Nickel Deficiency Symptoms Are Influenced by Foliar Zn:Ni or Cu:Ni Concentration Ratio

The occurrence of nickel (Ni) deficiency of pecan [Carya illinoinensis (Wangenh.) K. Koch] in orchards is an increasingly common problem. There is uncertainly regarding the primary cause of the problem, as orchard soils have plenty of Ni. The influence of essential micronutrients on the endogenous bioavailability of Ni is unknown and is a possible factor triggering Ni deficiency. This study examines the linkage between Ni deficiency and endogenous foliar concentration of Ni, zinc (Zn), and copper (Cu). This relationship was tested in a greenhouse study using ‘Desirable’ seedlings trees growing in an orchard soil known to cause Ni deficiency in potted trees. Amendment of the potting soil with various amounts of either Zn-sulfate or Cu-sulfate produced seedling trees possessing a variety of Zn:Ni and Cu:Ni concentration ratios in developing foliage, and growth/morphological symptoms expressing various degrees of Ni deficiency. Severity of Ni deficiency was unrelated to foliar Ni concentration, but strongly linked to foliar Zn:Ni or Cu:Ni ratios. Deficiency symptoms increased sigmoidally with increasing Zn:Ni or Cu:Ni ratio, and were correctable, regardless of the Zn:Ni or Cu:Ni ratio, in seedling trees by foliar applications of Ni-malate extracted from Alyssum biomass. Thus, Zn and Cu in foliage can function antagonistically to reduce the bioavailability of Ni and therefore trigger expression of Ni deficiency. Soil Zn or Cu supplements did not detectably affect foliar Ni concentration; thus, root uptake does not appear to be inhibited by Zn or Cu. It is concluded that Ni deficiency in pecan orchards is likely to be partially due to either Zn or Cu fertilization-induced reductions in the physiological availability of Ni, perhaps via either competitive inhibition or sequestration. It is also concluded that long-term or excessive Zn and/or Cu fertilization of crops potentially triggers Ni deficiency through endogenous antagonisms; thus, potentially threatening the sustainability of commercial pecan enterprises. INTRODUCTION Nickel (Ni) deficiency is an increasingly common problem in pecan [Carya illinoinensis (Wangenh.) K. Koch] orchards, especially in second-generation orchards where it is often manifested as a fatal orchard replant disease (Wood et al., 2003a, 2004, 2006); thus, supporting conclusions by Brown et al. (1987, 1990) that Ni is an essential nutrient element. The fundamental cause(s) of this relatively recent increase in incidence and severity of Ni deficiency is unknown, but appears linked to high soil Zn and/or Cu due to long-term excessive orchard fertilization (Wood et al., 2003a). Foliage often exhibits visible Ni deficiency symptoms even though the absolute concentration of Ni in foliage exceeds the apparent lower critical concentration; thus indicating that other nutrient elements affects endogenous bioavailability of Ni. Micronutrient interactions are common, especially in extreme situations of soil pH or composition (Kabata-Pendias, 2001); thus, triggering chemical stress linked to either antagonistic or synergistic effects on root uptake and/or cellular bioavailability. This study examines the relationship between Ni deficiency and endogenous foliar concentration of Ni, Zn and Cu, and finds that expression of visible symptoms of Ni deficiency by pecan depends on the Zn:Ni or Cu:Ni ratio within Proc. VI IS on Mineral Nutrition of Fruit Crops Eds.: M. Pestana and P.J. Correia Acta Hort. 868, ISHS 2010 164 symptomatic foliage. MATERIALS AND METHODS The relationship between the severity of Ni deficiency, as exhibited by visible morphological symptoms and the ratio of Ni to either Zn or Cu was studied using potted seedlings of pecan growing in soil containing different concentrations of either Zn or Cu. The study used seedlings, derived from open-pollinated ‘Desirable’ seed, growing in plastic pots (15 x 15 x 15 cm). The soil was pasteurized and was a Tifton loamy-sand soil. Treatment seedlings were maintained in a greenhouse. Such seedlings typically exhibit visible Ni deficiency symptoms at time of budbreak of the third growing season. Pots of seedlings were treated with different amounts of either Znor Cu-sulfate during the second dormancy season. Treated trees exhibited varying degrees of Ni deficiency during the third growing season. Treatments were supplemental Zn or Cu at one of seven concentrations (i.e., 0X, 0.5X, 1X, 2X, 4X, 8X, and 16X; where in the case of Cu: X = 0.312 g CuSO4·5H2O per pot (1.25 mmoles Cu per pot); and for Zn, X = 3.594 g ZnSO4·7H2O per pot (12.5 mmoles Zn per pot). Zn and Cu salts were dissolved in 400 ml of deionized water and applied as a soil drench about four weeks before budbreak. The study was structured as two experiments, one with Zn and one with Cu. Experimental design for each study consisted of the metal-sulfate salt at seven concentrations, replicated as a completely randomized design, with eight replicates, and with single-seedlings as experimental units (n = 56 per study). Subsequent watering was such that there was no mass flow of water through the pot’s soil, thus avoiding leaching of added salts. Individual seedlings were rated for degree of Ni deficiency in late April, ≈ 4 weeks after budbreak, and leaflets (three middle leaflet pairs of the three middle compound leaves) were sampled for ICP-MS analysis of Ni, Zn, and Cu (monitoring masses of Ni, Zn and Cu). Foliage collection used zirconium oxide ceramic scissors to avoid contaminating samples with metals. Samples were dry-ashed, dissolved in 20% HNO3, diluted with 2% HNO3, and further diluted with 2% HNO3 for ICP-MS analysis. Nickel deficiency was verified by randomly treating 50% of all seedlings exhibiting Ni deficiency with two foliar sprays of Ni the following spring, and leaving 50% without Ni treatment. Treatments were foliar sprays to leaf-drip with Ni-malate (at ≈ 100 μg ml) the following spring ≈ 7 and 14 d post budbreak. The Ni-malate was an aqueous extract of a pulverized Nienriched Alyssum biomass-a Ni hyperaccumulator (Wood et al., 2006). RESULTS AND DISCUSSION Seedlings growing in soils exposed to different amounts of Znor Cu-sulfate exhibited different concentrations of Zn and Cu, and different degrees of Ni deficiency, depending on Zn or Cu treatment, in the developing canopy displayed the following spring. The study produced seedling exhibiting visible morphologically-based Ni deficiency symptoms ranging from no symptoms (Class 1) to severely rosetted and dwarfed seedlings (Class 9). Ni deficiency severity class is based on the following scale derived for identification of Ni deficiency symptoms (Wood et al., 2004; Nyczepir et al., 2006): 1 = no Ni associated morphological distortions of leaflets or leaves (i.e., normal); 2 = 1 to 25% of leaflets on the seedling exhibiting Ni deficient morphological distortions (i.e., slightly blunted); 3 = 26 to 50% of leaflets exhibiting some degree of Ni associated morphological distortions; 4 = >50% of leaflets exhibiting morphological distortions; 5 = #4, plus leaflet cupping; 6 = #5, plus necrosis of leaflet tips; 7 = #6, plus necrosis of leaflet margins, crinkled leaflets, and dwarfed leaflets; 8 = #7, plus dwarfed shoots; 9 = #8, plus rosetting; and 10= #9, plus tree death. Figure 1 illustrates an example of expressed Ni deficiency by pecan seedlings. Zinc:Ni Ratio Zinc-treated seedlings, depending on amount of Zn to which soils supporting seedlings were exposed, exhibited foliar Ni concentrations between ≈ 0.06 to 2.5 μg g dw (dry weight) with essentially no linkage (R = 0.007) between severity of Ni deficiency and foliar Ni concentration (Fig. 2A). Foliar Zn concentration ranged from ≈ 50 to 1550 μg g