John M. DeLong

Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds

Abstract. The occurrence of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated fatty acids, is considered a useful index of general lipid peroxidation. A common method for measuring MDA, referred to as the thiobarbituric acid-reactive-substances (TBARS) assay, is to react it with thiobarbituric acid (TBA) and record the absorbance at 532 nm. However, many plants contain interfering compounds that also absorb at 532 nm, leading to overestimation of MDA values. Extracts of plant tissues including purple eggplant (Solanum melongena L.) fruit, carrot (Daucuscarota L.) roots, and spinach (Spinacia oleracea L.) leaves were assessed for the presence of MDA and other non-MDA compounds absorbing at 532 nm. A method described herein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA. The reliability and efficiency of this spectrophotometric method was assessed by altering the relative ratios of exogenous MDA additions and/or extracts of red cabbage (Brassica oleracea L.) leaves containing interfering compounds and then measuring MDA recovery. Reliability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques. Results indicated that over 90% of exogenously added MDA could be recovered through the improved protocol. If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 96.5%. Interfering compounds were not detected in vegetables such as lettuce (Lactuca sativa L.) and spinach which had low or negligible concentrations of anthocyanidin derivatives. Comparisons between the TBARS method presented here and two currently accepted protocols indicated that the new modified method exhibits greater accuracy for quantifying TBA-MDA levels in tissues containing anthocyanins and/or other interfering compounds. This modified protocol represents a facile and rapid method for assessment of lipid peroxidation in virtually all plant species that contain interfering compounds.

Oxygen concentration affects chlorophyll fluorescence in chlorophyll-containing fruit

Abstract The effect of lowering O2 concentration on chlorophyll fluorescence was continuously monitored in apple (Malus x domestica Borkh.), pear (Pyrus communis L.), banana (Musa L. Cavendish subgroup), kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson), mango (Mangifera indica L.), and avocado (Persea americana Mill.) fruit, using a large surface-area sensor. In all of the six fruit, there were specific O2 concentrations at which the Fo and Fv/Fm chlorophyll fluorescence values suddenly increased and decreased, respectively. When the O2 concentrations were increased, the Fo and Fv/Fm signals returned to their previous values. Since the O2 concentrations at which this phenomenon occurred were close to known low O2 thresholds for these fruit, it may be that chlorophyll fluorescence can rapidly and non- destructively determine the lowest acceptable O2 concentration for stored chlorophyll-containing plant products.

The influence of crop-load, delayed cooling and storage atmosphere on post-storage quality of 'Honeycrisp'™ apples

Summary The influence of crop-load, delayed cooling and storage environment on fruit quality and disorder, and on the incidence of rot was investigated on 7 year-old ‘Honeycrisp’ apple (Malus domestica Borkh.) trees on ‘Malling 26’ rootstocks at three different sites during 2003 and 2004. Fruit from non-thinned (control) trees, or trees thinned to three, six or nine fruit cm–2 trunk cross-sectional area (TCSA) were harvested and: (1) immediately cooled over 24 h to 3°C [commercial (Comm)] then stored at 3°C in refrigerated air (RA) or in a controlled atmosphere (CA; 2.5 kPa O2, 1.0 – 1.5 kPa CO2); or (2) held at 20°C for 6 d, followed by 1 d at 3°C [= 7 d pre-storage warming treatment = delayed cooling (DC)] then stored at 3°C under RA or in CA storage. After 3 and 6 months of storage, fruit mass (g), firmness (N), colour (% red), soluble solids content (SSC; %), titratable acidity (TA; mg malic acid equivalents 100 ml–1 juice), “greasiness” (0–3 scale), the extent of disorders such as soft scald (%) and low temperature breakdown (LTB; %), as well as the incidence of rot (%) were assessed. In general, as crop-load increased fruit mass, firmness, colour, SSC and TA decreased regardless of the effects of DC and storage treatment. Crop-load did not influence fruit “greasiness” and had an inconsistent and less-pronounced effect on soft scald, LTB and rot. The 7 d pre-storage warming treatment reduced the incidence of soft scald and LTB, irrespective of crop-load, but had little influence on other fruit quality measurements. Although the storage environment did not consistently alter quality, CA-stored ‘Honeycrisp’ apples tended to be more acidic, less “greasy” and develop less soft scald. Overall, the data indicate that the highest fruit quality was attained by thinning ‘Honeycrisp’ trees as close to three fruit cm–2 TCSA as possible, by subjecting newly-harvested fruit to a 7 d pre-storage warming period of 20°C, followed by CA storage at 2.5 kPa O2, 1 – 1.5 kPa CO2 at 3°C.

Comparison of a new apple firmness penetrometer with three standard instruments

Abstract The performance of a newly developed firmness penetrometer (fruit quality tester — FQT), was evaluated over two growing seasons with poststorage apples against the Effegi, Magness-Taylor (MT) and electronic pressure tester with the speed control engaged (EPT-SC). The FQT was operated in the: (a) standard (FQT-Std); (b) MT (FQT-MT); and (c) EPT (FQT-EPT) modes with the latter two simulating the operation of the MT and EPT-SC devices, respectively. ‘Cortland’, ‘McIntosh’ and ‘Northern Spy’ fruit were stored for 3–14 months in controlled atmosphere (CA) conditions and were then held at room temperature for 1–7 days, following which firmness was measured by four operators with each of the six instruments or modes. The ten cultivar/storage/poststorage fruit lots were designated as either hard or soft if the poststorage firmness average for the lot was ≥ or

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

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 temperature2) 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 (day2 temperature2) 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.

Fruit mass, colour and yield of ‘Honeycrisp’™ apples are influenced by manually-adjusted fruit population and tree form

Summary The influence of three manually-adjusted crop loads [3, 6 and 9 fruit cm–2 trunk cross-sectional area (TCSA)] on ‘Honeycrisp’™ apple (Malus domestica Borkh) quality and canopy volume were compared with unadjusted control trees. The crop-load treatments were applied at three sites over two consecutive seasons at 50 days after full bloom (DAFB). Sixteen trees at each site were selected annually, with the treatments being applied in a randomisedcomplete-block design. Fruit mass and fruit colour (%) were used to assess quality at harvest, while yield was determined by the mass (kg) of fruit cm–2 TCSA. The canopy volume (CV) for each tree was calculated following harvest and used as a covariate in determining its influence on fruit size and colour. As crop load decreased, harvested fruit mass and % red colour increased. For similar crop-load adjustments, larger tree CVs (in m3) resulted in greater fruit mass. Crop load and fruit-canopy density (fruit m–3 CV) were more highly correlated with fruit mass than with % fruit colour. The highest fruit quality occurred at the three and six fruit cm–2 TCSA. In addition, manually adjusting the crop-load at 50 DAFB did not eliminate the disposition of ‘Honeycrisp’™ towards biennial bearing in Nova Scotia’s climate.

The trend toward lower oxygen levels during apple (Malus × domestica Borkh) storage

Summary Advances in research and technology in recent years in the field of controlled atmosphere (CA) storage have led to the development of many new protocols that recommend lower oxygen (O2) levels than those historically deemed “safe”. As some of these protocols and technologies have been adopted by commercial CA storage rooms, especially in the apple industry, it is now opportune to review the relationships between O2 levels, respiration, fermentation, and fruit quality retention. Lowering O2 levels during apple storage slows respiration, reduces ethylene biosynthesis, and slows fruit maturation and senescence. In addition, low O2 influences the expression of genes associated with cellular energy consumption.When O2 levels become limiting, aerobic mitochondrial respiration is inhibited and plant tissues engage in the energetically less-efficient fermentation pathway. The accumulation of fermentation-related volatiles may lead to off-flavours and odours. At low levels, or for short durations, fermentation is generally not a problem and may even be commercially beneficial. Plants that are limited in their ability to ferment succumb to low-O2 stress more quickly than those that can readily ferment anaerobically. Increasing market pressure to reduce chemical residues in apple, as well as in all fruits and vegetables, is encouraging researchers, CA system manufacturers and suppliers, and some commercial storage operators to explore alternative methods to improve storage procedures.The future of CA storage may reside in a better understanding of what constitutes the lower O2 limit for metabolism and energy demands, improved product-response sensing capabilities, and the application of more dynamic forms of CA storage technology.

Determination of optimal harvest boundaries for Honeycrisp™ fruit using a new chlorophyll meter

DeLong, J., Prange, R., Harrison, P., Nichols, D. and Wright, H. 2014. Determination of optimal harvest boundaries for Honeycrisp™ fruit using a new chlorophyll meter. Can. J. Plant Sci. 94: 361-369. In this study, a new chlorophyll measurement tool [the delta absorbance (DA) meter] was used to develop an optimal harvest maturity model for Honeycrisp™ fruit. Apples from nine commercial orchards in the Annapolis Valley, Nova Scotia, Canada, were sampled over 11 consecutive weekly harvests during the 2010, 2011 and 2012 growing seasons. At each harvest, a sample of fruit was measured for its DA (IAD) values, firmness, titratable acidity (TA),% soluble solids content (SSC), red skin coloration and internal core ethylene. Following approximately 3 mo of storage at 3.5°C, samples were removed and assessed for disorder incidence. The optimal harvest period was identified by aligning all “at harvest” IAD values, fruit quality measurements and “post-storage” disorder data with the corresponding harvest week. Then, the IAD values associated with the harvests having high commercial fruit quality and the least collective expression of disorders, delineated the optimal harvest boundaries. As IAD units declined during fruit maturity, the upper boundary value of 0.59 was deemed “when to begin” harvest, while the lower boundary value of 0.36 was deemed “when to end” harvest for long-term storage. The use of the DA model approach for optimal harvest delineation is potentially applicable to all commercial apple cultivars, but should be developed for each within a distinct growing region.

Effect of fruit maturity on the incidence of bitter pit, senescent breakdown, and other post-harvest disorders in ‘Honeycrisp’tm apple

Summary This study examined the effect of harvest date (fruit maturity) on the occurrence of post-harvest disorders in ‘Honeycrisp’tm apple (Malus × domestica Borkh.). Fruit were sampled from six commercial orchards in the Annapolis Valley, Nova Scotia, Canada over two growing seasons and seven (in 2008) or nine harvest dates (in 2009). After 3 months storage in refrigerated air at 3.5ºC without delayed-cooling, soft scald, low temperature breakdown (LTB; also called soggy breakdown), bitter pit, and senescent breakdown developed in the fruit and the incidence of each varied with harvest date. Bitter pit developed in immature fruit and declined with harvest date, whereas senescent breakdown was non-existent in immature fruit and increased with harvest date. Minimum bitter pit plus senescent breakdown (combined) occurred at the mid-point of the harvest period (at approx. week-5), when internal ethylene was just beginning to appear in the fruit. An increase in fruit size (to ≥ 250 g) appeared to increase the occurrence of these two disorders. This study has shown that there is an optimum maturity (harvest date) in ‘Honeycrisp’tm apples, when the fruit are of sufficient size and colour to meet market requirements, with minimum risk of manifesting the two calcium-related disorders, bitter pit and senescent breakdown. The incidence of these two disorders may increase if fruit are picked earlier (bitter pit) or later (senescent breakdown), especially in larger fruit (≥ 250 g).

QTL analysis of soft scald in two apple populations

The apple (Malus×domestica Borkh.) is one of the world’s most widely grown and valuable fruit crops. With demand for apples year round, storability has emerged as an important consideration for apple breeding programs. Soft scald is a cold storage-related disorder that results in sunken, darkened tissue on the fruit surface. Apple breeders are keen to generate new cultivars that do not suffer from soft scald and can thus be marketed year round. Traditional breeding approaches are protracted and labor intensive, and therefore marker-assisted selection (MAS) is a valuable tool for breeders. To advance MAS for storage disorders in apple, we used genotyping-by-sequencing (GBS) to generate high-density genetic maps in two F1 apple populations, which were then used for quantitative trait locus (QTL) mapping of soft scald. In total, 900 million DNA sequence reads were generated, but after several data filtering steps, only 2% of reads were ultimately used to create two genetic maps that included 1918 and 2818 single-nucleotide polymorphisms. Two QTL associated with soft scald were identified in one of the bi-parental populations originating from parent 11W-12-11, an advanced breeding line. This study demonstrates the utility of next-generation DNA sequencing technologies for QTL mapping in F1 populations, and provides a basis for the advancement of MAS to improve storability of apples.