Sonali R. Padhye

Evaluating Calibrachoa (Calibrachoa *hybrida Cerv.) Genotype Sensitivity to Iron Deficiency at High Substrate pH

Floriculture crop species that are inefficient at iron uptake are susceptible to developing iron deficiency symptoms in container production at high substrate pH. The objective of this study was to compare genotypes of iron-inefficient calibrachoa (Calibrachoa 3hybrid Cerv.) in terms of their susceptibility to showing iron deficiency symptoms when grown at high vs. low substrate pH. In a greenhouse factorial experiment, 24 genotypes of calibrachoa were grown in peat:perlite substrate at low pH (5.4) and high pH (7.1). Shoot dry weight, leaf SPAD chlorophyll index, flower index value, and shoot iron concentration were measured after 13 weeks at each substrate pH level. Of the 24 genotypes, analysis of variance (ANOVA) found that 19 genotypes had lower SPAD and 18 genotypes had reduced shoot dry weight at high substrate pH compared with SPAD and dry weight at low substrate pH. High substrate pH had less effect on flower index and shoot iron concentration than the pH effect on SPAD or shoot dry weight. No visual symptoms of iron deficiency were observed at low substrate pH.Genotypes were separated into three groups using k-means cluster analysis, based on the four measured variables (SPAD, dry weight, flower index, and iron concentration in shoot tissue). These four variables were each expressed as the percent reduction in measured responses at high vs. low substrate pH. Greater percent reduction values indicated increased sensitivity of genotypes to high substrate pH. The three clusters, which about represented high, medium, or low sensitivity to high substrate pH, averaged 59.7%, 42.8%, and 25.2% reduction in SPAD, 47.7%, 51.0%, and 39.5% reduction in shoot dry weight, and 32.2%, 9.2%, and 27.7% reduction in shoot iron, respectively. Flowering was not different between clusters when tested with ANOVA. The least pH-sensitive cluster included all four genotypes in the breeding series ‘Calipetite’. ‘Calipetite’ also had low shoot dry weight at low substrate pH, indicating low overall vigor. There were no differences between clusters in terms of their effect on substrate pH, which is one potential plant iron-efficiency mechanism in response to low iron availability. This experiment demonstrated an experimental and statistical approach for plant breeders to test sensitivity to substrate pH for iron-inefficient floriculture species. Floriculture species differ in susceptibility to developing micronutrient disorders, particularly iron and manganese toxicity or deficiency, depending on the efficiency at which micronutrients are taken up by plant roots and the solubility of micronutrients as a function of pH (Albano and Miller, 1998; Argo and Fisher, 2002). The solubility of inorganic Fe decreases 1000-fold for each unit increase in pH (Lindsay, 1979). Decreased solubility results in low levels of water-extractable iron in soilless substrates when pH is above 6 (Peterson, 1981). Appearance of iron deficiency in iron-inefficient species such as calibrachoa (Calibrachoa ·hybrida) develops at high substrate pH levels (pH > 6.4) and often requires supplemental applications of chelated iron fertilizer (Fisher et al., 2003). Cultivars of iron-efficient floriculture species have been shown to differ in their tendency to accumulate excess iron/manganese at low substrate pH (Albano and Miller, 1998; Harbaugh, 1995). Marigold (Tagetes erecta L.) cultivars developed different degrees of ‘‘leaf bronzing’’ resulting from toxic iron levels in mature leaves after high micronutrient concentrations were applied to the substrate (Albano and Miller, 1998). Susceptible cultivars of pentas (Pentas lanceolata Benth.) developed lower leaf necrosis at substrate pH less than 5.5, which was correlated with high tissue iron levels (Harbaugh, 1995). Cultivars of agronomic crop species grown at high pH and in calcareous soils are also known to differ in susceptibility to iron deficiency (Fr€oechlich and Fehr, 1981; Gao and Shi, 2007; Marschner, 1995; Norvell and Adams, 2006). Typical symptoms of iron deficiency include interveinal chlorosis of young shoots and reduced shoot growth during early stages and can progress to severe stunting and shoot tip death in later stages (Marschner, 1995; R€omheld, 1987). Symptoms of iron deficiency are well documented for floriculture species, with photos of iron deficiency for a range of floriculture species including calibrachoa published by Argo and Fisher (2002), Gibson et al. (2007), and others. Strategies for evaluating agronomic crop species for sensitivity to iron deficiency include growing cultivars in noncalcareous and calcareous soils and measuring differences in shoot chlorosis, growth, and yield (Fr€oechlich and Fehr, 1981; Graham et al., 1992; Hintz et al., 1987; Niebur and Fehr, 1981). Fr€oechlich and Fehr (1981) used percent reduction in plant height and yield to compare soybean (Glycine max L.) cultivars grown in calcareous vs. noncalcareous soils. Gao and Shi (2007) used hierarchical cluster analysis to group peanut (Arachis hypogaea L.) cultivars by sensitivity to iron chlorosis based on leaf SPAD chlorophyll content, physiologically ‘‘active’’ leaf iron at flowering stage, and pod yield. Genotypic differences in iron efficiency has not been studied in calibrachoa, which often shows iron deficiency symptoms at high substrate pH or low iron fertilizer level (Wik et al., 2006). The objective of this study was to compare 24 genotypes of calibrachoa for their sensitivity to showing iron deficiency symptoms (reduced shoot growth, chlorophyll content, tissue iron concentration, and flower number as well as chlorosis and necrosis on new shoots) when grown at high vs. low substrate pH. Twenty of the genotypes were commercial cultivars from four breeding companies, in addition to four experimental genotypes. Eleven genotypes were propagated from seed and the remainder from vegetative cuttings. We hypothesized that differences in sensitivity may be related to the tendency for a genotype to increase pH and thereby reduce iron solubility, and/or higher demand for iron (milligrams iron per plant, from either a high required iron concentration per unit dry weight, or high vigor in terms of dry weight gain). Received for publication 15 June 2016. Accepted for publication 3 Oct. 2016. We thank PanAmerican Seed, USDA-ARS Floriculture and Nursery Research Initiative no. 583607-8-725, and industry partners of the Floriculture Research Alliance at the University of Florida (floriculturealliance.org) for supporting this research. We also thank James Colee from the University of Florida’s Department of Statistics for providing statistical consulting. Corresponding author. E-mail: pfisher@ufl.edu. 1452 HORTSCIENCE VOL. 51(12) DECEMBER 2016 In a greenhouse factorial experiment, seedling plugs and rooted liners of each genotype were transplanted into 11.4-cmdiameter containers and grown for 13 weeks in a soilless peat:perlite substrate at low (initial 5.4) and high (initial 7.1) substrate pH, with analysis of final substrate pH and substrate-electrical conductivity, leaf SPAD chlorophyll content, total shoot dry weight, tissue iron concentrations, and visual indexes of iron chlorosis symptoms and flower number. Materials and Methods Experimental design The experiment was a 24 genotype by two substrate pH factorial using a randomized complete block design with eight blocks (one replicate per block). Genotypes were grown in plastic azalea containers at one plant per container, and each treatment replicate was an individual container (384 total containers). Blocks were divided evenly between two adjacent, identical greenhouses at one block per greenhouse bench. Greenhouse benches were oriented north to south. Plant materials and propagation On 30 Jan. 2014, 24 genotypes of ironinefficient calibrachoa were transplanted from 128-count seedling trays into 11.4-cm (4.5-inch)-diameter azalea containers (500 cm; Poppelman Plastics US LLC, Claremont, NC) at one plant per container. Twenty genotypes were from six commercial breeding series (‘Aloha Kona’, ‘Cabaret’, ‘Calipetite’, ‘Crave’, ‘Kabloom’, and ‘Minifamous’) in addition to four experimental genotypes. Eleven genotypes were propagated by seed and 13 by vegetative tip cuttings. Vegetatively propagated genotypes included genotypes of ‘Aloha Kona’ (‘Canary Yellow’, ‘Milk and Honey’, ‘True Blue’, and ‘Dark Red’), ‘Cabaret’ (‘Deep Blue’, ‘Bright Red’, ‘Deep Yellow’, and ‘White 2015’), ‘Calipetite’ (‘Red’, ‘Blue’, ‘Yellow’, and ‘White’), and ‘Minifamous Pink 2014’. Seedpropagated genotypes included genotypes of ‘Kabloom’ (‘Blue’, ‘Deep Pink’, ‘Denim’, ‘Red’, ‘White’, and ‘Yellow’), ‘Crave Sunset’, and the remaining experimental genotypes (‘E113’, ‘E144’, ‘E153’, and ‘E15597’). Growing conditions and data collection The substrate was (v/v) 80% Canadian Sphagnum peat (Sun Gro Horticulture, Agawam, MA) with long fibers and little dust (Von Post scale 1–2; Puustjarvi and Robertson, 1975) and 20% coarse perlite with preplant fertilizer (in g·m 41.7N, 15.3P, 63.4K, 111.2Ca, 83.4S, 4.2Mg, 0.08B, 0.15Cu, 0.08Fe, 0.54Mn, 0.15Mo, and 0.46Zn) but with no initial liming agent. Nutrients were derived from ammonium nitrate, ammonium phosphate, calcium nitrate, boric acid, copper sulfate, iron ethylenediaminetetraacetic acid (EDTA), magnesium nitrate, manganese sulfate, potassium nitrate, sodium molybdate, and zinc sulfate. Hydrated dolomitic limestone [Graymont Western Lime, Inc., Eden, WI, 97% Ca(OH)2·MgO of which 92% passed through a 45-mm mesh and had an acid neutralizing value of 140 calcium carbonate equivalents (CCEs)] was incorporated at rates of 1.1 and 2.0 kg·m for initial substrate pH levels of 5.4 and 7.1, respectively. Substrate pH levels of 5.4 and 7.1 were considered low and high, respectively, according to the pH range (5.8 to 6.2) recommended for adequate nutrient availability for most bedding plant species by Peterson (1981). Plants at low pH (5.4) were expected to have adequ