Uptake and translocation of pharmaceutically active compounds by olive tree (Olea europaea L.) irrigated with treated municipal wastewater.

Introduction: The use of treated municipal wastewater (TWW) represents a relevant opportunity for irrigation of agricultural crops in semi-arid regions to counter the increasing water scarcity. Pharmaceutically active compounds (PhACs) are often detected in treated wastewater, posing a risk to humans and the environment. PhACs can accumulate in soils and translocate into different plant tissues, reaching, in some cases, edible organs and entering the food chain. Methods: This study evaluated the uptake and translocation processes of 10 PhACs by olive trees irrigated with TWW, investigating their accumulation in different plant organs. The experiment was conducted in southern Italy, in 2-year-old plants irrigated with three different types of water: freshwater (FW), TWW spiked with 10 PhACs at a concentration of 200 µg L-1 (1× TWW), and at a triple dose (3× TWW), from July to October 2021. The concentration of PhACs in soil and plant organs was assessed, collecting samples of root, stem, shoot, leaf, fruit, and kernel at 0 (T0), 50 (T1), and 107 (T2) days of irrigation. PhACs extraction from soil and plant organs was carried out using the QuEChERS method, and their concentrations were determined by high-resolution mass spectrometry coupled with liquid chromatography. Results: Results of uptake factors (UF) showed a different behavior between compounds according to their physicochemical properties, highlighting PhACs accumulation and translocation in different plant organs (also edible part) in 1× TWW and 3× TWW compared to FW. Two PhACs, carbamazepine and fluconazole, showed interactions with the soil-plant system, translocating also in the aerial part of the plant, with a translocation factor (TF) greater than 1, which indicates high root-to-leaf translocation. Discussion: Findings highlight that only few PhACs among the selected compounds can be uptaken by woody plants and accumulated in edible parts at low concentration. No effects of PhACs exposure on plant growth have been detected. Despite the attention to be paid to the few compounds that translocate into edible organs, these results are promising for adapting wastewater irrigation in crops. Increasing knowledge about PhACs behavior in woody plants can be important for developing optimized wastewater irrigation and soil management strategies to reduce PhACs accumulation and translocation in plants.

Carbon isotope discrimination and water use efficiency in interspecific Prunus hybrids subjected to drought stress.

In C3 plants, carbon isotope composition (δ13C) is influenced by isotopic effects during diffusion from the atmosphere to the chloroplasts and carboxylation reactions. This work aimed to demonstrate if δ13C of leaf soluble carbohydrates (δ13Cleaves) and of dry matter from new-growth shoots (δ13Cshoots) of Prunus plants subjected to a period of water deficit was related to water use efficiency (WUE). For this purpose, three interspecific Prunus hybrids rootstocks (6-5, 7-7 and G × N) were gradually subjected to drought and then rewatered. Soil water content (SWC) decreased from 26.1 to 9.4% after 70 days of water shortage, when plants reached values of predawn leaf water potential (LWP) ranging from -3.12 to -4.00 MPa. Gas exchange, particularly net photosynthetic and transpiration rates, differed among the three hybrids, leading to different values of WUE. After 70 days of drought, a significant δ13C increase of 5.86, 4.28 and 4.99‰ was observed in 6-5, 7-7 and G × N, respectively. Significant correlations between δ13C and other parameters (substomatal CO2/atmospheric CO2 ratio, stomatal conductance and stem water potential) were found in all hybrids. The rewatering phase caused a recovery of the physiological status of the plants. The isotope composition of δ13Cshoots was correlated with the average WUE measured during the whole experiment. δ13Cleaves and δ13Cshoots were positively related (r = 0.87; p < 0.001). The isotopic signature was a reliable screening tool to identify Prunus genotypes tolerant to drought stress. The results suggest the possibility of using δ13C as an integrated indicator of level of drought stress in plants subjected to prolonged stress conditions.

Soil management type differentially modulates the metabolomic profile of olive xylem sap.

In conventional olive growing, frequent soil tillage strongly reduces the complexity and diversity of the agro-ecosystem. Here, a metabolomic analysis was carried out on the xylem sap (XS) of olive plants (Olea europaea L.) from a grove located in Southern Italy (Basilicata region). The orchard has been divided in two plots that have been managed for 18 years with two different systems: a) 'sustainable management' (Smng), with no-tillage, fertigation and internal C-inputs (spontaneous weeds and pruning residues), and b) an adjacent rainfed 'conventional management' (Cmng), that included soil tillage and mineral fertilization. The XS was extracted from olive shoots in two sampling times (ST1: May; ST2: October) using a Sholander pressure chamber, and its metabolome analyzed by ultra-high performance liquid chromatography (UHPLC) coupled to a hybrid quadrupole-time-of-flight mass spectrometer (QTOF-MS). The discriminating compounds were 94 at ST1 and 119 at ST2, and 35 of them were in common between the two sampling times. The majority of the discriminating metabolites (73 on 94 at ST1, and 109 on 119 at ST2) were found at higher concentration in the XS of Smng plants, compared to that of Cmng ones. Most of the discriminating metabolites found in XS (about 80%, both at ST1 and ST2) were involved in plant secondary metabolism, mainly for plant chemical defense, growth regulation and signal transduction. The most prevailing class of compounds included terpenoids, phytohormones, alkaloids, sterols/steroids, retinols/retinoids, tocopherols and carotenoids. For the first time, we have demonstrated that the XS of a tree crop significantly responds to a shift of soil management. Generally, the plants of the Smng plot showed an up-regulated secondary metabolism. The results of our study encourage the use of a set of sustainable agricultural practices in a productive orchard, in order to enhance plant physiological status, increase yield quantity/quality, safeguard the environment and ameliorate human health.

Evaluation of the possible persistence of potential human pathogenic bacteria in olive orchards irrigated with treated urban wastewater.

Under suitable conditions, low-quality, treated urban wastewater (TWW) is an additional water resource for irrigation in water-scarce environments but its use in agriculture requires a careful monitoring of a range of hygiene parameters, including human pathogenic bacteria (HPB). DNA-based microbiological analyses on soil, xylem sap, and leaves surface (phyllosphere) were carried out in an olive (Olea europaea L.) grove located in Southern Italy (Basilicata region). The experimental grove has been managed in two plots for 18 years. The experimental plot (WWtr) was drip irrigated daily with TWW (2800 m3 ha-1 year-1), while the control plot (RFtr) was rainfed. The results of the 16S-rRNA-based metagenomic analysis demonstrated that the phyllosphere had the lowest number of potential HPB (6), compared to soil (22) and xylem (26) compartments. Gammaproteobacteria, including potential HPB, like Pseudomonas and Acinetobacter spp., were significantly higher in WWtr soil and xylem sap, compared to RFtr. A similar trend was observed for Burkholderia spp. (Betaproteobacteria) and Mycobacterium spp. (Actinobacteria). The Firmicutes Enterococcus, Staphylococcus and Streptococcus spp. were more abundant in WWtr xylem sap. The pathogenic Clostridium perfringens was found higher on WWtr leaves (relative abundance 7.17 in WWtr and 1.33 in RFtr) and Enterococcus faecalis in WWtr xylem sap (93.22 in WWtr and 7.08 in RFtr). On the basis of the results obtained, the irrigation with TWW can be considered a realistic and safe agronomic practice in Mediterranean orchards, and an opportunity for farmers and consumers.

Correlations between morpho-anatomical changes and radial hydraulic conductivity in roots of olive trees under water deficit and rewatering.

The effects of prolonged drought were studied on olive (Olea europaea L.; drought-sensitive cultivar Biancolilla and drought-tolerant cultivar Coratina) to examine how morpho-anatomical modifications in roots impact on root radial hydraulic conductivity (Lpr). Two-year-old self-rooted plants were subjected to a gradual water depletion. The levels of drought stress were defined by pre-dawn leaf water potentials (Ψw) of -1.5, -3.5 and -6.5 MPa. After reaching the maximum level of drought, plants were rewatered for 23 days. Progressive drought stress, for both cultivars, caused a strong reduction in Lpr (from 1.2 to 1.3 × 10(-5) m MPa(-1) s(-1) in unstressed plants to 0.2-0.6 × 10(-5) m MPa(-1) s(-1) in plants at Ψw = -6.5 MPa), particularly evident in the more suberized (brown) roots, accompanied with decreases in stomatal conductance (gs). No significant differences in Lpr and gs between the two olive cultivars were observed. Epifluorescence microscopy and image analyses revealed a parallel increase of wall suberization that doubled in white stressed roots and tripled in brown ones when compared with unstressed plants. In drought-stressed plants, the number of suberized cellular layers from the endodermis towards the cortex increased from 1-2 to 6-7. Recovery in Lpr during rewatering was correlated to the physical disruption of hydrophobic barriers, while the time necessary to obtain new mature roots likely accounted for the observed delay in the complete recovery of gs. Radial hydraulic conductivity in olive roots was strongly influenced by soil and plant water availability and it was also modulated by structural root modifications, size, growth and anatomy. These findings could be important for maintaining an optimal water status in cultivated olive trees by scheduling efficient irrigation methods, saving irrigation water and obtaining yield of high quality.

Fruit calcium accumulation coupled and uncoupled from its transpiration in kiwifruit.

Accumulation of Ca in several fleshy fruit is often supposed to depend, among others, by climatic variables driving fruit transpiration. This study tests the whole causal chain hypothesis: VPD → fruit transpiration → Ca accumulation. Also there are evidences that relationship between fruit transpiration and Ca content is not always clear, hence the hypothesis that low VPD reduces the fraction of xylemic water destined to transpiration was tested by examining the water budget of fruit. Attached fruits of Actinidia deliciosa were subjected to Low (L) and High (H) VPD. Their transpiration was measured from early after fruit-set to day 157 after full bloom (DAFB). Fruits were picked at 70, 130 and 157 DAFB for Ca and K determinations and for water budget analysis. Cumulative transpired water was ∼ 70 g and ∼ 16 g H2O f(-1) in HVPD and LVPD, respectively. Calcium accumulated linearly (R(2) = 0.71) with cumulative transpiration when VPD was high, while correlation was weaker (R(2) = 0.24) under LVPD. Under low VPD the fraction of xylem stream destined to transpiration declined to 40-50%. Results suggest that Ca accumulation is coupled to cumulative transpiration under high VPD because under that condition cumulative transpiration equals xylem stream (which carry the nutrient). At LVPD, Ca gain by fruit is uncoupled from transpiration because ∼ 60% of the xylemic water is needed to sustain fruit growth. Results will apply to most fruits (apples, tomatoes, capsicum, grapes etc.) since most suffer Ca deficiency disorders and grow in changing environments with variable VPD, also they could be supportive for the implementation of fruit quality models accounting also for mineral compositions and for a reinterpretation of certain field practices aimed at naturally improve fruit Ca content.

Hydraulic resistance of developing Actinidia fruit.

BACKGROUND AND AIMS: Xylem flows into most fruits decline as the fruit develop, with important effects on mineral and carbohydrate accumulation. It has been hypothesized that an increase in xylem hydraulic resistance (RT) contributes to this process. This study examined changes in RT that occur during development of the berry of kiwifruit (Actinidia deliciosa), identified the region within the fruit where changes were occurring, and tested whether a decrease in irradiance during fruit development caused an increase in RT, potentially contributing to decreased mineral accumulation in shaded fruit. METHODS: RT was measured using pressure chamber and flow meter methods, the two methods were compared, and the flow meter was also used to partition RT between the pedicel, receptacle and proximal and distal portions of the berry. Dye was used as a tracer for xylem function. Artificial shading was used to test the effect of light on RT, dye entry and mineral accumulation. KEY RESULTS: RT decreased during the early phase of rapid fruit growth, but increased again as the fruit transitioned to a final period of slower growth. The most significant changes in resistance occurred in the receptacle, which initially contributed 20 % to RT, increasing to 90 % later in development. Dye also ceased moving beyond the receptacle from 70 d after anthesis. The two methods for measuring RT agreed in terms of the direction and timing of developmental changes in RT, but pressure chamber measurements were consistently higher than flow meter estimates of RT, prompting questions regarding which method is most appropriate for measuring fruit RT. Shading had no effect on berry growth but increased RT and decreased dye movement and calcium concentration. CONCLUSIONS: Increased RT in the receptacle zone coincides with slowing fresh weight growth, reduced transpiration and rapid starch accumulation by the fruit. Developmental changes in RT may be connected to changes in phloem functioning and the maintenance of water potential gradients between the stem and the fruit. The effect of shade on RT extends earlier reports that shading can affect fruit vascular differentiation, xylem flows and mineral accumulation independently of effects on transpiration.

Fruit transpiration in kiwifruit: environmental drivers and predictive model.

BACKGROUND AND AIMS: In most fruit crops, storage quality varies greatly between regions and seasons, causing significant commercial loss. Understanding the sources of this variability will contribute to the knowledge of fruit developmental physiology and may also benefit commercial fruit production via altered managements that reduce it or forecasts that predict it. A causal-chain relationship is proposed to help elucidate the sources of variability in fruit storage quality: the weather →(i)→ fruit transpiration →(ii)→ fruit calcium →(iii)→ fruit storage quality. This paper explores the first link of this hypothesis, →(i)→, for Hayward kiwifruit using field measurements of fruit transpiration rate and concurrent meteorological recordings. The aims are to identify the key environmental variables driving fruit transpiration and develop a predictive fruit transpiration model. METHODOLOGY: Fruit transpiration was determined hourly over several 24-h periods by recording weight loss of detached fruit, on Days 23, 35, 49, 65, 94 and 140 after full bloom. Meteorological records were made every 15 min throughout the season at an adjacent regional weather station. A model of fruit transpiration was developed in which the usual meteorological variables (radiation, temperature, windspeed and relative humidity) were incorporated in a Fick's Law transpiration flux equation. PRINCIPAL RESULTS: Fruit transpiration rate (i.e. the molar flux density, mmol cm(-2) h(-1)) varied diurnally and decreased during the season. The dominant fruit variable governing transpiration rate was skin conductance and the dominant environmental variables were relative humidity and temperature. Radiation and windspeed were not significantly influential. CONCLUSIONS: The model provides a good fit to the fruit transpiration rate measurements regardless of the time of day/night or the stage of fruit development. The model allows reasonably accurate and continuous predictions of fruit transpiration rate throughout fruit development based on standard meteorological recordings. It also allows estimates of cumulative fruit transpiration throughout the season.

An external heat pulse method for measurement of sap flow through fruit pedicels, leaf petioles and other small-diameter stems.

The external heat ratio method is described for measurement of low rates of sap flow in both directions through stems and other plant organs, including fruit pedicels, with diameters up to 5 mm and flows less than 2 g h(-1). Calibration was empirical, with heat pulse velocity (v(h)) compared to gravimetric measurements of sap flow. In the four stem types tested (Actinidia sp. fruit pedicels, Schefflera arboricola petioles, Pittosporum crassifolium stems and Fagus sylvatica stems), v(h) was linearly correlated with sap velocity (v(s)) up to a v(s) of approximately 0.007 cm s(-1), equivalent to a flow of 1.8 g h(-1) through a 3-mm-diameter stem. Minimum detectable v(s) was approximately 0.0001 cm s(-1), equivalent to 0.025 g h(-1) through a 3-mm-diameter stem. Sensitivity increased with bark removal. Girdling had no effect on short-term measurements of in vivo sap flow, suggesting that phloem flows were too low to be separated from xylem flows. Fluctuating ambient temperatures increased variability in outdoor sap flow measurements. However, a consistent diurnal time-course of fruit pedicel sap flow was obtained, with flows towards 75-day-old kiwifruit lagging behind evaporative demand and peaking at 0.3 g h(-1) in the late afternoon.

Osmotic regulation in leaves and roots of olive trees during a water deficit and rewatering.

We evaluated the osmotic adjustment capacity of leaves and roots of young olive (Olea europaea L.) trees during a period of water deficit and subsequent rewatering. The trials were carried out in Basilicata (40 degrees 24' N, 16 degrees 48' E) on 2-year-old self-rooted olive plants (cv. 'Coratina'). Plants were subjected to one of four drought treatments. After 13 days of drought, plants reached mean predawn leaf water potentials of -0.45 +/- 0.015 MPa (control), -1.65 +/- 0.021 (low stress), -3.25 +/- 0.035 (medium stress) and -5.35 +/- 0.027 MPa (high stress). Total osmotic adjustment increased with increasing severity of drought stress. Trees in the high stress treatment showed total osmotic adjustments ranging between 2.4 MPa at 0500 h and 3.8 MPa at 1800 h on the last day of the drought period. Osmotic adjustment allowed the leaves to reach leaf water potentials of about -7.0 MPa. Active osmotic adjustment at predawn decreased during the rewatering period in both leaves and roots. Stomatal conductance and net photosynthetic rate declined with increasing drought stress. Osmotic adjustment in olive trees was associated with active and passive osmotic regulation of drought tolerance, providing an important mechanism for avoiding water loss.

Antioxidant defences in olive trees during drought stress: changes in activity of some antioxidant enzymes.

The effects of drought on the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POD), indoleacetate oxidase (IAAox) and polyphenol oxidase (PPO) were studied in 2-year old Olea europaea L. (cv. 'Coratina') plants grown under high temperatures and irradiance levels and gradually subjected to a controlled water deficit. After 20 d without irrigation, mean predawn leaf water potential fell from -0.37 to -5.37 MPa, and decreases in net photosynthesis and transpiration occurred. The activities of SOD, APX, CAT and POD increased in relation to the severity of drought stress in both leaves and roots. In particular, a marked increase in APX activity was found in leaves of plants at severe drought stress. CAT activity increased during severe water deficit conditions in leaves and fine roots. The patterns of POD and IAA oxidase activity ran in parallel and showed increases in relation to the degree of drought. In contrast, PPO activity decreased during the progression of stress in all the tissues studied. The results show that the ability of olive trees to up-regulate the enzymatic antioxidant system might be an important attribute linked to drought tolerance. This could limit cellular damage caused by active oxygen species during water deficit.

Lipoxygenase activity and proline accumulation in leaves and roots of olive trees in response to drought stress.

The olive tree (Olea europaea L.) is commonly grown in the Mediterranean basin and is able to resist severe and prolonged drought. Levels of proline (PRO) and malondialdehyde (MDA), and the lipoxygenase (LOX) activity were determined in 2-year-old olive plants (cv. 'Coratina') grown in environmental conditions characterized by high temperatures and high photosynthetic photon flux density levels and gradually subjected to a controlled water deficit for 20 days. Before and during the experimental period, leaf and root samples were collected and analysed for PRO and MDA. The levels of PRO increased in parallel with the severity of drought stress in both leaves and roots. Significant increases of LOX activity and MDA content were also observed during the progressive increment of drought stress in both leaf and root tissues. Measurements of transpiration and photosynthetic rate, stomatal conductance and substomatal CO(2) concentration were carried out during the experiment. The accumulation of PRO indicates a possible role of PRO in drought tolerance. The increases of MDA content and LOX activity show that the water deficit is associated with lipid peroxidation mechanisms.