2012 Prediction of Internal Browning Risk in ENVY

Prediction of internal browning risk in Scilate
apples
Brookfield PL, Rowan D, Hedderley D, Robertson J, McGhie T, Hunt M,
Seymour S, Hall M, Friend A, Diack R, Oliver M, Johnston J
November 2012
A CONFIDENTIAL report prepared for
Enza Limited
Brookfield PL, Robertson J, Oliver M

Plant & Food Research, Hawkes Bay
Johnston J, Hall M
Plant & Food Research, Auckland
Rowan D, McGhie T, Hunt M, Hedderley D

Plant & Food Research, Palmerston North
Seymour S, Friend A, Diack R

Plant & Food Research, Motueka
SPTS No. 7773

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PUBLICATION DATA
Brookfield PL, Rowan D, Hedderley D, Robertson J, McGhie T, Hunt M, Seymour S, Hall M, Friend A, Diack R, Oliver
M, Johnston J.November 2012. Prediction of internal browning risk in Scilate apples. A confidential report prepared for:
ENZA Limited. Plant & Food Research Data: Milestone No. 47292. Contract No. 28199. Job code: P/274053/01. SPTS
No. 7773.
This report has been prepared by The New Zealand Institute for Plant & Food Research Limited
(Plant & Food Research), which has its Head Office at 120 Mt Albert Rd, Mt Albert, Auckland.
This report has been approved by:
Jason Johnston
Scientist/Researcher, Postharvest Team
Date: November 2012
Jocelyn Eason
Science Group Leader, Postharvest and Whole Foods
Date: November 2012
Contents
Executive summary i
1 Introduction 1
2 Materials and methods 2

2.1 Experimental design 2
2.2 Assessments 2
2.3 Analysis 3
3 Results and discussion 4

3.1 Physiological relationships 4
3.2 Metabolite relationships 10
4 Conclusions and recommendations 14
5 References 15
6 Acknowledgements 15
Appendix 1 16
Executive summary
Prediction of internal browning risk in Scilate apples
Brookfield PL, Rowan D, Hedderley D, Robertson J, McGhie T, Hunt M, Seymour S, Hall M, Friend A,
Diack R, Oliver M, Johnston J
November 2012. SPTS No. 7773
Scilate apples are susceptible to the development of internal browning (IB) but management of
disorder risk is complicated by varying and unpredictable susceptibility of fruit. The ability to
identify at-risk fruit would enable more appropriate and targeted handling according to the
predisposition of fruit. The aim of this project is to evaluate the potential for predicting IB risk by
evaluating physiological and metabolite characteristics in relation to disorder incidence in
Scilate apples.
For 2011, fruit were sampled from each of three orchards in Hawkes Bay at three stages during
commercial harvest and cool-stored in air or CA (3%O2: 1%CO2) after a 10-day delay with and
without 1-MCP treatment, or in CA after a 2-day delay without 1-MCP treatment. Fruit were
assessed for incidence and severity of internal browning after 15 weeks of coolstorage at 0.5C
and 7 days at 20C. For 2012, fruit were harvested from 16 orchards (eight in Hawkes Bay and
eight in Nelson) and cool-stored in air or in CA (2% O2: 2% CO2) after 2 and 10 days of
coolstorage in air. Fruit were assessed for incidence and severity of disorders after 12 weeks of
coolstorage at 0.5C and 7 days at 20C.
Although 2011 data supported the potential role of at-harvest respiration as a physiological
indicator of at-risk lines, prediction based just on respiration did not account for a majority of the
variation in IB incidence and this measure was not validated as a significant predictor of IB risk
in the 2012 results . None of the physiological relationships from the 2012 study were significant
in themselves for orchards in Hawkes Bay, and none that were significant for Nelson orchards
have shown consistency over seasons/orchards. These results reinforce the need to explore
new metabolite based approaches to identify at-risk orchards.
Analysis of 2011 metabolite data discriminated the prevalence of internal browning among
orchards based on the relative concentrations of five compounds closely associated with the
disorder, and of particular interest was the association of increased pentose sugars at harvest in
fruit from orchards with higher IB incidence after storage. These associations from the 2011
data and the potential for new ones need to be examined in the 2012 data for the 16 orchards.
Given the implication of oxidation of phenolic metabolites being involved, the direct analysis on
these secondary metabolites using LCMS is a priority and will be carried out and made
available to Enza when completed.
Given the lack of any robust physiological or simple industry metrics, we recommend a
continuation of using orchard historical incidence as a means for predicting future risk. We also
recommend caution over the use of secondary technologies that exacerbate browning in high-
risk orchards, such as CA and 1-MCP, until robust handling protocols are developed. A low
incidence of browning still occurs in air storage, suggesting that a strategy of minimising usage
of secondary technology will not completely solve the problem. Future studies could aim to link
the storage responses in this study with industry information on cultural factors for the 16
The New Zealand Institute for Plant & Food Research Limited (2012) Page i
This report is confidential to ENZA Limited
Prediction of internal browning risk in Scilate apples. November 2012 SPTS No. 7773
orchards, and also aim to validate metabolite-based prediction approaches once the 2012
metabolite analysis is complete.
For further information please contact:
Jason Johnston
The New Zealand Institute for Plant & Food Research Ltd
Plant & Food Research Mt Albert
Private Bag 92 169
Victoria Street West
Auckland 1142
NEW ZEALAND
Tel: +64-9-925 7000
Fax: +64-9-925 7001
Email: jason.johnston@plantandfood.co.nz
The New Zealand Institute for Plant & Food Research Limited (2012) Page ii
Prediction of internal browning risk in Scilate apples. November 2012 SPTS No. 7773
1 Introduction
Scilate apples are susceptible to the development of internal browning (IB) and postharvest
practices for maintaining fruit quality need specific protocols to minimise the risk of disorder
expression. At harvest, fruit predisposition to the disorder varies depending on orchard and
seasonal effects. In some cases, the disorder has been found in air-stored fruit but it has more
often been associated with controlled atmosphere storage (CA). Fruit sensitivity declines with
longer time after harvest. Coolstorage in air for up to three weeks is needed before application
of CA to minimise the risk of increasing incidence of the disorder in CA (Brookfield et al. 2010).
There is also the need for protocols for the application of 1-MCP alone or in combination with
CA as there is a good incentive to use these technologies for superior fruit quality maintenance
of Scilate apples (Brookfield et al. 2011).
Despite the awareness of postharvest opportunities for minimising disorder expression, the
management of internal browning risk in Scilate apples is complicated by the varying and
unpredictable susceptibility of fruit on an orchard and seasonal basis. The ability to identify at-
risk fruit would enable more appropriate and targeted handling according to the predisposition of
fruit. However, a more fundamental understanding of factors leading to fruit susceptibility and to
disorder expression is needed in order to develop methods for prediction of IB risk. Internal
browning is thought to be ultimately related to development of an imbalance between oxidative
and reductive processes leading to loss of membrane function and cell death (Franck et al.
2007). Treatment with the antioxidant diphenylamine (DPA) provides a measure of protection
against the disorder in Braeburn apple (Mattheis and Rudell 2008). Nevertheless, it has been
difficult to develop a useful understanding of the manifestation of such disorders as they involve
multiple pathways and complex responses. A new approach that has the potential to improve
understanding is the application of rapidly developing analytical platforms such as Liquid and
Gas Chromatography Mass Spectrometry (LC- and GC-MS) techniques for metabolic profiling
(Hertog et al. 2011). Characterisation of metabolic changes associated with disorder
development could lead to identification of biomarkers useful for disorder risk identification and
management (Rudell et al. 2009).
The aim of this project is to evaluate the potential for predicting IB risk by evaluating
physiological and metabolite characteristics in relation to disorder incidence in Scilate apples.
Our approach has been to first profile changes in fruit at harvest and during disorder
development. Analyses were carried out on samples from the previous season (Brookfield et al.
2011) whereby fruit from three different orchards were exposed to five different postharvest
treatments that resulted in a range of IB incidence. This was necessary to identify targets from
the metabolite analysis that could be validated with a more targeted dataset applied to the wider
study of 16 Scilate orchards carried out in 2012.
The New Zealand Institute for Plant & Food Research Limited (2012) Page 1
Prediction of internal browning risk in Scilate apples. November 2012. SPTS No. 7773
2 Materials and methods
2.1 Experimental design
2011: Fruit were sampled from each of three orchards in Hawkes Bay at three stages during
commercial harvest and cool-stored in air or CA (3%O2: 1%CO2) after a 10-day delay with and
without 1-MCP treatment, or in CA after a 2-day delay without 1-MCP treatment (Brookfield
et.al. 2011). At each harvest and after 10, 20 and 40 days of storage, 3 replicates of 8 fruit were
sampled from each orchard x treatment combination for physiological and metabolite analyses.
Physiological assessments at harvest (detailed below for 2012 assessments) included fruit
weight, red colour coverage, foreground colour (Enza foreground colour chart for Envy apple
where 1 = pale and 8 = dark), background colour, firmness, soluble solids concentration,
internal ethylene concentration, ethylene production and respiration rate. At 10, 20 and 40 days
of coolstorage fruit firmness, internal ethylene concentrations, ethylene production and
respiration rates were assessed. At each assessment, measurements were carried out on each
fruit immediately after removal from coolstorage. Using the same fruit for metabolite sampling, ~
8 plugs of 9mm diameter were obtained from an equatorial slice of ~ 6mm thickness from each
fruit. The tissue from each 8-fruit replicate was immediately snap-frozen in liquid nitrogen and
stored at -80C for later analyses. The incidence of internal browning was assessed from 3
crates of fruit (~70 fruit per crate) for each combination of orchard x harvest x storage treatment
after ~ 15 weeks of storage at 0.5C and 7 days at 20C (Brookfield et al. 2011).
2012: Fruit were harvested from 16 orchards (eight in Hawkes Bay on 2nd April and eight in
Nelson on 16th April) during commercial harvest and cool-stored in air or in CA (2% O2: 2% CO2)
after 2 and 10 days of coolstorage in air. At each orchard three crates of fruit were harvested
from each of three rows of trees and for each row, one crate was allocated to each of the three
treatments. A further 54 fruit (sampled as six fruit per crate during filling of crates) were
allocated as two sets of nine fruit per row and used for physiological and metabolite
assessments at harvest (assessed at 20C the following day) and after 10 days of coolstorage
in air (each orchard sample assessed immediately after removal from coolstorage). All storage
samples were labelled and placed in coolstorage in air at 0.5C on the day of harvest. Fruit
were moved into a commercial CA store after 2 or 10 days of delay as required. The CA
atmosphere was adjusted from 3% O2: 1% CO2 to 2% O2: 2% CO2 on 8th May, resulting in 5
and 4 weeks at the initial atmosphere for Hawkes Bay fruit in the 2 and 10 day delay
treatments, and 3 and 2 weeks at the initial atmosphere for Nelson fruit in the 2 and 10 day
delay treatments. The incidence of disorders for each combination of orchard and postharvest
treatment was assessed after 12 weeks of coolstorage and 7 days at 20C.
2.2 Assessments
2.2.1 At harvest
The following measurements were made using 27 fruit per orchard:
1. Individual fruit weight (g) was recorded on an electronic balance (Mettler Toledo
PG5002-S Delta Range)
2. Background colour was assessed using the ENZA Background colour chart for
Scilate/Envy apples, where 1=green and 8=yellow
3. Red colour coverage was assessed visually as percentage of the apple surface with red
colour and red colour intensity was assessed using the ENZA foreground colour chart
for Envy apples where 1=pale and 8=dark
4. Respiration (ml/kg.h) was determined for each of three nine-fruit samples per orchard
by withdrawing a 1-ml sample of headspace gas after sealing each sample in a 8500-ml
container for one hour and measuring CO2 using an infra-red CO2 transducer
(Servomex Autotech Engineering, UK) with nitrogen as the carrier gas
5. Internal ethylene concentration (IEC; l/L) was measured from a 1-ml sample of gas
drawn from the cortical cavity of each fruit and analysed by flame ionisation gas
chromatography (Hewlett Packard 6890 Series gas chromatograph fitted with a glass
activated alumina column)
6. Flesh firmness (kgf) was assessed using a fruit texture analyser (GSS, model GS14,
South Africa) fitted with an 11.1-mm diameter probe. Two measurements per fruit were
made on pared surfaces on opposite sides of the fruit
7. Soluble solids (%) was assessed from juice expressed during the firmness test using an
Atago Smart-1 automatic refractometer
8. Starch pattern index was assessed by staining the cut half of each apple with iodine
solution and recording the amount of staining according to the ENZA starch pattern
index for apples (SPI) 0-7 where 0=fully stained and 7=no staining
9. Fruit density (g/cm3) was determined for individual fruit from fruit weight and estimation
of volume by displacement in water on a balance
10. Metabolite samples were obtained by taking ~8 plugs of cortex tissue from an equatorial
slice of each fruit (9 fruit x 3 samples per orchard), immediately freezing in liquid
nitrogen and storing at -80C until analysis.
11. Metabolite analysis was carried out on frozen, milled apple tissue (ca. 200 mg) following
the method of Roessner et al. (2000).
2.2.2 Ten days after harvest
The following measurements were made using 27 fruit per orchard:
1. Dry matter content (%) was assessed for individual fruit from tissue wedges by
calculating the difference between fresh sample weight and weight after fan oven drying
at 65C for 24 h
2. IEC, SPI and metabolites were assessed as described above
2.2.3 After storage
Fruit were inspected visually for external disorders followed by at least three equatorial cuts per
fruit for internal assessment. For assessment of IB, the severity was rated according to cut
surface area affected as either none, 1-10%, 11-25% or >25%.
2.3 Analysis
The main effects and their interactions on physiological measures and incidence of IB were
analysed using generalised linear mixed models. The relationships of physiological measures
with incidence of IB were examined using partial least squares analysis (2011 data) or by
Pearson correlation coefficients (2012 data). The relationships of metabolites derived from GS-
MS analysis with incidence of IB for the 2011 dataset were examined using standard partial
least squares-discriminant analysis (PLS-DA) and by screening of standardised regression
coefficients to identify which compounds might be important predictors of IB incidence in
relation to the main effects (orchard, harvest, treatments).
3 Results and discussion
3.1 Physiological relationships
3.1.1 2011
The incidence of IB varied between orchards, harvests and storage treatments. The treatment
effects were broadly similar for each harvest (harvest x treatment not significant) and allowing
for different overall levels of disorder, for each orchard (orchard x treatment not significant) with
the highest incidence occurring in the 2-day delayed CA treatment. The orchard differences in
disorder incidence (averaged across harvests and treatments) were 5%, 6% and 18% for
Griffiths CG202, Griffiths M106/M9, and Evenden orchards, respectively, indicating that
Evenden orchard was the more highly susceptible orchard in this study (Figure 1).
Figure 1. Internal browning incidence in Scilate apples from three orchards (Griffiths CG202, Griffiths
M106/M9 and Evenden) harvested three times during maturation and cool-stored in air, or CA (3%O2: 1%CO2)
after a 2-day delay or in CA after a 10-day delay without and with 1-MCP treatment. Fruit were assessed after
~ 15 weeks of storage at 0.5C and 7 days at 20C.
Partial least squares analysis (PLS) was used to find whether any measures of physiological
characteristics at harvest related to the incidence of IB that developed during storage. Using the
2-day delayed CA data as the maximum disorder expression treatment, the PLS resolved two
significant dimensions, accounting for 90% of the variance in the proportion of fruit with internal
browning and between 48% and 98 % of the variance in the physiological measures at harvest.
Looking at how the at-harvest physiological measures load on the two significant PLS
dimensions and how the proportion of fruit with IB correlates with that, the two strongest
predictors were at-harvest fruit respiration rate and firmness; higher incidence of IB was
associated with higher fruit respiration rate and firmness. Higher ethylene production was a
relatively weaker predictor of higher IB (Figure 2).
BgCol
Red
FgCol LogIEC
SS
PLSDim2
C2H4CO2
1 Logit
Wt FF 1
IB
1
PLSDim1
Figure 2. Relationship between physiological characteristics measured at-harvest and incidence of internal
browning after storage for three orchards in 2011. Two significant dimensions of a partial least squares
analysis are shown. The Scilate apples used in this analysis were stored in controlled atmosphere (3%O2:
1%CO2) following a 2-day delay after harvest. Internal browning incidence is represented by logit IB.
The fruit physiological characteristics were weight (Wt), foreground colour (Fg Col), background colour
(Bg Col), red colour coverage (Red), internal ethylene concentration (Log IEC), soluble solids (SS), ethylene
production (C2H4), respiration rate (CO2) and firmness (FF).
Combining the at-harvest, 10-day, 20-day and 40-day physiological data to predict IB after
storage (2-day delayed CA) the first three significant dimensions accounted for 99% of the
variance in the proportion of fruit with IB. High firmness at all time points was highly associated
with IB, as was high respiration rate at harvest. Lesser predictors were high initial ethylene
production and respiration rate at 10 days. Respiration rate at later times was not associated
with IB much at all (Figure 3). Analysis combining data across all treatments rather than just the
2-day delayed CA treatment for at-harvest and for the 10-, 20- and 40-day measurements gave
a similar outcome, although with less variation in IB incidence accounted for than when using
the 2-day delayed CA results (data not presented).
Analysis of physiological changes during the early postharvest period when sensitivity to IB is
high indicated there was a significant transient rise in respiration rate associated with fruits
exposed to CA that did not occur in fruits held in air (Figure 4). These CA treatments were
associated with higher incidence of IB after storage. Although fruit from all three orchards in this
study experienced an early elevation in respiration rates in association with early exposure to
CA, it was the orchard (Evenden) with highest at-harvest respiration rates that also had the
highest incidence of IB after storage (Brookfield et al. 2011). These results appear to support
the outcome derived by PLS indicating that orchards with high at-harvest fruit respiration rates
could be at higher risk of IB. Although these 2011 data support the potential role of at-harvest
respiration as a physiological indicator of at-risk lines, prediction based just on respiration did
not account for a majority of the variation in IB incidence (data not presented). Also, the
relationship would need support from validation over more orchards than the three used in this
study to be considered useful for managing IB risk on an orchard basis.
0.5 0.5
C2H4
CO2
Wt Wt Wt CO2Wt
LogIEC
LogIEC C2H4
LogIEC
Wt C2H4 Logit Wt FFFF
FFLogit
PLSDim2
PLSDim3
CO2FF
LogIEC FF IB LogIEC CO2FF IB
FFFF
CO2
C2H4 C2H4
0.5 0.5 0.5 Wt CO2 0.5
Harvest Wt Harvest C2H4
CO2 LogIEC
LogIEC
10d 10d
C2H4
C2H4
CO2 LogIEC
20d 0.5 20d 0.5
PLSDim1 PLSDim1
40d 40d
Figure 3. Relationship between physiological characteristics and incidence of internal browning after
storage for three orchards in 2011. The three most significant dimensions of a partial least squares analysis
are shown, incorporating physiological characteristics at harvest, and after 10, 20 and 40 days of storage.
Internal browning incidence (logit IB) was for fruit from the 2-day delay controlled atmosphere (3%O2:
1%CO2) treatment. Fruit physiological characteristics were weight (Wt), internal ethylene concentration
(Log IEC), ethylene production (C2H4), respiration rate (CO2) and firmness (FF).
4.0
Air
Respirationrate(ml/kg.hr)
3.0
Air+MCP
2.0
2daydelay
+CA
1.0
10daydelay
+CA
0.0
10day+CA+
10 20 40 MCP
Daysincoolstorage
Figure 4. Respiration rate (mls/kg.hr) of Scilate apples after 10, 20 and 40 days of coolstorage.
Fruit were cool-stored in air, or CA (3%O2: 1%CO2) after a 2-day delay or in CA after a 10-day delay without
and with 1-MCP treatment. Measurements were made on cold fruit immediately following removal of fruit
from coolstorage. Data are averaged over fruit from three orchards picked on three harvest dates during
maturation.
3.1.2 2012
Averaged over storage treatments, the incidence of IB was higher in Nelson (36%) than in
Hawkes Bay (6%). Averaged over orchards, incidence was much lower in air (2%) than in CA,
as expected, with 2-day delayed CA being 47% and 10-day delayed CA being 36%. The
difference in incidence between the 2-day and 10-day delayed CA treatments was much smaller
in 2012 than 2011, with a much larger reduction in incidence occurring for the 10-day delay
treatment in 2011. This suggests that the 2012 crop not only had a higher predisposition for
developing the disorder, but also required more severe amelioration strategies with regards to
timing of application of secondary postharvest technologies such as CA. Orchard differences
where expression was highest (2-day delayed CA) varied from 12 to 39 % in Hawkes Bay and
from 44% to 90% in Nelson (Figure 5). The shorter initial period of CA at 3%O2: 1%CO2 may
have contributed to the higher incidence of IB in Nelson fruit. However, even in air storage there
was a higher incidence of IB in Nelson (6%) than in Hawkes Bay fruit (0%), indicating a higher
biological susceptibility of Nelson fruit in the 2012 season. The incidence of browning in air
storage, while low, highlights that the disorder cannot be solely attributed to CA and 1-MCP
treatments (Brookfield et al. 2011), instead these latter conditions seem to exacerbate a
condition that expresses in low-temperature air storage.
The severity of disorder as a proportion of orchard incidence follows a similar pattern between
regions and orchards as for total incidence (Figure 5). However severity differed between
treatments; although % IB increased between 10- and 2-day delayed CA as expected. This was
accompanied by a notable increase in the proportion of fruit with high disorder severity (Figure
5). Analysis of the data using a weighted severity score (1, 2 and 3 for low middle and high
severity rating respectively) had the effect of increasing differences between orchards and
between treatments compared with what was revealed using incidence alone (data not
presented).
2ddelay+CA
100%
80%
60%
%IB
40%
20%
0%
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
HB NL
10ddelay+CA
100%
80%
60%
%IB
40%
20%
0%
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
HB NL
Air
100%
80%
60%
%IB
40%
20%
0%
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
HB NL
Figure 5. Internal browning incidence (%IB) in Scilate apples from eight orchards in Hawkes Bay (HB) and
eight orchards in Nelson (NL) in 2012. Fruit were harvested from each orchard once during commercial
harvest and cool-stored in air, or CA after a 2-day or 10-day delay. The CA atmosphere was adjusted from 3%
O2: 1% CO2 to 2% O2: 2% CO2 on 8th May, resulting in 5 and 4 weeks at the initial atmosphere for Hawkes
Bay fruit in the 2 and 10 day delay treatments, and 3 and 2 weeks at the initial atmosphere for Nelson fruit in
the 2 and 10 day delay treatments. Fruit were assessed after 12 weeks of coolstorage and 7 days at 20C.
The severity of internal browning according to cut surface area affected is shown as 1-10% (blue), 11-25%
(orange) or >25% (green).
Relationships between fruit physiological characteristics at harvest (Appendix I) and IB after

storage on an orchard basis were examined by correlation coefficients using data for the 2-day
delayed CA treatment (maximum expression). These relationships were also examined for the
air treatment which had much lower expression of the disorder but was not influenced by the
effects of CA (Table 1). Scatter plots of the relationships were examined to check for influence
on the correlations of outlying orchard data values. This was important especially for those
relating to density, foreground colour and background colour which were consistent in being
negative for both growing regions (Table 1). However, except for respiration (see following) the
distribution of values in the scatter plots indicated that the correlations were a fair representation
of the relationship, not unduly affected by wayward values for certain orchards (data not
presented). For fruit stored in CA only, one factor was significant at harvest (respiration), but this
correlation was strongly affected by one orchard (Nelson 7) having very low respiration in
relation to IB compared with other orchards and with this orchard removed, this correlation (-
0.25) was not significant (Table 1 A and B). The correlation with fruit density was near
significant for Nelson orchards, but was negative (rather than positive as implicated in the 2011
study (Brookfield et al. 2011). Looking at correlations on an orchard basis for air-stored fruit,
percentage red colour coverage and firmness of fruit were significantly correlated with IB
(incidence and severity weighted incidence) for orchards in the Nelson region where IB
incidence and severity were higher (Table 1 C and D).
Table 1. Correlation coefficients of relationships between physiological characteristics at harvest and

occurrence of internal browning after storage of Scilate apples. Fruit were harvested from eight orchards in
Hawkes Bay (HB) and in Nelson (NL) and assessed after 12 weeks of coolstorage plus 7 days at 20C.
Internal browning was assessed for incidence and severity with severity weightings of 1, 2 and 3 applied for
low, middle and high severity based on the cut surface area of affected tissue. Correlations were performed
on an orchard basis for fruit stored in controlled atmosphere following a 2-day delay (A and B) or in air (C
and D), and according to % incidence (A and C) or weighted severity incidence (B and D). Significant
correlations are in bold, and near significant correlations in bold italics.
A Wgt Density Red% Fgnd Bgnd Log IEC Resp FF SS SPI
HB -0.22 -0.05 -0.64 -0.28 -0.18 0.43 0.37 0.10 0.13 0.33
NL 0.36 -0.74 0.41 -0.48 -0.48 -0.20 -0.53 0.22 -0.22 -0.54
HB&
NL -0.13 -0.23 0.19 -0.01 -0.47 -0.13 -0.48 0.19 -0.03 -0.05
B
HB -0.23 -0.06 -0.65 -0.24 -0.10 0.4 0.44 0.05 0.17 0.34
NL 0.34 -0.73 0.41 -0.52 -0.41 -0.17 -0.54 0.23 -0.18 -0.50
HB&
NL -0.11 -0.26 0.24 -0.04 -0.45 -0.14 -0.52 0.20 -0.02 -0.08
C
HB 0.18 -0.57 -0.38 -0.14 -0.20 0.334 0.34 -0.57 -0.06 0.42
NL 0.41 -0.41 0.88 -0.19 -0.15 0.07 0.06 0.73 0.02 -0.59
HB&
NL 0.03 -0.24 0.61 0.03 -0.31 -0.05 -0.18 0.47 0.02 -0.23
D
HB 0.20 -0.53 -0.31 -0.21 -0.20 0.38 0.32 -0.50 -0.07 0.40
NL 0.33 -0.35 0.88 -0.14 -0.05 0.13 0.10 0.76 0.06 -0.51
HB&
NL 0.01 -0.20 0.64 0.05 -0.24 -0.02 -0.14 0.52 0.05 -0.22
None of the relationships from the 2012 study were significant in themselves for orchards in
Hawkes Bay, the only individual relationships of note being in Nelson air-stored fruit (virtually
no internal browning occurred in air-stored fruit from Hawkes Bay). It is possible that
multivariate models of physiological measures may account for sufficient variation in IB on an
orchard basis to assist prediction of orchard risk. However, the lack of consistent significant
relationships over seasons/orchards for these measures to date supports the need to explore
relationships at a more fundamental level in order to identify at-risk orchards more reliably.
3.2 Metabolite relationships

Stresses are imposed on the fruit during development, harvesting and postharvest storage that
alter their metabolism. This provides the potential to identify biological markers of change
related to disorder risk that could be developed as a tool for fruit risk assessment. An initial
analysis of metabolite associations and IB incidence was carried out on the entire 2011 dataset
(i.e. inclusive of at-harvest, 10-, 20- and 40-day samples and in relation to the effects of
orchard, harvest, storage treatment and time in storage during the first 40 days) in order to
assess global changes in metabolite patterns associated with the main effects. PLS analysis
provided an 8-dimensional solution which accounted for between 39% and 95% of the variation
in the concentrations (log values) of 65 compounds determined by GCMS (data not presented).
This relatively complex analysis was then refined to identify patterns of metabolites in fruit at
harvest that were more specifically associated with orchard differences in IB incidence. Looking
at the at-harvest GCMS measures of compounds in relation to IB incidence on an orchard basis
following 2-day delayed CA storage (orchard IB incidence averaged over harvests was Evenden
37%, CG202 16% and M106/M9 12%), the PLS analysis provided a two-dimensional solution
that accounted for 86% of the harvest date trend (changes associated with harvest) and 80% of
that associated with differences in IB incidence between orchards. It also accounted for
between 2% and 98% of the variation in concentration (log value) of the compounds depending
on compound, indicating that some, as expected, are more relevant than others.
The first dimension (horizontal) relates mostly to changes associated with harvest maturity (later
harvest is more positive) and the second dimension (vertical) relates mostly to orchard
differences in IB (higher IB is more negative) (Figure 6A). Plotting the loadings of metabolite
compounds against these dimensions and the correlations of the compounds with these
dimensions indicated that for the first dimension, compounds annotated as sugar disaccharides
were associated with early harvest and decreased with later harvest while some hexose sugars
were associated with later harvest (Figure 6B). For the second dimension relating to the level of
orchard IB, higher orchard prevalence of IB was associated with higher levels of a pentose
sugars and with certain organic acids; lower orchard IB was associated with inositol and with
several compounds of unknown identity (Figure 6B).
A
Dim1&2 0.5
Aminoacid
cyclito
disaccharide
hexitol
hexose
misc
0.5 0.5
unknown
organicacid
pentose
phosphate
0.5
Figure 6. Relationship between metabolites measured at-harvest and incidence of internal browning after
storage for three orchards in 2011. Partial least squares analysis (PLS) was used. Fruit were harvested from
each orchard three times during maturation and cool-stored in controlled atmosphere (3%O2: 1%CO2) after a
2-day delay. Fruit were sampled for metabolite analysis at harvest and for incidence of internal browning
after ~ 15 weeks of storage at 0.5C and 7 days at 20C. On the horizontal dimension, later harvest is more
positive and on the vertical dimension higher orchard prevalence of internal browning is more negative (A).
The location of metabolites on these dimensions indicates their association with harvest and orchard
prevalence of internal browning (B).
Although the PLS indicated that particular classes of compounds were not clearly linked with IB,
several individual compounds were quite highly associated. This was explored further by
looking at standardised regression coefficients to identify which compounds were the strongest
predictors of IB (Figure 7). Those that were strongly associated with orchard prevalence
( 0.1) were the pentose sugar pentose 2 and malic acid (higher concentration associated
with higher disorder incidence), unknown metabolite M138T10.23, 4-aminobutyric acid and a
disaccharide ds0 (higher concentration associated with lower disorder incidence).
Figure 7. Standardised regression coefficients of relationships between compounds and their

concentrations in relation to harvest maturity (Harvest) and orchard prevalence of internal browning
(Orchard Prev) in Scilate apple. Individual compounds are plotted but for simplicity they are ordered and
labelled according to group.
Plotting the orchard means of the most important metabolites relating to the orchard incidence
of IB following 2-day delayed CA illustrates how the concentration patterns of certain
metabolites could be associated with disorder risk on an orchard basis (Figure 8). These results
are encouraging, given that the metabolite patterns associated with IB found in this preliminary
data set were derived from just three orchards.
Pentose2 Malic acid Unknown (M138T10.23)
8.1
6.1 4.2
4.1
6.0 8.0 4.0
3.9
5.9
3.8
7.9
5.8 3.7
M106/M9
M106/M9
M106/M9
Cg202
Cg202
Cg202
Evendon
Evendon
Evendon
4 aminobutyric DS0
7.4
2.9 7.3
7.2
7.1
7.0
2.8
6.9
6.8
6.7
2.7
M106/M9
M106/M9
Cg202
Cg202
Evendon
Evendon
Figure 8. Concentrations (log 10 transformed) of five metabolites at harvest closely associated with
incidence of internal browning in Scilate apples after storage as affected by orchard and harvest. Fruit were
harvested three times from each orchard and samples for metabolite analysis were taken at each harvest.
The GCMS metabolite analysis provides a number of associations between metabolites and
fruit physiology (Figure 6) which need to be validated in further experiments, in particular using
samples collected from the 16 orchards from 2012.
Of particular interest is the association of increased pentose sugars with a higher incidence of
IB suggesting increased operation of the pentose pathway, an alternative pathway for the
metabolism of sugars which is used for the production of phenolic compounds. Oxidation of
phenolic metabolites is widely implicated in the occurrence of oxidative disorders and tissue
browning. The direct analysis on these secondary metabolites using LCMS is a priority for the
research on metabolites of Scilate apples.
4 Conclusions and recommendations
This research showed a high predisposition of Scilate apples for developing internal browning
in 2012, especially for orchards from the Nelson region. When comparing the browning
incidence for 2-day and 10-day delay CA treatments, the 10-day delay treatment was less
effective at reducing browning in 2012 than 2011. This suggests that the disorder may have
been more difficult to manage through postharvest interventions in 2012. From a research
perspective, this diversity of responses provided a robust platform for a prediction study across
two seasons.
Although 2011 data supported the potential role of at-harvest respiration as a physiological
indicator of at-risk lines, prediction based just on respiration did not account for a majority of the
variation in IB incidence. None of the physiological relationships from the 2012 study were
significant in themselves for orchards in Hawkes Bay, and at-harvest respiration was not
validated by the 16 orchard study as a measure of sufficient precision for predictive purposes.
The lack of consistent significant relationships over seasons/orchards for these measures to
date reinforce the newer approach used in this study to explore relationships at a more
fundamental level in order to identify at-risk orchards.
Analysis of 2011 metabolite data discriminated the prevalence of internal browning among
orchards based on the relative concentrations of five compounds closely associated with the
disorder, and in particular, the association of increased pentose sugars in fruit from orchards
with a higher incidence of IB. These associations will be validated in the related PFR funded
project on metabolomics for tissue collected from 16 orchards in 2012. Analysis on phenolic
metabolites using LCMS will be a priority for continued research on metabolites, with results
made available to Enza on completion.
In terms of risk management strategies for the 2013 season we recommend:
Continuation of the strategy of historical orchard incidence as a means for identifying

future orchard risk, on the basis that industry and physiological indices show limited
potential for improved risk assessment.
Caution of the use of secondary technologies such as CA (and 1-MCP; Brookfield et al.
2011) for high-risk fruit until robust handling protocols are developed.
Continue a research approach of finding robust risk factors for the disorder by aligning
industry-collected information on orchard cultural factors with the browning results in
this study, on the basis that the study provides data for orchard responses for a
consistent set of storage conditions. Industry out-turn data are less precise as
differences in out-turn could be due to differences in supply chain management or
orchard predisposition. However, industry out-turn information for these orchards would
also be useful to place the present results in a commercial context. We also recommend
that a metabolite prediction study be validated once the metabolite results for 2012 are
available.
5 References
Brookfield PL, Seymour S, Robertson J, Diack R, Hedderley D. 2010. Management of disorder
risk in Scilate apples. Report to ENZA Limited. Plant & Food Research SPTS No. 4688.
Brookfield PL, Robertson J, Johnston J, Farrell M. Hedderley D, Oliver M, Dayatilake D. 2011.

Postharvest handling affects occurrence of internal browning in Scilate apples Report to ENZA
Limited. Plant & Food Research SPTS No. 6179.
Franck C, Lammertyn J, Ho QT, Verboven P, Verlinden B, Nicolai BM.2007. Browning disorders

in pear fruit. Postharvest Biol.Technol, 43, 1-13.
Hertog M, Rudell D, Pedreschi R, Schaffer R, Geeraerd A, Nicolai B, Ferguson I. 2011. Review.

Where systems biology meets postharvest. Postharvest Biology and Technology 62: 223-237.
Mattheis JP and Rudell. 2008. Diphenylamine metabolism in Braeburn apples stored under
conditions conducive to the development of internal browning. J. Agric. Food Chem. 2008, 56,
33813385.
Roessner, U., C. Wagner, et al. (2000). "Simultaneous analysis of metabolites in potato tuber by
gas chromatography-mass spectrometry." Plant J. 23: 131-142.
Rudell D R, Mattheis JP, Hertog MLATM. 2009. Metabolomic change precedes apple superficial
scald symptoms. J. Agric. Food Chem. 57, 8459-8466.
6 Acknowledgements
We thank Enza Scilate apple growers for access to their orchards to sample fruit and Simon
Thurstfield and his team at Apollo Pac Ltd for assisting with postharvest treatment and storage
of fruit. We acknowledge the contribution of the Plant & Food Future Science Project
Metabolomics that covered the metabolite analyses.
Appendix 1
At-harvest physiological characteristics of Scilate apples harvested from eight orchards in
Hawkes Bay and eight orchards in Nelson in 2012.
Wgt Density Red Fgnd Bgnd IEC Resp FF SS SPI

Orchard (g) (g/cm3) % (1-8) (1-8) (l/l) ml/kg.h (kgf) (%) (0-7)
Atharvestmeans
HB1 281 0.891 85 6.0 5.0 5.1 12.0 10.3 15.0 4.3
HB2 261 0.883 77 5.9 4.4 3.9 12.0 9.8 13.9 4.4
HB3 250 0.903 77 5.8 4.4 8.1 12.8 10.9 15.1 4.7
HB4 255 0.890 77 6.1 4.6 3.5 10.9 10.7 14.6 3.9
HB5 251 0.897 81 5.8 4.8 6.0 11.6 11.1 14.2 3.7
HB6 249 0.902 82 5.8 4.6 2.2 10.7 11.4 13.8 3.3
HB7 273 0.892 85 5.8 4.2 2.3 10.7 10.4 13.3 3.5
HB8 215 0.907 81 5.8 4.4 0.6 11.4 10.8 13.1 2.6
NL1 234 0.904 79 6.0 4.3 0.6 7.4 9.9 12.9 3.8
NL2 243 0.899 78 6.0 4.8 3.3 12.2 10.7 15.3 4.7
NL3 251 0.886 84 5.9 4.3 3.9 10.5 11.3 14.3 4.1
NL4 250 0.893 89 5.9 4.7 5.3 11.2 11.9 14.7 3.9
NL5 264 0.894 85 5.7 4.5 2.5 11.1 11.1 14.6 3.9
NL6 254 0.904 79 6.0 4.4 5.7 12.5 10.4 14.3 4.4
NL7 249 0.886 77 5.9 3.8 2.0 3.0 10.0 13.7 3.6
NL8 253 0.900 86 6.1 3.8 1.2 11.8 11.1 13.5 2.6
1
Wgt = weight, Fgnd = foreground colour, Bgnd = background colour, IEC = internal ethylene
concentration, Resp = respiration rate, FF = flesh firmness, SS = soluble solids, SPI = starch pattern index

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Prediction of internal browning risk in Scilate

A CONFIDENTIAL report prepared for

Brookfield PL, Robertson J, Oliver M

Rowan D, McGhie T, Hunt M, Hedderley D

Seymour S, Friend A, Diack R

SPTS No. 7773

This report has been approved by:

2 Materials and methods 2

3 Results and discussion 4

4 Conclusions and recommendations 14

For further information please contact:

The following measurements were made using 27 fruit per orchard:

2.2.2 Ten days after harvest

The following measurements were made using 27 fruit per orchard:

2. IEC, SPI and metabolites were assessed as described above

2.2.3 After storage

Relationships between fruit physiological characteristics at harvest (Appendix I) and IB after

Table 1. Correlation coefficients of relationships between physiological characteristics at harvest and

3.2 Metabolite relationships

Figure 7. Standardised regression coefficients of relationships between compounds and their

In terms of risk management strategies for the 2013 season we recommend:

Continuation of the strategy of historical orchard incidence as a means for identifying

Brookfield PL, Robertson J, Johnston J, Farrell M. Hedderley D, Oliver M, Dayatilake D. 2011.

Franck C, Lammertyn J, Ho QT, Verboven P, Verlinden B, Nicolai BM.2007. Browning disorders

Hertog M, Rudell D, Pedreschi R, Schaffer R, Geeraerd A, Nicolai B, Ferguson I. 2011. Review.

Wgt Density Red Fgnd Bgnd IEC Resp FF SS SPI

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