Screening Citrus Rootstocks and Related Selections in Soil and Solution Culture for Tolerance to Low-iron Stress

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William S. CastleUniversity of Florida, IFAS, Horticultural Sciences Department, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850

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James NunnalleeUniversity of Florida, IFAS, Horticultural Sciences Department, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850

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John A. MantheyU.S. Citrus and Subtropical Products Laboratory, USDA, ARS, 600 Avenue S, NW, Winter Haven, FL 33881

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A broad range of plant selections across the orange subfamily Aurantioideae were screened in solution and soil culture for their tolerance to low iron (Fe) stress. Young seedlings grown in soil were transferred to tubs of +Fe nutrient solution, which was later replaced after a brief period with a –Fe solution. Over several trials, ≈20 white root tips were harvested periodically from the plants in each tub and assayed for their ability to reduce Fe3+. The procedure was miniaturized to determine if a fewer number of root tips could be assayed to screen individual plants and to estimate the required sample size. For solution screening, seven root tips were estimated to be adequate for representing a single plant. Seedlings of a few selections were also grown in small containers of soil amended with 0% to 5.9% CaCO3. The results in solution and soil culture were consistent with each other and with previous assessments of the various selections. Based on a summary of the solution and soil responses, the citrus selections were grouped in descending order of Fe3+ reduction rates as Volkamer lemon/Rangpur/sour orange selections/Citrus macrophylla > mandarins and mandarin hybrids > citranges > citrumelos > trifoliate orange. Of the citrus relatives tested in solution culture only, those in the genera Glycosmis, Citropsis, Clausena, and Murraya had high Fe reduction rates with good seedling growth and new leaves developed a light yellow color or showed no loss of greenness. Other citrus relatives in the genera Severinia, Atalantia, and Fortunella and most somatic hybrids had low seedling vigor and produced too few root tips to be properly assessed. The results are useful because of the breadth of selections screened, the identification of various citrus relatives as potential sources of low-Fe stress tolerance in breeding new rootstocks, and the apparent positive relationship between the Fe3+ reduction responses, soil screening responses, and field experiences with carbonate-induced Fe chlorosis responses.

Abstract

A broad range of plant selections across the orange subfamily Aurantioideae were screened in solution and soil culture for their tolerance to low iron (Fe) stress. Young seedlings grown in soil were transferred to tubs of +Fe nutrient solution, which was later replaced after a brief period with a –Fe solution. Over several trials, ≈20 white root tips were harvested periodically from the plants in each tub and assayed for their ability to reduce Fe3+. The procedure was miniaturized to determine if a fewer number of root tips could be assayed to screen individual plants and to estimate the required sample size. For solution screening, seven root tips were estimated to be adequate for representing a single plant. Seedlings of a few selections were also grown in small containers of soil amended with 0% to 5.9% CaCO3. The results in solution and soil culture were consistent with each other and with previous assessments of the various selections. Based on a summary of the solution and soil responses, the citrus selections were grouped in descending order of Fe3+ reduction rates as Volkamer lemon/Rangpur/sour orange selections/Citrus macrophylla > mandarins and mandarin hybrids > citranges > citrumelos > trifoliate orange. Of the citrus relatives tested in solution culture only, those in the genera Glycosmis, Citropsis, Clausena, and Murraya had high Fe reduction rates with good seedling growth and new leaves developed a light yellow color or showed no loss of greenness. Other citrus relatives in the genera Severinia, Atalantia, and Fortunella and most somatic hybrids had low seedling vigor and produced too few root tips to be properly assessed. The results are useful because of the breadth of selections screened, the identification of various citrus relatives as potential sources of low-Fe stress tolerance in breeding new rootstocks, and the apparent positive relationship between the Fe3+ reduction responses, soil screening responses, and field experiences with carbonate-induced Fe chlorosis responses.

Citrus trees on many commercial rootstocks do not perform well in high-carbonate soils (Campbell, 1991; Castle, 1987; Castle et al., 2004; Cooper and Peynado, 1953, 1954; Ferguson et al., 1990; Hamze et al., 1986; Hodgson, 1967; Sagee et al., 1992; Sudahono and Rouse, 1994; Wutscher, 1979). Such rootstocks are limited by their inability to sufficiently extract micronutrients, including iron (Fe), that are rendered largely unavailable in these kinds of soils (Korcak, 1987; Manthey et al., 1994a). This limitation particularly applies to Poncirus trifoliata and its hybrids, which include some of the world's most popular rootstocks like Troyer and Carrizo citranges and Swingle citrumelo (Castle, 1987; Wutscher, 1979). The international significance of this problem is evident in the continued reporting of trials and germplasm releases concerning rootstocks specifically investigated for tolerance to high-carbonate soils (Tagliavini and Rombolá, 2001; Wei et al., 1994).

In the United States, citrange and citrumelo rootstocks are the mainstays of the Florida and California citrus industries, but both states also have areas of high-carbonate soils (Castle et al., 1993; Ferguson et al., 1990). Trees can be grown in those questionable sites if the grower is willing to choose other rootstocks with less desirable traits (Castle et al., 1992). Two examples are sour orange and rough lemon rootstocks, which are both well adapted to calcareous soils. Trees on sour orange produce excellent quality fruit, but are susceptible to citrus tristeza virus; those on rough lemon are high-yielding, but produce poor-quality fruit and are susceptible to citrus blight. Other management options for high-carbonate soils such as application of chelates are often expensive, so there is a strong incentive to develop new rootstocks (Castle et al., 2004; Grosser et al., 2004).

We began a screening project to search for superior citrus rootstocks suitable for calcareous sites based on the measurement of root Fe+3 reduction rates (Castle and Manthey, 1998). This method was selected because of its potential to minimize various interfering conditions that can occur in other screening methods mostly related to Fe chemistry (Cooper and Peynado, 1954; Hamze et al., 1986; Korcak, 1987; Manthey et al., 1993, 1994a; Pestana et al., 2005; Sagee et al., 1992; Sudahono and Rouse, 1994). For example, when using soil, pH affects the chemical state of Fe, and both pH and air-drying influence bicarbonate content, and the buffering system in solution studies affect Fe availability and uptake (Korcak, 1987).

Plants can respond to low-Fe stress through several inducible mechanisms, including, among others, electron release at the root surface (Briat and Lobréaux, 1997; Manthey et al., 1994b). By measuring a fundamental plant response, Fe reduction rate, in the simple test environment of a complete nutrient solution minus Fe, other possibly complicating factors could be minimized and the results would be more broadly applicable.

Our first screening project involved primarily common rootstocks and other citrus selections (Castle and Manthey, 1998). The selections ranked in terms of Fe3+ reduction rates in the same general order as their rankings developed from field-based and other screening trials, i.e., the lemon-type rootstocks such as Rangpur and Volkamer and rough lemon had the highest Fe reduction rates, the mandarins had intermediate rates, and the citranges and citrumelos along with trifoliate orange had the lowest rates. However, some selections ranked well below their expected ranking from field observation; also, measurements of Fe+3 reduction rates along with growth and leaf chlorosis suggested that the selections fell into several categories based on a composite of the three variables that improved the ranking procedure.

Two questions remaining after our first efforts were: 1) could the procedure be miniaturized so that individual plants might be rapidly and efficiently evaluated, thus enabling high-throughput screening for citrus breeders, a clear advantage in plant breeding; and 2) how do the solution culture and soil screening methods compare given that soil is a more natural environment and may produce results more closely linked to field experience? Thus, our objectives were to expand the range of material screened for tolerance to low-Fe stress to include additional rootstocks, citrus relatives, and other selections to confirm our previous results, develop a miniaturized procedure, and compare solution culture with soil screening.

Materials and Methods

Plant material.

All plants were either seedlings or cuttings grown in containers with a peat-based medium before their use in these experiments (Table 1). The seedlings were grown in the spring, except as noted, in a temperature-controlled greenhouse with natural light and were fertilized regularly with a tap water mix of a water-soluble 20N–20P–20K plus micronutrients fertilizer.

Table 1.

Plant materials for low-iron stress screening.

Table 1.

Nutrient solution screening, Summer and Fall 1998.

Uniform seedlings ≈10 cm tall with four to six leaves were removed from their containers and washed free of the medium. They were transferred to 11-L plastic tubs filled with a nutrient solution of 1.3 mm Ca(NO3)2, 1.0 mm KNO3, 0.8 mm MgSO4, 0.1 mm K2HPO4, 0.56 μM ZnSO4, 6.7 μM MnSO4, 0.24 μM CuSO4, 0.2 μM Na2MoO4, 33.0 μM boric acid, H3BO3, and 35 μM FeHEDTA. The solutions were prepared with tap water containing less than 0.15 mg·L−1 (2.7 μM) Fe. There were two tubs (replications) of each selection and each tub held 28 plants suspended in the lid in 3-cm diameter, equally spaced holes and kept in place with a soft foam stopper. Some screening runs included single 22-L tubs holding 54 plants of one selection. Plants were grown in the +FeHEDTA solution for ≈10 d and then the solution was replaced with one excluding Fe. The solution was changed approximately every 2 to 3 weeks at which time the initial nitrogen (N) level had declined not more than 20% as determined in previous studies. Each solution change was amended with 0.8 g (small tubs) or 1.6 g (large tubs) of Banrot® fungicide (Grace-Sierra Crop Protection, Milpas, CA). Solution pH was ≈8.0 and weekly monitoring showed that it varied less than 0.2 units during a run. A typical run was 6 to 10 weeks.

Screening runs using these standard procedures were conducted in the summer and fall, often in the same or a similar greenhouse as where the seedlings were grown. The tubs for all solution screenings were arranged in a completely randomized design on a bench and located to minimize any environmental effects within the greenhouse. One aquarium-style aeration stone was placed in each tub and connected to a small air compressor. Aeration was provided for 20 min every hour throughout the 24-h day. Clamps were used so that aeration was a gentle stream of bubbles in each tub.

Approximately 20 to 30 white root tips, each 1.0 to 1.5 cm long, were harvested weekly among all the plants in each tub. The same plants were not used at each harvest. The root tips were placed in 300-mL Fleaker beakers (Pyrex®, Lowell, MA) containing a solution of 2-(4-morpholino)-ethane sulfonic acid buffer, Ca(NO3)2, KNO3, MgSO4, and 0.2 mm bathophenanthrolinedisulfonic acid (BPDS); 0.3 mm FeHEDTA was added to the solution and root Fe3+ reduction rates assayed in a darkened room at 33 °C in a water shaker bath (Castle and Manthey, 1998; Manthey et al., 1993). The amount of Fe2+(BPDS)3 formed was measured at 2-h intervals over a 6-h period on a spectrophotometer (Turner Model 340; Biomolecular, Inc., Reno, NV) at 536 nm. Compensation for microlocation-specific Fe3+ reduction by sources other than roots was accomplished by spectrophotometric use of a blank assay mixture attached to each Fleaker beaker. New roots generally appeared within the 10-d period when the plants were still in +Fe solution; thus, baseline Fe3+ reduction rates were measured when the plants were being transitioned from the +Fe to the –Fe solution. Assays continued until a peak value was identified. At the completion of each assay, the root tips were retrieved and oven-dried at 70 °C. Fe3+ reduction rates were expressed as μM Fe3+/h·g−1 root dry weight.

Fresh weights of all plants were measured before placing them in the tubs and at the end of a run; also, two tubs each of Volkamer lemon, Cleopatra mandarin, and Swingle citrumelo seedlings in +Fe solution were included in the summer run to verify good growth when Fe was not limiting. The development of Fe deficiency symptoms was monitored by measuring changes in leaf greenness with a SPAD-502 chlorophyll meter (Minolta Camera Co., Osaka, Japan) (Monge and Bugbee, 1992). Five plants were randomly selected in each tub. One fully expanded lower leaf selected at the beginning of a run and the most recent fully expanded upper leaf were measured biweekly on the same plants. All SPAD data were the mean of five readings/leaf.

Modified nutrient solution screening, summers of 1999 and 2001.

A screening experiment was conducted in 1999 with eight tubs each of Volkamer lemon, Carrizo citrange, and x639 rootstocks. Four tubs (replicates) of each rootstock were assigned for measurement of Fe3+ reduction rates by the standard method described previously using 20 to 30 root tips at each sampling date. To assess the potential for scaling down measurements to individual plants, four tubs were used to collect 10 root tips from one plant in each tub. Each root tip was assayed in a 1.5-mL microcentrifuge tube. Adequate roots were generally produced from individual plants for multiple harvests, but occasionally another plant was selected, but the same length of root tip was collected at each harvest. The root tips from single plants were pooled when dried and their mean weight used for calculating Fe reduction rates. The outcomes of this trial were used to develop an estimate of sample size for the 2001 assays using the root tips of single plants.

The 1999 experiment was repeated in 2001 with Volkamer lemon, Carrizo citrange, and Swingle citrumelo with two tubs/treatment. The standard procedure was compared with the modified one; however, in the latter treatment, nine root tips were collected and assayed individually from each of four plants to examine among-plant variability within tubs.

Additional rootstock selections were screened in the 2001 trial using the standard procedure. The somatic hybrid selections in this trial were first grown in +Fe standard nutrient solution or one amended with 0.25 mm (NH4)2SO4 to observe their root growth response to extra N and to potentially promote root acidification. There was no apparent effect resulting from N source and rate. Adequate root growth occurred only after 30 d at which time the somatic hybrid plants were transferred to –Fe solutions.

Soil screening, 1999.

Immokalee fine sand (sandy, siliceous, hyperthermic Arenic Haplaquod) soil was collected at a depth of 0 to 15 cm from an uncultivated site and amended with CaCO3 at the rates of none, 1.25%, 2.50%, 5%, or 10% by weight [rates suggested by T.A. Obreza based on previous field experience (Obreza, 1995)]. The soil was wetted to field capacity. The amended soil was used to fill 2.5-L plastic pots. After a 30-d equilibration period, samples taken from a subset of pots were shown to contain 0.4%, 1.4%, 2.2%, 4.2%, and 5.9% CaCO3, respectively, using an acetic acid procedure (Loeppert et al., 1984). Additional sampling indicated that no changes occurred in soil CaCO3 concentration during the screening run. Factorial treatments were formed from combinations of the five CaCO3 rates and 12 rootstocks. After equilibration, six single-pot replications of each rootstock selection were planted and arranged in a completely randomized design on greenhouse benches in early summer. A row of buffer plants was placed at the edges of each bench. All plants were irrigated as needed and fertilized monthly with a 30.4N–30.4P–30.4K–6.1Mg–0.8Cu–0.1B–0.3Zn–.5Mn nutrient solution with Ca(NO3)2 as the N source and no Fe. Leaf greenness was measured as described previously along with plant fresh weights at the beginning and end of the run and biweekly plant heights.

Statistical procedures.

The standard method nutrient solution experiments and the experiment in soil were conducted in a completely randomized design. Data were analyzed by SAS PROC GLM (SAS Institute, Inc., Cary, NC) and comparison of means was with Tukey's honestly significant difference test. The soil experiment treatments were factorially arranged and analyzed as such; no interactions occurred, thus mean separation was by Tukey's test. The sample size estimate was determining by assessing root tip and plant-to-plant variability and calculating when they were approximately equal using PROC GLM and PROC MIXED analyses to estimate variance components.

Results and Discussion

Nutrient solution screening, Summer and Fall 1998.

The baseline Fe3+ reduction rates for plants before their transfer to the –Fe solution were similar among all selections and to those reported in our previous work (Table 2; Castle and Manthey, 1998). After transfer to the –Fe solution, peak values in the summer and fall trials confirmed the consistently high Fe3+ reduction rate of Volkamer lemon, a commercial rootstock that was significantly different from all others in the summer trial except Chinese Glycosmis and Carrizo citrange. Other selections with relatively high Fe3+reduction rates were Orange jessamine (Murraya paniculata), a citrus relative, and a new Cleopatra mandarin trifoliate orange hybrid from Spain (Forner et al., 2000). Among those with the lowest reduction rates were Swingle and F80-8 citrumelos and Chinese box orange (Severinia buxifolia). Overall, selections tested previously repeated their general relationship of Fe reduction rates: Volkamer lemon and mandarin types > citranges > citrumelos > trifoliate orange (Castle and Manthey, 1998). Selections not tested previously were mostly citranges. Their performance ranged from a high reduction rate (Carrizo) to intermediate (Uvalde, C-32, Kuharske, and Benton) suggesting that expectations of poor response to low Fe stress because of their trifoliate orange parent may not be justified depending on the particular citrange. We found a similar result for mandarin rootstocks (Castle and Manthey, 1998).

Table 2.

Iron (Fe) reduction rates (n = 2) for Citrus and related selections in solution culture.

Table 2.

Among the citrus relatives tested, Chinese Glycosmis, Orange jessamine, a second species of Citropsis, and Chinese wampee had Fe3+ reduction rates from ≈7 to 16. Other citrus relatives, including the kumquat species, Indian bael fruit, calamondin, Chinese box orange, and Ceylon Atalantia, showed little response with Fe3+ reduction rates 5.0 or less (Table 2).

A large number of selections, in particular somatic hybrids, were dropped from the trial because of poor root production. Sour orange and Rangpur also had low Fe3+ reduction rates apparently because the buffered assay solution prevented plants of those selections from expressing their ability to acidify their growth solutions as reported previously (Castle and Manthey, 1998). Acidification is the mechanism that apparently explains the known tolerance of those rootstocks to low-Fe stress (Castle and Manthey, 1998; Manthey et al., 1994a).

1998 leaf greenness and plant growth.

There were essentially no changes in leaf greenness of lower leaves present at the start of the trial regardless of Fe3+ reduction rate (Table 3). Changes in the color of young (upper) leaves, where Fe stress symptoms normally appear, were generally related to Fe3+ reduction rates, i.e., those selections with the highest Fe3+ reduction rates showed the least yellowing, but with some exceptions such as Chinese Glycosmis (Table 3). There were few statistical differences among the selections, but most values were less than 1 indicating some yellowing occurred in each selection. The upper leaves of the Sour orange + Benton citrange, Aegle marmelos (Indian bael fruit), and Cleopatra mandarin × trifoliate orange became slightly greener as the trial progressed. The upper leaves of Chinese Glycosmis and a somatic hybrid, Cleo + Flying Dragon TF, had the most change. Those results are supported by comparing the ratio of lower to upper leaves initially and at the end of the trial (Table 3). Like with the lower leaves, the initial lower:upper leaf greenness ratios among selections were not significantly different (data not given). At the end of the trial, plants of Clausena lansium (Wampee), F80-8 citrumelo, and Cleopatra mandarin + Flying Dragon trifoliate orange hybrid had high ratios because of considerable yellowing of the upper leaves, but overall, there were few differences among selections.

Table 3.

Leaf greenness (SPAD) readings (n = 2) among selections tested in the Summer and Fall 1998 solution culture trials.

Table 3.

We did not examine the relationship of Fe reduction rate to leaf yellowing in these experiments in which plants were grown in solution culture with virtually no Fe. However, in conjunction with our first study (Castle and Manthey, 1998), we analyzed the nutrient composition of plant parts before and after growth in the –Fe solution. The unreported results suggested that most selections with high Fe reduction rates had higher initial leaf Fe contents than those with low rates and they showed some evidence of Fe redistribution while growing in the –Fe solution.

Plants of most selections weighed ≈1 to 3 g initially and grew at different rates (Table 4). Final fresh weights ranged from 2.8 to 21.5 g/plant. Differences in initial size were accommodated by calculating relative growth rates which ranged from less than 1 to 6.1. Indian bael fruit (Aegle marmelos) plants grew the most vigorously, although they were not significantly different from the Volkamer lemon plants in the –Fe solution. Plants of Volkamer lemon, Swingle citrumelo, and Cleopatra mandarin grew relatively well in the +Fe solution, thereby serving as a general indicator of satisfactory growth among all plants. The Volkamer plants had high Fe3+ reduction rates and grew vigorously in the –Fe solution but less so in the +Fe solution. That particular result was observed in some of our other unreported studies and is an unexplained response. Perhaps some selections are more sensitive to HEDTA than others. The similar growth of the Cleopatra mandarin seedlings in both solutions combined with its Fe3+ reduction responses supports its intermediate ranking in regard to low-Fe stress tolerance. The Swingle citrumelo plants, which had the lowest Fe3+ reduction rate, grew significantly better in the +Fe solution indicating its lower tolerance to Fe stress.

Table 4.

Changes in plant fresh weight (n = 2) in the Summer and Fall 1998 iron (Fe) reduction trials conducted in –Fe solution culture except as noted.z

Table 4.

Modified nutrient solution screening, summers of 1999 and 2001.

The Fe3+ reduction rates of pooled samples of 20 to 30 root tips (standard method) among three selections did not differ when compared with the mean of individual root tips harvested from single plants and assayed (Table 5). Analysis of variance components showed that among the selections, plant-to-plant variability exceeded within-plant variability (Table 6). Using the estimates of those two sources of variability and calculating when tip-to-tip and plant-to-plant variability were equal, a sample size of seven root tips was adequate to represent one plant. A second run of the modified procedure confirmed the first results. The differences in Fe3+ reduction rates among selections were as observed previously, but there was no difference between methods (Table 7).

Table 5.

Root tip iron (Fe) reduction rates in solution culture among citrus selections and methods.z

Table 5.
Table 6.

Analysis of variance and variance components for sample size estimate.

Table 6.
Table 7.

Root tip iron (Fe) reduction rates (n = 2) among citrus selections and assay methods.

Table 7.

Among the selections tested using the standard method, Volkamer lemon plants had the highest Fe3+ reduction rate; C. macrophylla, C-35, and Carrizo citranges were intermediate; and Swingle citrumelo, Kinkoji, Rangpur, sour orange, and the somatic hybrids had the lowest rates. The consistently low rates with the latter may be a physiological anomaly associated with being tetraploid. Somatic hybrids have not grown well in the nutrient solution used in these trials and have always had low Fe3+ reduction rates regardless of the parents. Those hybrids did not respond to additional N or a change in N source. Their root production was generally weak.

Soil screening, 1999.

Initial plant fresh weight was 3.4 to 7.3 g with some significant differences among selections, but not CaCO3 treatments (Table 8). After 14 weeks, fresh weight relative growth rates (FW-RGR) varied among rootstocks and decreased with increasing CaCO3 level in the soil, but with no interaction between those two treatment factors. Cleopatra mandarin and sour orange plants had the highest FW-RGR and Swingle citrumelo and trifoliate orange plants had the lowest values. There were no statistical differences among virtually all the remaining selections, which had RGR values between 12.5 and 16.5. Plant relative growth rate expressed as height (HT-RGR) was highly correlated (r = 0.89***) with FW-RGR and HT-RGR likewise decreased as CaCO3 soil content increased. Leaf greenness values of the lower and upper leaves were not different initially (data not given) and increased from 1.0 to 2.0 indicating the development of lighter green color in upper leaves of most selections after 14 weeks; however, the ratios of Swingle citrumelo and trifoliate orange plants were significantly higher because the upper leaves turned yellow, especially at the higher CaCO3 rates. Leaf greenness ratios increased as CaCO3 soil content increased.

Table 8.

Plant growth and changes in leaf greenness (n = 6) among rootstocks in the Summer 1999 iron nutrition trial conducted in soil amended with CaCO3.

Table 8.

The difference in FW-RGR among selections was larger than it was among CaCO3 rates. That result along with the absence of a plant × CaCO3 interaction suggests that the range in CaCO3 rates may have been too narrow to separate the selections for their tolerance to calcareous conditions (Obreza, 1995); and RGR differences among the selections were more a reflection of their inherent vigor rather than responses to CaCO3.

A summary of the solution and soil culture results confirms and expands the general relationships among citrus types established in our previous study and in other studies (Table 9; Castle et al., 1993, 2004, 2006; Wutscher, 1979; Wutscher and Olsen, 1970). Volkamer lemon was very consistent in its tolerance to low-Fe stress in soil and solution culture, which matches its reported performance as a rootstock in the field (Castle, 1987; Wutscher, 1979). As such, it would be a reliable stress-tolerant control selection in screening trials. Also in the top group was C. macrophylla, another species likely to have citron in its genetic makeup as does Volkamer lemon (Barrett and Rhodes, 1976). Citron was a high performer in our earlier trials (Castle and Manthey, 1998). Mandarins and mandarin hybrids formed the next group in descending order. Cleopatra mandarin has also been a consistently good performer and is well known for its suitability for use in calcareous soils (Castle, 1987; Chapman, 1968; Wutscher, 1979). It appeared to confer tolerance to the Cleo × trifoliate orange hybrids tested in this trial, including x639, but not to the somatic hybrid with Flying Dragon trifoliate orange.

Table 9.

Summary of relative plant selection responses.z

Table 9.

Citranges and citrumelos formed the middle group in that order. Six citranges were tested and did not differ from each other, but Carrizo in all trials displayed the best tolerance within the group with moderately high Fe reduction rates in solution and good growth in soil culture. Swingle and F80-8 citrumelos had similar Fe reduction rates as the citranges, but almost always exhibited greater leaf chlorosis. Trifoliate orange is relatively intolerant of low-Fe stress as consistently demonstrated in our trials. Like Volkamer lemon, it would be useful as a control selection in screening trials, but as the poor tolerance representative.

The citrus relatives, and other species of Citrus not mentioned here, constituted a large portion of the selections reported here. Their results provided taxonomic insights and suggest the possibility of different mechanisms of plant response. In Swingle's classification, there are two tribes in the orange subfamily, Aurantioideae (Swingle and Reece, 1967). The subtribe, Clauseninae, has three genera, Glycosmis, Clausena, and Murraya. The species we tested from each of those genera had among the highest Fe3+ reduction rates in solution culture and should be tested in soil. In every instance, the species grew well, but leaf yellowing was common. They may be a source of genetic material, including ferretin-type genes (Briat and Lobréaux, 1997; Briat et al., 1999; Goto and Yoshihara, 2001) that could be used to introduce low-Fe stress tolerance into commercial species and in breeding new selections. Indian bael fruit (Aegle marmelos) seedlings produced an opposite response. They had a relatively low Fe3+ reduction rate, but grew vigorously and remained green. In the other tribe, Citreae, of the orange subfamily, several genera of subtribe 2, Citrinae, that includes Citrus, were tested in solution culture. Chinese box orange (Severinia buxifolia) seedlings hardly grew and had low Fe3+ reduction rates. We were unable to produce useable seedlings of Ceylon Atalantia, but the two species of Citropsis (Gillet's cherry-orange and West African cherry-orange) grew well, had among the highest Fe3+ reduction rates, and showed little to no leaf yellowing. Thus, Citropsis gilletiana and articulata may also be good genetic sources of low-Fe stress tolerance, particularly because they are one of the most closely related species to Citrus among all selections evaluated.

Somatic hybrids (tetraploids) were overall poor performers despite having, in many instances, one parent known to be relatively tolerant to low-Fe stress. Most of those hybrids were slow-growing and did not produce enough roots in solution culture for screening; thus, it may be that their true potential was not accurately assessed. Likewise, the citrus relatives of the Fortunella genus (kumquat) and the Procimequat hybrid were of moderate to low vigor with generally low Fe3+ reduction rates.

Results from our previous study (Castle and Manthey, 1998) and those reported here show that among a broad range of Citrus selections, low-Fe stress tolerance (i.e., a combination of plant growth, expression of Fe deficiency symptoms, and solution versus soil culture responses) is generally best among selections with a Citrus medica (citron) base and sour orange and related selections. Good sources of tolerance also appear to exist within several genera of the citrus relatives. Mandarin selections and some citranges have relatively high tolerances. Low tolerance occurs among sweet oranges, citrumelos, and trifoliate orange. In many instances, however, there are exceptions and so sweeping generalizations are not appropriate.

Observations made with Rangpur and sour orange suggest that these rootstocks may exhibit rhizosphere acidification as their primary response to Fe deficiency (Castle and Manthey, 1998; Manthey et al., 1994a; Treeby and Uren, 1993). Rangpur has been previously shown to express a high level of Fe deficiency-induced Fe3+ reduction (Manthey et al., 1994a); likewise, sour orange, at times, expressed similarly induced rates of Fe3+ reduction (Manthey, unpublished data). However, observations of plants grown in hydroponic solutions suggested that induced Fe3+ reduction for these two rootstocks did not simultaneously occur with root acidification, but rather, the two responses occurred separately. Whether this occurs with soil-grown roots is unknown. However, these abilities to acidify the root environment as well as to express an inducible Fe3+ reduction ability may explain why those selections are so well adapted to calcareous soils. Rhizosphere acidification increases Fe3+(free) solubility at the root surface 1000 times per decrease in pH unit, thus making Fe3+ reduction by either enzymatic or nonenzymatically catalyzed reactions far more rapid than at neutral or alkaline pH (Manthey et al., 1994b). Additionally, roots of a selection that is responsive to low Fe stress, growing in an acidified and reduction-rich microenvironment, may have sharply contrasting microbial and chemical ecologies, than non-responding selections growing in high carbonate soils (Manthey et al., 1994b). These possibilities may explain the variability in plant Fe3+ reduction, growth, and leaf greenness responses we recorded for other selections, including those related to sour orange, e.g., Chinotto, Taiwanica, Kinkoji, and Smooth Flat Seville.

We conclude that measuring Fe3+ reduction rate is a useful screening tool because of the generally good relationship between those plant responses in solution culture and soil and their combined positive relationship to known carbonate-induced Fe chlorosis responses of plants grown in soil under field conditions. However, plant responses were not always consistent and may be an expression of one or more tolerance mechanisms. It is also critical to be aware that some important aspects of Fe tolerance are not captured using this method; thus, this technique could exclude important tolerant genotypes.

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  • Castle, W.S. 1987 Citrus rootstocks 361 399 Rom R.C. & Carlson R.F. Rootstocks for fruit crops John Wiley and Sons New York, NY

  • Castle, W.S., Bowman, K.D., Graham J.H. Jr & Tucker, D.P.H. 2006 Florida rootstock selection guide Univ. Fla. Coop. Ext. Pub., SP-248

  • Castle, W.S., Grosser, J.W., Gmitter, F.G., Schnell, R.J., Ayala-Silva, T., Crane, J.H. & Bowman, K.D. 2004 Evaluation of new citrus rootstocks for ‘Tahiti’ lime production in southern Florida Proc. Fla. State Hort. Soc. 117 174 181

    • Search Google Scholar
    • Export Citation
  • Castle, W.S. & Manthey, J.A. 1998 Screening citrus rootstocks for iron-deficiency tolerance Fruits 53 375 381

  • Castle, W.S., Pelosi, R.R., Youtsey, C.O., Gmitter F.G. Jr, Lee, R.F., Powell, C.A. & Hu, X. 1992 Rootstocks similar to sour orange for Florida citrus trees Proc. Fla. State Hort. Soc. 105 56 60

    • Search Google Scholar
    • Export Citation
  • Castle, W.S., Tucker, D.P.H., Krezdorn, A.H. & Youtsey, C.O. 1993 Rootstocks for Florida citrus 2nd Ed Univ. Fla. Coop. Ext. Pub., SP-42

  • Chapman, H.D. 1968 The mineral nutrition of citrus 127 289 Reuther W., Batchelor L.D. & Webber H.J. The citrus industry Vol. 2 Univ. Calif Berkeley, CA

  • Cooper, W.C. & Peynado, A. 1953 A comparison of sour orange and Cleopatra mandarin seedlings on salty and calcareous soils Proc. Rio Grande Valley Hort. Soc. 7 95 101

    • Search Google Scholar
    • Export Citation
  • Cooper, W.C. & Peynado, A. 1954 Screening citrus seedlings for tolerance to calcareous soils J. Rio Grande Valley Hort. Soc. 8 100 105

  • Ferguson, L., Sacovich, N. & Roose, M. 1990 California citrus rootstocks Univ. Calif. Pub, 21477

  • Forner, J.B., Forner, M.A., Alcaide, A., Verdejo-Lucas, S. & Sorribas, F.J. 2000 New hybrid citrus rootstocks released in Spain Proc. Intl. Soc. Citricult. 1 58 61

    • Search Google Scholar
    • Export Citation
  • Goto, F. & Yoshihara, T. 2001 Improvement of micronutrient contents by genetic engineering—Development of high iron content crop Plant Biotechnol. 18 7 15

    • Search Google Scholar
    • Export Citation
  • Grosser, J.W., Medina-Urrutia, V., Ananthakrishnan, G. & Serrano, P. 2004 Building a replacement sour orange rootstock: Somatic hybridization of selected mandarin + pummelo combinations J. Amer. Soc. Hort. Sci. 129 530 534

    • Search Google Scholar
    • Export Citation
  • Hamze, M., Ryan, J. & Zaabout, N. 1986 Screening of citrus rootstocks for lime-induced chlorosis tolerance J. Plant Nutr. 9 459 469

  • Hodgson, R.W. 1967 Horticultural varieties of citrus 431 591 Reuther W., Webber H.J. & Batchelor L.D. The citrus industry Vol. 1 Univ. Calif Berkeley, CA

  • Korcak, R. 1987 Iron deficiency chlorosis Hort. Rev. (Amer. Soc. Hort. Sci.) 9 133 186

  • Loeppert, R.H., Hallmark, C.T. & Koshy, M.M. 1984 Routine procedure for rapid determination of soil carbonates Soil Sci. Amer. J. 48 1030 1033

  • Manthey, J.A., McCoy, D.L. & Crowley, D.E. 1993 Chelation effects on the iron reduction and uptake by low-Fe stress tolerant and intolerant citrus rootstocks J. Plant Nutr. 16 881 893

    • Search Google Scholar
    • Export Citation
  • Manthey, J.A., McCoy, D.L. & Crowley, D.E. 1994a Stimulation of rhizosphere iron reduction and uptake in response to iron deficiency in citrus rootstocks Plant Physiol Biochem. 32 211 215

    • Search Google Scholar
    • Export Citation
  • Manthey, J.A., Crowley, D.E. & Luster, D.G. 1994b Biochemistry of metal micronutrients in the rhizosphere CRC Press Boca Raton, FL

  • Monge, D.A. & Bugbee, B. 1992 Inherent limitations of non-destructive chlorophyll meters: A comparison HortScience 27 69 71

  • Obreza, T.A. 1995 Soil CaCO3 concentration affects growth of young grapefruit trees on Swingle citrumelo rootstock Proc. Fla. State Hort. Soc. 108 147 150

    • Search Google Scholar
    • Export Citation
  • Pestana, M., de Varennes, A., Abadía, J. & Faria, E.A. 2005 Differential tolerance to iron deficiency of citrus rootstocks grown in nutrient solution Sci. Hort. 104 25 36

    • Search Google Scholar
    • Export Citation
  • Sagee, O., Hasdai, D., Hamou, M. & Shaked, A. 1992 Screenhouse evaluation of new citrus rootstocks for tolerance to adverse soil conditions Proc. Intl. Soc. Citricult. 1 299 303

    • Search Google Scholar
    • Export Citation
  • Sudahono, D.H.B. & Rouse, R.E. 1994 Greenhouse screening of citrus rootstocks for tolerance to bicarbonate-induced iron chlorosis HortScience 29 113 116

    • Search Google Scholar
    • Export Citation
  • Swingle, W.T. & Reece, P.C. 1967 The botany of citrus and its wild relatives 190 430 Reuther W., Webber H.J. & Batchelor L.D. The citrus industry Vol. 1 Univ. Calif Berkeley, CA

    • Search Google Scholar
    • Export Citation
  • Tagliavini, M. & Rombolá, A.D. 2001 Iron deficiency and chlorosis in orchard and vineyard ecosystems Eur. J. Agron. 15 71 92

  • Treeby, M. & Uren, N. 1993 Iron deficiency stress responses amongst citrus rootstock. Z. Pflanzenemahr Bodenk 56 75 81

  • Wei, L.C., Ocumpaugh, W.R. & Loeppert, R.H. 1994 Plant growth and nutrient uptake characteristics of Fe-deficiency chlorosis susceptible and resistant subclovers Plant Soil 165 235 240

    • Search Google Scholar
    • Export Citation
  • Wutscher, H.K. 1979 Citrus rootstocks. Hort. Rev (Amer. Soc. Hort. Sci.) 1 237 269

  • Wutscher, H.K. & Olsen, E.O. 1970 Leaf nutrient levels, chlorosis, and growth of young grapefruit trees on 16 rootstocks grown on calcareous soil J. Amer. Soc. Hort. Sci. 95 259 261

    • Search Google Scholar
    • Export Citation

Contributor Notes

The technical assistance of James Baldwin is greatly appreciated.

To whom reprint requests should be addressed; e-mail bcastle@ufl.edu.

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  • Barrett, H.C. & Rhodes, A.M. 1976 A numerical taxonomic study of affinity relationships in cultivated Citrus and its close relatives Syst. Bot. 1 105 136

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    • Export Citation
  • Briat, J.F. & Lobréaux, S. 1997 Iron transport and storage in plants Trends Plant Sci. 2 187 193

  • Briat, J.F., Lobréaux, S., Grignon, N. & Vansuyt, G. 1999 Regulation of plant ferritin synthesis: How and why Cell. Mol. Life Sci. 56 155 166

  • Campbell, C.W. 1991 Rootstocks for the Tahiti lime Proc. Fla. State. Hort. Soc. 104 28 30

  • Castle, W.S. 1987 Citrus rootstocks 361 399 Rom R.C. & Carlson R.F. Rootstocks for fruit crops John Wiley and Sons New York, NY

  • Castle, W.S., Bowman, K.D., Graham J.H. Jr & Tucker, D.P.H. 2006 Florida rootstock selection guide Univ. Fla. Coop. Ext. Pub., SP-248

  • Castle, W.S., Grosser, J.W., Gmitter, F.G., Schnell, R.J., Ayala-Silva, T., Crane, J.H. & Bowman, K.D. 2004 Evaluation of new citrus rootstocks for ‘Tahiti’ lime production in southern Florida Proc. Fla. State Hort. Soc. 117 174 181

    • Search Google Scholar
    • Export Citation
  • Castle, W.S. & Manthey, J.A. 1998 Screening citrus rootstocks for iron-deficiency tolerance Fruits 53 375 381

  • Castle, W.S., Pelosi, R.R., Youtsey, C.O., Gmitter F.G. Jr, Lee, R.F., Powell, C.A. & Hu, X. 1992 Rootstocks similar to sour orange for Florida citrus trees Proc. Fla. State Hort. Soc. 105 56 60

    • Search Google Scholar
    • Export Citation
  • Castle, W.S., Tucker, D.P.H., Krezdorn, A.H. & Youtsey, C.O. 1993 Rootstocks for Florida citrus 2nd Ed Univ. Fla. Coop. Ext. Pub., SP-42

  • Chapman, H.D. 1968 The mineral nutrition of citrus 127 289 Reuther W., Batchelor L.D. & Webber H.J. The citrus industry Vol. 2 Univ. Calif Berkeley, CA

  • Cooper, W.C. & Peynado, A. 1953 A comparison of sour orange and Cleopatra mandarin seedlings on salty and calcareous soils Proc. Rio Grande Valley Hort. Soc. 7 95 101

    • Search Google Scholar
    • Export Citation
  • Cooper, W.C. & Peynado, A. 1954 Screening citrus seedlings for tolerance to calcareous soils J. Rio Grande Valley Hort. Soc. 8 100 105

  • Ferguson, L., Sacovich, N. & Roose, M. 1990 California citrus rootstocks Univ. Calif. Pub, 21477

  • Forner, J.B., Forner, M.A., Alcaide, A., Verdejo-Lucas, S. & Sorribas, F.J. 2000 New hybrid citrus rootstocks released in Spain Proc. Intl. Soc. Citricult. 1 58 61

    • Search Google Scholar
    • Export Citation
  • Goto, F. & Yoshihara, T. 2001 Improvement of micronutrient contents by genetic engineering—Development of high iron content crop Plant Biotechnol. 18 7 15

    • Search Google Scholar
    • Export Citation
  • Grosser, J.W., Medina-Urrutia, V., Ananthakrishnan, G. & Serrano, P. 2004 Building a replacement sour orange rootstock: Somatic hybridization of selected mandarin + pummelo combinations J. Amer. Soc. Hort. Sci. 129 530 534

    • Search Google Scholar
    • Export Citation
  • Hamze, M., Ryan, J. & Zaabout, N. 1986 Screening of citrus rootstocks for lime-induced chlorosis tolerance J. Plant Nutr. 9 459 469

  • Hodgson, R.W. 1967 Horticultural varieties of citrus 431 591 Reuther W., Webber H.J. & Batchelor L.D. The citrus industry Vol. 1 Univ. Calif Berkeley, CA

  • Korcak, R. 1987 Iron deficiency chlorosis Hort. Rev. (Amer. Soc. Hort. Sci.) 9 133 186

  • Loeppert, R.H., Hallmark, C.T. & Koshy, M.M. 1984 Routine procedure for rapid determination of soil carbonates Soil Sci. Amer. J. 48 1030 1033

  • Manthey, J.A., McCoy, D.L. & Crowley, D.E. 1993 Chelation effects on the iron reduction and uptake by low-Fe stress tolerant and intolerant citrus rootstocks J. Plant Nutr. 16 881 893

    • Search Google Scholar
    • Export Citation
  • Manthey, J.A., McCoy, D.L. & Crowley, D.E. 1994a Stimulation of rhizosphere iron reduction and uptake in response to iron deficiency in citrus rootstocks Plant Physiol Biochem. 32 211 215

    • Search Google Scholar
    • Export Citation
  • Manthey, J.A., Crowley, D.E. & Luster, D.G. 1994b Biochemistry of metal micronutrients in the rhizosphere CRC Press Boca Raton, FL

  • Monge, D.A. & Bugbee, B. 1992 Inherent limitations of non-destructive chlorophyll meters: A comparison HortScience 27 69 71

  • Obreza, T.A. 1995 Soil CaCO3 concentration affects growth of young grapefruit trees on Swingle citrumelo rootstock Proc. Fla. State Hort. Soc. 108 147 150

    • Search Google Scholar
    • Export Citation
  • Pestana, M., de Varennes, A., Abadía, J. & Faria, E.A. 2005 Differential tolerance to iron deficiency of citrus rootstocks grown in nutrient solution Sci. Hort. 104 25 36

    • Search Google Scholar
    • Export Citation
  • Sagee, O., Hasdai, D., Hamou, M. & Shaked, A. 1992 Screenhouse evaluation of new citrus rootstocks for tolerance to adverse soil conditions Proc. Intl. Soc. Citricult. 1 299 303

    • Search Google Scholar
    • Export Citation
  • Sudahono, D.H.B. & Rouse, R.E. 1994 Greenhouse screening of citrus rootstocks for tolerance to bicarbonate-induced iron chlorosis HortScience 29 113 116

    • Search Google Scholar
    • Export Citation
  • Swingle, W.T. & Reece, P.C. 1967 The botany of citrus and its wild relatives 190 430 Reuther W., Webber H.J. & Batchelor L.D. The citrus industry Vol. 1 Univ. Calif Berkeley, CA

    • Search Google Scholar
    • Export Citation
  • Tagliavini, M. & Rombolá, A.D. 2001 Iron deficiency and chlorosis in orchard and vineyard ecosystems Eur. J. Agron. 15 71 92

  • Treeby, M. & Uren, N. 1993 Iron deficiency stress responses amongst citrus rootstock. Z. Pflanzenemahr Bodenk 56 75 81

  • Wei, L.C., Ocumpaugh, W.R. & Loeppert, R.H. 1994 Plant growth and nutrient uptake characteristics of Fe-deficiency chlorosis susceptible and resistant subclovers Plant Soil 165 235 240

    • Search Google Scholar
    • Export Citation
  • Wutscher, H.K. 1979 Citrus rootstocks. Hort. Rev (Amer. Soc. Hort. Sci.) 1 237 269

  • Wutscher, H.K. & Olsen, E.O. 1970 Leaf nutrient levels, chlorosis, and growth of young grapefruit trees on 16 rootstocks grown on calcareous soil J. Amer. Soc. Hort. Sci. 95 259 261

    • Search Google Scholar
    • Export Citation
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