Determination of the optimal pre-storage delayed cooling regime to control disorders and maintain quality in Honeycrisp TM apples

Journal of Horticultural Science & Biotechnology (2009) 84 (4) 410 414 Determination of the optimal pre-storage delayed cooling regime to control disorders and maintain quality in Honeycrisp TM apples By J. M. DELONG 1 *, R. K. PRANGE 1, W. C. SCHOTSMANS 2, D. S. NICHOLS 1 and P. A. HARRISON 1 1 Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, 32 Main St., Kentville, Nova Scotia, B4N 1J5, Canada 2 Institute for Food and Agricultural Research and Technology, Av. Alcalde Rovira Roure, 191, E-25198 Lleida, Spain (e-mail: DeLongJ@agr.gc.ca) (Accepted 16 January 2009) SUMMARY Several pre-storage time (0, 1, 2, 4, and 6 d) and temperature (3.5º, 10º, 15º, 20º, 25º, and 30ºC) delayed cooling (DC) treatments were tested on harvested Honeycrisp TM apples to determine which combination was optimal for reducing soft scald and low temperature breakdown (LTB), while maintaining the highest fruit quality [i.e., firmness, minimal mass loss, titratable acidity (TA), soluble solids content (SSC), absence of rot, and minimal skin greasiness] after 4 months of refrigerated air (RA) storage. Fruit were harvested from three separate Annapolis Valley (Nova Scotia) orchard sites in 2006 and in 2007. Multiple linear regression and surface response curves showed that fruit firmness and SSC generally increased throughout the DC treatment, but were affected curvilinearly by temperature, reaching a maximum at approx. 15ºC, then declining. Loss of fruit mass was positively related to a (day temperature 2 ) interaction, indicating that it increased synergistically the longer and the warmer the DC treatment. Fruit acidity was affected only by temperature, with the highest TA values at approx. 15ºC, then declining at higher DC temperatures. Multiple logistic regression and surface responses demonstrated that the incidence of soft scald declined curvilinearly the longer and the warmer the DC treatment, while LTB declined curvilinearly with increasing DC temperature only. A positive (day 2 temperature 2 ) interaction indicated that fruit greasiness increased non-linearly as the duration and temperature of DC increased. Collectively, these results show that both soft scald and LTB were suppressed or eliminated by a DC regime of 25ºC for 1 2 d, or 30ºC for 1 d, without incurring a major reduction in fruit quality. The apple (Malus domestica Borkh.) cultivar Honeycrisp TM has become popular in the fresh markets of North America. Its unique texture, flavour, crispness, and long storage-life command premium prices for growers, which is helping to revitalise the apple industry in those areas where it is grown. However, Honeycrisp TM fruit can develop soft scald, as well as several other quality-related storage problems [e.g., bitter pit, watercore, low temperature breakdown (LTB), skin punctures, and decay], which vary in severity in each growing region. These problems threaten the commercial viability of this profitable cultivar (Evans, 2001; Greene and Weis, 2001; Schwallier, 2001; DeLong et al., 2004). In Nova Scotia, soft scald is the most frequently observed storage disorder of Honeycrisp TM, followed by LTB, which is also known as soggy or internal breakdown (DeLong et al., 2004; 2006). It is not unusual to observe a 30% incidence of these disorders after several months of storage (Prange et al., 2003; DeLong et al., 2004). Both of these disorders are exacerbated by low storage temperatures (i.e., < 2.5ºC; Watkins et al., 2004) or by rapid cooling of fruit after harvest (DeLong et al., 2004). Therefore, current Honeycrisp TM storage protocols usually recommend a storage temperature of 3ºC or 3.5ºC (Schwallier, 2001; Nichols et al., 2004; Watkins et al., 2004). In addition, several recent reports indicate that a *Author for correspondence. delay of 7 d at 10º 20ºC prior to controlled atmosphere (CA) or refrigerated air (RA) storage reduces the incidence of soft scald and LTB (Watkins and Nock, 2003; DeLong et al., 2004; 2006; Watkins et al., 2004). Despite adoption of these delayed cooling (DC) regimes, some fruit still develop these disorders (DeLong and Prange, unpublished data). Therefore, there is a need to determine the optimal pre-storage DC combination of temperature and time that minimises the development of soft scald and LTB, without compromising other fruit quality characteristics. For Honeycrisp TM, the most important quality attributes after storage are firmness, titratable acidity (TA), sugar content [i.e. soluble solids content (SSC)], and the extent of greasy and rotted fruit (Greene and Weiss, 2001; DeLong et al., 2004). The objective of this study was, therefore, to ascertain the pre-storage DC time and temperature combination that conferred the highest post-harvest quality in Honeycrisp TM apple fruit. MATERIALS AND METHODS Orchard sites and tree selection Three separate Honeycrisp TM grower sites were selected in 2006, and in 2007, in the Annapolis Valley, Nova Scotia, Canada (45º03'00N, 64º46'00W). The orchard systems were established in 1996 and 2000 on Malling 26 and 7a rootstocks, respectively. One year-old

J. M. DELONG, R. K. PRANGE, W. C. SCHOTSMANS, D. S. NICHOLS and P. A. HARRISON 411 feathered trees were planted at a density of 1,000 1,500 trees ha -1 and were trained as spindle bush or vertical axis forms. Harvest, delayed cooling, and storage Fruit were harvested on 25 September 2006, and on 24 September and on 2 and 3 October 2007, which coincided with the timing of commercial harvest. Apples from each site were then combined at random, prior to treatment application, in order to minimise the potential influence of trees on the variables measured. In each year, ten 30-apple samples (including the untreated control) of Honeycrisp TM fruit were placed in one of several time temperature DC regimes within 6 h of harvest. The DC treatments consisted of 10º, 15º, or 20ºC for 2, 4, or 6 d (2006) and 20º, 25º, or 30ºC for 1, 2, or 4 d (2007). The untreated control for both years was immediate cold storage at 3.5ºC (no DC). Immediately following the various DC treatments, the loss of fruit mass was calculated, then all fruit were stored in RA at 3.5ºC for 4 months. Measurements on stored fruit After 4 months of RA storage, 30 fruit were removed from each DC treatment and warmed to 23ºC. Fruit firmness (N) was measured on the red and green sides of individual apples using a fruit quality tester (Geo-Met Instruments, New Minas, NS, Canada), having the timelimit window set at > 0.1 s and < 1.0 s (DeLong et al., 2000). The mass (g) of each fruit was measured on a digital balance (Sartorius, Göttingen, Germany). Soluble solids content (%) and TA were measured on each composite 30-apple juice sample with a hand-held refractometer (Atago Co., Tokyo, Japan) and by titration of 2 ml apple juice with 0.1 M NaOH, respectively. Titratable acidity was expressed as mg equivalents of malic acid 100 ml -1 juice (DeEll and Prange, 1998). The incidence of soft scald, LTB, and rotted fruit was assessed as 0 (absent) or 1 (present), then calculated as a percentage of fruit showing the disorder, or rot, in the 30- apple sample. Epidermal greasiness was evaluated on a scale of 0 (none), 1 (slight), 2 (moderate), or 3 (severe and unmarketable). Experimental design and statistics The DC experiment was planned as a randomised complete design, with an incomplete factorial combination of the four temperatures (2006: 3.5º, 10º, 15º, and 20ºC; 2007: 3.5º, 20º, 25º, and 30ºC), and four durations (2006: 0, 2, 4, and 6 d; 2007: 0, 1, 2, and 4 d). The experiment was replicated at the three different grower sites each year. The influence of the independent variables: days [L (linear)]; temperature (L); days (L) temperature (L) interaction; day 2 [quadratic]; temperature 2 ; temperature (L) days 2 ; days (L) temperature 2 ; and days 2 temperature 2, were tested on each continuous, dependent variable (Y; i.e., firmness, mass loss, SSC, and TA) by multiple linear stepwise regression on the combined 2006 and 2007 data. The stepwise procedure identified the significant independent variables (P 0.10), while non-significant effects were not included in the final regression equations. In some cases, significant independent (X) variables were dropped from the final regression model if their partial R 2 (coefficient of determination) values contributed < 5% to the overall model R 2. Multiple logistic stepwise regression (SAS Institute, 2004) was performed on those variables with binomial (i.e., 0, 1) responses (e.g., LTB, soft scald, greasiness, rot) to identify significant main and interaction effects, as stated above. Surface responses demonstrating the influence of days and temperature (i.e., linear, quadratic, and their interactions) were plotted as three-dimensional graphs using SigmaPlot graphical software (SigmaPlot Ver. 11; Systat Software, San Jose, CA, USA). For graphical presentation, the binomial data were transformed to ln (i.e., natural log) to smooth and demonstrate treatment effects more clearly. All statistical analyses were performed using the linear regression (Proc Reg) and logistic regression (Proc Logistic) procedures of the SAS Institute (2004) software. RESULTS AND DISCUSSION Fruit quality variables having a continuous, normal distribution [i.e., firmness (Figure 1A), mass loss (Figure 1B), SSC (Figure 1C), and TA (Figure 1D)], were influenced linearly and/or curvilinearly by days, temperature, or their interactions. For firmness (Figure 1A), SSC (Figure 1C), and TA (Figure 1D), negative curvilinear temperature effects occurred, indicating that these quality variables increased, plateaued, then decreased as DC temperatures increased. The highest values for these measurements occurred at approx. 15ºC. Firmness (Figure 1A) and SSC (Figure 1B) were also positively related to the duration of the DC treatments. Fruit mass loss (Figure 1B) was positively related to a (day temperature 2 ) interaction (accounting for most of the variation in the model) indicating that a synergistic influence on mass loss occurred the longer and the warmer the DC treatment (Figure 1B). Studies reporting the influence of DC (usually one temperature for one time duration) on Honeycrisp TM fruit quality do not generally show changes in firmness, SSC, or TA (DeLong et al., 2004; Watkins et al., 2004; Watkins and Nock, 2003). However, in one recent study, TA dropped by 5 9% in DC apples compared with the control (DeLong et al., 2006). In this study, Honeycrisp TM TA declined by 14% to 25% when the highest levels at 15 o C were compared with corresponding values at 25 o C or 30 o C (Figure 1D; Table I). Thus, higher pre-storage DC temperatures conferred a greater reduction in TA levels after 4 months of RA storage. The fruit mass loss data (Figure 1B; Table I) demonstrated that the duration of elevated pre-storage temperature can have a substantial effect on weight loss in the stored product. The goal of the DC technique is to expose fruit to the time and temperature regime which best controls disorders and maintains the highest quality, with minimal loss of mass. A 1.45% mass loss (e.g., 4 d at 30 o C) potentially translates into thousands of dollars lost in fruit tonnage. In this study, soft scald and LTB were eliminated with minimal loss of fruit mass by a DC regime of 1 d at 30 o C (Table I). For those disorders having binomial responses (i.e., 0, 1), logistic multiple regression analysis and surface responses demonstrated that the incidence of soft scald declined curvilinearly the longer and the warmer the DC treatment (Figure 1E), while LTB declined curvilinearly

412 Post-storage fruit quality in Honeycrisp apple FIG. 1 Three-dimensional surface responses of temperature (ºC) (X1) and time TM (d) (X2) on Honeycrisp apple following 4 months of refrigerated air storage. Panel A, firmness [firmness (N) = 71.57 + 0.75 (days) 0.01 (temp)2]; Panel B) mass loss [mass loss (%) = 0.018 + 0.004 (days temp) + 0.0003 (days temp2)]; Panel C, soluble solids content [SSC (%) = 12.35 + 0.11 (days) 0.001 (temp)2]; Panel D, titratable acidity [TA (mg malic acid 100 ml 1 juice) = 478.92 + 6.63 (temp) 0.26 (temp)2]; Panel E, soft scald [soft scald = 1.15 + 0.133 (temp) 0.009 (temp)2 0.04 (days)2]; Panel F, low temperature breakdown [LTB = 2.82 + 0.20 (temp) 0.01 (temp)2]; Panel G, greasiness [greasiness = 0.67 0.01 (days temp) + 0.0002 (days2 temp2)]. Mass loss was measured after the DC treatment and prior to storage. Soft scald, LTB, and greasiness values (0, 1) were transformed by ln to smooth the surface responses (Panels E G). The regression equations were calculated on non-transformed data.

J. M. DELONG, R. K. PRANGE, W. C. SCHOTSMANS, D. S. NICHOLS and P. A. HARRISON 413 TABLE I Honeycrisp TM apple quality and development of disorders following several delayed cooling treatments applied immediately after harvest and 4 months of refrigerated air storage at 3.5ºC Fruit Quality Incidence of the disorder Temp. (ºC) Time (d) Firmness (N) SSC (%) TA Mass loss (%) Soft Scald (%) LTB (%) Greasiness (%) 3.5 0 70.8 12.2 497 0.00 32 10 30 10 2 72.8 12.7 513 0.10 30 7 20 4 73.4 12.6 517 0.08 18 13 24 6 73.6 12.8 510 0.25 9 12 38 15 2 74.9 12.9 541 0.28 11 10 39 4 73.2 12.7 559 0.52 10 8 23 6 75.7 12.8 525 0.81 11 3 26 20 1 67.6 11.7 469 0.21 20 3 37 2 71.1 12.2 516 0.46 11 2 33 4 71.4 12.4 496 0.89 10 2 35 6 74.2 12.5 518 1.32 0 2 50 25 1 67.5 11.8 482 0.29 2 0 39 2 67.5 11.7 474 0.59 2 1 23 4 68.7 11.9 476 1.18 1 0 26 30 1 66.7 11.7 478 0.40 0 0 23 2 67.2 11.7 457 0.76 0 0 27 4 67.3 11.9 421 1.45 0 0 62 Each mean consists of 1 or 2 years of data with 90 or 180 observations, respectively. mg malic acid equivalents 100 ml 1 juice. Mass loss was measured after the DC treatment and before refrigerated air (RA) storage. Soft scald, LTB and greasiness values represent the percentage of fruit displaying the disorder. only with increasing DC temperature (Figure 1F). Interestingly, a positive (days 2 temperature 2 ) interaction influenced fruit greasiness (Figure 1G), indicating the presence of a synergistic curvilinear influence as the duration and temperature of the DC treatment increased. In general, fruit that manifested greasiness were largely in category 1 ( slightly greasy ) and would be acceptable as fresh market apples. Interestingly, the DC treatment of 30ºC for 1 d or 2 d did not show an increased incidence of greasy fruit. At 30ºC for 4 d, however, nearly three-times more apples were greasy. Also, apples exposed to a DC regime of 20ºC for 6 d manifested greasiness on 50% of the fruit evaluated (Table I). Thus, the longer the duration of the DC treatment at temperatures above 15ºC (e.g., 20º, 25º, or 30ºC), the greater the likelihood of exacerbating the development of greasiness. Although the amount of rotted fruit was influenced by a DC (days temperature) interaction, its incidence was 7 % for any time temperature treatment combination (data not shown). The incidence of rot in Honeycrisp TM can be high, causing significant economic loss. Concern over the potential increase in rotten apples due to DC treatment has been raised by commercial operators (Scotian Gold Cooperative, Coldbrook, NS, Canada; Glooscap Apples, Kentville, NS, Canada; personal communications) and by researchers (Greene and Weis, 2001; DeLong et al., 2004). The data from this study show that the incidence of rotted fruit was not markedly influenced by any of the pre-storage warming periods tested, supporting a recent report in which Honeycrisp TM fruit was DC-treated for 6 d at 20ºC (DeLong et al., 2006). Soft scald and LTB can cause high post-storage losses in Honeycrisp TM fruit, being particularly troublesome in those geographic areas that are more susceptible to its development (e.g., northeast and mid-west regions of North America). In the last few years, three time temperature studies on pre-storage DC regimes to control these two disorders in Honeycrisp TM have been published. Unfortunately, they have been limited to one temperature (e.g. 10º, 15º, or 20ºC) and to one duration (6 or 7 d) (Watkins et al., 2004; DeLong et al., 2004; 2006). While these regimes often reduce the development of Honeycrisp TM storage disorders, the optimal pre-storage time and temperature combination that eliminates soft scald and LTB was not discovered. Data from this study indicate that, for Honeycrisp TM apples from Nova Scotia, 30ºC for 1 d can eliminate both soft scald and internal browning disorders. Surface response analyses (Figure 1E, F) also showed that these disorders were suppressed more strongly at higher rather than at lower DC temperatures and with shorter exposure intervals (e.g., 30ºC for 1 d reduced the disorders more than 15ºC for 6 d; Table I). The cellular causes of soft scald and LTB are presently unknown. Local seasonal observations indicate that some fruit are pre-disposed to these disorders in the field, and express them during the first 2 to 3 months in storage, and thereafter (data not shown). The environmental and genetic factors that induce susceptibility and trigger the development of soft scald and LTB in tissues are also not known. How DC suppresses the development and expression of these disorders therefore remains elusive. It may be that the water loss associated with a period of DC acts as an evaporative carrier, dissipating volatile compounds which are associated with the disorder(s) (DeLong et al., 2004; Wills et al., 1968). Past work has shown that water loss from McIntosh fruit is associated with a reduction in senescent breakdown and LTB (Blanpied, 1981), and a reduction in brown core (Lidster, 1990). In this study, the elimination of soft scald and LTB was associated with a loss in mass (presumably synonymous with water loss) of 0.4%, which was facilitated by a DC regime of 30ºC for 1 d (Table I). It is also known that fruit warming mitigates chilling-related browning disorders by reducing the concentration of toxic substances that can accumulate at lower temperatures (e.g., at 2º 3ºC; Alwan and Watkins, 1999; Watkins et al. 1995). Thus, active respiratory processes occurring during the warming

414 Post-storage fruit quality in Honeycrisp apple period may catabolise the toxic substances that are required for disorder development. Soft scald and LTB appear to be more problematic in Honeycrisp TM fruit the further East the growing region in North America. For example, New England and Nova Scotia generally have a higher incidence of the disorders than the Pacific northwest. Interestingly, western regions that delay-cool fruit to control soft scald and LTB, report success with lower temperatures of shorter duration (e.g., 1 2 d at 10ºC) than those observed in this study and what has been observed in the past (DeLong et al., 2004; 2006; Watkins et al., 2004). It may be that, in cooler growing regions, the effect of environmental triggers on genetic expression predisposes fruit towards an accumulation of those metabolic precursors required for expression of the disorders. Or it may be that warmer temperatures simply facilitate degradation of the metabolic precursors that are fundamental for the disorder to appear during storage. Whatever the relationship between the growing environment and disorder development, it appears that regional differences have a large effect on the degree of incidence, and on whether soft scald and LTB are major post-harvest concerns in Honeycrisp TM fruit. Fortunately, pre-storage DC regimes appear to eliminate expression of these disorders. CONCLUSIONS This work shows that treating Honeycrisp TM apple fruit with a DC regime consisting of 25ºC for 1 2 d, or 30ºC for 1 d, strongly suppresses (or entirely eliminates) the occurrence of both soft scald and LTB, while causing the least loss of fruit mass. While the 25ºC or 30ºC treatments may cause some reduction in TA, the potential economic benefits of a period of DC far outweigh the reduction in acidity, or in any other fruit quality attribute measured in this study. The authors thank the Annapolis Valley cooperating growers Peter Sanford, Blake Sarsfield, and Doug Nichols for providing the Honeycrisp TM fruit trees used in this project. Financial and technical support are gratefully acknowledged from the Nova Scotia Fruit Growers Association, the Nova Scotia Department of Agriculture Technology Development 2000 Program, and Agriculture and Agri-Food Canada, through the ACAAF Councils, Agri-Futures NS and PEI Adapt. The authors thank Dr. Kenneth McRae for statistical advice, and Mr. Charles Embree and Ms. Dela Erith for reviewing the manuscript. Contribution No. 2356 of the Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada. REFERENCES ALWAN, T. F. and WATKINS, C. B. (1999). Intermittent warming effects on superficial scald development of Cortland, Delicious and Law Rome apple fruit. Postharvest Biology and Technology, 16, 203 212. BLANPIED, G. D. (1981). 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