This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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2 Environmental and Experimental Botany 103 (2014) Contents lists available at ScienceDirect Environmental and Experimental Botany jo ur nal home p ag e: Multiple abiotic stresses occurring with salinity stress in citrus J.P. Syvertsen a,, F. Garcia-Sanchez b a University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, FL 33850, USA b CEBAS, CSIC, Murcia, Spain a r t i c l e i n f o Article history: Received 4 June 2013 Received in revised form 26 September 2013 Accepted 30 September 2013 Keywords: Flooding stress Water use efficiency Drought Osmotic stress Nutrient imbalances Pests and diseases a b s t r a c t Citrus, one of the most important fruit crops in the world, is sensitive to many environmental stresses including salt stress. The negative effects of stresses often lead to poor tree growth and reductions in fruit yield and quality. Under natural conditions, citrus trees often experience multiple stresses at the same time so there are direct and indirect interactions between salinity and almost all physical abiotic stresses that include flooding, drought, nutrient deficiency, high irradiance, high temperature, and high atmospheric evaporative demand. In addition, salinity stress also has direct effects on roots predisposing trees to biotic environmental stresses including attack by root rot, nematodes and bacterial disease. The agronomical and physiological responses of citrus exposed to two or more stress factors, can differ depended on stress intensity or duration. Since citrus leaf Cl accumulation has been linked to water use, for example, other environmental factors including high CO 2 concentration, lowered temperature and high relativity humidity which decrease leaf transpiration, can improve the salt tolerance. Citrus rootstocks known to be tolerant to root rot and nematode pests, can become more susceptible to these biotic stresses when irrigated with high salinity water. Root pests can, in turn, affect the salt tolerance of citrus roots and may increase salt uptake. Moderate salinity stress, however, can reduce physiological activity and growth allowing citrus seedlings to survive cold stress and can even enhance flowering after the salinity stress is relieved. In this review, we discuss the currently available information about the effects of salinity in citrus trees from an agronomic and physiological point of view, and how these responses interact with other abiotic/physical and biotic environmental factors. Short-term potential benefits of moderate stresses including salinity, will also be discussed Elsevier B.V. All rights reserved. 1. Introduction Although citrus is grown in many areas of the world, it is a sub-tropical crop that is not tolerant of freezing temperature or poorly drained soils. According to United Nations Conference on Trade and Development, in 2007 there were 140 citrus producing countries, producing 115,650,545 tons. Around 70% of the world s total citrus production is grown in the Northern Hemisphere, in particular countries around the Mediterranean and the United States, although Brazil is also one of the largest citrus producers. Citrus is most often grown in warm climates with well-drained soils in areas that are subject to water deficit and that often require supplemental irrigation. Even when irrigation water is of good quality, the use of fertilizers and other agro-chemicals raises the likelihood of salts building up in the soil causing salinity stress (Syvertsen et al., 1989). In the Mediterranean basin, hot dry summers lead to annual drought stress conditions. Irrigation water from rivers or aquifers Corresponding author at: University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, FL 33850, USA. Tel.: address: jmsn@ufl.edu (J.P. Syvertsen). can often be of low quality from excessive concentrations of soluble salts (Cl and/or Na + ) with an electrical conductivity greater than 3 ds m 1, the critical level for citrus production (Garcia-Sanchez et al., 2002a). Relative to many crop plants, citrus trees have been classified as a salt-sensitive crop (Maas, 1993; Storey and Walker, 1999) because saline irrigation water reduces citrus tree growth and fruit yield relatively more than in many other crops (Grieve et al., 2007; Prior et al., 2007). In Mediterranean climates, winter rainfall can lead to flooding stress especially in areas with poorly drained soil. Thus, drought, salinity and flooding stresses can occur annually in Mediterranean citrus orchards. Environmental factors are seldom all at optimal levels so some form of environmental stress on plants is the rule rather than an exception. Depending on climate, soil conditions and irrigation water quality, salinity stress often occurs along with poor soil drainage, flooding, high temperature and evaporative demand, drought, boron toxicity, and/or nutrient deficiencies and imbalances. In addition, it is anticipated that the concentration of CO 2 in the atmosphere will double by the end of the century (IPCC, 2007), which will affect crop responses to all stresses including the salinity. In this article, we have chosen to focus on a multiple of stresses, both abiotic and biotic, that occur with salinity stress /$ see front matter 2013 Elsevier B.V. All rights reserved.

3 J.P. Syvertsen, F. Garcia-Sanchez / Environmental and Experimental Botany 103 (2014) in citrus. Understanding these interactions with salinity and other environmental stresses can provide insights into cultural practices or modified environmental conditions that can improve production. There are direct interactions between salinity, water deficit, irradiance, leaf temperature, and atmospheric evaporative demand that are impossible to separate in the field. Thus, physiological mechanisms underlying interactions with salinity or with any other environmental stresses can only be studied in controlled environments where individual stresses and their simple two-way interactions can be described. In addition, plant responses to simultaneous stresses are highly complex and responses can be different from responses to individual stresses. Rather than being additive, the presence of an abiotic/biotic stress can have the effect of reducing or enhancing susceptibility to salinity, and vice versa. All plants also undergo continuous exposure to various biotic stresses in their natural environment. Long lived citrus trees suffer different pests and diseases through time causing mortality of newly planted trees and also a decline and yield loss of mature trees. Phytophthora root rot, nematodes and citrus greening (Huanglong Bing, HLB) result in the losses of millions of trees per year around the world. Controlled environment studies on stress are most often carried out in a pest free environment so that potentially complicating interactions between biotic and abiotic stresses can be avoided. Thus, there are only a few studies on salinity and biotic stresses from pests and diseases. There was an extensive review on salinity effects in citrus (Levy and Syvertsen, 2004) but here we also elaborate on abiotic interactions between salinity and important pests, including root weevil larvae, root rot, mycorrhizae, nematodes and HLB. Short-term potential benefits of moderate stresses including salinity, will also be discussed. 2. Effect of NaCl stress in citrus Salinity affects all plant physiological responses and production in three ways: osmotic stress, toxic ion stress and nutritional imbalances. Dissolved salts in the nutrient solution exert an osmotic effect that reduces the availability of free (unbound) water through physical processes that impair water extraction from soil by roots. This situation is analogous to drought stress that lowers plant water potential. Growth and yield of all plants is reduced by decreased leaf water potential (Maas, 1986). The effect of osmotic stress is different when stress increases gradually avoiding salt shock and allowing the plant to adjust, compared to the situation when osmotic stress in the soil solution increases abruptly (Levy and Syvertsen, 2004). When salinity stress is gradual, the osmotic effect is practically negligible as the downward osmotic adjustment in citrus leaves is very effective at maintaining turgor (Garcia-Sanchez and Syvertsen, 2006). High leaf Cl and Na + accumulation can reduce the osmotic potential allowing citrus leaves to reduce leaf water potential with no alteration of leaf turgor potential thus avoiding leaf dehydration (Perez-Perez et al., 2007). Such responses are passive osmotic adjustment from the concentration of salt ions since energetic cost is not required to decrease leaf osmotic potential. On the other hand, those salt tolerant citrus rootstocks which limit the translocation of the toxic ions Cl and Na + into the leaves, can acclimate to osmotic stress in the root zone by closing stomata and reducing leaf transpiration (E leaf ; Syvertsen and Smith, 1983; Nieves et al., 1991) even in the absence of water deficits. In this way, the water loss by leaf transpiration and water uptake by the roots is balanced. In contrast, when salinity stress occurs abruptly, osmotic stress can occur from a rapid increase in salinity of the soil solution that can result from high saline irrigation water, excessive fertilization or when a light rain leaches accumulated salts into the root zone (Levy and Syvertsen, 2004). Such osmotic effects, which can also result from a rapid drought stress in well drained soils, can increase abscisic acid (ABA), ethylene production and leaf abscission (Gomez-Cadenas et al., 1998). Following such a shock, the leaf typically separates at the abscission zone between the lamina and the petiole. The petiole may remain green and attached to the stem for some time. After such events, leaf analysis of the abscised leaves may not reveal an increase in Cl or Na + content. It is possible that the ethylene rise was first induced by the osmotic component of salinity and then sustained by Cl accumulation. The addition of ABA to the nutrient solution before exposure to salt stress, can reduce ethylene release and leaf abscission (Gomez-Cadenas et al., 2002; see also Section 3.6). In non-salinized plants, ABA reduced net gas exchange in leaves whereas in salinized plants, the ABA treatment slightly increased both stomatal conductance (g s ) and net assimilation of CO 2 (A CO2 ). These results suggest a protective role for ABA in citrus under salinity stress. Interactions with leaf and root ABA are apparently operative in determining differences between tetraploid and diploid responses to water deficit stress (Allario et al., 2013) and to salinity (see Section 3.7 below). The effect of salinity on plant growth is not only related to osmotic effects, but growth reductions in citrus also can be related to a gradual accumulation of toxic levels of Cl, Na + or boron (B) in leaves (Levy and Syvertsen, 2004). Exactly how the salts exert their toxicity remains unknown. Salts may build up in the apoplast and dehydrate the cell, they may build up in the cytoplasm and in the chloroplast inhibiting enzymes involved in carbohydrate metabolism and photosynthetic process (Munns and Tester, 2008). Nevertheless, it is well known that Cl and/or Na + toxicity reduce the net CO 2 assimilation in citrus trees where non-stomatal factors could have a marked influence in the photosynthesis reduction. In some experiments, it has been reported that despite that stomatal conductance (g s ) lowered concomitantly with A CO2, values of substomatal CO 2 concentration (C i ) are maintained or even increased in stressed plants (Perez-Perez et al., 2007; Garcia-Sanchez et al., 2007). These results have been also supported by the damages in leaf anatomy or changes in the chlorophyll a fluorescence, which indicate possible impairment in the electron transport chain in salinized citrus plants (Lopez-Climent et al., 2008). In several plant species, oxidative stress has been also shown to be one of the causes of damage produced by salinity. However, in citrus trees, the antioxidant machinery appears to be sufficient to avoid high levels of damage caused by active oxygen species at moderate to low stress levels (Arbona et al., 2003). Oxidative stress responses as a consequence of salt stress can be diminished by pre-treatments of roots with the oxidants hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) (Tanou et al., 2009a). These oxidants elicited long-lasting systemic primer-like antioxidant defense responses in the leaves of 5-month-old sour orange (Citrus aurantium L.) plants grown in the absence or presence of 150 mm NaCl for 16d. Such a build-up of the antioxidant status by bio-chemical treatments may be useful in protecting plants against subsequent salt stress. Proteomic studies revealed overlapping roles of H 2 O 2 and NO in increasing salt tolerance and in the acclimation of citrus plants to salinity (Tanou et al., 2009b). Pre-exposure of roots to H 2 O 2 or NO could ensure plant survival in a high salt environment, and supports the notion that interaction between H 2 O 2 and NO production may be locally and systematically sensed in citrus plants (Tanou et al., 2012). These findings may provide useful targets for genetic modification following biotechnological approaches, ultimately aiming to improve citrus response to salinity stress. Since soil solution Cl and Na + are transported in the nutrient solution by the transpiration stream, they remain in the plant after transpired water has evaporated. In most plant species, the accumulation of Na + is often a greater concern than the accumulation Cl. Citrus and other fruit tree crops (e.g. peaches) are relatively

4 130 J.P. Syvertsen, F. Garcia-Sanchez / Environmental and Experimental Botany 103 (2014) unique in this respect since Cl accumulation in citrus leaves is usually a greater concern than Na + accumulation. This depends on specific rootstocks, however, as Na + toxicity can be important in those citrus rootstocks that are good at excluding Cl. This is especially true in salinity experiments that do not incorporate sufficient calcium to the nutrient solution (Ca + ; Rengel, 1992). In addition, field experiments with different rootstocks with contrasting ability to exclude Cl, Na + or both, have pointed out that Cl is more toxic than Na + (Banuls et al., 1997) when leaf Cl concentration reach 0.7% by dry weight (Ferguson and Grattan, 2005). This threshold concentration depends on environmental condition (e.g. drought, evaporative demand), chemical composition of nutrient solution and duration of salt exposure. Leaf Cl concentration level under well watered saline conditions in the greenhouse, can be as high as 3.5% after 5 months in young Valencia trees grafted on Carrizo without showing visible toxicity symptoms (Grosser et al., 2012). Since citrus rootstocks have a great influence on the amount of Cl and/or Na + accumulated in the foliage of grafted trees, salt tolerance in citrus has been linked to the exclusion of toxic ions from the shoot (Garcia-Sanchez et al., 2002b). Although the Cl and Na + exclusion mechanisms in the different rootstocks remain unresolved, leaf transpiration, ion sequestration in root tissues (Camara et al., 2004) and/or compartmentation at the tissue and cell levels are involved (Moya et al., 1999; Balal et al., 2012; Rodriguez-Gamir et al., 2012). The sequestration of Na + in root tissue vacuoles and its immobilization by cell walls are key mechanisms contributing to the ability of Swingle citrumelo roots to maintain lower levels of Na + in leaves than in leaves on rough lemon (RL) roots (Gonzalez et al., 2012). Among common commercial citrus rootstocks, sour orange (C. aurantium L.) is considered a good Cl and Na + excluder, mandarin Cleopatra (Citrus reticulata Blanco) is a good Cl excluder, citrange Carrizo (Citrus sinensis Poncirus trifoliata) and Swingle citrumelo (Citrus paradisi P. trifoliate) are Na + excluders, and Citrus Macrophylla (Citrus macrophylla wester) and RL (Citrus Jambhiri L.) rootstocks are both Cl and Na + accumulators (Storey and Walker, 1999; Nieves et al., 1991; Gonzalez et al., 2012). Despite the importance of Cl accumulation in classifying the relative salt tolerance in citrus rootatocks (Levy and Syvertsen, 2004), Cl is not more metabolically toxic than Na + in citrus leaves. Rootstocks like Poncirus trifoliata and its hybrid citranges exclude Na + more efficiently than Cl. Thus, Cl continues passing to the leaves and becomes the more significant toxic component of the saline solution (Munns and Tester, 2008). Nonetheless, Cl toxicity, rather than Na + toxicity and/or the concomitant osmotic perturbation, appears to be the primary factor involved in the molecular responses of citrus plant leaves to salinity (Brumos et al., 2009). In addition, membrane transporter genes are differentially regulated in the tolerant (Cl excluder) and the sensitive (Cl accumulators) rootstocks. Citrus manage Cl homeostasis as a nutrient rather than a toxic ion such as Na + (Brumos et al., 2010). Both sensitive and tolerant rootstocks actively accumulate Cl in the shoot when it is provided in the low milimolar range (4 5 mm), exceeding the critical requirement by one order of magnitude in the tolerant rootstocks and by two orders of magnitude the sensitive rootstocks. These rootstocks modulate Cl uptake according to nutrient availability, and the regulation of root-to-shoot Cl transport (Garcia-Sanchez et al., 2000) and reduced net Cl loading into the root xylem appear to be the determining factors in chloride exclusion (Brumos et al., 2010). Citrus responses to salinity, of course, also depend on other environmental factors, some of which may enhance the leaf Cl /Na + accumulation and others may decrease their concentration. Salinity may cause nutrient deficiencies or imbalances in many plant species, due to the competition of Na + and Cl with nutrients such as K +, Ca 2+, Mg 2+ and NO 3 acting on biophysical and/or metabolic components of plant growth (Grattan and Grieve, 1992). In citrus, salinity can causes leaf nutrient deficiencies and this idea is fully developed in Section 3.8. Ruiz et al. (1997) observed that in addition to the osmotic and toxic effects of the salinity, an imbalance of essential nutrients may also contribute to the reduction in citrus plant growth under saline condition. For example, when Na + /Ca 2+ ratio is high in the leaves, the Na + can displaces Ca +2 in the cell membrane altering their integrity (Greenway and Munns, 1980). 3. Salinity interactions with abiotic physical environmental factors 3.1. Boron toxicity When irrigation water is scarce, citrus growers tend to use poor quality water, including effluent from industry or domestic use. Although wastewater treatment can remove many contaminants and organic matter, salt ions and boron (B) concentration often remain high in this water (Levy and Syvertsen, 2004). Boron, Na + and Cl concentrations in reclaimed water often exceed the recommended level for grapefruit in Spain (Pedrero et al., 2013). Boron is an essential nutrient element but B can also be toxic to plants especially for citrus since which is very sensitive to high B in irrigation water. A concentration of B in the irrigation water that exceeds 1 mg l 1 can negatively affect the yield of most traditional Mediterranean crops including citrus. Trees can start to show B toxicity symptoms when leaf concentrations of B exceed 200 mg kg 1 dry weight (Obreza and Morgan, 2008; Levy and Syvertsen, 2004). In citrus trees, transpiration rate and exposure time affects B distribution in plant tissues so B accumulates mainly in old leaves causing a decrease of A CO2 and injury to the leaf anatomy (Papadakis et al., 2004a,b). In Verna lemon trees, citrus Macrophylla and Cleopatra mandarin rootstocks seem to be more tolerant of high B than sour orange and Carrizo citrange (Gimeno et al., 2012a,b). The relative boron tolerance was not linked to the B concentration reaching the leaves, however, but rather to a combination of rootstock-dependent physiological, biochemical and anatomical responses that determined B tolerance. High concentrations of boron are often associated with saline soils and frequently crops are exposed to both B and salinity stresses simultaneously (Gupta et al., 1985). There is little information about the combined effects salinity and boron toxicity in plants but there probably is not a unique citrus response to combinations of salinity and boron toxicity. Cooper et al. (1952) reported that in citrus seedlings supplied with saline irrigation water at 8 ds m 1 plus 6 mg l 1 of B, a lower level of B accumulated than in control leaves. This reduced B accumulation in leaves in the presence of salinity could have been the result of the reduced rates of transpiration. In broccoli cultivars, a combination of B and NaCl can trigger a hydric response involving aquaporins, together with changes in nutrient transport and plasma membrane stability these responses differ from responses when B and NaCl were applied individually (Rodriguez-Hernandez et al., 2013). Interactive effects on B and NaCl stress responses have been clearly established in several plant species but results are inconsistent and variably indicate antagonistic or synergistic interactions even within the same plant species (Wimmer and Goldbach, 2012). Contradictory reports on salt/b interactions are related to B supply levels and concomittant differences in predominant B-uptake pathways (Wimmer and Goldbach, 2012) Soil texture The soil media not only provides mechanical support for the tree, but also oxygen, water and all the required nutrients to support root growth. Thus, soil type is a major influence on the growth

5 J.P. Syvertsen, F. Garcia-Sanchez / Environmental and Experimental Botany 103 (2014) of citrus roots and trees and the quality of their fruit. In the Mediterranean, citrus trees can be grown on a wide range of soils, including deep sandy loam, loam and clay loam (Agusti, 2000). However, citrus does not grow well in very heavy, clayey, sandy, alkaline or water logged soils (Srivastava and Singh, 2009). Soil ion exchange capacity, soil texture, depth to water table, and/or the effect of soil matric potential can influence the availability of Na + and Cl ions in the soil solution and, consequently, influence salinity tolerance (Villagra and Cavagnaro, 2005). Thus, soil texture can interact with salinity stress and can affect the response of crops to salinity. In an experiment with Carrizo citrange rootstock seedlings grown in a greenhouse in three different types of substrates, a well-drained Candler sand soil, a Floridana sandy clay soil and one commercial soilless substrate of peat/perlite/vermiculite potting media, seedlings from Candler sand had the highest salt tolerance and those grown in Floridana soil had the lowest salt tolerance (Garcia-Sanchez and Syvertsen, 2009). The highest salt tolerance that occurred in seedlings grown in Candler soil was linked to the low leaf Cl concentration as consequence of low soil clay content (1%) which increased the leaching fraction and decreased the salt ion accumulation in the soil solution. Soil texture and root penetrability also can influence in the rates of Cl uptake and transport from root to shoot by changes in root permeability and increases in passive ion fluxes. Seedlings of Etrog citron and Rangpur lime growing in sand had a higher leaf Cl concentration than those in solution culture (Storey, 1995). In addition to the soil physical characteristics, its chemical properties can play a relevant role in tolerance to salinity. The severity of the effect of Cl accumulation on growth can also depend on the quantities of other nutrient ions in leaves. Those soils which favor a greater accumulation of certain nutrients such as Ca 2+ and K +, could mitigate the negative effects of the toxicity of the Cl and Na + (Garcia-Sanchez and Syvertsen, 2009). On the other hand, in certain substrates, plant growth is more limited by the substrate than the effect of salinity. To determine soil aeration effects on citrus salinity stress, we grew seedlings of Cleopatra mandarin and Carrizo citrange in four different substrates including perlite, clay-loam soil, river wash sand and aerated solution culture (Gimeno et al., 2010). The salt treatment reduced plant growth in both solution culture and sand but not in perlite or clay-loam soil which both supported the lowest plant growth. Thus, comparative studies of crop responses to salinity and relative salt tolerance should consider the potential contributions of soil type and growth substrate High temperature and evaporative demand Citrus leaves growing in full sun on the outside of the canopy can experience midday temperatures that exceed air temperature by as much as 9 C (Syvertsen and Albrigo, 1980). Leaf temperatures up to 45 C not only enhance respiratory rates and exceed optimum temperature for photosynthesis, but also lead to large vapor pressure differences (VPD) between leaves and air. Although citrus leaf stomata are sensitive to evaporative demand (Jifon and Syvertsen, 2003a), large VPDs can increase the transpirational demand from leaves but g s is negatively related to VPD (Syvertsen and Lloyd, 1995). Thus, leaf water use efficiency (WUE = A CO2 /E leaf ) under high temperatures is decreased by both A CO2 depletion and E leaf increase. Decreasing VPD by lowering leaf temperature or by increasing humidity can increase g s and A CO2 without increasing total water use thus, improve WUE. Leaf Cl accumulation has been linked to water use in citrus (Moya et al., 2003). In other crops like tomato, it has been observed that misting leaves with high quality water can improve tolerance to soil salinity by decreasing the accumulation of toxic ions (Romero-Aranda et al., 2002). Since salinity stress is usually greater for sun-exposed than for shaded leaves, additional shade can improve salinity tolerance. Artificial shade screens (Jifon and Syvertsen, 2001) or foliar sprays of kaolin clay particle film (Jifon and Syvertsen, 2003b) during the warmest season can reduce citrus leaf temperature and improve WUE. However, when 2-year-old Valencia orange trees grafted on Carrizo citrange were grown under a 50% shade cloth, their salt tolerance was not improved because the Carrizo citrange trees under shade had a largest leaf Na + concentration even though their leaf Cl was reduced (Garcia- Sánchez et al., 2006b). Thus, when using agronomic strategies which facilitate a reduction of leaf Cl concentration by decreasing the leaf temperature, it must be borne in mind that other negative aspects of salinity such as Na + toxicity could be enhanced (Syvertsen et al., 2010) Salinity and flooding stress In Mediterranean citrus areas, very heavy winter and spring time rainfall frequently occurs which can induce flooding especially in areas with poorly drained soils (Kijne, 2006). Flooding affects soils by altering soil structure, depleting O 2, accumulating CO 2, inducing anaerobic decomposition of organic matter, and increasing availability of Fe and Mn (Kozlowski, 1997). Such changes can reduce root hydraulic conductance (Syvertsen et al., 1983) perhaps by the down regulation of the expression of PIP aquaporins (Rodriguez-Gamir et al., 2012) and the increase of Fe and Mn reaching toxic level in roots (Gimeno et al., 2012a,b). This can lead to decreased A CO2, water and nutrient uptake and carbohydrates levels resulting in decreases in root and shoot growth (Garcia-Sanchez et al., 2007; Arbona et al., 2009). In coastal areas, flooding can also occur with saline water intrusion from the sea (Bianchette et al., 2009). Growth reductions from flooding with saline water can be greater than flooding with fresh water since flooding with saline water has an osmotic effect and can cause a rapid increase of Cl and Na + transport to the shoot (Craig et al., 1990). Winter time A CO2 of lemon (Citrus limon) leaves was more inhibited by flooding with saline water than by fresh water due to the higher leaf Na + concentration in saline flooded leaves (Velikova et al., 2012) Elevated CO 2 Growing trees at elevated CO 2 levels of twice ambient, can enhance A CO and growth as much as 40% over that at ambient concentrations of CO 2 and elevated CO 2 can compensate for P deficiency (Syvertsen and Graham, 1999). The CO 2 -enhanced growth of citrus seedlings (Koch et al., 1986) and trees (Idso et al., 1996) is relatively great compared to other C3 species. Enhanced growth of sour orange trees at elevated CO 2, however, resulted in reduced leaf N apparently due to either growth dilution (Idso et al., 1996) or to preferential reallocation of N preferentially to roots (Saxe et al., 1998). At the same time as growth enhancement occurs, high CO 2 decreases g s and leaf transpiration. Thus, elevated CO 2 almost always leads to higher WUE as it disconnects rapid tree growth from high water use (Syvertsen and Levy, 2005). Growing citrus under conditions of elevated CO 2 therefore, offers a tool to study mechanisms of salinity tolerance while separating plant growth from plant water use. Tolerance to the salinity has been linked to tree growth rate so many vigorous rootstocks that produce fast growing trees that use relatively high amount of water also tend to have poor salt tolerance (Castle et al., 1993). However, experiments from Moya et al. (2003), and Garcia-Sanchez and Syvertsen (2006) reported that Cl absorption in the relatively salt sensitive Carrizo citrange (C. sinensis P. trifoliata) and in the salt tolerant Cleopatra mandarin (C. reticulata) was linked to their water use. Growing plants at elevated CO 2 usually increases growth. If salt uptake is coupled with water uptake, then leaves grown at elevated CO 2 should

6 132 J.P. Syvertsen, F. Garcia-Sanchez / Environmental and Experimental Botany 103 (2014) have lower salt concentrations than leaves grown at ambient CO 2 (Ball and Munns, 1992). On the other hand, if ion accumulation is related to growth, then growing plants in elevated CO 2 should increase ion accumulation. In a greenhouse studies of salinity tolerance of four contrasting citrus rootstock seedlings, Rangpur lime (Citrus limonia), Cleopatra mandarin, sour orange and sweet orange (C. sinensis), grown under ambient (370 ppm) and elevated CO 2 (700 ppm), all species increased their growth and WUE in response to elevated CO 2 (Syvertsen et al., 2000). The relative salt tolerant species, Rangpur lime, had the lowest leaf Cl and Na + concentration but under elevated CO 2, these concentrations and growth parameters were unaffected. In the other species, the salinityinduced accumulation of Na + in leaves was less when seedlings were grown at elevated CO 2 than at ambient CO 2, implying that the lower Na + accumulation was linked to increased WUE. The accumulation of leaf Cl in salinized sour orange was greater at elevated CO 2 than at ambient CO 2 implicating a link between growth and Cl accumulation in leaves. Leaf Cl concentrations in Cleopatra mandarin and Sweet orange, however, were less at elevated CO 2 than at ambient conditions. Thus, the decreases in Cl accumulation at elevated CO 2 in these two rootstocks were related to the increase in WUE. These contrasting patterns of Na + and Cl imply these ions accumulate by different transport pathways and relationships between ion accumulation and water use in Citrus is species-dependent. Garcia-Sánchez et al. (2006a) also showed that at elevated CO 2, leaf Cl and Na + concentration in the citrus rootstocks Cleopatra mandarin and Carrizo citrange was linked with WUE and not with growth rate Drought stress Drought stress in citrus trees reduces g s, E leaf and net A CO2 implying that the stomata are important regulators of gas exchange (Arbona et al., 2005). However, such responses do not mean that there is a direct physiological relationship among these parameters, since despite the significant correlation between g s and A CO2, increases in A CO2 can occur with no change or even a slight increase in C i (Garcia-Sánchez et al., 2006b). This implied that g s was not the dominant limitation to A CO2 but rather the non-stomatal mesophyll conductance was the major limitation (Farquhar and Sharkey, 1982). However, even in species like citrus where patchy stomatal closure is not considered an important issue (Syvertsen and Lloyd, 1995), the use of calculated C i to describe non-stomatal limitations on A CO2 should be interpreted with caution as changes in mesophyll conductance can affect CO 2 diffusion and its concentration at the chloroplasts (Flexas et al., 2004). Therefore, the mesophyll conductance limitation of A CO2 of drought stressed citrus leaves should not be ruled out. Fruit quality and yield are reduced by long-term periods of drought stress, but such reductions depend, of course, on duration and severity of the stress. Mild drought stress can improve fruit quality (Hockema and Etxeberria, 2001). Moderate drought can retard growth and increase fruit abscission; fruit that reach maturity have low juice content and inferior quality (Carr, 2012). Leaf abscission was observed in field-grown trees when pre-dawn leaf water potentials reached 2.75 MPa (Ribeiro and Machado, 2007). Synthesis of abscisic acid (ABA) in the root is the primary sensitive signal to water stress which control the stomata closure and modifying levels of ethylene, which is the hormonal activator of leaf abscission (Gomez-Cadenas et al., 1996). In severe drought stressed Cleopatra mandarin, ABA was increased 8 and 24 fold in the leaves and roots, respectively, leading to stop the leaf transpiration and provoking the leaves abscission. This was a protective response avoided further drought stress and thus, could interact with salinity. Pretreatment with ABA can reduce leaf abscission and can increases salt tolerance in citrus (Gomez-Cadenas et al., 1998, 2002). Plants have developed various mechanisms to withstand drought such as preferentially allocating growth to roots resulting in increased root-shoot ratios, producing fewer and smaller leaves, concentrating solutes in leaves lowering osmotic potential, or increasing activity of oxidative stress enzymes in leaf cells (Garcia-Sanchez et al., 2010). All of these responses have been described in citrus but do not always occur simultaneously as some are scion-rootstock dependent (Perez-Perez et al., 2008a,b; Garcia-Tejero et al., 2010). For instance, in citrus leaves, osmotic adjustment by the regulation of leaf proline appears to be different from many other plant species since heterogeneous responses indicate that proline accumulation can depend on genotype, intensity and duration of the stress (Garcia-Sanchez et al., 2007; Perez-Perez et al., 2009). It has been assumed that low soil moisture (drought) in saline soil would aggravate salt toxicity, further impeding root growth, which might strongly restrict the acquisition of water from the subsoil and thus the ability of the plants to withstand drought stress (Srivastava and Singh, 2009). However, citrus seedlings suffering drought stress were less affected when seedlings were preconditioned with saline water than when not preconditioned (Perez-Perez et al., 2007). The previous salt stress maintained a better leaf water status during drought stress after osmotic adjustment and the rapid accumulation of Cl and Na + in leaves. However, high levels of salt ions impeded the recovery of leaf water status and photosynthesis after re-irrigation with non-saline water since translocation of Cl and/or Na + from roots to leaves continued in salinized plants. Therefore, recovery of salinized citrus trees does not end after leaching with good quality water since Cl and Na toxicity from roots can continue. Further investigations are needed about how recovery of citrus occurs in Mediterranean climates after a period of summer salinity stress to avoid tree decline even after sufficient rains have arrived Salinty tolerance and ploidy level There has been a series of interesting papers discussing the effects of ploidy level and related changes in gene expression on salinity tolerance in citrus. Tetraploid (4 ) citrus plants are typically smaller than corresponding diploid (2 ) plants and leaves, stems and roots of (4 ) citrus are typically thicker and more succulent than 2 (Syvertsen et al., 2000). Such differences are reflected in lower water use and root conductivity in 4 than in 2 and physiological difference also contributed to greater salt tolerance in 4 than in 2 (Saleh et al., 2008). At the beginning of this experiment, 4 plants were significantly smaller when compared to their corresponding 2 so 4 plants undoubtedly used less water when salt stressed. In a similar subsequent experiment under well watered conditions, however, tetraploid seedlings were more sensitive to salt stress than diploid, as 4 accumulated more toxic ions and were more affected by salinity than 2 (Mouhaya et al., 2010). The 4 leaves accumulated more Cl than 2 leaves and the maximum quantum yield of PSII was reduced more in 4 than in 2. Thus, salt stress responses Saleh et al. (2008) were apparently complicated by drought stress. Physiological differences between responses to salinity stress were associated with subtle changes in gene expression which may be at the origin of the phenotypic differentiation of 4 citrus when compared with 2 (Allario et al., 2011). In a greenhouse study, three allotetraploid zygotic hybrids from a cross of two allotetraploid somatic hybrids ( tetrazyg ) rootstock selections were able to exclude Na + and Cl ions from Valencia leaves better than most other rootstocks while maintaining good growth with no phytotoxic symptoms (Grosser et al., 2012). These three tetraploid hybrid rootstock selections certainly merit further evaluation of salinity tolerance and horticultural performance in the field.

7 J.P. Syvertsen, F. Garcia-Sanchez / Environmental and Experimental Botany 103 (2014) Allario et al. (2013) also investigated the water deficit tolerance of 2 and 4 clones of Rangpur lime (RL, C. limonia)rootstocks grafted with 2 Valencia (V) sweet orange (C. sinensis) scions. Valencia on 4 RL was much more tolerant to water deficit than V/2 RL as V/4 RL leaves had lower stomatal conductance and greater abscisic acid (ABA) content. Root ABA content was also higher in V/4 RL and was associated to a greater expression of drought responsive genes. Tetraploidy modified the expression of genes in Rangpur lime citrus roots to regulate long-distance ABA signaling and adaptation to drought stress and undoubtedly salt stress. Further investigations are now needed to better understand the differential regulation of genes in autotetraploid roots compared with diploid roots (Allario et al., 2013) Fertilization and nutrient imbalances Almost all plant crops must be fertilized to achieve maximum productivity. If crops are grown on low fertility soils, they can appear to be more salt tolerant than those grown with adequate fertility. This is because that soil fertility, not salinity, is the primary factor limiting plant growth. Thus, proper fertilizer applications can increase yields whether the soil is saline or not, but proportionately more so if soil is non-saline (Maas, 1993). Low soil fertility may activate a series of physiological and biochemical adjustments that may limit plant growth. Slower growing plants use less water so are exposed to less salinity than faster growing plants. In addition, in a saline habitat, the presence of NaCl alters the nutritional balance of plants, resulting in high ratios of Na + /Ca 2+, Na + /K 2+, Na + /Mg 2+, Cl /NO 3 and Cl /H 2 PO 4 (Grattan and Grieve, 1992), which can cause reductions in growth. To avoid these nutrient imbalances, irrigation water, soil solution and leaves need to be assayed and management strategies formulated accordingly. In citrus, it has been reported that 10 mm KNO 3 has high enough nitrate to act as a chloride and sodium antagonist (Cerezo et al., 1999; Iglesias et al., 2004). However, high KNO 3 concentration in the nutrient solution could also have negative effects on plant growth from the osmotic effect in the root medium. Although 17 mm KNO 3 in the nutrient solution decreased leaf Cl and Na + and increased leaf K + and N concentration in 2-year-old potted Fino 49 lemon trees, salt tolerance was not improved as consequence of the decreasing leaf osmotic potential (Gimeno et al., 2009a,b). Raveh and Levy (2011) did not find a beneficial effect of an increase the amount of KNO 3 fertilization in grapefruit trees. Similar to the Cl and NO 3 relationship, K + is known to have a negative interaction with Na +. An increase in Na + in soil solution can reduce K + uptake by the plant (Grattan and Grieve, 1992). In comparison with NaCl in citrus, however, both CaCl 2 and KCl increased leaf Cl concentration although elevated leaf Ca 2+ concentration ameliorated the negative effects of Cl toxicity by reducing leaf abscission (Romero-Aranda et al., 1998). 4. Salinity interactions with biotic stresses 4.1. Root rot Phytophthora root rot in citrus occurs as the result of root infection by the pathogenic fungi Phytophthora citrophthora and/or Phytophthora nicotianae. As the disease progresses the foliage turns yellow and twigs die back. Phytophthora root rot usually starts at the deeper roots, since these are more likely to be wet, and the fungus is more active in wet conditions. Trees may become more dependent on surface roots, and are therefore more sensitive to drought stress from drying out (Graham et al., 2012). The rootstock and the scion can affect the susceptibility of the scion-rootstock to root rot and root weevil larvae (Shaked et al., 1984). Salinity stress may inhibit plant defense mechanisms against Phytophthora (Afek and Sztejnberg, 1993) and decrease root regeneration under pathogen pressure. In citrus, high salinity did not stimulate growth of the pathogen in vitro so the increase in disease under saline conditions probably was not attributable to a direct effect of salt on the fungus but rather to a reduction in the resistance of the host. High salinity apparently causes reduced accumulation of the phytoalexin, 6,7-dimethoxycoumarin and therefore, increases susceptibility of citrus plant tissues to invasion by the fungus (Sulistyowati and Keane, 1992). In greenhouse experiments, irrigation with high salinity water predisposed citrus rootstocks to attack by a group of root pathogens (Combrink et al., 1996). Rootstock seedlings of Troyer citrange (C. sinensis P. trifoliata), Carrizo citrange, Volkamer lemon (Citrus volkamerianna) and Rough lemon were most affected by the treatment consisting of three root pathogens in combination (Phytophthora sp., Fusarium solani, and Tylenchulus semipenetrans) under saline conditions. Growth of these seedlings was significantly less when subjected to both the group of pathogens and salinity stress together than when seedlings were subjected to either the pathogens or salt stress alone. The ability of Phytophthora to tolerate high levels of salinity could significantly diminish the resistance of Phytophthora tolerant rootstocks under saline conditions (Blaker and MacDonald, 1986) especially since saturated soils and salinity stress frequently occur together Nematodes Nematodes are microscopic worms that live in the soil and attack roots. The most common nematodes affecting citrus trees are citrus nematodes (T. semipenetrans) (Duncan, 2005). Although all varieties of citrus are attacked, some rootstocks such as trifoliate orange are highly resistant to citrus nematode attack. Others such as Troyer and Carrizo citrange are moderately tolerant and some are highly susceptible as sweet orange. Feeding on the roots does not kill the citrus tree but the capacity of the root to carry water and nutrients is impeded and yields are noticeably reduced early in the life of the tree (Duncan, 2005). The citrus nematode can reduce the salt tolerance of citrus roots and increase Cl uptake (Willers and Holmden, 1980). Leaf Cl levels of severely infected trees varied between 1.75 and 2.00% compared to only % in less infected trees under the same conditions. This was true for both salinity tolerant rootstocks and salinity sensitive rootstocks. Nematodes increased the Cl concentration more than three fold in leaves but decreased the Cl concentration in roots (Mashela and Nthangeni, 2002). Thus, nematode infection modified the allocation patterns of Cl within citrus trees. Soil salinity apparently caused a breakdown in root chemical defenses (Dunn et al., 1998) and increased the susceptibility of citrus roots to attack by the citrus nematode (Mashela et al., 1992b). In addition, intermittent salinity stress increased the nematode population densities more than continuous irrigation with saline water (Mashela et al., 1992a) Mycorrhizae Arbuscular mycorrhizal fungi (AMF) can establish symbiotic associations with 70 90% of land plant species (Wang and Qiu, 2006). Mycorrhizal symbionts receive about 20% of the host s photosynthates in citrus plants grown in high-p soil and in exchange, facilitate nutrient and water uptake by their hosts (Peng et al., 1993). AMF can increase the host plant s ability to cope with several abiotic stresses, including salinity, drought, high temperature and heavy metals (Miransari, 2010). Although, salinity significantly inhibited mycorrhizal colonization in citrus trees, AMF association improve plant growth and root morphology by decreasing

8 134 J.P. Syvertsen, F. Garcia-Sanchez / Environmental and Experimental Botany 103 (2014) leaf Na + concentration, improving the ionic balance as leaf K + and Mg 2+ concentrations and the K + /Na + ratio are increased, and enhancing the activate enzymatic and non-enzymatic antioxidant defenses to control ROS formation caused by salinity (Wu et al., 2010a,b). Citrus roots are relatively dependent on vesicular arbuscular mycorrhizae (VAM) colonization, especially under conditions of low soil phosphorus (P) concentration or in previously sterilized soils (Krikun and Levy, 1980). VAM can increase P and plant growth particularly in arid soils (Jeffries et al., 2002) or under saline conditions and thus, alleviate salinity stress. However, VAM throughout the root system can also increase the concentrations of Cl in leaves and roots of Sweet orange and sour orange seedlings irrigated with high salinity water (Graham and Syvertsen, 1989). This increase could not be attributed to increased transpiration in the VAM plants. Salinized VAM plants of Carrizo citrange and sour orange accumulated more Cl in leaves than non-mycorrhizal plants but Cl was higher in non-mycorrhizal roots of Sweet orange and Carrizo citrange than in VAM roots (Hartmond et al., 1987). On the other hand, leaf Na + concentrations were not affected by VAM. Salt tolerance also can depend on the species of fungi. It have been reported that Glomus versiforme is the best effective mycorrhizal fungus in alleviating salt stress of trifoliate orange and G. etunicatum is the lowest effective mycorrhizal fungus (Zou and Wu, 2011) HLB and stress tolerance Huanglongbing (HLB) is the most important citrus disease that affects all varieties of citrus and is now found in all counties where commercial citrus is produced (Manjunath et al., 2008; Salifu et al., 2012). HLB, also known as citrus greening, is caused by a fastidious, phloem-limited bacterium that is transferred from tree to tree by a small phloem-feeding insect called the Asiatic citrus psyllid. Either the physical presence of the bacteria population or some form of bacterial toxin disrupts phloem function in leaves causing starch to accumulate, roots to decline and limiting the tree s ability to take up nutrients. Initial visible symptoms of HLB include yellowing of leaves from excessive starch, root decline, premature fruit drop, and small green misshapen fruit that contain bitter juice with no economic value. The rate of spread of HLB is affected by the size of the psyllid population and by tree age because the psyllids prefer new leaf growth (Brlansky et al., 2011). Young trees, which are more vigorous as compared to mature trees, produce more leaf flushes and thereby, are more susceptible to psyllid feeding and disease transmission. In the case of mature trees, the disease spreads more slowly (Gottwald, 2010). If the rate of infection in a particular location is relatively high at the time the disease is first discovered, a policy of eradication of symptomatic trees may result in destruction of the entire orchard. Since HLB affects carbohydrate allocation, tree growth and yield, HLB interacts with environmental stresses in a variety of ways. There is no question that stressed or declining trees succumb to HLB more rapidly than previously healthy trees. Just as rootstocks affect tree growth vigor, water relations and nutrient uptake, there appears to be some relationship between susceptibility to HLB and certain rootstock types (Albrecht and Bowman, 2011) and scion varieties (Stover et al., 2012). When the psyllid vector is controlled well by frequent pesticide applications, well-nourished trees and trees receiving luxurious amounts of nutrients applied to leaves appear to be able to survive the presence of the bacterium. Ultimately, the survival of citrus will depend on the development of tolerant genotypes of rootstocks and scions but in the meantime, it is important to understand interactions between HLB and other environmental stresses. 5. Potential benefits of moderate stress-drought and salinity By definition, a stress is negative. Under some conditions, however, a moderate level of stress can become beneficial. Other than the nutritional benefits from moderate applications of fertilizer salts, salinity is usually not beneficial for citrus in the long run. Since citrus can produce profitable yield using proper cultural practices and salt tolerant cultivars, there may be some short-term benefits from moderate salinity including increased chilling/freezing tolerance, reduced water use and increased flowering. Drought stress and cool winter time temperatures can enhance flower induction (Chica and Albrigo, 2013) and cold hardiness (Yelenosky, 1979). Apparent drought stress from moderate salinity at levels of mm of NaCl applied for 2 months, can reduce growth and total plant transpiration but enhanced cold hardiness of Sweet orange and Cleopatra mandarin seedlings (Syvertsen and Yelenosky, 1988). This occurred even though osmotic potential and leaf proline concentration did not change significantly. Thus, moderate salinity stress under greenhouse conditions can substitute for cool temperature-induced freeze tolerance in seedlings by reducing physiological activity and growth. However, young grapefruit trees on different rootstocks with high Cl content were more susceptible to freeze injury than those with low Cl (Peynado, 1982). Du Plessis (1985) suggested that reduced transpiration caused by salinity stress could potentially be a benefit for reducing the accumulation of soil salinity since the lower water uptake should increase the leaching fraction. This implies that an increase in the leaching fraction occurs when irrigating with saline water scheduled at the same frequency as non-saline water. However, soil salinity usually increases proportionally to the salinity in the irrigation water and thereby reduces growth and yield. Just as drought stress combines with cool wintertime temperatures to enhance flower induction (Melgar et al., 2010; Chica and Albrigo, 2013), it is possible that moderate salinity stress will also increase flowering. In a warm, wet climate with inadequate chilling or drought stress to maximize flower induction, controlled salinization might offer a substitute to induce flowering in citrus. Such a practice is used to induce flowering of litchee (Litchi chinensis) in Thailand (E. Tomer, personal communication, 2001) and low levels of salinity can increase flowering in several varieties of avocado (Persea sp.; Downton, 1978) and pear fruit trees (Pyrus sp.; Okubo et al., 2000). 6. Conclusions and future perspective There are many things citrus growers can do to ameliorate problems associated with abiotic stresses from choosing the best rootstock and scion cultivars to appropriately managing irrigation and fertilizer application methods (Levy and Syvertsen, 2004). Salinity tolerance is a whole plant phenomenon that requires an appreciation of citrus rootstock/scion interactions in the field. Such relationships are complicated by interactions between physical environmental factors and also with biotic pests and diseases. There are direct and indirect interactions between salinity, leaf water relations, pests and diseases that almost always have synergistic effects on citrus. To help citrus growers cope with stress problems, researchers should study the underlying mechanisms of stress tolerance and understand their modes of interaction with biotic stresses. An understanding of mechanisms and interactions of stress and disease tolerance, will also benefit researchers developing new rootstocks and scions with improved stress and disease tolerance. Not all stress effects are negative, however, as moderate short-term low temperature and osmotic stress can reduce physiological activity and growth allowing citrus seedlings to survive