Article pubs.acs.org/JAFC
Influence of Cultivar and Fertilizer Approach on Curly Kale (Brassica oleracea L. var. sabellica). 1. Genetic Diversity Reflected in Agronomic Characteristics and Phytochemical Concentration Marie Groenbaek,*,† Sidsel Jensen,† Susanne Neugart,§ Monika Schreiner,§ Ulla Kidmose,† and Hanne Lakkenborg Kristensen† †
Department of Food Science, Faculty of Science and Technology, Aarhus University, Kirstinebjergvej 10, Aarslev DK-5792, Denmark Department Quality, Leibniz-Institute of Vegetable and Ornamental Crops Grossbeeren/Erfurt e.V., Theodor-Echtermeyer-Weg 1, 14979 Grossbeeren, Germany
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ABSTRACT: The objectives were to investigate if genetic diversity among field-grown traditional and F1 hybrid kale cultivars was reflected in different agronomic characteristics and consequently glucosinolate (GLS) and flavonoid glycoside concentration. This study evaluated how nitrogen and sulfur supply and biomass allocation modified phytochemicals in two experiments with combinations of three cultivars and four N and two S application levels. Results showed less growth, and higher N concentration in the traditional cultivar ‘Tiara’ was associated with increased indole and total GLSs compared to traditional ‘Høj Amager Toftø’ and F1 hybrid ‘Reflex’ cultivars, which exhibited higher yield, lower N concentration, and different biomass allocation. S application increased total GLS concentration, whereas aliphatic GLS percentage decreased when N application increased. Decrease of six ‘Reflex’ GLSs besides quercetin glycosides and total flavonoid glycosides with increased N indicated higher N responsiveness for ‘Reflex’. In conclusion, differences in agronomic characteristics were reflected in diverse phytochemical composition. KEYWORDS: curly kale, cultivars, nitrogen, sulfur, glucosinolates, flavonoid glycosides
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INTRODUCTION Due to a characteristic phytochemical content, members of the Brassicaceae have been of special interest during almost half a century.1 The Brassica genus includes several varieties comprising curly kale (Brassica oleracea L. var. sabellica), red and white cabbage (B. oleracea L. var. capitata), broccoli (B. oleracea L. var. italic), Brussels sprouts (B. oleracea L. var. gemmifera), and cauliflower (B. oleracea L. var. botrytis), all known for the species phytochemical content.1 Kale ancestors can be traced back to landraces and wild kales grown on the Iberian peninsula and in the Black Sea region.2 Currently, the highly bred cultivars dominate commercially grown fields. However, several traditional cultivars have been studied to identify potential disease resistance, specific agronomic traits, and sensory properties or phytochemicals beneficial to health, such as glucosinolates (GLSs), flavonoids, and carotenoids.3−5 In general, differences in GLS composition and concentration are found in different B. oleracea varieties.4,6 Each variety rarely contains more than 12 different GLSs, although more than 120 different GLSs are known.1 Furthermore, GLS composition and concentration differences between cultivars have been identified within cauliflower, white cabbage, broccoli, Brussels sprouts, and kale.6,7 Some GLS breakdown products have been linked to the anticarcinogenic properties of Brassica vegetables.8 Moreover, Williams et al.9 associated GLSs with the bitter and pungent taste of cabbages, which was further investigated in the present experiments and reported by Jensen et al.10 Studies suggested different responses in GLS group concentration, for example, indole and aliphatic are influenced by growing conditions and genetic background, respectively.11,12 Nitrogen and sulfur © 2014 American Chemical Society
fertilizers’ effects on GLSs have been widely studied because fertilizers play a major role in GLS biosynthesis, and N and S are incorporated in GLS structure. In brief, several studies reported GLS concentration was generally increased by S fertilizer application in B. oleracea; however, N level, form, and application timing exhibited diverse responses, possibly due to different responses in primary metabolism induced by genotypic variations in N and S uptake and metabolization.12−17 If primary metabolism in relationship to plant growth and protein synthesis is reduced due to limited C, N, or S, a shift toward secondary metabolism of C-, N-, or S-containing phytochemicals, such as flavonols and glucosinolates, might occur.18−20 Consequently, genetic variability between traditional and F1 hybrid cultivars might be reflected in agronomic characteristics, N and S uptake besides plant N and S concentration, and biomass allocation to different plant parts, as well as secondary metabolism. Flavonoid health benefits depend on flavonoid structure.21,22 In curly kale, Schmidt et al.23 found 71 flavonoid glycosides based on the aglycones quercetin, kaempferol, and isorhamnetin, and Zietz et al.22 reported higher antioxidant activity in quercetin glycosides compared to the corresponding kaempferol glycosides in curly kale. Quercetin, kaempferol, and isorhamnetin aglycones exhibited anti-inflammatory effects,24 whereas quercetin glycosides showed enhanced absorbance in the human small intestine compared to quercetin aglycone.25 Studies revealed an inverse Received: Revised: Accepted: Published: 11393
June 30, 2014 October 22, 2014 October 22, 2014 October 22, 2014 dx.doi.org/10.1021/jf503096p | J. Agric. Food Chem. 2014, 62, 11393−11402
Journal of Agricultural and Food Chemistry
Article
Table 1. Effects of Available Nitrogen and Cultivar on Yield and Nutrients in Curly Kale Cultivars HAT, Reflex, and TIARA N (kg ha−1) 90 230 N effect 90 135 185 230 N effect 90 135 185 230 N effect
cv.
total yielda
edible yielda
stalk weighta
stalk heightb
leaf Nc
leaf Sc
stalk Nc
stalk Sc
total N uptaked
total S uptaked
HAT
50.1 ± 2.6e 75.4 ± 2.7 ***f 50.6 ± 3.6 59.1 ± 1.5 66.3 ± 1.4 68.3 ± 6.7 ** 45.1 ± 4.4 50.2 ± 5.5 54.0 ± 2.2 56.7 ± 3.1 *
13.7 ± 1.3 18.3 ± 2.7 NS 17.1 ± 1.1 22.2 ± 1.1 22.6 ± 0.7 23.7 ± 1.3 *** 14.2 ± 0.5 14.1 ± 3.3 15.3 ± 0.3 15.8 ± 2.7 NS
19.4 ± 1.9 34.7 ± 4.8 ** 19.0 ± 1.3 22.2 ± 2.3 24.6 ± 1.0 27.6 ± 1.3 *** 16.9 ± 3.2 21.1 ± 1.6 21.3 ± 0.9 25.4 ± 3.8 *
77 ± 6.7 104 ± 14.9 * 70 ± 5.1 75 ± 2.6 79 ± 3.8 90 ± 4.0 ** 69 ± 2.7 75 ± 4.9 80 ± 3.6 83 ± 8.3 *
23.6 ± 0.8 26.2 ± 0.9 * 23.9 ± 1.3 25.2 ± 2.4 27.9 ± 0.2 26.5 ± 2.9 NS 27.6 ± 3.1 32.0 ± 9.5 27.7 ± 1.5 32.1 ± 1.8 NS
7.7 ± 0.4 7.2 ± 0.5 NS 6.7 ± 0.6 6.6 ± 1.1 7.2 ± 0.7 6.3 ± 1.2 NS 8.8 ± 0.2 9.5 ± 2.1 8.5 ± 1.7 7.9 ± 0.4 NS
9.9 ± 1.8 14.8 ± 3.9 NS 9.7 ± 1.1 11.7 ± 0.8 12.7 ± 1.0 13.6 ± 1.6 * 15.0 ± 0.9 17.9 ± 3.2 19.6 ± 0.8 17.7 ± 2.4 NS
3.9 ± 0.3 3.4 ± 0.4 NS 4.4 ± 0.4 4.2 ± 1.0 4.2 ± 0.3 3.5 ± 0.8 NS 5.7 ± 0.2 6.2 ± 0.7 5.7 ± 0.4 5.0 ± 0.8 NS
168 ± 22.9 264 ± 35.6 * 169 ± 13.5 223 ± 28.0 254 ± 29.0 271 ± 27.3 ** 203 ± 31.6 238 ± 47.0 237 ± 5.9 300 ± 60.8 NS
57 ± 10.6 69 ± 10.0 NS 53 ± 7.4 63 ± 11.0 69 ± 11.3 66 ± 7.6 NS 68 ± 4.5 74 ± 11.5 71 ± 11.5 77 ± 16.5 NS
*** *** NS
*** *** NS
*** *** NS
*** *** NS
NS ** NS
** *** NS
* *** NS
*** NS NS
NS NS NS
Reflex
TIARA
significance N CV N × CV
NS NS NS
Tonne ha−1, bcm. cg kg−1dry matter. dkg ha−1. e± standard deviation. f*, p ≤ 0.05; **, p < 0.01; ***, p < 0.001 in a general linear model among N level and cultivar means (n = 3) within each column. NS, not significant. a
cultivars ‘Halvhøj Ekstra Moskruset Tiara’ (TIARA) accession 1833 and ‘Høj Amager Toftø’ (HAT) accession 4085 (both NordGen germplasm collection)28 were sown on April 4, 2011, and propagated in an unheated greenhouse. Plants were transferred to the field on May 5, 2011, with a row/plant distance of 50/60 cm, respectively. Plots of 3 × 5 m2 with the two outermost rows as guard rows to eliminate border effects were arranged in a complete randomized block design with three replication of plots (n = 3) per fertilizer level and cultivar (see below). An insect net was applied over the plots and used for pest management to reduce herbivory until August 16, 2011. Weeding was conducted manually and mechanically once during the experimental trial. The trials included two separate experimental regimes. Experiments were performed as follows: In experiment I, Reflex, HAT, and TIARA were grown under 30 kg S ha−1 with 90, 135, 185, or 230 kg N ha−1 available in the field (36 plots in total with 30 kg S and 185 kg N ha−1 being standard conditions); and under experiment II, Reflex had either 4 or 30 kg S ha−1 available combined with 90, 135, or 185 kg N ha−1 accessible in the field (18 plots in total). Fertilizer levels applied were adjusted according to the levels in the field measured before planting. S fertilizer was dispersed just prior to planting, and approximately half the total N fertilizer dose was distributed just after planting. The other half was given 6 weeks later. Granulate kieserite (MgSO4, 20% S, K+S Kali GmbH, Germany) and granulate calcium ammonium nitrate (NH4NO3+Ca, 27% N, Hydro Agri Danmark A/S, Denmark) were used as S and N sources, respectively. On October 10, 2011, 10 plants from each replicate plot were harvested, including the entire stalk and leaves (corresponding to 3.1 m2), and total yield was assessed. Four of 10 plants from each plot were evaluated with respect to stalk height from plant base at soil surface to the plant top. Yield characteristics were estimated as follows: edible yield as usable leaf weight (stem diameter < 7 mm) and stalk weight without leaves, stems, and top. Entire plants were maintained at a temperature of 1 °C and 100% relative humidity in black plastic bags until further analysis within 4 days. The plants were handled carefully to avoid tissue disruption and thereby loss of GLSs. The described experiments correspond to analyses 2, 3, and 4 reported by Jensen et al.10 from which sensory evaluation of the different fertilization approaches was assessed. Plant Analyses. Representative samples of approximately 200 g of separated leaves and stalks from each plot were oven-dried at 80 °C to a constant weight, and dry matter content (DM) was calculated. Total leaf and stalk N and S concentrations were analyzed from the oven-dried material. VDLUFA methods were used to determine plant N and S.29,30
relationship between N availability and flavonol or total phenolic concentrations in leaf mustard (Brassica juncea), tomato (Lycopersicon esculentum), and Arabidopsis thaliana.26,27 Moreover, a report on eight different field-grown curly kale cultivars showed relatively high flavonoid concentrations in traditional old cultivars in contrast to lower concentrations in hybrids.5 However, the influence of the genetic variation on flavonoid glycoside and GLS concentrations associated with fertilizer strategies, agronomic characteristic, and biomass allocation in traditional and F1 hybrid cultivars of curly kale has not been reported. Trials conducted under field growth conditions with less control over biogeochemical environments compared to, for example, greenhouse experiments are necessary in the testing of agronomic practices, particularly when a crop stand is examined with relevance to growers and consumers. On the basis of testing genetic variation between traditional and F1 hybrid kale cultivars to optimize fertilizer management based on genotype, the objectives of this study were as follows: (i) investigate whether GLS and flavonoid glycoside concentration and composition differed markedly among cultivars; (ii) determine if varying N supplies in relationship to agronomic characteristic and biomass allocation modified GLS and flavonoid glycoside composition and concentration; and (iii) investigate field S and N application effects on GLS concentration and composition.
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MATERIALS AND METHODS
Chemicals. Glucoiberin, progoitrin, glucoraphanin, gluconapin, sinigrin, 4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin were obtained from C2 Bioengineering ApS (Karlslunde, Denmark); glucotropaeolin was from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany); and acetic acid, barium acetate, calcium glycerophosphate hydrate, methanol, sulfatase, and sodium hydroxide were obtained from Sigma-Aldrich (Steinheim, Germany). Field Experiment. The field site was located at the Department of Food Science in Aarslev, Denmark (55°18′N, 10°27′E) on a sandy loam (Typic Agrudalf) soil containing 1.35% organic C, 33% coarse sand, 38.8% fine sand, 12.6% silt, and 13.4% clay. Inorganic N and S levels in the field before planting were 10 and
