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Using Non-Destructive Methods to Correlate Chilling Injury in Nectarines with Fruit Maturity G. Hale, J. Lopresti,D. Stefanellia and R. Jones Department of Primary Industries Knoxfield Centre Private Bag 15, Ferntree Gully DC Victoria 3156 Australia

L. BonoraDepartment of Fruit Tree and Woody Plant Sciences University of Bologna Viale Fanin 46, 40127 Bologna Italy

Keywords: stone fruit, firmness, maturity stages, ethylene, cold storage Abstract

Nectarine physiological maturity at harvest may have an important effect on the development and expression of chilling injury during cold storage. Prior to cold storage, two non-destructive instruments, an acoustic firmness sensor (AWETA), and a vis/NIR DA-meter, were used to classify ‘August Fire’ nectarines into maturity stages based on both fruit firmness and ethylene production. Both non-destructive parameters were also correlated with Effegi penetrometer firmness. DA-meter readings were found to be strongly correlated to physiological maturity of nectarine as expressed by the rate of fruit ethylene production (r2=0.74), and only moderately correlated with penetrometer fruit firmness (r2=0.52). Acoustic firmness measurements were strongly correlated with penetrometer firmness (r2=0.76). Three significantly distinct fruit maturity classes specific to this cultivar were identified based on measured ethylene production and fruit firmness measured by AWETA. Chilling injury (CI) severity increased during cool storage at 5°C with fruit maturity having a significant effect on CI expression after 34 days storage. The main symptoms observed were flesh bleeding, browning and a reduction in expressible juice. Chilling injury severity was found to be dependent on fruit maturity as assessed by both non-destructive methods with mature, post-climacteric fruit being less susceptible. Maturity classification using both the DA-meter and AWETA firmness sensor may allow prediction of CI development during cold storage.

INTRODUCTION

Chilling injury is a common physiological disorder of peaches and nectarines. Symptoms are usually expressed during ripening after cold storage and can include flesh or pit cavity browning (internal browning), flesh reddening (bleeding), lack of juiciness (mealiness), and a rubbery texture (leatheriness) (Lurie and Crisosto, 2005). Severity of chilling injury is highly cultivar dependent, but length of cold storage period, storage temperature, and fruit maturity can also have a significant effect on expression (Crisosto et al., 1999). The relationship between physiological maturity and chilling injury expression has not been fully determined. Fernández-Trujilo et al. (1998) found that immature fruit, measured using skin colour and firmness, were more susceptible to chilling injury than riper fruit. On the other hand, Lurie et al. (2011) showed that mature fruit classified using time-resolved spectroscopy were more susceptible to chilling injury. Campos-Vargas et al. (2006) determined that there was no link between severity of chilling injury and fruit maturity based on physiological parameters including skin ground colour, flesh firmness, soluble solids content, respiration, and ethylene production.

Research to date has been hampered by the difficulty in accurately classifying nectarines or peaches into different physiological maturity classes, and by the inability to track the same fruit at different levels of the peach/nectarine supply chain. With the recent development of non-destructive technology to measure physiological maturity and fruit a Dario.Stefanelli@dpi.vic.gov.au

Proc. 7th International Postharvest Symposium Eds.: H. Abdullah and M.N. Latifah Acta Hort. 1012, ISHS 2013

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firmness at harvest, the potential exists to accurately categorize individual fruit into a maturity class, and to determine the effect of maturity on chilling injury expression after cold storage (Ziosi et al., 2008). In this study, ‘August Fire’ (Prunus persica L. Batsch), a late-season yellow-fleshed nectarine, was used to determine whether classification into maturity classes based on non-destructive measurements is an effective method for elucidating the relationship between fruit maturity and susceptibility to chilling injury.

MATERIALS AND METHODS

‘August Fire’ nectarines were harvested at export maturity from eight year-old trees within a commercial orchard in the Swan Hill district in south-eastern Australia. Mean fruit mass (120.1±1.1 g SD) and firmness (flesh elasticity) of 700 fruit were measured non-destructively using a laboratory model acoustic firmness sensor – AFS (AWETA, Nootdorp, Netherlands). Physiological maturity of each fruit was determined using a portable vis/NIR DA-meter (Sinteleia, Bologna, Italy). Firmness and maturity were assessed on opposite cheeks of each fruit and the average value calculated. All measurements were conducted at a fruit temperature of 20°C. One hundred representative fruit were selected to establish DA-meter maturity classes based on ethylene emission, and to correlate AWETA impact firmness (IF) measurements with Effegi penetrometer readings. Forty-eight fruit were individually placed in sealed 1-L jars and a 1.0-ml gas sample removed and injected into a Shimadzu GC-14B packed-gas chromatograph (Column = Packed Alumina SS 80/100 180 cm; 140°C; Inj/Det = 180°C, Shimadzu, Kyoto, Japan). Fruit were left to incubate for at least one hour at 20°C prior to a second gas sample being removed and injected into the GC. Destructive fruit firmness was then determined for all 100 fruit using a hand-operated Effegi penetrometer equipped with an 8 mm diameter Magness-Taylor probe and mounted on a hand-operated drill press.

The remaining 600 hundred fruit were treated with a post-harvest fungicide dip (Spin Flo™, Active constituent: 500 g/L Carbendazim) at a rate of 75 ml/100 L water prior to cold-storage at 5°C in unsealed MAP (Lifespan™). Two hundred fruit were removed after 15, 23 and 34 days, respectively, and ripened for 48 h at 20°C. After ripening, fruit quality measurements included soluble solids content (°Brix), titratable acidity (g/L), expressible juice (% w/w), and Effegi penetrometer firmness (kgf). Fruit were then assessed for severity of chilling injury expressed as flesh browning, flesh bleeding and texture. Flesh browning and bleeding was rated on a five-point scale where 1=none, 2=trace (0-5% of flesh), 3=slight (5-25% of flesh), 4=moderate (25-50% of flesh) and 5=severe (>50% of flesh) while texture was measured on a scale of 1=very juicy, 2=moderately juicy, 3=slightly juicy, 4=mealy and 5=no juice (dry).

The effect of cold storage period and fruit maturity class on chilling injury severity was determined using GenStat statistical software (Version 13.1, Lawes Agricultural Trust, 2010). A two-way analysis of variance (ANOVA) for unbalanced designs was conducted as the number of fruit within each maturity class was not equal. Means were predicted using a regression model that included maturity class and storage period factors, and an interaction term. A least significant difference (LSD) test at a 1% significance level (p<0.001) was then used for comparison of chilling injury severity means calculated for each maturity stage and storage period. Correlations between fruit maturity and firmness measurements were determined by fitting exponential functions using the standard curve module in GenStat.

RESULTS AND DISCUSSION

Determination of Fruit Maturity Stages

‘August Fire’ nectarine ethylene emission at 20°C was well correlated (r2=0.74) with DA-meter readings confirming that chlorophyll a content, as measured by difference of absorbance just beneath the fruit skin, is a reasonable predictor of fruit physiological ripening stage (Fig. 1). For this cultivar the rate of ethylene emission remained low above a DA-meter value of 1.0 indicating that fruit was pre-climacteric. The rate of ethylene

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production increased exponentially below a DA-meter reading of 0.8. Three significantly distinct physiological maturity stages specific to this cultivar were then created based on measured ethylene emissions (Fig. 2).

The correlation between fruit physiological maturity, as measured indirectly using the DA-meter, and Effegi penetrometer firmness was significant (p<0.001) with 52% of the variation in fruit firmness explained by the DA-meter value (Fig. 3). The shape of the curve indicates that low and high DA values are associated with soft/ripe and hard/unripe fruit, respectively.

A stronger correlation (r2=0.76) was found between non-destructive acoustic firmness measurements (AWETA IF) and Effegi penetrometer firmness (Fig. 4). Fruit firmness was assessed on both the ‘August Fire’ calibration sample (n=100) and on a separate fruit population from another orchard (n=120), and data combined to increase the range of firmness values. For this cultivar AWETA IF values below 70 correspond to penetrometer readings of <4 kgf that are associated with ripe fruit while IF values >73 correspond to hard, unripe fruit (Crisosto et al., 2004). A significant decrease in fruit firmness between approximately 2 and 6 kgf as measured with the Effegi penetrometer was associated with relatively small changes in AWETA IF values (IF=66-73).

Chilling Injury and Maturity Classes

Based on ‘August Fire’ maturity stages established using the DA-meter and AWETA IF, fruit removed from cold storage after 15, 24 and 34 days were classified as pre-climacteric, climacteric or post-climacteric prior to ripening and destructive quality assessment (Table 1). Statistically significant differences in mean bleeding, browning and texture were found between maturity classes after 34 days cold storage at 5°C even though severity of chilling injury symptoms was relatively low in all circumstances during the study (Table 2). On the other hand, there was no significant effect of maturity class on chilling injury severity after 15 and 23 days cold storage at 5°C (data not shown).

After 34 days cold storage and ripening for 48 hours nectarines classified with the DA-meter as post-climacteric had significantly lower mean severity scores for flesh bleeding, browning and texture than fruit classified as either pre-climacteric or climacteric. A significant difference was found in mean expressible juice between all fruit maturity classes, with expressible juice highest in the most mature fruit. The quantity of juice extracted from fruit flesh is an indirect measure of flesh mealiness and chilling injury (Crisosto et al., 2004).

After 34 days cold storage there was no significant difference in flesh bleeding severity between fruit from the different maturity stages. For flesh browning and texture, fruit classified as post-climacteric by AWETA had significantly lower chilling injury severity than pre-climacteric fruit, but not climacteric fruit. Expressible juice was also significantly higher in post-climacteric fruit in comparison to the other two maturity classes.

Interestingly, there was no effect of maturity classification method (AWETA or DA-meter) on mean chilling injury severity for pre-climacteric and climacteric fruit. On the other hand, in post-climacteric fruit there was only a significant effect on chilling injury severity when fruit were classified by DA-meter. Chilling injury symptoms in post-climacteric fruit as determined by DA-meter were between 0.5 and 1.0 lower than equivalent fruit classified using AWETA IF. A possible explanation for this is that the AWETA IF is less sensitive to changes in firmness as measured by Effegi penetrometer in ripe fruit. Thus fruit classified as ripe by AWETA is likely to contain a wider spread in fruit maturities than fruit classified by DA-meter.

CONCLUSIONS

Chilling injury severity in ‘August Fire’ nectarine, a moderately-susceptible late season cultivar, was shown to be dependent on fruit maturity as classified non-destructively using a DA-meter and an acoustic firmness sensor (AWETA). Fruit that was physiologically more mature, corresponding to DA-meter readings of <0.5 and AWETA

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IF readings of <70, were less susceptible to chilling injury. Fernández-Trujillo et al. (1998) also found a correlation between higher fruit ethylene production, and fruit maturity, and lower chilling injury severity.

Fruit were successfully classified at harvest using both non-destructive techniques but classification of post-climacteric, softer fruit was more accurate based on DA-meter measurement, when correlated with rates of ethylene emission. Although a strong sigmoidal relationship was found between AWETA IF values and Effegi penetrometer readings (r2=0.76), measurement error in AWETA IF readings <65 increased with softer fruit as measured by penetrometer (Infante et al., 2011; De Ketelaere et al., 2006). Measurement error in softer fruit probably resulted from the different physical fruit properties measured by the two techniques. Acoustic firmness measurements are related to fruit flesh elasticity while the Effegi penetrometer measures the maximum force required for tissue failure (Garcia-Ramos et al., 2005). As fruit softens the correlation between these two physical parameters weakens and direct comparison becomes difficult (Valero et al., 2007).

In this study fruit with AWETA IF readings of between 70 and 73 were classified as climacteric yet the potential Effegi penetrometer firmness values within this maturity class ranged from approximately 4 to 6 kgf. This relatively large variation in fruit firmness may explain why the effect of fruit maturity on chilling injury severity was not as apparent when fruit were classified using AWETA IF in comparison to the DA-meter.

DA-meter readings were well correlated to physiological maturity of nectarine as expressed by the rate of ethylene production. Maturity classification using both the DA-meter and AWETA firmness sensor may allow prediction of chilling injury development during cold storage. Future work should focus on verifying these classification methods using peach and nectarine cultivars with a higher-susceptibility to chilling injury along with consideration of early to late season fruit.

ACKNOWLEDGEMENTS

This research was part of Premium Fruit, a Victorian Department of Primary Industries (DPI) and Horticulture Australia Ltd. (HAL) funded project (#08278; Victorian Stone-fruit Industry). The authors would also like to thank Bret Henderson (DPI), the grower and his staff.

Literature Cited Campos-Vargas, R., Becerra, O., Baeza-Yates, R., Cambiazo, V., González, M., Meisel,

L., Orellana, A., Retamales, J., Silva, H. and Defilippi, B.G. 2006. Seasonal variation in the development of chilling injury in O’Henry peaches. Sci. Hort. 110:79-83.

Crisosto, C.H., Garner, D., Crisosto, G.M. and Bowerman, E. 2004. Increasing ‘Blackamber’ plum (Prunus salicina Lindell) consumer acceptance. Postharvest Biol. Technol. 34:237-244.

Crisosto, C.H., Mitchell, F.G. and Zhiguo, J. 1999. Susceptibility to chilling injury of peach, nectarine and plum cultivars grown in California. Postharvest Biol. Tech. 34(6):1116-1118.

De Ketelaere, B., Howarth, M.S., Crezee, L., Lammertyn, J., Viane, K., Bulens, I. and De Baerdemaker, J. 2006. Postharvest firmness changes as measured by acoustic and low-mass impact devices: a comparison of techniques. Postharvest Biol. Tech. 41:275-284.

Fernández-Trujilo, J.P., Cano, A. and Artes, F. 1998. Physiological changes in peaches related to chilling injury and ripening. Postharvest Biol. Tech. 13:109-119.

Garcia-Ramos, F.J., Valero, C., Homer, I., Ortiz-Canavate, J. and Ruiz-Altsent, M. 2005. Non-destructive fruit firmness sensors: a review. Spanish J. Agri. Research 3(1):61-73.

Infante, R., Contador, L., Rubio, P., Mesa, K. and Meneses, C. 2011. Non-destructive monitoring of flesh softening in the black-skinned Japanese plums ‘Angeleno’ and ‘Autumn beaut’ on-tree and postharvest. Postharvest Biol. Tech. 61:35-40.

Lurie, S., Vanoli, M., Dagar, A., Weksler, A., Lovati, F., Eccher Zerbini, P., Spinelli, L.,

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Torricelli, A., Feng, J. and Rizzolo, A. 2011. Chilling injury in stored nectarines and its detection by time-resolved reflectance spectroscopy. Postharvest Biol. Tech. 59:211-218.

Lurie, S. and Crisosto, C. 2005. Chilling Injury in peach and nectarine. Postharvest Biol. Tech. 37:195-208.

Valero, C., Crisosto, C.H. and Slaughter, D. 2007. Relationship between non-destructive firmness measurements and commercially important ripening fruit stages for peaches, nectarines and plums. Postharvest. Biol. Tech. 44:248-253.

Ziosi, V., Noferini, M., Fiori, G., Tadiello, A., Trainotti, L., Casadoro, G. and Costa, G. 2008. A new index based on vis spectroscopy to characterize the progression of ripening in peach fruit. Postharvest Biol. Tech. 37:195-208.

Tables Table 1. AWETA (IF) and DA-meter value ranges used to classify ‘August Fire’

nectarine into three maturity classes.

Table 2. Effect of physiological fruit maturity and firmness on chilling injury expression

in nectarine after storage at 5°C for 34 days and ripening for 48 hours at 20°C. n=173 for each chilling injury symptom.

1 Means separation at 1% level (P<0.001).

Fruit maturity class DA-meter AWETA (IF)

Pre-climacteric (Hard) >1.0 >73Climacteric (Medium firmness) 0.5 - 1.0 70 - 73Post-climacteric (Soft) <0.5 <70

Maturity/ Firmness class DA-meter AWETA (IF)

Pre-climacteric (Hard) 2.4 a1 2.3 aClimacteric (Medium firmness) 2.4 a 2.4 aPost-climacteric (Soft) 1.3 b 2.3 a

Pre-climacteric (Hard) 2.1 a 2.2 aClimacteric (Medium firmness) 2.1 a 2.0 abPost-climacteric (Soft) 1.1 b 1.9 b

Pre-climacteric (Hard) 2.3 a 2.4 aClimacteric (Medium firmness) 2.2 a 2.3 abPost-climacteric (Soft) 1.6 b 2.1 b

Pre-climacteric (Hard) 36.3 a 35.2 aClimacteric (Medium firmness) 38.5 b 36.4 aPost-climacteric (Soft) 44.7 c 39.1 b

Flesh texture (mean score)

Expressible juice (g/g fresh weight)

Flesh bleeding (mean score)

Flesh browning (mean score)

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Figures

Fig. 1. Correlation between measured DA-meter reading and ethylene emission at 20°C

for ‘August Fire’ nectarine (n=48).

Fig. 2. DA-meter fruit maturity classes based on mean ethylene emission at 20°C for

‘August Fire’ nectarine (n=48). Bars represent the standard error (SE) of each class mean. All classes were significantly different (P<0.05) based on an unpaired t-test comparing class means.

DA Value

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Eth

yle

ne e

mis

sion

(uL

*kg-1

*hr-1

)

0

2

4

6

8

10

r2 = 0.74; p<0.001r2=0.74; P<0.001

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.0-0.49 0.5-0.99 1.0-1.4

DA Index Classes

Eth

ylen

e e

mis

sio

n (

nl

l-1

h-1

g F

W-1

)

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Fig. 3. Relationship between Effegi penetrometer firmness values and DA-meter readings

measured at harvest for ‘August Fire’ nectarine (n=100).

Fig. 4. Relationship between Effegi penetrometer firmness values and AWETA firmness

readings measured at harvest for ‘August Fire’ nectarine harvested from two orchards (n=220).

0.0

2.0

4.0

6.0

8.0

10.0

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

DA-meter value (Arbitrary units)

Eff

egi

pen

etro

met

er r

ead

ing

(kg

/cm

2)

r2=0.52; P<0.001

0.0

2.0

4.0

6.0

8.0

10.0

45 50 55 60 65 70 75 80 85

AFS impact firmness (Arbitrary units)

Eff

egi

pen

etro

met

er r

ead

ing

(kg

/cm

2)

r2=0.76; P<0.001

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