NA has been shown to be an important divalent metal chelator and is involved in metal transport and homeostasis in plants.Previous evidence also showed over expression of genes involved in NA synthesis increased nickel tolerance in tobacco and Arabidopsis thaliana.Liao et al. revealed that NA and histidine have the highest binding constants for Cu2+ in chicory and tomato.Thus, the observed up-regulation of NA and histidine in leaves is a possible Cu2+ detoxification mechanism for lettuce plants. Phytochelatins and metallothioneins have been shown to play an important role detoxifying excess Cu2+.PCs are synthesized from reduced glutathione in a transpeptidation reaction.In addition, GSH is involved in a plethora of cellular processes, including defense against ROS,and sequestration of heavy metals.Glycine and glutamate are the main constituents of GSH. The observed elevated levels of glycine may indicate GSH and PCs are upregulated to detoxify excess Cu2+. Pidatala et al. also observed that glycine and glutamate increased in response to Pb. Increasing Tolerance. In addition to chelating copper, lettuce plants must employ other strategies to increase their tolerance to higher copper levels. The level of spermidine and putrescine, which are important polyamines, were elevated in all treated plants . Previous studies showed that putrescine and spermidine play an important role in plant stress response to diverse environmental stresses by acting as antioxidants to scavenging free radicals.Therefore, elevated polyamines may contribute to enhanced tolerance of lettuce to copper. In this study,hydroponic nft channel ethanolamine levels were increased in all NP treated plants. Rajaeian et al. suggested that EA increased salt tolerance of tobacco plants by stimulation of antioxidative responses.

Kogan et al. showed pretreatment with ethanolamine enhanced the tolerance of Helianthus annuus L. to salt stress.Elevated K+ is another possible important tolerance mechanism. Increased K+ in plants can lower ROS production by reducing activity of NAD oxidases and maintaining photosynthetic electron transport.K+ has been implicated in regulating plant stress responses. Guard cells take up mainly K+ .It has been demonstrated that K+ triggers the stomata to open. In guard cells of open stomata, K+ was 2−4 times higher, malic acid 6 times higher, and citric acid 3 times higher, compared to closed stomata.It has been repeatedly hypothesized that organic acid synthesis would accompany stomatal opening.The accumulated K+ in guard cells may promote stomata opening. Increased stomatal opening/ transpiration is expected to promote photosynthesis and thereby increase plant growth.Borowski and Michalek showed that foliar application of potassium salts to spinach leaves resulted in more intensive gas exchange in leaves and, as a consequence of that, increased leaf yield. Antioxidant Defense. As reported before, Cu generates ROS in cells through the Fenton reaction. Our study also showed ROS was triggered by Cu2 NPs . ROS scavenging enzymes and antioxidant molecules are a common plant response to ROS stress.Phenolic acids and ascorbic acid are important low molecular antioxidants.Previous studies indicate ROS stress increases accumulation of antioxidant molecules.Up-regulated low molecular weight antioxidants can serve as scavengers of free radicals to protect plants from oxidative damage.Interestingly, our results showed the levels of three phenolic compounds and dehydroascorbic acid, which are important antioxidant molecules, were significantly decreased in all nanopesticidetreated lettuce leaves . GABA levels also decreased. It is possibly that biosynthesis of these metabolites was activated in response to ROS stress induced by Cu at an early stage of defense. However, since the stress was sustained for one month, this induced the imbalance between ROS and the antioxidant defense system.

Therefore, the antioxidant system was impaired due to the continuously generated ROS and limited ability to regulate them.Exposure to copper-based nanopesticides is likely to increase. For lettuce, exposure via foliar application, as intended, did not result in visible leaf damage. In fact, in several cases leaf biomass increased significantly. Cu2 nanopesticides can clearly enter stomata, even when aggregated. We demonstrated that Cu was translocated to the roots, although almost all the Cu mass was accumulated in leaves. Despite no visible damage, metabolomics revealed some significant changes in levels of amino acids, organic acids, carbohydrates and other important metabolites, particularly in leaves. The effect in roots was much smaller. The plants may be up-regulating some of these metabolites to increase the tolerance of plant to Cu2 nanopesticide. Metabolomics can be used as a sensitive and powerful tool to understand the response of plants to nanoparticles at a molecular level. However, it is not clear if the observed metabolic changes were entirely induced by Cu ions or if NPs also contributed. Future work should address how best to use Cu ions as a control at the same level of bio-availability, to better distinguish the contribution of nano-Cu from that of the Cu ions to the observed metabolomics changes.Micro-irrigation has become the optimal standard for irrigation and fertigation of horticultural crops in Australia, due to increased water scarcity and higher costs of fertilizers over the last decade. Intensive fertigation schedules have been developed to increase yield and quality of many permanent horticultural crops, including mandarin. This combines drip irrigation and fertigation to deliver water and nutrients directly to the roots of the crop, with the aim of synchronizing the applications with crop demand and maintaining the desired concentration and distribution of ions and water in the soil . The overall aim of these interventions is to develop an irrigation and nutrient management program that increases yield and fruit quality, while reducing leaching. The fundamental principle of drip fertigation is to apply water and nutrients regularly to a small volume of soil at a low application rate and at a high frequency to closely meet crop demand .

However, the potential for movement of water and mineral nutrients, especially nitrogen , below the root zone and into the ground- and then surface-waters using these approaches is still high. This is due to a number of factors: amount and intensity of precipitation, the large amounts of water and nutrients being applied, the limited capacity of roots to take up these nutrients, and to the ability of irrigators to manage drainage and hence leaching. Citrus is one of the important horticultural crops being grown under intensive fertigation systems in Australia. The vast majority of citrus plantings are oranges , with the rest split between mandarins , lemons and limes , and grapefruit . About 75% of the Australian citrus industry is located in the Murray-Darling Basin, utilising the lighter-textured free-draining soils adjacent to the Murray, Darling and Murrumbidgee rivers,nft growing system and thus potential off-site effects of poorly managed fertigation may have wider implications. Irrigated horticulture has, in general, been identified as the major source of nitrogen in drainage waters in the Murray Darling Basin . A significantly high nitrate level has been reported in drainage water and soil solution under grapevines in the Murray Darling Basin. These values are significantly higher than the Australian environmental trigger value for nitrate . Leaching of nitrates from soils under perennial horticulture may pose a potential threat to groundwater. The main sources of nitrate in mandarin production are mineral fertilizers. Nitrate is removed from the soil by plant uptake or through decomposition by micro-organisms in the process of denitrification. In well-aerated soils typical of this region, denitrification is often negligible because of a lack of favourable conditions . Nitrate, being an anion, moves freely in these mineral soils, and hence has the potential to leach into groundwater and waterways if fertigation is not well scheduled . Several researchers have reported substantial leaching of applied N in citrus cultivation under field conditions . Syvertsen and Jifon found that N leaching was higher under weekly fertigated orange trees than under daily or monthly fertigated trees. Syvertsen and Sax reported that increasing the number of fertigation events could significantly reduce N leaching. However, they observed 38–52% leaching of N from fertilizer, and the nitrogen use efficiency ranging between 25% and 44% in Hamlin orange trees. Other researchers have reported that nitrate accumulates toward the boundary of the wetted volume for most combinations of drip emitter discharge, input concentrations, and volumes applied. These studies suggest that there is a need for efficient tools, capable of describing and quantifying nitrate leaching, as well as nitrate uptake by crops, which in turn would help in designing and managing drip irrigation systems and achieving a high N fertilizer use efficiency, thereby limiting the export of this nutrient as a pollutant to downstream water systems. In addition to nitrate leaching, salinity is also an important factor influencing the sustainability of the citrus production worldwide, as citrus species are relatively salt sensitive. The reported value of the average threshold electrical conductivity of saturation extract and slope for oranges are 1.7 dS m 1 and 16%, respectively . Salt damage is usually manifested as leaf burn and defoliation, and is associated with accumulation of toxic levels of Na+ and/or Cl in leaf cells. Under drip irrigation there are many factors influencing the distribution of soil water and salts, and hence the water use efficiency , such as water quality, dripper discharge rate , irrigation water depth , and irrigation frequency . Simulation models have been valuable research tools in studies involving complex and interactive processes of water flow and solute transport through the soil profile, as well as the effects of management practices on crop yields and the environment .

HYDRUS-2D has been used extensively in evaluating the effects of soil hydraulic properties, soil layering, dripper discharge rates, irrigation frequencies, water quality, and timing of nutrient applications on wetting patterns and solute distribution . Although these studies demonstrate well the importance of numerical modelling in the design and management of irrigation and fertigation systems for various crops, most studies involving salinity and nitrate leaching are based on either an analysis of hypothetical scenarios, or are carried out for annual crops. Hence, there is a need to carry out modelling studies for perennial horticultural crops such as mandarin, using experimental results from field studies involving modern irrigation systems such as drip. The objectives of the present investigation were to evaluate water, salt , and nitrate movement in soil below young mandarin tree using HYDRUS-2D, and to evaluate various irrigation and fertigation strategies for controlling deep drainage and nitrate leaching, whilst maintaining soil salinity below the threshold for mandarin. This approach will help us understand the best irrigation and fertigation management practices to be adopted in future practical applications, with the goal to increase root water and nutrient uptake.The field experiment was conducted at the Dareton Agricultural and Advisory Station , located in the Coomealla Irrigation Area, 3 km from Dareton and 10 km from Went worth in New South Wales . The research station forms part of the Sunraysia fruit growing district of NSW and Victoria located in the Murray Darling Basin. An experimental site with an intensive fertigation system, consisting of various mandarin varieties budded onto a number of root stock varieties , was established in October 2005. The trees were planted at a spacing of 5 m-2 m. The actual monitoring and measurements were initiated in August 2006. The trees were managed and fertilized following current commercial practices, although the amounts of applied fertilizer varied. The soils of the site are alkaline , with red sandy loam from the surface to 90-cm depth, and loam below . The total organic carbon content is very low in the first 30 cm, and below 0.25% in the remainder of the root zone. The climate is characterized as dry, with warm to hot summers and mild winters. The total rainfall during the experimental period from 21 August 2006 to 20 August 2007 was 187 mm , which was slightly below average for the area.Mild frost conditions occur during the winter months. Weather data were collected from an automated weather station located within the research station.The HYDRUS-2D software package was used to simulate the transient two-dimensional movement of water and solutes in the soil. This program numerically solves the Richards’ equation for variably-saturated water flow, and advection–dispersion equations for both heat and solute transport. The model additionally allows specification of root water uptake, which affects the spatial distribution of water, salts and nitrate between irrigation cycles. The solute transport equation considers the advective–dispersive transport in the liquid phase, as well as diffusion in the gaseous phase.