Blueberry estimated harvest from seven new cultivars: Fruit and anthocyanins

Food Chemistry 139 (2013) 44–50

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Blueberry estimated harvest from seven new cultivars: Fruit and anthocyanins Jessica Scalzo a,⇑, David Stevenson b, Duncan Hedderley c a

The New Zealand Institute for Plant & Food Research Limited (PFR), Hawke’s Bay, Private Bag 140, Havelock North 4157, New Zealand Plant & Food Research, Ruakura, Private Bag 3123, Waikato Mail Centre, Hamilton 3240, New Zealand c Plant & Food Research, Food Industry Science Centre, Batchelar Road, Palmerston North 4474, New Zealand b

a r t i c l e

i n f o

Article history: Received 22 November 2012 Received in revised form 28 January 2013 Accepted 30 January 2013 Available online 9 February 2013 Keywords: Vaccinium spp. Fruit weights Fruit harvest Yield Anthocyanins Polyphenols

a b s t r a c t This study compares the yields, weights and anthocyanin contents of fruit from a group of seven new cultivars released from the New Zealand blueberry breeding programme and selected for the longest possible combined harvest season. The measured factors were primarily influenced by cultivar, and seasonal variations had relatively minor effects. The late-ripening cultivars ‘Velluto Blue’ and ‘Centra Blue’ had the highest fruit yields, anthocyanin contents and estimated total anthocyanin harvestable from a given area. ‘Blue Moon’ and ‘Sky Blue’ had the largest fruit sizes. The early-ripening cultivars ‘Blue Bayou’, ‘Blue Moon’ and ‘Sunset Blue’ had the lowest anthocyanin contents. The yield, fruit size and total anthocyanin content results obtained from any single year were highly correlated with the average of the three years, which makes pursuing the evaluation for these traits from a single year and at an early stage of plant development a practical proposition. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cultivated blueberries (Vaccinium corymbosum, V. corymbosum hybrids and V. virgatum) are commercially produced in New Zealand and in the last few years the total area planted with blueberries increased from 239 ha in 2000 to 522 ha in 2009 (Hort Research, 2000; Plant & Food Research 2009). In New Zealand, V. corymbosum is commercially known as the ‘‘northern highbush’’ blueberry, V. corymbosum hybrids are known as ‘‘southern highbush’’ blueberry and V. virgatum is known as ‘‘rabbiteye’’ blueberry. Within the Vaccinium species internationally, there is a large range of cultivars, but relatively few are available in New Zealand. The cultivars that are readily available from nurseries may come from overseas breeding programmes, where they have been selected for environments that may be considerably different from that in New Zealand. Plant & Food Research (PFR) started a blueberry breeding programme in the 1980s with the aim of producing blueberry cultivars that were tailored to New Zealand conditions. Breeding objectives were specific agronomic and fruit traits such as high yield, disease resistance, winter chilling adaptability, seasonality range, hand-harvestable fruit of large size and light blue coloured fruit. A blueberry plantation of well-adapted cultivars has the potential ⇑ Corresponding author. Address: Plant & Food Research Hawke’s Bay, Private Bag 1401, Havelock North 4157, New Zealand. Tel.: +64 06 9758908; fax: +64 06 9758881. E-mail address: [email protected] (J. Scalzo). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.01.091

to produce a good crop for many years and thus planting the right genotype is crucial. Growing two or more different cultivars in the same commercial block is a popular choice among New Zealand growers, to satisfy pollination requirements and to extend the production window. There are reported differences in plant yields among blueberry cultivars, in seasonality changes of the yield and in plant management techniques affecting yield and fruit size (Celik, 2009; Finn, Strik, & Wagner, 2009; Gaskell, 2009; Lyrene & Williamson, 1997). Blueberry fruit available from the market come from cultivars that have been selected for specific agronomic and fruit traits such as high yield and large fruit size and not necessarily for high concentrations of health-promoting phytochemicals in the fruit. For this reason, it was important to measure the phytochemical content and composition of the fruit from the new cultivars. Blueberry fruit are believed to be good for health because of their anthocyanins and other polyphenolic compounds. Anthocyanins provide blueberries with their characteristic colour and have been shown to contribute to the antioxidant capacity of berryfruit (Connor, Finn, McGhie, & Alspach, 2005; Connor, Luby, Tong, Finn, & Hancock, 2002; Currie et al., 2006; Deighton, Brennan, Finn, & Davies, 2000; Ehlenfeldt & Prior, 2001; Kalt, McDonald, Ricker, & Lu, 1999; Kalt et al., 2001; Moyer, Hummer, Finn, Frei, & Wrolstad, 2002; Prior et al., 1998; Proteggente et al., 2002; Wang, Cao, & Prior, 1997). The health value of anthocyanins has been reviewed (Beattie, Crozier, & Duthie, 2005; Kong, Chia, Goh, Chia, & Brouillard, 2003) and as well as antioxidant capacity (Stintzing, Stintzing, Carle, Frei, & Wrolstad, 2002; Wang et al., 1997), anthocyanins are

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reported to have a role in improving circulation (Matsumoto et al., 2005), preventing stroke (Keli, Hertog, Feskens, & Kromhout, 1996), providing benefits to vision (Lee et al., 2005; Matsumoto, Nakamura, Tachibanaki, Kawamura, & Hirayama, 2003; Matsumoto et al., 2005), and their anti-inflammatory and anti-oxidative effects are extensively reported (Ghosh, McGhie, Zhang, Adaim, & Skinner, 2006; Lyall et al., 2009; Wang et al., 1999). The anthocyanins present in blueberry are galactosides, glucosides and arabinosides of the anthocyanidins delphinidin, cyanidin, petunidin, peonidin and malvidin, and their concentrations vary greatly between genotypes (Scalzo, Miller, Edwards, Meekings, & Alspach, 2009; Wang, Chen, Camp, & Ehlenfeldt, 2012a). Additionally, these glycosides may also be acylated (Wu & Prior, 2005). Blueberries are the richest sources of the more hydrophobic malvidins and petunidins among a wide selection of fruits and vegetables (Wu et al., 2006). Blueberries are also rich in delphinidin-3-galactoside and petunidin-3-glucoside (Kader, Rovel, Girardin, & Metche, 1996). Research shows that there is interest in the anthocyanin content of blueberry fruit and that changes are to be expected between fruit of different cultivars and between seasons (Connor, Luby, Tong, Finn, & Hancock, 2002; Scalzo et al., 2009; Wang et al., 2012a, 2012b). There is, however, little knowledge of how much anthocyanin can be harvested from a blueberry plantation, i.e., which cultivars have the best combination of high anthocyanin content and high yield. In this study, a combination of seven new cultivars released by the PFR blueberry breeding programme in New Zealand was considered. Altogether, the new seven cultivars in combination offer an extended production window, with an early harvest in mid November (late spring in New Zealand) and the last harvest around the beginning of April (mid autumn). We investigated and compared plant yields, fruit weights, total anthocyanin contents and polyphenol composition of the fruit for all the seven cultivars and over three years. Sources of the observed variations are discussed. 2. Materials and methods 2.1. Chemicals Solvents and general chemicals were obtained from local suppliers. Reference standards were obtained as follows: cyanidin-3-glucoside from Extrasynthese (Genay, France), chlorogenic acid and rutin from Sigma–Aldrich. 2.2. Plants and fruit sampling The blueberry plants under evaluation were planted in the ground in winter 2007 (June–July) at the Ruakura Research Centre – New Zealand (37°480 S 175°170 E) and grown in soil modified with additional organic material at pH 4.3. Details of the species and type of the seven cultivars and harvest dates are shown in Table

1. The site has a mean annual rainfall of about 1200 mm, with moderate temperatures (min./max. 0 °C/29 °C). Berries were harvested from plants growing in a replicated plot trial of 20 plants (four plots  five plants of each cultivar) and for three consecutive years (summers 2009–2010, 2010–2011 and 2011–2012). In this work, summer seasons are indicated as follows: 2009– 2010 = 2010, 2010–2011 = 2011 and 2011–2012 = 2012. For the yield assessments, fully ripe fruit were collected from each plant, starting when about 50% of the fruit per plant were estimated to be blue. Multiple harvests were required to collect the total yield per plant. Total yield recording started in year 3 after planting. Little crop was produced in years 1 and 2 and this was removed from the plants to promote growth. For the fruit weight determination, 50 uniform fully ripe fruit per plot were selected from the harvest at the stage when 50% of fruit were blue, and the mean fruit weight recorded within 2 h of harvesting. For the total anthocyanin content and the polyphenol assessments, a subsample of 100 g of uniform fruit was selected from the harvest at the stage when 50% of fruit were blue and stored at 20 °C until analysed. 2.3. HPLC analysis of blueberry anthocyanins 2.3.1. Sample preparation Approximately 15 g of frozen fruit was weighed accurately into a 50-ml screw-capped and graduated centrifuge tube and then stored frozen until needed. After thawing at room temperature for 30 min, ethanol/1% v/v formic acid was added to around the 37 ml mark, then the mixture homogenised with an Ultra Turrax T25 Basic homogeniser (IKA Labortechnik Asia, Selangor, Malaysia. After homogenisation into particles no more than 2 mm across, the tubes were sonicated in an ultrasonic bath for 30 min and finally centrifuged (1500 rpm/15 min) in a bench-top centrifuge with a swing-out rotor. The total volume of extract + pellet was recorded then the extract was sub-sampled and diluted 1:5 with aqueous 1% v/v formic acid for HPLC analysis. Preliminary trials showed that one extraction was sufficient to solubilise all the polyphenols, but a further extraction was required to remove the solvent trapped in the fruit tissue. We corrected for this effect by measuring the volume of the extract + fruit tissue, before centrifugation and sampling. 2.3.2. HPLC analysis Analyses were carried out on a Shimadzu 20-series analytical high performance liquid chromatograph (HPLC) with a column oven, auto-sampler, vacuum solvent degas module and diode-array detector. The column used was a 150  2 mm, Synergi Polar-RP, 4l particle size, 80 Å pore size, fitted with a Security-Guard 3  2 mm Polar RP guard cartridge (Phenomenex, Auckland, New Zealand). Flow rate was 0.6 ml/min and column temperature 50 °C. Solvents were (A) methanol and (B) 2% aqueous formic acid, and the initial mobile phase was 12% A and 88% B. The time pro-

Table 1 Harvest dates used to compare genotypic and seasonal variation of blueberry fruit. Fruit were harvested at the 50%-ripe stage, i.e., when 50% of the fruit on the plant were blue. Cultivars are listed in order of ripening from early to late (mean ripening date across three years). Cultivar

Species

Pedigree

Blueberry type

2009–2010

2010–2011

2011–2012

‘Blue Bayou’ ‘Sunset Blue’ ‘Blue Moon’ ‘Dolce Blue’ ‘Sky Blue’ ‘Velluto Blue’ ‘Centra Blue’

Vaccinium corymbosum hybrid V. corymbosum V. corymbosum V. virgatum V. virgatum V. virgatum V. virgatum

‘Reka’  ‘Island Blue’ ‘Reka’  B7-8-1 ‘Nui’  B7-8-1 ‘Centurion’  ‘Rahi’ ‘Centurion’  ‘Rahi’ ‘Maru’  ‘Briteblue’ ‘Centurion’  ‘Rahi’

SHB NHB NHB RE RE RE RE

2 December 2009 2 December 2009 10 December 2009 5 February 2010 31 January 2010 14 February 2010 22 February 2010

1 December 2010 7 December 2010 7 December 2010 22 January 2011 26 January 2011 11 February 2011 1 March 2011

1 December 2011 1 December 2011 13 December 2011 31 January 2012 3 February 2012 21 February 2012 8 March 2012

SHB, southern highbush; NHB, northern highbush; RE, rabbiteye.

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gramme of pump B concentration was set up as 86% at 2.5 min, 78% at 5 min, 62% at 8.5 min, 37% at 10.50 min, 30% from 10.5 to 12.5 min, returning to 88% at 12.75 min and staying at that concentration until the end of the run at 15 min. Sample injection volume was 10 ll. UV/visible spectra were recorded from 250 to 600 nm in 1.2-nm steps. Quantification of anthocyanins was carried out at 520 nm, in comparison with standard solutions of cyanidin glucoside. Chlorogenic acid and quercetin glycosides were quantified at 350 nm compared with standards of chlorogenic acid and rutin, respectively. Results were expressed as mg cyanidin glucoside equivalent, chlorogenic acid, or rutin equivalents/100 g fruit. Concentrations in the HPLC samples were converted to fruit equivalents using dilution factors, fruit weight and extract volume.

2.3.3. Confirmation of identity of compounds To assist identification of compounds observed during HPLC runs, some samples were rerun on the same HPLC, with the addition of a Shimadzu LCMS 2020, single quadrupole mass spectrometer, fitted with an electrospray (ESI) interface. Instrument parameters used were the manufacturer’s default settings. MS scans were carried out in both positive and negative mode in the same run, using a mass range of 140–650 Da. Confirmation of identity was achieved both though comparison of expected and observed molecular ion masses and ‘‘neutral losses’’ of sugars during ionisation to leave aglycones or partially glycosylated ions. For example, cyanidin glucoside exhibited a positive molecular ion at 449 Da and an ion at 287 Da, corresponding to loss of the glucose residue (162 Da) to leave the cyanidin fragment ion. Compounds were identified by comparison with previously reported data (Borges, Degeneve, Mullen, & Crozier, 2010; Lohachoompol, Mulholland, Srzednicki, & Craske, 2008).

2.4. Statistical design The field trial was established as a randomised complete block design with four replications of five plants each. Data were analysed using linear mixed effects models, testing effects of year (i.e., harvest season), cultivar and their interaction (fixed effects) against the variability between blocks, plot within block, and block by year and plot by year interactions (random effects). Data were log transformed to stabilise the variance; the means presented have been back-transformed. The least significant differences for the log-scale data have also been back-transformed to give least significant ratios (LSR); two means are significantly different if the larger is more than LSR times the smaller. The analysis was done with GenStat (version 14, 2011, VSNi Ltd, Hemel Hempstead, UK).

3. Results and discussion 3.1. Fruit yield, fruit weight and total anthocyanin content (ACY) Little crop was produced in years 1 and 2 and this was removed from the plants early in the season to improve their growth and establishment of root systems. The yield in the first year (2010) was lower than that in years 4 and 5, for most of the cultivars; however, it did appear to give an indication of future yield potential. 3.1.1. The fruit yield The fruit yield showed significant variation with year, cultivar and the interaction of these two factors (Table 2A). The F ratios for year and cultivar, however, are both much higher than that for the interaction year  cultivar, suggesting that the cultivars reacted similarly to seasonal climate variations. The F ratios also suggest that seasonal variations were much greater than variations between cultivars, although the seasonal variations were confounded by the marked increase in fruit yield per plant over the three years in all cultivars except ‘Dolce Blue’ and ‘Sky Blue’. A yield increase with year was to be expected, since the plants were still growing at a strongly rate. Significant differences in fruit yield were found between the three years when cultivars were individually compared, and among cultivars within each year. The cultivars with the highest mean yield over the three years were ‘Velluto Blue’ and ‘Centra Blue’ (Table 3), but the former’s fruit yield increased only slightly (by 7%) as the plants matured. In contrast, ‘Centra Blue’ had the second highest mean yield, but increased by 77% as the plant matured, suggesting that this cultivar may produce the highest yields over the longer term. The cultivars with the lowest mean yields were ‘Blue Moon’ and ‘Blue Bayou’. ‘Blue Moon’ was consistently the cultivar with the lowest yield within each year. 3.1.2. The individual fruit weight The individual fruit weight varied significantly with cultivar and the year  cultivar interaction, with cultivar being the major influence (Table 2A). This suggests that seasonal yield variations primarily result from changes in the number of fruit, rather than their size. ‘Blue Moon’ and ‘Sky Blue’ had the highest mean fruit weights and no significant variation was found between years (Table 3). The cultivar with the lowest average fruit weight was ‘Blue Bayou’, and its fruit size was consistently small across the years. 3.1.3. The total anthocyanin content The total anthocyanin content varied significantly with cultivar and the year  cultivar interaction (Table 2A). The F ratio for year  cultivar was relatively lower than that for cultivar, meaning that although there were some variations in the pattern of anthocyanin content variation from year to year, they were smaller than

Table 2 The analysis of variance results for the variables yield, fruit weight, total anthocyanin content (A) and polyphenol components of blueberries (B). A. Fixed effects

DF

Year (Y) Cultivar (C) YC B. Fixed effects

Year (Y) Cultivar (C) YC

Plant yield

2 6 12 DF

2 6 12

Fruit weight

ACY

F-value

P-value

F-value

P-value

F-value

P-value

51.5 21.8 6.5

<0.001 <0.001 <0.001

8.5 82.1 5.8

0.003 <0.001 <0.001

1.78 77.21 7

0.181 <0.001 <0.001

TM

AA

CA

Q

F-value

P-value

F- value

P-value

F-value

P-value

F-value

P-value

12.64 72.65 6.7

<0.001 <0.001 <0.001

12.94 31.99 16.39

<0.001 <0.001 <0.001

309.79 51.33 17.22

<0.001 <0.001 <0.001

1366.46 45.82 7.95

<0.001 <0.001 <0.001

ACY, total anthocyanin content; TM, malvidin; AA, acylated anthocyanins; CA, chlorogenic acid; Q, quercetin glycosides.

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Table 3 Genotypic and seasonal variation of yield, fruit weight and total anthocyanin content (ACY) of blueberries. Cultivars are listed in order of ripening from early to late (mean ripening date across three years). Plant yield (g/plant)

‘Blue Bayou’ ‘Sunset Blue’ ‘Blue Moon’ ‘Dolce Blue’ ‘Sky Blue’ ‘Velluto Blue’ ‘Centra Blue’ Mean

Fruit weight (g)

ACY (mg/100 g)

2010

2011

2012

Mean

2010

2011

2012

Mean

2010

2011

2012

Mean

885 1560 783 2432 1884 5433 4000 2425 a

1667 2655 986 2553 2924 5742 4831 3051 b

1750 3170 1146 2455 2061 5835 7080 3357 b

1434 a 2461 b 972 a 2480 b 2290 b 5670 c 5304 c

1.2 2.5 3.2 1.6 2.9 2.8 1.8 2.3 b

1.0 2.1 3.0 1.7 2.8 2.2 1.8 2.1 a

1.2 2.3 3.0 1.7 2.7 2.4 2.7 2.3 b

1.1 2.3 3.1 1.7 2.8 2.5 2.1

172 111 147 313 217 242 260 198 a

183 110 191 231 185 262 215 191 a

157 83 157 385 239 277 288 205 a

170 b 101 a 164 b 303 e 213 c 260 d 252 d

a d e b e d c

Means in the same column with the same letter are not significantly different (least significant difference test on log-transformed data, a = 0.05). In this work, summer seasons are indicated as follows: 2009–2010 = 2010, 2010–2011 = 2011 and 2011–2012 = 2012.

the general trends for cultivars. The cultivar with the highest mean anthocyanin content over the three years of evaluation was ‘Dolce Blue’ (303 mg/g) (Table 3). ‘Dolce Blue’ fruit had the highest anthocyanin content of these cultivars in 2010 and 2012, but not in 2011. ‘Sunset Blue’ fruit had the lowest mean anthocyanin content (101 mg/g) and were the lowest in each year. The cultivars appear to have responded differently to weather variations within each season. The fruit from the first three cultivars listed in Table 3 were harvested in December and had markedly lower anthocyanin contents than the remaining four cultivars, which were harvested in January–March. Examination of weather records for the three growing seasons (not shown) detected no significant differences in either temperature or solar radiation intensity between December and January–March. There was, however, a markedly lower temperature and solar intensity all through the 2012 season, which had no detectable effect on the anthocyanin contents of the fruit for the seven cultivars considered in this study (Table 3). If anything, this parameter was slightly higher in 2012. It therefore appears that the observed differences are related to fundamental differences between the early ripening V. corymbosum cultivars and the later ripening V. virgatum cultivars. Blueberry cultivars with high yield are desirable and always popular with growers, whereas cultivars with large fruit are particularly advantageous to the majority of New Zealand growers who hand-harvest their crop. Blueberry cultivars with high anthocyanin contents can have an advantage in marketing to consumers because anthocyanins have been reported to have health benefits, as previously discussed. This study agrees with previous reports (Clark, Howard, & Talcott, 2002; Connor, Luby, & Tong, 2002; Connor, Luby, Tong, Finn, et al., 2002; Pranprawit, Molan, Heyes, & Kruger, 2009; Scalzo et al., 2009; Wang et al., 2012a, 2012b; You et al., 2011) in finding considerable variation in fruit anthocyanin contents between cultivars (Table 3). The fruit yield also varies, independently of anthocyanin content, between cultivars and between years, and it generally increases with plant maturity (Finn et al., 2009). There is little knowledge of whether the anthocyanin content in blueberry increases with the plant age, similarly to the plant yield; however, there is reported evidence that it varies between years (Connor, Luby, Tong, Finn, et al., 2002; Wang et al., 2012b). Our results suggest that, in the New Zealand environment, total fruit anthocyanin content does not significantly increase as blueberry plants mature.

3.2. Polyphenol composition Compositional analysis of the polyphenols revealed that malvidin (as the sum of the three glycosidic forms) was consistently the major anthocyanin component, comprising 30–47% of the mean total anthocyanin content (Table 4). The most variable anthocyanin

Table 4 Relative content of polyphenol components of blueberries expressed as the percentage of each polyphenol relative to the total anthocyanin content. Percentages are calculated using the mean values over the three years of the trial. Cultivars are listed in order of ripening from early to late (mean ripening date across the three years). Cultivar

TM%

AA%

CA%

Q%

‘Blue Bayou’ ‘Sunset Blue’ ‘Blue Moon’ ‘Dolce Blue’ ‘Sky Blue’ ‘Velluto Blue’ ‘Centra Blue’

42 38 36 47 37 45 30

10 11 25 1 4 1 1

8 15 6 16 14 28 21

24 23 17 6 10 8 8

TM, malvidin; AA, acylated anthocyanins; CA, chlorogenic acid; Q, quercetin glycosides.

components were the acylated anthocyanins (AA) (Cho, Howard, Prior, & Clark, 2004, 2005), present in low percentages in rabbiteye blueberry fruit, and the anthocyanins chlorogenic acid (CA) and quercetin (Q) (in the form of 2–3 different glycosyl derivatives) also exhibited significant variability (Table 2B). Total malvidin (TM) showed significant variation with year, cultivar and their interaction (Table 2B). The F ratio for cultivar was much higher than that for the other main factors and the differences in cultivar content are shown in Table 5. Similarly to TM, AA showed significant variation for all the main factors, with cultivar being the predominant factor (Table 2B). Significant differences in AA were found between the three years when cultivars were individually compared, and among cultivars within each year (Table 5), with ‘Blue Moon’ being the cultivar with the highest AA content in each of the three years. CA was more abundant in V. virgatum cultivars than in V. corymbosum. AA and Q were predominant in early-ripening cultivars such as ‘Blue Bayou’, ‘Sunset Blue’ and ‘Blue Moon’, rather than in the late-ripening rabbiteye cultivars. CA showed significant variation with year, cultivar and their interaction (Table 2B). The F ratio for year, however, was much higher than those for cultivar and the interaction of year  cultivar. Large and significant differences in CA were found between the three years when cultivars were individually compared, and among cultivars within each year (Table 5). Similarly to CA, Q showed significant variation for all the main factors, and the F ratio for year was much higher than those for cultivar and the interaction of year  cultivar (Table 2B). In particular in year 2010, the Q content was the lowest in each cultivar, compared with values in 2011 and 2012. In summary, CA, AA and Q showed much larger seasonal variations than the variation in total anthocyanin content, but these are relatively minor components, so the importance of these variations is much less than that of the anthocyanins.

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Table 5 Content of four polyphenol components (mg/100 g) of blueberries, with means for each cultivar and each year. Cultivars are listed in order of ripening from early to late (mean ripening date across three years). TM

‘Blue Bayou’ ‘Sunset Blue’ ‘Blue Moon’ ‘Dolce Blue’ ‘Sky Blue’ ‘Velluto Blue’ ‘Centra Blue’ Mean

AA

CA

Q

2010

2011

2012

Mean

2010

2011

2012

Mean

2010

2011

2012

Mean

2010

2011

2012

Mean

70 40 55 132 71 89 58 69 a

76 42 68 123 77 150 73 81 b

68 33 54 179 91 120 100 81 b

71 c 38 a 59 b 143 e 80 c 117 d 75 c

9 9 37 3 5 3 3 6a

30 19 49 1 6 3 1 6a

22 9 36 21 18 0 17 10 b

18 d 11 cd 40 e 4b 8c 1a 4b

37 61 46 115 63 137 124 75 c

26 32 19 38 29 57 35 32 b

3 2 1 28 14 49 32 9a

14 b 15 b 10 a 50 d 29 c 72 e 52 d

13 9 9 5 7 8 9 8a

81 39 60 38 46 38 27 44 c

62 38 45 36 29 30 29 37 b

41 e 24 c 29 d 18 a 22 bc 21 ab 19 a

TM, malvidin; AA, acylated anthocyanins; CA, chlorogenic acid; Q, quercetin glycosides. Means in the same column with the same letter are not significantly different (least significant difference test on log-transformed data, a = 0.05). In this study summer seasons are indicated as follows: 2009–2010 = 2010, 2010–2011 = 2011 and 2011–2012 = 2012.

3.3. Estimated anthocyanin harvest per hectare and seasonality changes If blueberries were to be grown for processing, particularly for manufacture of high-anthocyanin polyphenol extract powders, then the most important factor would be the overall yield of anthocyanins per hectare. This factor might be optimised by high anthocyanin content berries, high fruit yield, and preferably both. To estimate the potential anthocyanin yield for each cultivar and each year, the average anthocyanin content per plant was multiplied by the plant density (Table 6). By comparing the mean values for the three years, ‘Velluto Blue’ was the cultivar with the highest estimated anthocyanin yield, at an estimated plant density of 4125 plants per hectare. Under these conditions, we estimated that the potential total anthocyanin harvest from ‘Velluto Blue’ fruit was 1.1-fold higher than that of ‘Centra Blue’, 1.9-fold higher than that of ‘Dolce Blue’, 3.1-fold higher than that of ‘Sky Blue’, 6.1-fold higher than those of ‘Sunset Blue’ and ‘Blue Bayou’, and 9.2-fold higher than that of ‘Blue Moon’. By comparing the estimated total harvest of anthocyanin in the last year of evaluation (2012), the mature plants of ‘Centra Blue’ were the ones producing the highest potential harvest, because of that cultivar’s high fruit yield in 2012. Modifying plant yield and fruit size by changing the plant density has been reported (Gaskell, 2009; Lyrene & Williamson, 1997; Strik & Buller, 2002): however, we have found no report relating the anthocyanin content of fruit from different cultivars to changes in plant density in the field. According to our results, late-ripening cultivars such as rabbiteye blueberries have higher anthocyanin contents than early ripening highbush blueberries (‘Sunset Blue’, ‘Blue Bayou’ and ‘Blue Moon’). As discussed above, these differences appear to relate to the different species involved, rather than to weather variation through the season. One might expect smaller fruit to contain more anthocyanins because these compounds are found only in the skin and small fruit have a relatively larger sur-

Table 6 Estimated total anthocyanin content (g/ha) harvestable for each blueberry cultivar planted with plant density of 4125 plants per hectare. Cultivars are listed in order of ripening from early to late (mean ripening date across three years).

‘Blue Bayou’ ‘Sunset Blue’ ‘Blue Moon’ ‘Dolce Blue’ ‘Sky Blue’ ‘Velluto Blue’ ‘Centra Blue’

2010

2011

2012

Mean over3 years

63 71 47 314 169 542 429

126 120 78 243 223 621 428

113 109 74 390 203 667 841

101 100 66 316 198 610 566

In this study, summer seasons are indicated as follows: 2009–2010 = 2010, 2010– 2011 = 2011 and 2011–2012 = 2012.

face area in relation to volume. Inspection of Table 3, however, shows this not to be the case: when comparing ‘Blue Bayou’, the smallest fruit, and ‘Blue Moon’, the biggest, their anthocyanin contents are very similar. 3.4. Correlation between traits and between years Evaluating a multitude of cultivars in test trials is common practice in breeding programmes and it is associated with considerable cost related to the cultivation and maintenance of large numbers of bushes and data collection from the trial. Amongst all the traits included in our work, determining the yield during the three years for all the cultivars was the most expensive part of the research. According to our results, the yield from a single year collected from all the cultivars was highly correlated to the average yield across the three years (Table 7). Therefore, assessing this trait from a single year of data should be mostly reliable for individual cultivars. Our 21 yield measurements show only two outliers, ‘Sky Blue’, the yield of which decreased markedly between 2011 and 2012, and ‘Dolce Blue’ the yield of which in year 2012 was slightly lower than that of year 2011. In addition, the relative fruit yields of the cultivars under evaluation appear to be reasonably predictable from the third year of cultivation (i.e., the first year of meaningful yield determination). Similar results were obtained previously: (Finn et al., 2009), who monitored the harvest of different cultivars over a 3–9 year time frame, found that the harvest in years 4–6 was strongly correlated to the cumulative yield over all years and could predict genotypic differences. Similarly to the yield, the single-year results obtained for fruit weights and total anthocyanin contents were highly correlated to the averages across the three years (Table 7), which makes evaluation of these traits from a single year and at an early stage of plant development reasonably achievable. It therefore appears that it would be practical to select promising crosses from a breeding programme in the third year of plant growth, with a low probability of missing high-performing plants that happened to perform unusually poorly in year 3. As an example, ‘Centra Blue’ had by far the Table 7 Correlations between individual year results for blueberry cultivars and mean results across the three years for cultivars. Correlation

Plant yield 2010–2012

Fruit weight 2010–2012

ACY 2010–2012

2010 2011 2012

0.977 0.987 0.958

0.967 0.987 0.923

0.984 0.883 0.988

ACY, total anthocyanin content; in this study, summer seasons are indicated as follows: 2009–2010 = 2010, 2010–2011 = 2011 and 2011–2012 = 2012.

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Table 8 Correlation of polyphenol components of blueberries with the total anthocyanin content in each year and mean correlation across three years. Significant correlations are highlighted in bold. Anthocyanin in same year

ACY 2010

ACY 2011

ACY 2012

Mean ACY level 2010–2012

TM AA CA Q

0.822 0.549 0.768 0.598

0.894 0.402 0.614 0.220

0.969 0.109 0.760 0.497

0.914 0.550 0.824 0.536

ACY, total anthocyanin content; TM, malvidin; AA, acylated anthocyanins; CA, chlorogenic acid; Q, quercetin glycosides. In this study, summer seasons are indicated as follows: 2009–2010 = 2010, 2010–2011 = 2011 and 2011–2012 = 2012.

highest yield of the cultivars in year 5 (2012), and this was a considerable increase over its year 3 yield. Its year 3 yield (2010), however, was still the second highest, so it would still have been selected as a high yielding cultivar, based solely on year 3 results. Only the promising selections from year 3 would need to be further monitored, thereby freeing up most of the plots in which new crosses could then be planted. The correlation of the individual polyphenol compounds with the total anthocyanin contents in each year and over the three-year evaluation showed some significant differences (Table 8). Only TM showed consistently positive and significant correlations with ACY, each year and over the three-year period. CA had high positive correlations with ACY, but not in 2011. Overall, TM and CA were highly and positively correlated to the total anthocyanin content over the three years of evaluation. The correlations between AA and ACY, and Q and ACY, were negative over the entire evaluation, but not significantly so. 4. Conclusions Year effect was the main factor that influenced fruit yield and polyphenols such as CA and Q. As the plants matured, the average total yield per plant increased. The average CA was significantly reduced in 2012, while the Q content was the lowest for all the cultivars in 2010. The FW and ACY did not significantly change between the years of evaluation. Although there were some variations of fruit weight and in the pattern of anthocyanin content from year to year, they were much smaller than the general trends for cultivars. The yield, fruit size and total anthocyanin contents obtained from any single year were highly correlated to the average across the three years, which makes pursuing the evaluation for these traits from a single year and at an early stage of plant development a practical proposition. This finding could permit large savings in time and resources during selection of new blueberry cultivars, by cutting the time required from 5 to 3 years. Traditionally, blueberries have been bred primarily to optimise the production and consumer attributes of taste and appearance for fresh eating fruit. Little or no attention has been paid to optimising polyphenol content for perceived heath attributes, or for making polyphenol extracts for use in formulated functional foods. We have introduced a different approach by additionally selecting for a high polyphenol content and particularly for anthocyanins that have potential health benefits. If blueberries were to be grown for processing, particularly for manufacture of high-anthocyanin polyphenol extract powders, then the most important factor would be the overall yield of anthocyanins per hectare. Agronomic traits would still be important, but consumer attributes much less so. Our results show that for a given plant density, the total anthocyanin content harvestable from a blueberry cultivation varies greatly among cultivars. We estimated that the potential total anthocyanin harvest from rabbiteye cultivars was higher than those for southern highbush and northern highbush. For processing purposes, ‘Velluto Blue’ and ‘Centra Blue’ would be clearly

much better than the other cultivars studied, since each has both a high fruit yield and high anthocyanin content.

Acknowledgements The authors would like to acknowledge Dawei Deng for the polyphenol analysis, Judith Rees, Carolyn Edwards and Shirley Miller for assisting with the fruit harvest.

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