Materials and Methods

‘Honeycrisp’ apple fruit (Malus ×domestica Borkh) were obtained from trees planted in 2000, grafted on M.9 rootstock at the Cornell Orchard (Ithaca, NY, USA) on 3 Sep [harvest 1 (H1)] and 13 Sep [harvest 2 (H2)] 2014. The dates represent the commercial harvest window for the cultivar at this location. All fruit from six trees were harvested and bulked to obtain the maximum number of fruit per I_AD value category, and they were then separated based on I_AD values using a using a DA meter (TR Turoni Srl, Forli, Italy). The I_AD value of each fruit was measured on the blushed and nonblushed side, and the average was taken to sort into categories of 0 to 0.20, 0.21 to 0.40, 0.41 to 0.60, 0.61 to 0.80, 0.81 to 1.00, 1.01 to 1.20, 1.21 to 1.40, and 1.41 to 1.60. The categories with sufficient fruit numbers to allow measurement of the IEC at harvest, sampling during storage, and evaluation of disorder incidence were chosen for further experimentation.

The IEC of five fruit per category was measured by taking gas samples from each apple using a 1-mL syringe. Gas samples were injected into a Hewlett-Packard 5890 series II gas chromatograph (Hewlett-Packard, Wilmington, DE, USA) equipped with a flame ionization detector and an alumina column (1.5 m × 3 mm). Gas analyses were conducted isothermally at 160 °C. Nitrogen carrier, air, and H₂ flows were 45, 400, and 45 mL·min^–1, respectively. The injector and detector were kept at 230 and 245 °C, respectively.

The remaining fruit were stored in air at 0.5 or 3 °C. The IEC of five fruit per category and the temperature was measured as described earlier at 1, 2, 3, 4, 6, 8, 10, 14, and 18 weeks while still cold. At week 18, all remaining fruit were cut equatorially at least five times from the calyx end to the stem end of the fruit. External and internal disorders were calculated based on the percentage of the total fruit assessed.

Results and Discussion

The distribution of I_AD values at the two harvest dates showed a shift toward lower values (less chlorophyll concentrations) at H2 compared with H1 (Fig. 1).

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The IECs of fruit across all categories with at least five fruit were measured (Table 1). The IECs at H1 were low, averaging 0.220 µL·L^–1. There was a single fruit in the 0.41- to 0.60-unit range in which the IEC exceeded 1 µL·L^–1 (Table 1). At H2, the average IEC was 1.14 µL·L^–1, with several fruit with ethylene concentrations exceeding 1 µL·L^–1.

Table 1. The average and range of internal ethylene concentrations of ‘Honeycrisp’ fruit in each index of absorbance difference value category at harvests 1 and 2.

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The categories with sufficient fruit numbers for sampling over time ranged from 0.61 to 1.60 units at H1 and 0.41 to 1.40 units at H2. During storage, the IECs of the fruit kept at 0.5 °C remained low until week 3, with maximum concentrations reached at ∼6 to 8 weeks, before leveling off or declining (Fig. 2A and B). Increases in the IEC were earlier in fruit from H2 than H1. There was little differentiation in IECs among I_AD value categories at 0.5 °C. At 3 °C, however, the increase in the IECs of the fruit was faster and at greater concentrations that at 0.5 °C (Fig. 2A and B). Also, the differences among I_AD value categories at 3 °C were more pronounced, with generally greater IECs in the 1.21-to-1.40 category than in the others.

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Greater IECs in the least mature fruit (higher I_AD values) were not expected. However, this effect has also been found in ‘Gala’ fruit in which harvest maturity interacted with the effect of cold temperature on stimulation of ethylene production (Cai et al. 2023). The relationships between ethylene production of fruit with I_AD values warrant further investigation.

Separation of fruit into I_AD value categories revealed effects of fruit maturity and storage temperature with greater insight (Fig. 3). Soft scald is a major disorder of ‘Honeycrisp’ apples stored at 0.5 °C. The incidence of the disorder is decreased by increasing the storage temperature with or without conditioning of the fruit at 10 °C for 7 d (Al Shoffe et al. 2020; Moran et al. 2020; Watkins et al. 2004). Soft scald also tends to be associated with later harvests (Watkins et al. 2004; Ehsani-Moghaddam and DeEll 2013). In our experiment, soft scald incidence was high in fruit only for H2 stored at 0.5 °C and was clearly associated with lower I_AD values (Fig. 3). Soggy breakdown, a low-temperature disorder that is affected by the same factors as soft scald, was detected only in fruit in the 0.41-to-0.6 category, being 29% and 6% at H1 and H2, respectively.

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In contrast, bitter pit is a major disorder of ‘Honeycrisp’ apples at a warmer storage temperature such as 3 °C and it is further exacerbated by conditioning (Al Shoffe et al. 2020). However, in fruit from susceptible orchard blocks, the disorder will occur at all storage temperatures. Consistent with these observations, bitter pit incidence was high at each harvest date and at both storage temperatures (Table 2), with incidences at 3 °C being approximately twice that at 0.5 °C and tending to be associated with less mature fruit at each harvest date (Fig. 3). The susceptibility of fruit to bitter pit is usually higher in less mature fruit and at warmer storage temperatures, but is often associated with low Ca concentrations and ratios of Ca with other minerals (Ferguson and Watkins 1989). However, bitter pit incidence decreases as fruit maturity increases, as shown in ‘Cox’s Orange Pippen’ (Perring 1986) and in ‘Honeycrisp’ (Prange et al. 2011) apples. Interestingly, this decrease in bitter pit incidence occurs while Ca concentrations are also decreasing as a result of increasing fruit size, indicating that factors other than maturity are involved in the susceptibility of fruit to the disorder (Perring 1984; Watkins 2025).

Table 2. The incidences of soft scald, bitter pit, superficial scald, and core browning in ‘Honeycrisp’ apples harvested 10 d apart and stored at 0.5 or 3 °C for 18 weeks plus 7 d at 20 °C.

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Superficial scald is rarely reported in ‘Honeycrisp’ apples (Al Shoffe et al. 2023). However, it was found in H1 fruit stored at either 0.5 °C or 3 °C, and incidence was not associated consistently with I_AD values (Fig. 3). Although the disorder is thought to be a CI, less mature fruit stored at low temperatures tend to be the most susceptible (Lurie and Watkins 2012). We do not have an explanation for its occurrence in the fruit used in our experiment.

Core browning occurred only in fruit stored at 0.5 °C. At H1, the incidence was strongly affected by I_AD value, with less mature fruit having an increasingly higher disorder incidence. At H2, the relationship with I_AD values was less pronounced (Fig. 3). In ‘Honeycrisp’, the disorder is associated with storage temperatures close to 0.5 °C (Al Shoffe et al. 2021), and in general is regarded as a low-temperature disorder of apple fruit (Watkins and Mattheis 2019).

To visualize the data and explain the variation in the data, PCA, which reduces the dimensionality of the data while preserving as much variance as possible, was undertaken. The analyses revealed that PC1 accounts for 45% of the variation, whereas PC2 explains 24% (Fig. 4). The eigenvectors revealed that the IECs, bitter pit, soft scald, and soggy breakdown were correlated with PC1 at –0.5, –0.5, 0.5, and 0.4, respectively. However, core browning and superficial scald were correlated with PC2 with r = –0.7 and 0.4, respectively. Eigenvalues for PC1 and PC2 were 2.6 and 1.4, respectively, and were significant at P < 0.0001. Our analyses reinforce the results shown in Fig. 3. Harvest date did not influence the IEC, soft scald, soggy breakdown, or core browning. However, H1 fruit had greater bitter pit and superficial scald compared with those at H2 (Fig. 4A). The IEC, bitter pit, and core browning were greater in fruit with higher I_AD values, whereas soft scald development increased at lower I_AD values. Superficial scald and soggy breakdown, however, showed inconsistent patterns in relation to the I_AD values (Fig. 4B). When fruit were categorized by storage temperature, soft scald, soggy breakdown, and core browning were higher at 0.5 °C, whereas bitter pit, superficial scald, and the IEC were greater at 3 °C (Fig. 4C).

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In most studies, the evaluation of I_AD values as an additional measure of fruit maturity is based on bulked fruit samples (DeLong et al. 2014, 2020; Doerflinger et al. 2016; Moran et al. 2020; Mostofi and DeEll 2024; Nyasordzi et al. 2013; Serra et al. 2016). To compare the results based on fruit categorization by I_AD values, we also determined disorder incidences in the population if bulked samples were used to investigate maturity and storage effects. For each harvest date and storage temperature, the percentage of each disorder in each category was recalculated based on the proportion of fruit in each one (Table 2).

The results of our study confirm the usefulness of fruit categorization by I_AD values to allow a more granular understanding of the effects of fruit maturity on the susceptibility of fruit to physiological disorders. Similar approaches to date have been taken for superficial scald (Farneti et al. 2015) and soft scald (Al Shoffe et al. 2018; Moran et al. 2020). These approaches could improve the precision of studies of biochemistry and the development of different physiological disorders. Studies of this type can be confounded by a range of maturities of fruit in a typical bulked sample of fruit, which results in difficulties distinguishing susceptible and unsusceptible fruit.

Conclusion

In conclusion, this study highlights the potential of categorizing fruit by I_AD values as a tool to minimize variation of fruit within a population, and therefore increases the sensitivity of investigations into factors affecting storage performance. Both I_AD values and storage temperatures affect the IEC and incidences of physiological disorders of ‘Honeycrisp’ apples. This could be especially useful when studying changes at the biochemical level when minimizing the variation of fruit could reduce confounding factors associated with averaged fruit maturity.