Abstract
Citrus crops in the Mediterranean region are often grown in high pH calcareous soils, which promote nutrient imbalances, especially iron deficiency. ‘Navelina’ trees grafted on eight different citrus rootstocks were assessed in terms of their relative tolerance to these soils. To do so, leaf gas exchange and chlorophyll a fluorescence parameters were measured for 2 consecutive years in leaves without visible injury symptoms. Trees were grown on the rootstocks Carrizo citrange, hybrids of Cleopatra mandarin × Poncirus trifoliata no. 5 (F-A 5), 13 (F-A 13), and 16 (030116), Troyer citrange × common mandarin no. 18 (F-A 418), King mandarin × P. trifoliata no. 7 (F-A 517), 020324 (Troyer citrange × Cleopatra mandarin), and Volkamer lemon × P. trifoliata no. 64 (230164). gS and transpiration rate were clearly lower in leaves of shoots grafted on Carrizo citrange than in those on the other rootstocks assayed, but net photosynthetic flux did not differ. However, leaves of shoots on Carrizo citrange displayed a decline in their maximum quantum yield of PSII photochemistry [variable fluorescence/maximum fluorescence (Fv/Fm) ratio] and other chlorophyll parameters in the steady state such as photochemical quenching (qP) and the quantum efficiency of PSII photochemistry (ΦPSII) as well as an increase in nonphotochemical quenching (qN). Other rootstocks such as 030116, F-A 517 and, especially, F-A 5 showed the highest ΦPSII and Fv/Fm values, whereas F-A 5 had the lowest qN in the steady state. The photosynthetic characteristics observed in leaves on Carrizo citrange showed them to be the least tolerant to these calcareous conditions, whereas photosynthesis of ‘Navelina’ trees budded on F-A 5 were the most tolerant.
Citrus is the major fruit crop grown in the Mediterranean area, especially in the Valencia region in Spain, and is one of the most important fruit crops in the world. It is estimated that between 20% and 50% of fruit trees grown in the Mediterranean basin suffer from iron (Fe) deficiency (Jaegger et al., 2000). The most prevalent cause of Fe deficiency in this region is the presence of high levels of carbonate ions in calcareous soils, leading to a high pH and low availability of Fe and the condition known as lime-induced chlorosis (Pestana et al., 2005). The citrus trees planted in these soils often show signs of severe Fe deficiency or Fe chlorosis because of low Fe availability (Wallace, 1986). Iron deficiency affects the biochemistry, morphology, and physiology of the whole plant because Fe is an important cofactor of many enzymes, including those involved in the biosynthetic pathway of chlorophylls (Abadía, 1992; Larbi et al., 2006; Molassiotis et al., 2006).
In calcareous soils, citrus-growing success depends on availability of suitable rootstock that is tolerant of low Fe. Trifoliate orange [Poncirus trifoliata (L.) Raf.], sweet orange [C. sinensis (L.) Osb.], and Carrizo citrange (C. sinensis × P. trifoliata) are all susceptible to lime-induced chlorosis, whereas sour orange (C. aurantium L.) and Cleopatra mandarin (C. reshni Hort. ex Tan.) are more lime-tolerant (Castle, 1987; Hamzé et al., 1986; Pestana et al., 2005; Treeby and Uren, 1993). Within this context, a breeding program is being carried out at the Instituto Valenciano de Investigaciones Agrarias (IVIA; Valencia, Spain) in the search for better-performing citrus rootstocks in alkaline soils. As a result of this program, four new hybrid rootstocks are now available in Spanish citrus nurseries: Forner-Alcaide 5 (F-A 5) and F-A 13 (both C. reshni Hort. ex Tan. × P. trifoliata), F-A 418 [Troyer citrange (C. sinensis × P. trifoliata) × C. deliciosa Ten.], and F-A 517 (C. nobilis Lour. × P. trifoliata). These rootstocks have been tested in calcareous soils. ‘Navelina’ trees grafted on F-A 5 or F-A 13 yielded ≈40% more than trees on Carrizo citrange, whereas trees on F-A 5 or F-A 13 produced smaller but similar quality fruit than those on Carrizo (Forner et al., 2003; Forner-Giner et al., 2003).
Evaluation of growth and yield parameters may not be sufficient to rank citrus rootstocks according to their tolerance to Fe chlorosis (Pestana et al., 2003, 2005). In this respect, physiological parameters like leaf chlorophyll, CO2 gas exchange, and chlorophyll a fluorescence emission are known to be affected in leaves of trees that are not tolerant to Fe chlorosis or calcareous soils (Baveresco et al., 2006; Chouliaras et al., 2004; Larbi et al., 2006; Molassiotis et al., 2006; Morales et al., 2000).
The main objective of this study was to evaluate calcareous soil tolerance of ‘Navelina’ orange trees grafted on Carrizo citrange and seven new citrus rootstocks by assessing gas exchange and chlorophyll a fluorescence.
Materials and Methods
Plant material.
Measurements were taken for leaves of 13-year-old ‘Navelina’ orange trees, Citrus sinensis (L.) Osb. (selection Iniasel 7), grafted on Carrizo citrange and seven new citrus rootstocks obtained by J. Forner at the IVIA: F-A 5, F-A 13, F-A 418, F-A 517, 030116 (Cleopatra mandarin × P. trifoliata), 020324 (Troyer citrange × Cleopatra mandarin), and 230164 (C. volkameriana Ten. & Pasq. × P. trifoliata). The trees were planted in 1993 with nursery plants (1 year after grafting) and arranged in a complete randomized experimental plot. There were six trees for each rootstock type in the plot. Tree spacing was 2.5 × 6 m. The soil was sandy loam with 40% of CaCO3, 8% deactive calcium carbonate, and pH 8. A relative level of carbonate between 5% and 10% is considered high. Standard cultural practices were followed with drip irrigation and fertilizing without iron chelates, mechanical weed control between rows, and chemical control between trees. Drip irrigation frequency was adjusted according to the season of the year, from once per week in winter to 5 d per week in summer, with 40 L/tree per irrigation. Water pH was 7.6, electrical conductivity 1.4 dS·m−1, Cl– 134.8 mg·L−1, and NO3– 158.0 mg·L−1. Fertilization was applied from March to September in the following amounts: ammonium nitrate (33%) 2 kg/tree, monoammonium phosphate 0.5 kg/tree, and potassium nitrate 0.8 kg/tree. During these months, the percentage distribution was 10, 12, 15, 18, 20, 15, and 10 for ammonium nitrate; 10, 20, 15, 15, 15, 15, and 10 for monoammonium phosphate; and 7, 10, 13, 15, 25, 20, and 10 for potassium nitrate.
Gas exchange measurements.
Measurements were taken at the end of October during 2 consecutive years (2005 to 2006) on eight expanded spring leaves for each rootstock type in the field. A portable gas exchange system (CIRAS-2; PP Systems, Hitchin, U.K.) was used with a PLC 6(U) universal leaf autocuvette in closed-circuit mode (Larbi et al., 2006). All measurements were taken on attached mature leaves that were ≈6 months old without visible injury symptoms and with an apparent homogeneous green color. All measurements were performed at saturation irradiance (1000 μmol·m−2·s−1) between 1030 hr and 1130 hr, whereas leaf temperature ranged between 24 and 26 °C. The infrared method was used to determine leaf temperature. The gas exchange parameters determined at saturation irradiance were: transpiration rate (E) [mmol(H2O)·m−2·s−1], net photosynthetic flux (Pn) [μmol(CO2)·m−2·s−1], gS to water vapor [mmol(H2O)·m−2·s−1], and CO2 substomatal concentration (Ci) [μmol(CO2)·mol−1 (air)]. The enclosed portion of the leaf was 2.50 cm2 and the cuvette flow was 200 mL/min. The ambient CO2 concentration was maintained at 375 ppm and citrus leaves were chosen according to Larbi et al. (2006).
The environmental conditions during measurements were: temperature means, 24.1 °C (2005) and 24.9 °C (2006). Air relative humidity means were 65.8% (2005) and 64.2% (2006). No rainfall was recorded on the measurement days. These environmental conditions were similar on all measurement days.
Pigment analysis.
The concentration of Chl per area was estimated in the same attached leaves as those for which gas exchange measurements were taken using a SPAD portable apparatus (Minolta Co., Osaka, Japan). Strong positive correlations between SPAD readings and chlorophyll concentration were found in leaves on all rootstocks (r2 > 0.8).
Chlorophyll a fluorescence measurements.
Like in the case of gas exchange, chlorophyll a fluorescence (Chl F) was measured at ambient temperature (24 to 26 °C) in the field during the same time period on 2 consecutive years using a portable fluorometer (PAM-2001; Walz, Effeltrich, Germany). The same eight spring flush leaves for each rootstock used for previous measurements (four leaves per year) were darkened for 30 min before fluorescence measurement. The minimum (dark) fluorescence (Fo) was obtained after chlorophyll excitation with a weak beam from a light-emitting diode. Maximum fluorescence (Fm) was determined following a 600-ms pulse of saturating “white light.” The yield of variable fluorescence (Fv) was calculated as Fm – Fo. After 2 min of dark readaptation, actinic “white light” (430 μmol·m−2·s−1) was switched on and a saturating pulse (14,000 μmol·m−2·s−1) was applied at 60-s intervals for 13 min to determine: 1) maximum fluorescence yield during actinic irradiation (Fm′); 2) the level of modulated fluorescence during a brief interruption of actinic irradiation in the presence of far-red radiation (Fo′); and 3) the Chl F yield of fluorescence during the actinic illumination (Ft). Quenching resulting from nonphotochemical dissipation of absorbed light energy (qN) was calculated at each saturating pulse according to the equation qN = (Fm – Fm′)/(Fm – Fo′) (van Kooten and Snell, 1990). The coefficient for photochemical quenching, qP, which represents the fraction of open PSII reaction centers, was calculated as (Fm′ – Ft)/(Fm′-Fo′) (Schreiber et al., 1989). The quantum efficiency of PSII photochemistry, ΦPSII, closely associated with the quantum yield of noncyclic electron transport, was estimated from (Fm′ – Ft)/Fm′ (Genty et al., 1989). The fraction of photons absorbed by PSII, which was not used in photochemistry or dissipated in the PSII antenna (%X), was estimated from (Fv′/Fm′) (1 – qP) × 100 (Demmig-Adams et al., 1996).
Statistical analysis.
Data collected in the 2 consecutive years were tested for significance using analysis of variance using time and rootstocks as independent variables (statistical significance, P ≤ 0.05), but the time variable (year) did not account for a significant amount of variation. Means were compared using the least significant differences method at a 95% confidence level. Statistical analyses were performed using StatGraphics Plus for Windows, Version 2.1 (Statistical Graphics, Englewood Cliffs, NJ).
Results
Leaf chlorophyll.
The SPAD measurements were used as an equated estimate of leaf chlorophyll concentration, because there was a linear relationship between these two parameters (r2 > 0.8; Jifon et al., 2005). Leaves of shoots grafted on F-A 5, F-A 13, and 020324 had the highest Chl concentration. Leaves of shoots budded on 230164 and Carrizo citrange showed the lowest Chl concentration (Table 1).
Total chlorophyll and gas exchange parameters of leaves from ‘Navelina’ trees grafted on eight rootstocks grown in calcareous soil.z
Gas exchange measurements.
The leaves of shoots grafted on F-A 5 had the highest net photosynthetic flux (Pn), which differed significantly from leaves on 230164 and Carrizo citrange but not from those grafted on the other rootstocks (Table 1). Leaves of shoots budded on F-A 517 had the lowest Ci, whereas leaves of shoots grafted on the other rootstocks had significantly higher Ci with the exception of leaves of shoots budded on F-A 13. gS and transpiration (E) were significantly lower in Carrizo citrange than in the other rootstocks.
Changes in chlorophyll a fluorescence parameters.
Table 2 shows the Chl a fluorescence of dark-adapted parameters for the ‘Navelina’ spring flush leaves. The maximum quantum yield of PSII photochemistry estimated using the Fv/Fm ratio was the smallest in leaves on Carrizo citrange. The decline in this ratio was mostly the result of a decrease in the fluorescence parameter Fm, not of an increase in the minimum level of fluorescence, Fo. The higher Fv/Fm values from leaves on F-A 5 and 030116 than on the other rootstocks was the result of an increase in Fm.
Changes in Fo, Fm, and Fv/Fm at the steady state in dark-adapted leaves from ‘Navelina’ trees grafted on eight rootstocks grown in calcareous soil.z
An apparently steady state was reached after ≈4 min of actinic illumination for quantum yield (ΦPSII) of the PSII electron transport and the fraction of open PSII centers (qP) in leaves on all rootstocks (Fig. 1A–B). Two minutes into the kinetics study, the values of ΦPSII and qP for leaves on Carrizo citrange were the lowest. The steady state of the ΦPSII value for leaves on Carrizo citrange was ≈27% lower than ΦPSII value for leaves on F-A 5, 030116, and F-A 517.
Effects of rootstock on the kinetics of fluorescence parameters ΦPSII, the quantum efficiency of PSII photochemistry (A), qP, photochemical quenching (B), and qN, nonphotochemical quenching (C) of ‘Navelina’ trees grown in calcareous soil. Data are mean values and sd for n = 8. For all of measurements, sd of ΦPSII, qP, and qN was lower than 0.03, 0.04, and 0.02, respectively. Symbols denote Carrizo citrange (●), F-A 517 (○), F-A 13 (▼), 030116 (△), F-A 5 (■), 020324 (□), 230164 (◆), and F-A 418 (◇).
Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.280
Effects of rootstock on the kinetics of fluorescence parameters ΦPSII, the quantum efficiency of PSII photochemistry (A), qP, photochemical quenching (B), and qN, nonphotochemical quenching (C) of ‘Navelina’ trees grown in calcareous soil. Data are mean values and sd for n = 8. For all of measurements, sd of ΦPSII, qP, and qN was lower than 0.03, 0.04, and 0.02, respectively. Symbols denote Carrizo citrange (●), F-A 517 (○), F-A 13 (▼), 030116 (△), F-A 5 (■), 020324 (□), 230164 (◆), and F-A 418 (◇).
Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.280
Effects of rootstock on the kinetics of fluorescence parameters ΦPSII, the quantum efficiency of PSII photochemistry (A), qP, photochemical quenching (B), and qN, nonphotochemical quenching (C) of ‘Navelina’ trees grown in calcareous soil. Data are mean values and sd for n = 8. For all of measurements, sd of ΦPSII, qP, and qN was lower than 0.03, 0.04, and 0.02, respectively. Symbols denote Carrizo citrange (●), F-A 517 (○), F-A 13 (▼), 030116 (△), F-A 5 (■), 020324 (□), 230164 (◆), and F-A 418 (◇).
Citation: HortScience horts 44, 2; 10.21273/HORTSCI.44.2.280
Immediately after actinic irradiation, qN rose quickly and reached maximum values within 1 min (Fig. 1C). The maximum value was obtained for leaves of shoots grafted on Carrizo citrange. Nonphotochemical quenching declined until reaching steady-state values after 8 min of actinic irradiation with higher values for leaves on Carrizo citrange.
Discussion
In this article, we describe how rootstock affected the photosynthetic process in leaves from ‘Navelina’ trees grown on calcareous soil. In this respect, net photosynthetic values for leaves of shoots grafted on the eight different citrus rootstocks under study were similar to those reported in the literature (Calatayud et al., 2006; Syvertsen, 1987; Vu et al., 2002). Only leaves on F-A 5 rootstocks showed a higher Pn rate than those on Carrizo citrange, whereas the intercellular CO2 concentration (Ci) in leaves on Carrizo citrange was similar to the majority of other rootstocks. However, the gs and the E for leaves on Carrizo citrange were the lowest, as was chlorophyll content, when compared with the other citrus rootstocks assayed under the same conditions. Therefore, the lowest gs and E values for leaves of shoots grafted on Carrizo citrange did not decrease in Pn or Ci. This can be explained by the fact that, as occurs under water stress, only very critically low levels of gs affect Pn and Ci, which is in agreement with Flexas et al. (2004).
On the other hand, although Pn for leaves of shoots grafted on Carrizo citrange was not lower than for those grafted on the other rootstocks under study, fluorescence was impaired because leaves of shoots grafted on Carrizo citrange had a lower Fm, Fv/Fm ratio, ΦPSII, and qP, but higher qN than those on the other rootstocks. The same behavior has been reported for Troyer citrange, in which the Fv/Fm ratio decreased significantly with low Fe availability in calcareous soils (Pestana et al., 2005).
Maximum efficiency of photosystem II (Fv/Fm) is only slightly affected by Fe deficiency in orange tree leaves (Pestana et al., 2001). However, we show that the variable part of the fluorescence was clearly affected by Carrizo citrange rootstocks on citrus trees grown on calcareous soils. The Fv/Fm ratio was much lower in leaves of shoots grafted on this rootstock without changes in Fo as compared with leaves on the other rootstocks, especially F-A 5 and 030116, for which Fv/Fm values were the highest (Table 2). This decrease in the value of Fv/Fm in Carrizo citrange was the result of a decrease in Fm (maximum fluorescence yield in the dark-adapted leaves), suggesting that the photochemistry of PSII was affected (Calatayud et al., 2004). By contrast, under the same conditions, Fm leaves of shoots budded on F-A 5 and on 030116 remained unaffected. Similar changes have been observed in Fe-deficient leaves of peach and pear trees and grapevine (Baveresco et al., 2006; Morales et al., 2000; Nedunchezhian et al., 1997). Furthermore, ‘Navelina’ leaves of shoots grafted on Carrizo citrange displayed the lowest fraction of open PSII centers, qP, in the steady state (Fig. 1B). This would indicate an incomplete reoxidation of the QA acceptor and an increase in closed PSII centers during actinic irradiation (Calatayud et al., 2002). Leaves of shoots grafted on the other rootstocks had significantly higher qP values than on Carrizo citrange. The greater rate of closing of PSII centers in leaves on Carrizo citrange reduced the possibility of electron transport to photosystem I (Calatayud et al., 2004; Seaton and Walker, 1990). According to these qP values, efficiency of PSII centers (ΦPSII) value in the steady state was lowest in leaves of shoots grafted on Carrizo citrange. In addition, the incomplete reoxidation of the QA acceptor led to a greater fraction of reduced QA in Carrizo citrange and, consequently, an increase in nonphotochemical quenching at the expense of photochemical use of excitation energy, as shown by our results for qN values. Leaves of shoots grafted on Carrizo citrange and F-A 418 showed the highest photoprotective qN in the steady state (Fig. 1C). ‘Navelina’ leaves of shoots budded on the other rootstocks presented qN values that were significantly minor to Carrizo citrange. The complementary study of the fraction neither used in photochemistry nor dissipated in the antennae (%X) showed that Carrizo citrange had the highest %X as compared with the other rootstocks (data not shown). This fraction is the least desirable pathway because it may lead to oxidative damage (Calatayud et al., 2003; Demmig-Adams et al., 1996).
On the other hand, the study of nonphotochemical quenching (qN) during fluorescence kinetics showed that qN reached their maximum value within 1 min for leaves of shoots grafted on all rootstocks and leaves on Carrizo citrange had the highest qN value (Fig. 1C). Subsequently, qN values underwent a fast relaxation apart from Carrizo citrange, which had a slower ramp (Fig. 1C). This would be related to the fact that ‘Navelina’ leaves of shoots grafted on Carrizo citrange rootstocks grown in calcareous soil had lower stomatal opening and transpiration (gs and E values) than those budded on all the other rootstocks. This limitation also correlated with a reduction in energy conversion efficiency of PSII (ΦPSII) for Carrizo citrange during fluorescence kinetics, because its yield was significantly lower compared with the other rootstocks 2 min into the kinetics study (Fig. 1A). Moreover, the fraction of open PSII centers, qP, decreased, reaching ≈70% for all rootstocks after the first saturation. However, this situation quickly reversed and all rootstocks recovered, reaching 90% of open PSII centers in the following saturations, apart from Carrizo citrange, which could not surpass 73% (Fig. 1B). This could be the result of the incomplete reoxidation of QA, leading to the downregulation of photosynthesis in comparison with the other rootstocks under assay.
In conclusion, based on chlorophyll content, gas exchange, and the Chl a fluorescence characteristics, trees grafted on F-A 5 performed best under these calcareous soil conditions, whereas those on Carrizo citrange were poorly adapted. Trees on the other six hybrid rootstocks performed well. These results are consistent with previous field evaluations on F-A 5 and F-A 13 rootstocks.
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