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

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 Fe. 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 Fe 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 Fe 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 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 Fe 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 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 Received for publication 29 Dec. 2008. Accepted for publication 24 Mar. 2009. The technical assistance of James Baldwin is greatly appreciated. To whom reprint requests should be addressed; e-mail [email protected]. 638 HORTSCIENCE VOL. 44(3) JUNE 2009 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. 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 mM ZnSO4, 6.7 mM MnSO4, 0.24 mM CuSO4, 0.2 mM Na2MoO4, 33.0 mM boric acid, H3BO3, and 35 mM FeHEDTA. The solutions were prepared with tap water containing less than 0.15 mg L (2.7 mM) 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 Table 1. Plant materials for low-iron stress screening. Common name Scientific name Source Trials Summer 98 soln. Fall 98 soln. 1999 soil Soln. 1999 Mintube 1999 Soln. 2001 Minitube 2001 Benton citrange C. sinensis (L.) Osb. · Poncirus trifoliata (L.) Raf. USNGR x C-32 citrange C. sinensis Ruby · P. trifoliata USNGR x C-35 citrange C. sinensis Ruby · P. trifoliata USNGR x Calamondin C. madurensis Lour. DPI ARB EF-55 x Carrizo citrange C. sinensis · P. trifoliata Reed Bros. x x x x x Ceylon Atalantia Atalantia ceylanica (Arn.) Oliv. USNGR CRC 3287 One tub Chinese box orange Severinia buxifolia (Poir.) Ten. DPI ARB-11-1 x Chinese glycosmis Glycosmis pentaphylla Auct. DPI ARB-17.5-2 x Chinese wampee Clausena lansium (Lour.) Skeels USNGR CRC 3126 x Chinotto sour orange C. myrtifolia Raf. DPI ARB-PL-2 x Cleo + FDT C. recticulata Blanco Cleopatra + P. trifoliata Flying Dragon JG x Cleo · TF (52) C. reticulata · P. trifoliata Spain 30142 x Cleo · TF (51) C. reticulata · P. trifoliata Spain 3017 x x Cleo · TF (50) C. reticulata · P. trifoliata Spain 3015 One tub x Cleopatra mandarin C. reticulata DPI ARB 1-7 x x F80-8 citrumelo C. paradisi · P. trifoliata DPI ARB 19-24 x Gillet’s cherry-orange Citropsis gilletiana Swing. & M. Kell. USNGR CRC 3296 x Hamlin + rough lemon C. sinensis + C. jambhiri Lush. JG x Indian bael fruit Aegle marmelos (L.) Corr. USNGR x Kinkoji C. obovoidea Hort. ex Tak. DPI ARB-12-11 x x Kuharske citrange C. sinensis · P. trifoliata DPI x Macrophylla C. macrophylla Wester DPI x Marumi kumquat Fortunella japonica (Thunb.) Swing. DPI ARB-10-5 x Meiwa kumquat Fortunella crassifolia Swing. DPI ARB-10-4 x Milam + Kinkoji C. jambhiri hybrid + C. oboviodea JG One tub Nova + HB pummelo [C. reticulata · (C. paradisi · C. reticulata)] + C. maxima (Burm.) Merrill JG x x Nova + Ichangensis [C. reticulata · (C. paradisi · C. reticulata)] + C. ichangensis Swing. JG x Orange jessamine Murraya paniculata (L.) Jack, Malay DPI ARB-17.5-1 x Procimequat Fortunella hybrid DPI ARB-10-6 x Rangpur Citrus limonia Osb. DPI ARB-10-9 x x x Smooth Flat Seville C. aurantium hybrid Reed Bros. x Sour orange C. aurantium (L.) DPI ARB-13-12 x x x x Sour orange + Benton citrange C. aurantium + (C. sinensis · P. trifoliata) JG x Sour orange + Carrizo C. aurantium + (C. sinensis · P. trifoliata) JG x x Sour orange + Flying Dragon TF C. aurantium + P. trifoliata JG x Sour orange + PSL C. aurantium + C. limettioides Tan. JG x x Sour orange + Rangpur C. aurantium + C. limonia JG x Sour orange + TF (50-7) C. aurantium + P. trifoliata JG x Succari + Atalantia ceylanica C. sinensis + A. ceylonica JG x Sunki · Benecke TF C. sunki Hort. ex Tak. · P. trifoliata Benecke DPI x Swingle citrumelo C. paradisi Macf. · P. trifoliata DPI F/E23 19-24 x x Tachibana C. tachibana USDA ARB 9-6 x Taiwanica C. taiwanica Tan. & Shim. DPI x TF 50-7 P. trifoliata DPI ARB-12-7N x Troyer · mandarin (C. sinensis · P. trifoliata) · C. reticulata Spain 020418 One tub Uvalde citrange C. sinensis (L.) Osb. · Poncirus trifoliata USDA ARB-6-9 One tub Volkamer lemon Citrus volkameriana Ten. & Pasq. DPI F/E 23(5-6) x x x x x x x West African cherry-orange Citropsis articulata (Willd.) Swing. & M. Kell. USNGR x Willits citrange C. sinensis · P. trifoliata USDA One tub x639 C. reticulata Cleopatra · P. trifoliata Rubidoux DPI ARB-14-11 x USNGR = U.S. National Germplasm Repository for Citrus & Dates, Riverside, CA; DPI = Florida Dept. Agr. Consumer Services, Div. Plt. Industry, Winter Haven, FL; Reed Bros. = commercial seed supplier, Dundee, FL; JG = Dr. Jude Grosser, UF/IFAS, CREC, Lake Alfred; Spain = Dr. Juan Forner; USDA = Whitmore Foundation Farm, Leesburg, FL. See ‘‘Materials and Methods’’ for descriptions. HORTSCIENCE VOL. 44(3) JUNE 2009 639 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 aquariumstyle 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 Fe 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 Fe(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 microlocationspecific 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 Fe 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. Fe reduction rates were expressed as mM Fe/h g 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 Fe 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 Table 2. Iron (Fe) reduction rates (n = 2) for Citrus and related selections in solution culture. Selection 1998 Fe reduction rate (mmol Fe/h g)