Soil Science Society of America Journal 65:385-390 (2001)
© 2001 Soil Science Society of America
DIVISION S-4-SOIL FERTILITY & PLANT NUTRITION
HEDTA–Nitrilotriacetic Acid Chelator-Buffered Nutrient Solution for Zinc Deficiency Evaluation in Rice
Calvin L. Trostlea,
P.R. Bloomb and
D.L. Allanb
a Texas A&M Univ. Research & Extension Center, Route 3, Box 213AA, Lubbock, TX 79403
b Dep. of Soil, Water, & Climate, Univ. of Minnesota, 439 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108-6028
Corresponding author (c-trostle{at}tamu.edu)
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ABSTRACT
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Chelator-buffering methods with N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid (HEDTA) are used to elucidate Poaceae growth response to micronutrient metal activities including (Zn2+), but reliable hydroponic methods that maintain stable (Zn2+) for evaluating Zn deficiency in rice (Oryza sativa L.) have not been reported. The objective was to develop a chelator-buffered method that gauges rice growth response to (Zn2+) in an otherwise chemically stable environment. Using GEOCHEM-PC to estimate solution activities, an aerobic HEDTA–nitrilotriacetic acid (NTA) dual-chelator method was developed that imposed five (Zn2+) levels on cv. IR-36 seedlings for 21 d after transplanting (DAT) in a growth chamber. Control of pH 5.50 ± 0.05 using 3.0 mM 2-(4-morpholino)-ethanesulfonic acid (MES) combined with periodic adjustment was critical to preserving target (Zn2+). Solution treatments ranged from Zn deficient, where (Zn2+) = 10-10.0 M (0.25 µM total chelated Zn), to fully Zn sufficient where (Zn2+) = 10-8.8 M (4.00 µM total chelated Zn). Using 200.0 µM total chelated Fe(III), adequate Fe was maintained at (Fe3+) = 10-14.3 M. Phosphorous supply was controlled to prevent toxic P accumulation at low (Zn2+). With increasing (Zn2+), total biomass at 21 d ranged from 0.94 to 1.90 g plant-1. Shoot Zn responded to (Zn2+), not total chelated Zn2+, and roots responded similarly. Critical (Zn2+) for normal growth was 10-9.1 M, and leaf Zn-deficiency symptoms were observed at (Zn2+) 
10-9.4 M (28 mg Zn kg-1 shoot). The HEDTA-NTA method provides a rapid and reliable means for evaluating Zn deficiency tolerance in IR-36 via diagnostic visual and physical symptoms in response to a range of (Zn2+) levels.
Abbreviations: DAT, days after transplanting • HEDTA, N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid • ICP-AES, inductively coupled plasma atomic emission spectroscopy • MES, 2-(4-morpholino)-ethanesulfonic acid • NTA, nitrilotriacetic acid
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INTRODUCTION
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CHELATOR-BUFFERED NUTRIENT SOLUTIONS using HEDTA, often in excess of total micronutrient metal concentrations, have been used successfully for imposing Zn-deficient conditions due to low Zn activity, that is, (Zn2+), for species of the graminae Poaceae including maize (Zea mays L.) (Bell et al., 1991b; Parker, 1997), barley (Hordeum vulgare L.) (Bell et al., 1991a; Norvell and Welch, 1993), and wheat (Triticum aestivum L.) (Parker, 1997; Rengel and Graham, 1995a,b). Chelates of Zn–HEDTA have the necessary stability for Poaceae nutrition within a suitable pH range for plant growth (Norvell and Welch, 1993). Using computer chemical speciation programs such as GEOCHEM-PC (Parker et al., 1995b), solution (Zn2+) may be calculated, then correlated with plant response. Chelator HEDTA does not bind Fe3+ as tightly as other chelators such as EDDHA or DTPA (Chaney and Bell, 1987). This is particularly important for rice, which has minimal expression of the Fe(III) phytosiderophore uptake system common to other Poaceae (Marschner and Römheld, 1994).
With chelator-buffered nutrient solutions the activity of a specific cation may be varied, while other micronutrient cation activities remain unchanged (Parker et al., 1995a). In general, (Zn2+) = 10-10 to 10-11 M is required for the onset of Zn deficiency at total solution Zn concentrations of 1 µM. Many plants cannot adequately absorb Zn at (Zn2+) <10-10.5 M (Parker et al., 1995a). Solutions with HEDTA are useful for evaluating growth response to (Zn2+) ranging from sufficiency to deficiency in barley (Bell et al., 1991a; Norvell and Welch, 1993), maize (Bell et al., 1991b; Parker, 1997), and wheat (Parker, 1997; Rengel and Graham, 1995a,b). However, chelator buffering with HEDTA or other chelates has not worked well with rice mainly because of inadequate Fe nutrition in the presence of high chelator concentrations.
Hydroponic culture has been used to evaluate Zn and other nutrient deficiencies in rice, but these methods produced questionable results in part because (i) unnecessarily high concentrations of some nutrients (e.g., P) are used to maintain availability (Yoshida et al., 1976, p. 61–66.), (ii) the low Zn concentrations required for nutrient deficiency are not stable with time due to plant nutrient uptake, and (iii) Zn contamination may be great enough to result in near normal growth (Parker et al., 1995a; Bowen, 1986).
Low Fe availability may be a limitation for hydroponic rice. Iron occurs as Fe(III) in aerobic hydroponic culture, but rice is normally grown in flooded or strongly acid soils where Fe(II) is the dominant form. Rice produces few phytosiderophores (Mori et al., 1991), resulting in low Fe(III) acquisition. Thus rice culture requires high levels of free Fe(III) (unless difficult-to-maintain anoxic systems are used) and the use of chelators to maintain adequate soluble Fe in solution at lower pH where Fe(III) activity is greater. Regulating Fe nutrition in hydroponic solution is further complicated because plant Fe uptake from chelators is a function of both activity and total solution concentration (Bell et al., 1991a). Most nutrient solutions for Poaceae contain low total Fe(III), (e.g., 20 µM Fe(III) for barley; Norvell and Welch, 1993), but in rice much higher Fe(III) levels may be necessary in Zn experiments to compensate for strong chelation used to maintain low targeted (Zn2+). Thus in Zn nutrition experiments Bell et al. (1991b) used 158 µM Fe(III) at pH 5.9 for maize, and Rengel and Graham (1995a)(1995b) used 100 µM Fe(III) at pH 6.0 for wheat.
Yang et al. (1994) studied rice response to (Zn2+) using HEDTA- and DTPA-buffered nutrient solutions, each supplemented with the Fe2+-specific chelator ferrozine, 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine), to supply Fe(II) under oxidized conditions and in the presence of chelators that strongly bind Fe3+. A 50% oxidation of ferrozine Fe2+ was assumed (X. Yang, 1993, personal communication). Rice growth (cv. Shanyou 64) for 28 d after germination in 3 L of chelator-buffered nutrient solution was only 0.21 g plant-1 for normal growth down to 0.13 g plant-1 at the lowest (Zn2+), or about a 42% reduction in growth. The plants may not have received adequate N (not replenished), and shoot tissue Fe was borderline deficient (50–75 mg Fe kg-1 shoot) compared with Yoshida's (1981) critical value of 70 mg Fe kg-1 shoot. Also, shoot Cu (3–6 mg Cu kg-1 shoot) was below critical sufficiency levels of 6 mg Cu kg-1 shoot (Yoshida, 1981).
Since (Zn2+) and (Fe3+) are pH dependent, pH control is essential to maintaining critical target activities. The biological pH buffer MES has been used at 1 and 2 mM with several Poaceae to maintain stable pH near 6.1 (Bell et al., 1991a,b; Rengel and Graham, 1995a, b). Bugbee and Salisbury (1985) reported that 10 mM MES produced good pH control with little harmful effects on wheat due to MES levels or its associated Na salts. Miyasaka et al. (1988) observed decreased Zn uptake in wheat at 5 mM.
Our initial trials with rice indicated HEDTA was too strong at pH 5.5 to permit adequate Fe nutrition and chelator buffering of Zn and other micronutrient cations. Thus dual chelation using a second chelate with different (generally lower) cation affinities, in combination with HEDTA was tested. Preliminary hydroponic solutions combining HEDTA and NTA indicated good rice growth, adequate Fe nutrition, and control of Zn near deficient levels. The NTA chelator was chosen because it has lower binding constants with micronutrient metals than most other chelators (Norvell, 1991), which enabled us to increase total excess chelator while still maintaining Fe(III) solubility.
The objective was to develop a hydroponic method for imposing a range of (Zn2+) on rice from deficient to sufficient levels, which included appropriate conditions for total chelated Zn, free Zn (i.e., Zn2+), and Zn buffering capacity.
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MATERIALS AND METHODS
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Hydroponic System
Exterior surfaces of 10-L opaque polypropylene tubs (36 by 31 by 14 cm; Nalgene Co., Rochester, NY) were painted black and then covered with duct tape to exclude light from the nutrient solution. Tubs were fitted with 4.5-mm-thick black Plexiglas lids containing eight 20-mm holes for holding rice plants. A self-adhering foam strip (6.4 by 12.7 mm; Norton Co., Grandville, NY) was attached to the lid to form a seal with the tub. After use, the tub interior was rinsed with HCl and then deionized water to remove silica or other nutrients that may have adhered to the surface.
Chemical Reagents
All chemical reagents were analytical grade. Formula weights for HEDTA and NTA (Sigma Chemical Co., St. Louis, MO) and MES buffer (Fisher Biotech, Chicago, IL) did not include water of hydration in the labeled formula weight. Chelator formula weight was verified indirectly by analyzing for N content (Stalcup and Williams, 1955; Technicon Industrial Systems, 1974) and adjusting for moles of water of hydration. Uncorrected hydration in chelator formula weight would have reduced solution HEDTA by up to 2.3% and reduced solution NTA by up to 6.1%, thus resulting in a change in target activities of +20% for (Zn2+) and +7% for (Fe3+). The formula weight for FeCl3·6H2O was also verified by analyzing Fe content using inductively coupled plasma atomic emission spectroscopy (ICP-AES). Water used for all solutions was deionized then distilled to 1.5 x 10-4 S m-1.
Basal Nutrient Solution
Factors affecting solution activities, especially (Fe3+) and (Zn2+), such as pH, chelates, and changes in nutrient composition (e.g., nutrient uptake, contamination) were modeled with GEOCHEM-PC in a myriad of theoretical cases (Trostle, 1997). Macronutrient and micronutrient stock solutions were made separately and then mixed in the tub with other components directly added to the tubs (Trostle, 1997) (Table 1). Stock solutions were acidified with HCl to maintain solubility. Micronutrient stock solutions contained NTA to chelate microcations and maintain solubility and were stored in the dark. Iron solution was mixed separately with HEDTA to promote formation of the Fe(III)–HEDTA complex before adding directly to the tubs. Silicic acid was added to tub nutrient solutions before final dilution to 8 L and pH adjustment to 5.50. Table 1 gives the basal nutrient solution formulation. Cadmium was included in anticipation of eventual Cd work, but was not a necessary component of these solutions. Any solution constituents differing from the basal solution are reported below.
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Table 1. Total concentrations of the basal nutrient solution constituents for normal growth of rice. Concentrations were entered into GEOCHEM-PC for calculation of solution activities (pH 5.50)
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Solution pH was adjusted with 1 M NH4OH, which also replenished N in proportion to rice NH+4 uptake, thus ensuring solution activities, particularly (Fe3+) and (Zn2+), were maintained near their targeted activities. Solution P was maintained at 20 µM and added periodically rather than as a single large addition of P to the initial solution. This protects Zn-deficient plants from hyperaccumulation of P (Parker et al., 1992; Parker, 1997). Nutrient solutions were regularly sampled and analyzed for Zn using ICP-AES to determine any possible Zn contamination.
Rice Culture
Rice cv. IR-36 was chosen because it produces high biomass, is tolerant to Zn deficiency, and until recently was the most widely grown cultivar worldwide. Thus conditions that imposed Zn deficiency on IR-36 may be sufficient to produce Zn deficiency in other cultivars. The high growth rate helped gauge the ability of the nutrient solution to supply sufficient plant nutrients during periods of high growth.
Seeds were germinated for 
48 h in distilled water, then transferred to growth pouches containing 8 mL of the normal basal nutrient solution less Zn (see below) to which 12 mL distilled H2O was added (Trostle, 1997). Pouches were placed in a growth chamber (Model E15, Conviron, Inc., Pembina, ND) at 24°C and 16 h of light, and solution was replaced every other day. Eight seedlings of average size (leaf stage 3.0, 9–11 cm tall) were transplanted to each tub 9 d after germination (0 DAT), each stem supported between two cotton balls per lid hole so the crown protruded into the nutrient solution. Treatments (8 L tub-1) were placed in the growth chamber with 16-h photoperiod, 460 µmol m-2 s-1 photon flux density, and 27/24°C (day/night) in the leaf canopy. Tubs were rotated at each pH adjustment (see below) to mitigate chamber position effects.
Early trials determined a schedule for nutrient solution replacement, pH adjustment, and P addition in response to nutrient uptake during the increasingly rapid growth of rice after transplanting (Table 2). At the first nutrient solution change (10 DAT) eight plants for each treatment were culled to four by choosing a modal distribution of plants based on average size and health (apart from Zn deficiency). Trials with eight plants grown to 21 DAT (or four plants grown to 28 DAT) resulted in unacceptably large pH changes of -0.5 to -0.8 d-1 in 5 mM MES later in the growth cycle.
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Table 2. Schedule of nutrient solution changes, P addition, and pH adjustment for growth of rice up to 21 d after transplanting (DAT)
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Sensitivity analyses using GEOCHEM-PC and subsequent test solutions demonstrated the deleterious effect potential pH changes have on solution activities of critical nutrients, particularly (Fe3+) and (Zn2+), in the HEDTA-NTA system at a pH range of 5.1 to 5.7 (Fig. 1)
. These potential changes in (Fe3+) and (Zn2+) demonstrate the caution necessary in constructing and managing solutions that minimize changes in pH and hence enable experiments to best maintain target activities. The target of limiting change in log(Zn2+) to <0.1 was not achieved in the last 2 to 4 d of growth for the fast growing cultivar IR-36 in solutions at (Zn2+) 
10-9.4 M (Trostle, 1997). Thus targeted solution pH of 5.50 was adjusted twice daily the last few days of growth. All subsequent experimental work used 3.0 mM MES. The limited benefit of more pH buffering did not merit the added cost of MES, the most expensive component of the solution.
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Fig. 1. Calculated change in (Fe3+) and (Zn2+) vs. pH in a basal nutrient solution (pH 5.5) chelated with HEDTA and NTA with 200.0 µM total chelated Fe(III), 2.00 µM total chelated Zn, and 3.0 mM MES as calculated with GEOCHEM-PC
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Data Collection and Analysis
Leaves were examined during pH adjustment (Table 2) for Zn deficiency symptoms, and the severity estimated on a scale from 0 to 4: 0-no visible symptoms; 1—very light bronze to blackish leaf flecking; 2—bronze and blackish blotches on 1 to 10% of the leaf; 3—moderate or blotches >10%, with some short, stubby leaves; and 4—severe deficiency with leaf discoloration >50%, severe necrosis, and rolling of the leaf from the tip downward. At harvest (21 DAT) main culm leaf number, tiller number, and plant height from culm base to leaf tip of the longest leaf were measured. Plants were rinsed with distilled water then dried in a forced-air oven at 65°C for 3 d. Individual plant shoots and roots were separated and weighed. Ground tissue shoots or roots were ashed at 485°C for 12 h, digested with 10 mL of 2.5 M HCl for 2 h, then refluxed in the digestion crucible on a hotplate for 30 min to increase Fe and Mn recovery. Reflux increases measurable total plant Fe by 15 to 22% (Trostle, 1997). The filtrate was analyzed for metals and other nutrients via ICP-AES (Dahlquist and Knoll, 1978; Munter and Grande, 1981). Tissue N was analyzed using Kjeldahl digestion followed by steam distillation of NH+4 (Nelson and Sommers, 1973).
Plant response to deficient levels of (Zn2+) was determined by calculating ratios of both shoot weight and shoot Zn for each (Zn2+) level relative to sufficient (Zn2+) levels. One-way analysis of variance was calculated for plant growth parameters vs. solution chemical treatments (five levels x two replicates), and means separation was conducted using Fisher's protected least significant difference.
Preliminary Experiments
Four preliminary experiments-MES pH buffer level, total HEDTA and NTA chelator concentration, critical (Fe3+), and total chelated Fe(III)—were conducted using IR-36 in developing the above basal nutrient solution (Table 1), which would not limit rice growth yet maintained stable (Fe3+) and (Zn2+) (Trostle, 1997). Test ranges or targeted values for nutrient levels, targeted solution pH, and chelator conditions were established.
Growth response of IR-36 suggested critical (Fe3+) was near 10-14.3 M at pH 5.50, which was maintained in the basal solution with HEDTA = 182.8 µM, and NTA = 94.2 µM (70 µM chelator in excess of micronutrient metal cations). In the test for critical (Fe3+), solution (Zn2+) and shoot Zn were similar across all (Fe3+) levels in spite of increasing total solution Zn. This suggests that unlike the finding of Bell et al. (1991a) for barley, shoot Zn concentration of rice responds to free Zn2+, not total chelated Zn2+.
Management of Fe to provide adequate rice nutrition while maintaining stable low chelated (Zn2+) was the greatest difficulty encountered in developing a suitable chelator-buffered solution. Total Fe uptake during the (Fe3+) and total chelated Fe(III) preliminary experiments ranged from 0.3 to 0.7% of the added Fe(III). Replenishing solution Fe due to plant uptake was unnecessary (Bell et al., 1991a).
Critical (Zn2+) and Total Chelated Zinc Concentration
Further preliminary experiments indicated little further increase in IR-36 growth at (Zn2+) > 10-9.1 M. In early trials with IR-36 as well as other cultivars normal growth decreased 50% in the range of (Zn2+) = 10-10.0 to 10-9.7 M (Trostle, 1997). The primary objective of this experiment was to incorporate results of the preliminary experiments into solutions for IR-36 ranging from sufficient to deficient (Zn2+), which would identify critical (Zn2+) and gauge IR-36 response to deficient Zn. Critical activity for Zn2+ was defined by Kelling and Matocha (1990) as the activity below which reductions in growth may be expected due to low supply of the nutrient. In practice, the critical level occurs at some point across a limited range of decreasing activity.
A secondary objective was to evaluate the buffering of total chelated Zn and (Zn2+) in order to limit 
log(Zn2+) < 0.1 due to Zn uptake. Five solutions with (Zn2+) = 10-10.0, 10-9.7, 10-9.4, 10-9.1 (basal solution), and 10-8.8 M were tested. All solution components were the same as in the basal solution (Table 1) except HEDTA and Zn. Chelator HEDTA concentrations beginning at the lowest (Zn2+) were (in µM): 181.1, 181.1, 181.6, 182.8 (basal solution), and 184.5. The corresponding total added Zn values were (in µM): 0.25, 0.50, 1.00, 2.00 (basal solution), and 4.00.
Zinc deficiency symptoms were noted as they appeared, and their severity was recorded for each tub at harvest when other plant parameters were measured. Nutrient solution samples were checked periodically for Zn contamination using ICP-AES. Daily change in pH was recorded for each solution. Seeds of IR-36 were ground in a mill to pass a 425-µm screen, ashed, and then digested with HCl to determine seed Zn content. Total plant Zn uptake as a percentage of total solution Zn was determined. Relative shoot growth was determined among the five treatments compared with the basal solution at (Zn2+) = 10-9.1 M. The experiment was duplicated.
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RESULTS AND DISCUSSION
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IR-36 Response to Variation in (Zn2+)
Total biomass ranged from 1.90 g plant-1 at (Zn2+) = 10-8.8 M to 0.93 g plant-1 at (Zn2+) = 10-10.0 M (Fig. 2)
. Tiller number decreased from 16.3 tillers plant-1 to 7.8 tillers plant-1 at lowest (Zn2+). Across the tested range of (Zn2+) the decreased shoot and root growth at (Zn2+) = 10-10.0 M was significant (P < 0.001). Likewise, shoot Zn declined from 46.1 mg kg-1 at highest (Zn2+) to 17.5 mg kg-1 at lowest (Zn2+) (Table 3). Little additional growth occurred at shoot Zn > 35 mg kg-1, and shoot growth declined considerably at shoot Zn 20 mg kg-1. The root Zn response was similar to shoot Zn, declining from 62.3 mg kg-1 at highest (Zn2+) to 22.1 mg kg-1 at lowest (Zn2+). Reduced rice growth was evident just prior to the appearance of visual Zn deficiency symptoms at 8 DAT for (Zn2+) = 10-10.0 M. Similar Zn deficiency symptoms also appeared at 11 DAT for (Zn2+) = 10-9.7 M. Symptoms included dark-brown, bronze, and eventually blackish spots. Among the approximately nine main culm leaves per plant which developed, Leaves 4, 5, and 6 on the main culm had the highest incidence of visual Zn deficiency symptoms, often with a short, stubby appearance (Yoshida et al., 1973). The most severely affected leaves at (Zn2+) = 10-10.0 M rolled inward from the sides near the tip progressing toward the base of the leaf, which agrees with responses found by Karim and Vlamis (1962). Some tiller leaves were similarly affected. The greatest average number of symptomatic leaves per tub (four plants) was 13.5, and occurred at (Zn2+) = 10-10.0 M (despite few tillers) decreasing to 2.5 symptomatic leaves per tub at (Zn2+) = 10-9.4 M and no Zn-deficient leaves at (Zn2+) 10-9.1 M (Fig. 3)
. The appearance of Zn-deficient leaves at (Zn2+) = 10-9.4 M where shoot Zn = 27.8 mg kg-1 was unexpected. Critical shoot Zn concentrations in rice are typically 20 to 25 mg kg-1 in the tillering stage (Yoshida, 1981).
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Fig. 2. Response of IR-36 plant, shoot, and root dry mass vs. (Zn2+) at 21 d after transplanting in an HEDTA-NTA chelator-buffered system at pH 5.5. Mean ± 1 SD at each point represents eight values (duplicate trials, four plants per tub). Significance was determined at  = 0.05
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Table 3. Micronutrient metal response of IR-36 to variation in (Zn2+). For all solutions total chelated Fe(III) = 200.0 µM, (Fe3+) = 10-14.3 M, (Mn2+) = 10-6.25 M, (Cu2+) = 10-12.45 M, and target pH = 5.50. Results are the average of duplicate trials
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Fig. 3. Affected rice leaves per tub (four plants) vs. (Zn2+) at 21 d after transplanting in an HEDTA-NTA chelator-buffered system at pH 5.5. Each value is the mean of two trials. Mean tiller number represents eight plants and indicates higher number of leaves for high (Zn2+)
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Shoot Fe increased with Zn deficiency ranging from 175 mg kg-1 at (Zn2+) = 10-8.8 M to near 210 mg kg-1 at the two lowest (Zn2+) levels, whereas root concentrations were 400 mg kg-1, and they demonstrated no trend with (Zn2+). Shoot and root Mn increased with decreasing (Zn2+) similar to shoot Fe (Table 3). Shoot Mn appears to be high at 376 to 593 mg kg-1 tissue, but Yoshida (1981) reported that critical toxicity levels for Mn in the rice shoot at the tillering stage is 7000 mg kg-1, whereas Neue (1994) suggested the value at maximum tillering is 2000 mg kg-1. The increases in shoot and root Mn may have been due to the concentrating of nutrient due to Mn accumulation in slower-growing plants. Total Mn was fixed at 2.0 µM in all (Zn2+) solutions, and likewise calculated (Mn2+) = 10-6.25 M was the same for each solution. Shoot and root Cu concentrations showed no trend with (Zn2+), which is in contrast to reports that Zn and Cu compete for the same uptake carrier sites in the roots (Kochian, 1993). Total Cu was fixed at 0.5 µM, and (Cu2+) = 10-12.45 M for all (Zn2+) solutions.
Shoot P increased slightly to 4520 mg kg-1 at (Zn2+) = 10-10.0 M compared with other (Zn2+) levels (4050 ± 80 mg kg-1), which was insufficient to cause P toxicity (P > 10000 mg kg-1 tissue) (Parker, 1997). Root P, shoot and root Ca and Mg, and other elements were mostly unaffected by (Zn2+) (Trostle, 1997).
During the 21-d growth period, uptake of Zn ranged from 4.9% of total solution Zn at highest (Zn2+) to 14.5% of total solution Zn at (Zn2+) 10-9.7 M. No external Zn contamination was observed. Seed Zn reserves accounted for only 0.6% of total Zn uptake (high Zn treatment) to 3.4% (low Zn treatment) of total Zn uptake. The maximum change in (Zn2+) due to plant Zn uptake, a decrease in log(Zn2+) 0.07 (-15%), occurred at the two lowest Zn activities; thus Zn uptake did not substantially affect (Zn2+). We suggest a solution be modified (greater volume per plant) if activity varies by log(Zn2+) 0.1 due to plant Zn uptake.
Biological variability was examined by comparing plant biomass, shoot/root weight ratio, tiller number, and Zn uptake of each tub (four plants) for rice grown in the basal nutrient solution, (Zn2+) = 10-9.1 M, among eight trials including preliminary studies. All CV values ranged from 8.6% (shoot/root weight ratio) to 19.8% (root weight). Chaney et al. (1989) reported that 20% CV is typical for biological data. All tissue nutrient levels were comparable to published normal ranges (Yoshida, 1981; Neue, 1994).
The critical (Zn2+) for rice in this study was (Zn2+) 10-9.1 M, higher than most other reports for Poaceae. The lower rice growth rate of Yang et al. (1994) may have led to lower (Zn2+) demand, which could contribute to lower critical (Zn2+) values. Although plants were 7 d younger than in Yang et al. (1994), comparable normal plants in this study produced about eight times as much biomass per plant. Using rice cv. Shanyou 64, Yang et al. (1994) reported the onset of Zn deficiency in rice at (Zn2+) < 10-10.6 M. In light of the probable oxidation of Fe(II) in their ferrozine–HEDTA system, we suspect (Zn2+) values were higher. Remodeling their solutions with GEOCHEM-PC suggests the actual (Zn2+) values found by Yang et al. (1994) may be as high as 10-8.7 M (vs. 10-10.6 M reported). Compared with IR-36 rice, other critical (Zn2+) levels in HEDTA-buffered solutions are (Zn2+) = 10-11.0 to 10-10.2 M at 29 d for maize and wheat (Parker, 1997) and (Zn2+) = 10-9.4 M for maize at 18 d.
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CONCLUSIONS
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The HEDTA-NTA method for imposing sufficient to deficient (Zn2+) in rice produced a range of Zn deficiency symptoms and rice growth response to (Zn2+). Critical shoot Zn for IR-36 in the tillering stage (35 mg Zn kg-1) was higher than expected. From preliminary experiments adequate Fe nutrition at total Fe(III) = 200.0 or (Fe3+) = 10-14.3 M overcame the chelation needed to impose (Zn2+) sufficiently low to cause Zn deficiency without restricting growth due to Fe. In this oxidized system high chelated Fe(III) also permitted increased chelated Zn and hence improved (Zn2+) buffering against plant Zn uptake or possible contamination. Potential P toxicity at low (Zn2+) was avoided due to restricted P supply. Stable pH control near 5.5 was important for maintaining targeted (Zn2+) in the HEDTA–NTA system. This method, in contrast to pot studies or field soils, may not reflect all factors important in Zn uptake; however, it is a quick and effective method for evaluating Zn deficiency tolerance in rice.
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ACKNOWLEDGMENTS
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Seed was provided by the Texas Agric. Expt. Station, Beaumont, TX. This project was supported in part by the United States Department of Agriculture's Special Constraints program.
Received for publication January 21, 2000.
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ABSTRACT
INTRODUCTION
