Scientia Horticulturae 83 (2000) 127±137. Niklas Jeppsson *

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1 Scientia Horticulturae 83 (2000) 127±137 The effects of fertilizer rate on vegetative growth, yield and fruit quality, with special respect to pigments, in black chokeberry (Aronia melanocarpa) cv. `Viking' Niklas Jeppsson * BalsgaÊrd-Department of Horticultural Plant Breeding, Swedish University of Agricultural Sciences, FjaÈlkestadsvaÈgen 123-1, S Kristianstad, Sweden Accepted 6 May 1999 Abstract The effect of fertilizer rate on fruit quality parameters in the black chokeberry (Aronia melanocarpa [Michx] Ell.) cv. `Viking' was studied over three years. A combined N±P±K fertilizer was applied in different rates. Plant height, yield, berry weight, anthocyanin content, content of brown compounds, content of soluble solids, anthocyanin composition and total acidity were recorded for the different treatments. Increased application of fertilizers resulted in increased vegetative growth and yield whereas the content of anthocyanins and total acidity decreased. Mean berry weight, content of brown compounds, soluble solids and anthocyanin composition were unaffected. Correlations between the different parameters were calculated at single plant level. Positive correlations were found between plant height and yield, between plant height and browning index, and between anthocyanin content and total acidity. Negative correlations were found between plant height on one hand, and anthocyanin content and total acidity on the other hand. It was concluded that an increased fertilizer application results in increased growth and higher yield whereas pigment content and total acidity decrease. Maximum production of anthocyanins per plant was obtained with a medium high fertilizer rate (50 kg N/ha, 44 kg P/ha, 100 kg K/ha). # 2000 Elsevier Science B.V. All rights reserved. Keywords: Rosaceae; Aronia; Fertilizer; Yield; Fruit quality; Anthocyanin * Tel.: ; fax: address: niklas.jeppsson@hvf.slu.se (N. Jeppsson) /00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S (99)

2 128 N. Jeppsson / Scientia Horticulturae 83 (2000) 127± Introduction Black chokeberry (Aronia melanocarpa [Michx] Ell., Rosaceae), native to eastern North America, is a 0.5±3 m high shrub. The black berries are borne in clusters and ripen in early September in southern Sweden. Approximately, ha of this berry crop were reported to be in production in the 1980s in Russia for use in jam, juice and wine (Kask, 1987). At BalsgaÊrd, black chokeberry is being evaluated as a potential new crop for Sweden (Jeppsson, 1999). Anthocyanin-rich extracts are used as food colourants to improve the appeal of certain food products, and have partly replaced the formerly used azo-dyes. Commercial anthocyanin production is based mainly on grape skins but other sources such as elderberries (Sambucus nigra L.) are also used (Bridle and Timberlake, 1997) and others including the fruits of black chokeberry have been mentioned as a source of anthocyanin-rich extracts (Timberlake and Henry, 1988; Mazza and Miniati, 1993). The berries contain 725±800 mg anthocyanins/100 g fresh weight (Mazza and Miniati, 1993) and have a low tendency to browning, at least as compared to elderberry (Plocharski et al., 1989). The anthocyanins in black chokeberry consist mainly of cyanidin-3-galactoside and cyanidin-3- arabinoside, which are reported to constitute 64% and 29%, respectively (Oszmianski and Sapis, 1988), and are found in the peel as well as throughout the fruit flesh. Crop requirements must focus on production of high quality pigment if the fruits are intended as a source of natural food colourants. High berry yield is of course desired, whereas berry size is of little consequence for an industrial crop. Other important parameters are: (1) total anthocyanins in the juice, which should be as high as possible. Fertilizer application affects the anthocyanin formation in cranberry (Vaccinium macrocarpon Ait.) (Eaton, 1971; Eck, 1976) and apples (Saure, 1990; Raese and Drake, 1997); (2) content of brown compounds in the juice, which should be as low as possible since they may affect the appearance of fruit products negatively. I have found no data on effects of fertilizers on brown pigment content in any kind of fruit; (3) juice soluble solids which should be as low as possible since it determines the limit for the gain in volume decrease when producing fruit concentrates. In apples the soluble solids can decrease with increased nitrogen fertilizer rate (Raese and Drake, 1997), whereas no effect was seen in pears (Raese, 1997); (4) anthocyanin composition since it has been suggested to play a role in the stability of extracts (Starr and Francis, 1968), and (5) total acidity which should be high since anthocyanins are more stable in acid environments (Mazza and Miniati, 1993) and the stability of anthocyanins during processing is strongly influenced by the ph of the media (Chichester and McFeeters, 1970).

3 N. Jeppsson / Scientia Horticulturae 83 (2000) 127± This investigation was performed to (1) study whether pigment content and quality of the berries of black chokeberry cv. `Viking' is affected by fertilizer rate, (2) develop grower recommendations concerning fertilization of black chokeberry and (3) investigate associations among agronomical and fruit quality parameters. 2. Materials and methods 2.1. Experimental The trial was performed at BalsgaÊrd ± Department of Horticultural Plant Breeding (56807'N, 14810'E), Sweden. The trial site was an apple orchard until 1989, but without any nitrogen application from 1982 to Between 1989 and 1991 the field lay fallow and in 1991±1992 it was ploughed and treated with herbicides. The soil is classified as sandy with 2±4% of clay and a ph of 5.5, and contained 5.6 and 5.2 mg/100 g soil of plant-available P and K, respectively, in This is well below the values recommended of 8±12 and 14±16, respectively, for fruit orchards on sandy soils in the region. Black chokeberry cv. `Viking' rooted cuttings of even size were planted in October 1992, 1 m apart in rows that were 3.5 m apart. Four treatments (A, B, C and D), replicated in two blocks giving eight experimental units each comprising 10 plants, of which plants no. 1 and 10 were used as guard plants. Guard rows also surrounded the trial. Prior to planting, manure corresponding to 18 kg N/ha, 21 kg P/ha and 135 kg K/ha was applied. In 1993, manure in different rates by weight (times 1.0, 1.5, 2.0 and 3.0 for treatments A, B, C and D, respectively, with rate A corresponding to 30 kg N/ha, 45 kg P/ha and 150 kg K/ha) were applied. In 1994±1997, a commercial combined fertilizer was used. The same rates as in 1993 were used with rate A corresponding to 25 kg N/ha, 22 kg P/ha and 50 kg K/ha. The fertilizers were applied manually between April 16 and 22 each year. After application, the soil was not disturbed for five weeks. Thereafter weed control was performed, manually within rows and by harrowing between rows Weather Data were obtained for the Kristianstad ± EveroÈd airport approximately 25 km from the experimental site. In August, during the later stages in fruit development, the mean temperature was 188C, 188C and 208C for the years 1995, 1996 and 1997, respectively, whereas the precipitation was 12, 56 and 2 mm, respectively.

4 130 N. Jeppsson / Scientia Horticulturae 83 (2000) 127± Parameters evaluated Plant height was recorded after cessation of growth. Berries were harvested when visually estimated to be fully ripe (30±31 August 1995, 11±12 September 1996 and 11 September 1997). Total yield and berry size as the fresh weight of fifty berries, were measured. Berry samples for analysis were stored at 208C. Juice was produced from thawed berries with a garlic press to measure anthocyanins, brown compounds and soluble solids. Anthocyanins were measured by the ph differential method (Fuleki and Francis, 1968; Wrolstad, 1976) and expressed as cyanidin-3-galactoside using the molar absorbance of " ˆ and molecular weight of 445. Brown compounds were measured according to Wrolstad (1976): a juice sample diluted in distilled water was bleached with K 2 S 2 O 5 and browning was registered by absorbance at 420 nm and expressed as browning index. Absorbances were recorded with a Shimadzu UV PC spectrophotometer. Soluble solids were recorded with a digital refractometer (Atago, PR-100) and expressed as 8Brix. Anthocyanin composition was determined by thin layer chromatography (TLC) according to Nilsson and Trajkovski (1977) with modifications. Pigments were extracted in methanol (p.a.) with 1% (v/v) concentrated HCl (p.a. Merck) and separated on one-dimensional TLC using 0.1 mm cellulose plates (Merck) and 1- butanol (Kebo), acetic acid (Merck) and distilled water (90 : 15 : 30). The two spots obtained, here assumed to correspond to cyanidin-3-galactoside and cyanidin-3-arabinoside on basis of their relative size, were quantified in a CSscanner (Shimadzu CS 9001PC, supported with software v. 3.01). The relative content of cyanidin-3-galactoside to total anthocyanin content was estimated as the area of the largest spot divided by the sum of both areas. Acidity was measured by titration to ph 8.1 with 0.1 M NaOH and expressed as malic acid. The production of anthocyanins per plant [Acy(T)] was estimated as the anthocyanin content in the juice sample [Acy (c)] and the assumed (based on own unpublished observation) juice yield of 0.7 (1/kg): Acy T ˆ Acy c 0:7 Yield: 2.4. Statistical analysis of data Each of eight plants (no. 2±9) in an experimental unit was measured for height, berry weight, pigment content, browning index, soluble solids and total acidity in 1995±1997. Yield was based on eight plants in each experimental unit in 1995± 1996 and on the middle six plants in each experimental unit in Pigment composition was analysed on each of the middle six plants in each experimental unit in all three years. To test whether the neighbour treatment affected the

5 N. Jeppsson / Scientia Horticulturae 83 (2000) 127± outermost plants (in this case plants no. 2 and 9) in each experimental unit, the following statistical test was used: in each experimental unit the parameter values for the eight individual plants were ranked from the lowest to the highest. The rank values for plant no. 2 and 9 (R 2 and R 9 ) in each experimental unit were used to calculate A i : A i ˆ Abs R 2 4:5 Abs R 9 4:5 : The test quantity, T, was calculated as the sum of these A i. When testing eight experimental units over three years the critical value for T ˆ P24 iˆ1 A i is 109 at the 5% level. Testing eight experimental units for one year the critical value for T ˆ P8 iˆ1 A i is 40 at the 5% level. This means that there is a significant effect from neighbour treatment on the outermost plants when T is larger than or equal to this critical value. Average values for each experimental unit were then used to test the effects of block, year, treatment and the interaction year treatment using analysis of variance with Super ANOVA v Since the interaction year treatment was not significant, the analysis was repeated with exclusion of the interaction in the model. Duncan's Multiple Range Test (Super ANOVA v. 1.11) was used to test the significance between treatments and years. Associations between the different characters were determined at the individual plant level by Pearson correlation tests (SYSTAT v ). 3. Results Plants no. 2 and 9 in each experimental unit did not differ from the mean for any parameter except total acidity where T barely exceeded the critical value (111 vs. 109). The mean for each unit measurement was therefore based on all eight plants for all parameters. There were no significant effects from block for any of the parameters, but highly significant variation among years was found for all parameters except total acidity (Table 1). This was probably due to differences in mean temperature and precipitation among years, with August 1997 being very dry and having a mean temperature two degrees higher than the two previous years. In 1997, the lowest mean berry weight and a remarkable loss in yield (compared to 1996) were recorded, whereas the highest scores were obtained for both anthocyanin content and browning. Significant effects from treatments were found in height (p ˆ 0.021), yield (p ˆ 0.012), anthocyanin content (p ˆ 0.023) and total acidity (p ˆ 0.040). Both plant growth and yield were promoted by increased fertilizer rate whereas the anthocyanin production and the total acidity decreased (Table 2). There were no significant effects from treatments on either browning index (p ˆ 0.97), content

6 Table 1 The mean treatment values for plant height, yield, mean berry weight, anthocyanin content, browning index, soluble solids, anthocyanin composition and total acidity compared with Duncan Multiple Range Test for differences among years (in each column, entries with the same letters are not different at 5% level of significance) Height (cm) Yield (g) Mean berry weight (g) Anthocyanin content (mg/l) Browning index (l) Soluble solids (8Brix) Anthocyanin composition (%) a 440a 0.77a 730a 0.67a 17.6a 67.9a 0.72a b 3030b 0.85b 1030b 0.68a 16.3b 66.7b 0.67a c 1050c 0.61c 1530c 1.19b 17.8a 68.3a 0.70a Total acidity (g malic acid/100 g) 132 N. Jeppsson / Scientia Horticulturae 83 (2000) 127±137

7 Table 2 The mean three-year-values for height, yield, anthocyanin content and total acidity compared with Duncan Multiple Range Test for differences among treatments (in each column, entries with the same letters are not different at 5% level of significance) Treatment Height (cm) N. Jeppsson / Scientia Horticulturae 83 (2000) 127± Yield (g) Anthocyanin content (mg/l) Total acidity (g malic acid/100 g) A 65a 880a 1330a 0.75a 2.65a B 79ab 1260ab 1070ab 0.70ab 2.72a C 92b 1960b 1100ab 0.69ab 4.62a D 93b 1910b 880b 0.65b 3.58a Anthocyanin production (g/plant) of soluble solids (p ˆ 0.96) or anthocyanin composition (p ˆ 0.34). Treatment A differed significantly from C and D for both plant height and yield, whereas it differed significantly only from D for anthocyanin content and total acidity. Treatment C resulted in the highest (however, not significant) total anthocyanin production per plant in all three years. The increase in yield by increased fertilizer level did not result in any total increase in anthocyanin production since the crop produced less anthocyanin per weight unit. In 1995 and 1996, negative correlations were found between plant height and both anthocyanin content and total acidity (Table 3). For the same years, positive correlations were found between plant height and browning index and between anthocyanin content and total acidity. A positive correlation was found between plant height and yield in 1996 and All other correlations were either not Table 3 Correlation between those parameters among which significant correlations were found in at least one year Plant height and yield NS *** *** Plant height and anthocyanin content *** *** NS Plant height and browning index ** * NS Plant height and total acidity *** ** NS Yield and anthocyanin content NS ** NS Berry weight and browning index ** NS NS Berry weight and total acidity ** NS NS Anthocyanin content and browning index * NS NS Anthocyanin content and soluble solids NS NS * Anthocyanin content and total acidity *** *** NS Browning index and total acidity *** NS NS * Significantly different 5%, with Bonferroni test. ** Significantly different at 1%, with Bonferroni test. *** Significantly different at 0.1% level, with Bonferroni test.

8 134 N. Jeppsson / Scientia Horticulturae 83 (2000) 127±137 significant or significant only in one year. The drought in 1997 probably was the reason that most correlations recorded in 1995 and 1996 were not repeated in Discussion 4.1. Plant height and yield Both plant height and yield showed a simultaneous positive effect from increased fertilizer rate. By contrast, a negative relationship was found for yield and growth in cranberry, at least in the first year of cropping, which was explained as a result of competition (Eck, 1976) Anthocyanin content and soluble solids The negative effect recorded here on anthocyanin content from increased fertilizer rate in black chokeberry corresponds to previous investigations in apples; among the soil factors, available nitrogen is believed to be the most important and nitrogen application can lead to a decrease in colouration (Raese and Drake, 1997). In cranberry, Eaton (1971) suggested that increased application of nitrogen resulted in decreased content of red pigments, and Eck (1976) detected a significant decrease in anthocyanin content as a response to increased nitrogen application, in one year out of three. On the other hand, Davenport (1996) found no effect of nitrogen fertilizer level on total anthocyanin content in cranberry. Shading of the berries due to increased vegetative growth was suggested by Francis and Atwood (1961) to have caused the decrease in anthocyanin content in cranberry at high nitrogen fertilizer rate. Since there was a significant correlation between anthocyanin content and plant height in black chokeberry, shading could be responsible for the inverse relation between fertilizer rate and anthocyanin content. Sapers et al. (1986) reported an inverse relationship between fruit size and anthocyanin content in cranberries. However, the decrease in anthocyanin content with increased fertilizer rate in black chokeberry cannot be explained by dilution since fruit weight was not affected by treatment. In addition, the content of soluble solids was unaffected by treatment. These results agree with data from blackberry, where both the content of soluble solids and the content of total solids were unaffected by different nitrogen fertilizer rates (Alleyne and Clark, 1997) Browning and total acidity Browning, which is an important quality trait for any fruit crop, is partly caused by chemical reactions with phenolic compounds as a substrate. No effect from fertilizer rate on browning was found in the present experiment. By contrast, there

9 was a positive correlation between plant height and content of brown compounds in both 1995 and 1996, indicating that an increased fertilizer rate actually increases the content of brown compounds. The decrease in total acidity with increased fertilizer rate cannot be explained by a dilution effect since mean berry weight remained unaffected as well as the overall content of soluble solids (cf. anthocyanin content). By contrast, total acidity was not affected by nitrogen fertilizer rate in either apple or pears (Raese, 1997; Raese and Drake, 1997) Anthocyanin composition Differences in anthocyanin composition may affect pigment stability (Starr and Francis, 1968) as well as the appearance of the product. In the present investigation there was no effect from treatments on the relative area of the anthocyanin spots. Thus it could be concluded that there is no effect on anthocyanin composition from fertilizer rate. This is in agreement with a previous investigation on grape cultivars grown at different sites, which showed that the anthocyanin composition was cultivar specific, and not affected by environmental factors (Bakker and Timberlake, 1985). 5. Conclusion For the commercial cultivation of black chokeberry as raw material for food colourant production, grower recommendations must focus on the optimal production of anthocyanins per hectare. Yield increased with an increased fertilizer rate whereas anthocyanin content decreased. Even if the differences between treatments in anthocyanin production per plant were not significant, treatments A and D were significantly different for both yield and anthocyanin content. The resulting anthocyanin production was obtained for the two highest fertilizer rates (treatment C and D). The significant decrease in total acidity obtained with an increased fertilizer rate, however, points to the importance of keeping fertilization at a moderate level in order to favour a high content of organic acids. In addition by avoiding excessive application of fertilizers, the farmer makes an economic gain while, at the same time, the environment is protected from unnecessary fertilizer leakage. Overall it can be concluded that, in the present study, treatment C was the most beneficial from the growers point of view and could thus be recommended for black chokeberry production. Acknowledgements N. Jeppsson / Scientia Horticulturae 83 (2000) 127± Technical assistance was given by Pia Barnekow, Daniel Ehmann, Stefan Olsson and Fredrik AÊ berg. Jan-Eric Englund developed the model for testing the

10 136 N. Jeppsson / Scientia Horticulturae 83 (2000) 127±137 effect from neighbour treatment on the outermost plants. Hilde Nybom and Gustav Redalen gave valuable comments on the manuscript. Financial support was received from The Swedish Board for Technical Development and the Swedish Research Council for Forestry and Agriculture. References Alleyne, V., Clark, J.R., Fruit composition of `Arapaho' blackberry following nitrogen fertilization. HortScience 32, 282±283. Bakker, J., Timberlake, C.F., The distribution of anthocyanins in grape skin extracts of port wine cultivars as determined by high performance liquid chromatography. J. Sci. Food Agric. 36, 1315±1324. Bridle, P., Timberlake, C.F., Anthocyanins as natural food colours ± selected aspects. Food Chem. 58, 103±109. Chichester, C.O., McFeeters, R., Pigment degeneration during processing and storage. In: Hulme, A.C. (Ed.), The Biochemistry of Fruits and Their Products, vol. 2. Academic Press, London, pp. 707±725. Davenport, J.R., The effect of nitrogen fertilizer rates and timing on cranberry yield and fruit quality. J. Amer. Soc. Hort. Sci. 121, 1089±1094. Eaton, G.W., Effect of N, P and K fertilizer application on cranberry leaf nutrient composition, fruit color and yield in a mature bog. J. Amer. Soc. Hort. Sci. 96, 430±433. Eck, P., Relationship of nitrogen nutrition of `Early black' cranberry to vegetative growth, fruit yield and quality. J. Amer. Soc. Hort. Sci. 101, 375±377. Francis, F.J., Atwood, W.M., The effect of fertilizer treatments on the pigment content of cranberries. Proc. Amer. Soc. Hort. Sci. 77, 351±358. Fuleki, T., Francis, F.J., Quantitative methods for anthocyanins. 2. Determination of total anthocyanin and degradation index for cranberry juice. J. Food Sci. 33, 78±83. Jeppsson, N., Breeding black chokeberry (Aronia melanocarpa), an anthocyanin-rich crop. In: Proceedings of the Third International Symposium on Natural Colorants for Food, Nutraceuticals, Beverages, Confectionery and Cosmetics, 19±22 April 1998, Princetown, NJ, pp. 179±187. Kask, K., Large-fruited black chokeberry (Aronia melanocarpa). Fruit Varieties J. 41, 47. Mazza, G., Miniati, E., Anthocyanins in fruits vegetables and grains. CRC Press, Boca Raton, FL. Nilsson, F., Trajkovski, V., FaÈrgpigment hos arter och hybrider inom slaèktet Ribes. L. LantbrHoÈgsk. Medd., Uppsala, Ser. A. 282 (in Swedish with English summary). Oszmianski, J., Sapis, J.C., Anthocyanins in the fruits of Aronia melanocarpa (black chokeberry). J. Food Sci. 53, 1241±1242. Plocharski, W., Zbroszczyk, J., Lenartowicz, W., Aronia fruit (Aronia melanocarpa, Elliot) as a natural source of anthocyanin colourants. 2. The stability of the color of aronia juices and extracts. Fruit Sci. Rep. (Skierniewice) 16, 40±50. Raese, J.T., Cold tolerance, yield and fruit quality of `d'anjou' pears influenced by nitrogen fertilizer rates and time of application. J. Plant Nutrition 20, 1007±1025. Raese, J.T., Drake, S.R., Nitrogen fertilization and elemental composition affects fruit quality of `Fuji' apples. J. Plant Nutrition 20, 1797±1809. Sapers, G.M., Graff, G.R., Phillips, J.G., Deubert, K.H., Factors affecting the anthocyanin content of cranberry. J. Amer. Soc. Hort. Sci. 111, 612±617.

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