experiments were performed to evaluate the combined effects of ionic activity, pH, and contact time on the cadmium sorption in three different minerals, vermiculite, zeolite, and pumice, commonly employed as substrata in nurseries and recently considered for their potential use in remediation methods. The extent of cadmium sorption was vermiculite>zeolite>pumice, as shown by the Langmuir and Freundlich parameters, and it was highly dependent on mineral characteristics. The percentage of cadmium sorption in zeolite and vermiculite did not depend on cadmium concentration, while in pumice this percentage was positively correlated to the initial cadmium concentration. At low cadmium concentrations (30-120 microM), the metal sorbed on zeolite was mainly present in the nonexchangeable form (70%) at levels much higher than those found for vermiculite and pumice. The primary variable responsible for determination of cadmium mobility in these minerals was confirmed to be pH. The ionic concentrations of Hoagland nutrient solution were significantly modified by both pH and mineral composition, while the presence of cadmium caused no changes. With vermiculite and zeolite, the time-course of cadmium sorption was related to mineral composition to a greater extent than to cadmium concentration. While with pumice, the percentage of cadmium sorbed after 6 weeks was lower than with the other two minerals, and it was inversely correlated to the initial cadmium concentration.

Adsorption of nutrients and cadmium by different minerals: experimental studies and modelling.

This work analyses cadmium sorption by natural vermiculite, zeolite, and pumice, which have been recently considered for their potential use in remediation of contaminated soils. Batch experiments were performed to investigate the interactions between the mineral surfaces and the main nutritive cations of Hoagland solution. In addition to their ameliorative effect of reducing metal mobility, these minerals are able to interact to different extents with various components of the nutrient solution and can change the availability of essential nutrients in solution. Therefore, the minerals were also exposed to a range of cadmium doses to compare their different affinities for this element and to analyse the cadmium-nutrient interactions. The results showed that the nutrient solution composition was significantly modified by contact with zeolite and vermiculite, and to a minor extent with pumice. The ionic equilibrium between the solid and aqueous phases was attained at several time points depending on the cation. Ammonium and potassium ions were almost entirely absorbed by zeolite, while in vermiculite endogenous magnesium ions were exchanged with the liquid phase calcium ions. The amount of cadmium adsorbed on mineral surfaces equilibrated with the nutrient solution showed the following sequence zeolite>vermiculite>pumice, and the cation concentrations of the fluid phase were sensitive to cadmium addition.

Comparing morphological plasticity of root orders in slow- and fast-growing citrus rootstocks supplied with different nitrate levels.

BACKGROUND AND AIMS: Studies of the plasticity of functional root traits involved in resource acquisition have focused mainly on root length without considering such 'morphological components' as biomass allocation, specific root length, root fineness, and tissue density that affect root length. The plasticity of the above components in response to nitrate supply was studied in each root order of two co-generic citrus rootstocks, namely the fast-growing Citrus jambhiri 'Rough Lemon' (RL) and the slow-growing Citrus reshni 'Cleopatra Mandarin' (CM). METHODS: Morphological traits of individual root orders of CM and RL, grown at different nitrate levels (NO(3)-N at 0.1, 0.5, 1 and 10 mm) were examined by using an image-specific analysis system. KEY RESULTS: At high nitrate levels, the root length ratio, root mass ratio and, to a lesser degree, specific root length, root fineness and tissue density of tap and 1st-order laterals in both CM and RL were reduced. In 2nd-order laterals, however, the same treatment led to increased values of each morphological trait in CM but decreased values of the same traits in RL. At low nitrate supply, CM exhibited longer tap roots whereas RL developed longer 2nd-order laterals. These effects were due to root mass ratio and, to a lesser extent, specific root length. CONCLUSIONS: Biomass allocation was the main component of nitrate-induced changes in root length ratio. The 2nd-order laterals were more sensitive to nitrate availability than the tap root and 1st-order laterals. At low nitrate availability, RL displayed longer 2nd-order lateral roots and lower root plasticity than CM. This suggests a different root growth strategy among citrus rootstocks for adapting to nitrate availability: RL invests in 2nd-order laterals, the preferred zone for acquiring the nutrient, whereas CM responds with longer tap roots.

Cell wall immobilisation and antioxidant status of Xanthoria parietina thalli exposed to cadmium.

Total and cell wall-bound cadmium and the major antioxidants were measured in thalli of the lichen Xanthoria parietina (L.) Th. Fr. exposed to two Cd concentrations, namely 4.5 or 9.0 µm, in liquid medium during exposure periods of either 24 or 48 h. Total Cd in the thalli was within the range of previous field measurements and was proportional to the exposure concentration, but less than proportional with respect to exposure duration. More than half of the total Cd was immobilised by the cell wall. The adopted conditions of Cd stress caused: (i) no changes in dry weight and protein concentration; (ii) an increase in the level of ascorbic acid and a decrease in that of reduced glutathione, as well as an increase in guaiacol peroxidase activity; (iii) no changes or moderate decreases in the activities of superoxide dismutase, catalase, dehydroascorbate-, NADPH-dependent glutathione disulfide-, and monodehydroascorbate reductases and of ascorbate peroxidase; (iv) an increase of the level of thiobarbituric acid-reactive substances, assumed to reflect malondialdehyde formation arising from membrane lipid peroxidation. Thus, X. parietina might withstand realistic levels of Cd stress by: (1) intercepting the heavy metal at cell wall level, (2) the intervention of antioxidant metabolites, and (3) a moderate increase in guaiacol peroxidase activity.

Coumarin differentially affects the morphology of different root types of maize seedlings.

The effects of coumarin on the length, diameter, and branching density of different root types in maize seedlings (Zea mays L. cv. Cecilia) were investigated. The maize root system represents a useful model for morphological studies, as it consists of radicle, seminal, and nodal roots whose origin and development are quite different. Maize seedlings were grown in a hydroponic culture for 6 days, and then coumarin (at concentrations of 0, 25, 100, and 400 microM) was added to the nutrient solution. Coumarin inhibited root length, but effects differed depending on the root type. C 1/2 values, representing the coumarin concentration causing a 50% inhibition of the root length, were calculated by nonlinear regression. Six, 1, and 0.25 mM coumarin were sufficient to reduce the radicle, seminal, and nodal root lengths by 50%, respectively. At the highest coumarin concentration, the subapical root zone showed swelling. The degree recorded by average diameter was higher in nodal roots than in seminal and radicle roots. Furthermore, coumarin decreased the number of lateral roots and branching density more in the seminal than in the radicle roots. These results suggest the following order of sensitivity to coumarin: nodal > seminal > radicle roots. The observed spatial effects of coumarin could be ecologically significant, since taprooted species could benefit at the disadvantage of fibrous-rooted species and could modify community composition.

Spatial mapping of phosphorus influx in bean root systems using digital autoradiography.

A novel technique was developed to spatially map the phosphorus net influx capacity in intact root systems. The method is based on digital autoradiography and permits the quantification of phosphorus influx at high spatial resolution (2 mm). Roots of 18-d-old common bean plants were exposed to (32)P-labelled orthophosphate, quickly frozen, excised, lyophilized, scanned, and exposed to a storage phosphor screen. Plots of (32)P content versus root length (distance from the root tip or from the base of the root) were obtained for three different root classes: basal, basal laterals, and taproot laterals. Radioactivity detected by filmless autoradiography correlated well (r(2)=0.99) with measurements made by scintillation counting. Basal roots absorbed 2.5 times and 1.9 times more phosphorus than the taproot lateral and basal lateral root classes, respectively, in the first 20 mm from the root apex. External phosphorus markedly affected influx: roots averaged 5, 16, and 34 pmol P min(-1) in the apical 20 mm when exposed to 1, 5, and 10 microM P solutions, respectively. The spatial pattern of phosphorus influx along the root axes of the different root classes was rather homogeneous when measured on a root surface area basis. Phosphorus influx in the older segments of basal roots (those next to the hypocotyl) did not differ from the newer segments close to the root apex. However, a heterogeneous pattern was detected for basal roots when measured on a length basis, indicating that both root class and diameter constitute main factors controlling the spatial pattern of net influx.