from autooxidative processes that disrupt cell membranes and are brought about by depletion
of endogenous antioxidants such as ascorbic acid or a-tocopherol during low temperature storage
Meir and Bramlage, 1988. Free-radicals such as hydroxyl OH, hydroperoxyl HO
2
and super- oxide O
2 −
can attack and destroy lipid and protein macro molecular structures essential for
cellular function Anet, 1969; Halliwell and Gut- teridge, 1989; Stadtman, 1993 and subsequently
lead to scald symptom development. Extensive investigations in Australia Anet and Coggiola,
1974 and references cited therein indicated that scald results from the autoxidation of a volatile
sesquiterpene hydrocarbon a-farnesene 2,6,10- trimethyl-2,6,9,11-dodecatetraene in the fruit skin
Meigh and Filmer, 1969. Based on in vitro studies, oxidation of a-farnesene was hypothe-
sized to yield conjugated triene hydroperoxide free-radicals which injure the hypodermal cells
and give rise to the symptoms of scald Anet, 1969, 1972. However, Rowan et al. 1995 found
that in vivo only trace amounts of these were detectable; the major oxidation products that ac-
cumulated in apple peel were two 2,7,8,11-tetraen- 6-ol conjugated trienol isomers.
Scald development in ‘Granny Smith’, a culti- var very susceptible to scald, was correlated to the
oxidation of a-farnesene to conjugated triene hy- droperoxides Huelin and Coggiola, 1970; Filmer
and Meigh, 1971. Song and Beaudry 1996 found that 6-methyl-5-hepten-2-one MHO, a
volatile product of a-farnesene oxidation, caused a scald-like disorder when applied to peel tissue of
susceptible apple fruit. Scald can be induced by the conjugated triene CT products of a-far-
nesene oxidation, and the concentration and time of appearance of these products is related to the
severity of the disorder Anet, 1972. The scald inhibitor chemicals DPA and ethoxyquin are ef-
fective inhibitors of lipid peroxidation, and ethanol vapor which controls scald Scott et al.,
1995a; Wang and Dilley, 1996, 1997; Wang et al., 1997; Ghahramani and Scott, 1998a is a free-rad-
ical scavenger Halliwell and Gutteridge, 1989. Little et al. 1982 reported that scald of ‘Granny
Smith’ apples was largely controlled by an initial low O
2
stress followed by CA storage. Low O
2
could exert its action partly by increasing endoge- nous ethanol production Wang et al., 1997;
Ghahramani and Scott, 1998b. We have tested the concept that initial low O
2
stress prior to CA storage controls scald by inducing production of
ethanol and inhibiting a-farnesene synthesis and its oxidation to yield MHO. Initial low O
2
stress treatments were investigated to determine effec-
tive treatment regimens and to gain insight into the mechanism of scald control.
2. Materials and methods
2
.
1
. Initial low oxygen stress followed by CA storage
2
.
1
.
1
. Plant materials and ILOS treatments Preclimacteric ‘Law Rome’ apples were ob-
tained from the Michigan State University, Clarksville
Horticultural Experiment
Station MSU CHES and ‘Granny Smith’ apples were
obtained from a grower in southwest Michigan, about 2 weeks prior to the onset of autocatalytic
ethylene production. Forty to 60 fruit were placed in 20-l CA chambers in each of three replicates for
each cultivar and storage period evaluation. Two levels of initial low O
2
stress were employed: 0.5 and 0.25 O
2
for 2 weeks at 1°C. The control fruit did not receive low O
2
stress. The low O
2
stress atmospheres were generated with an air separator Permea™, Model 5, Permea, St Louis,
MO and distributed through a capillary flow board system for maintaining controlled atmo-
spheres in a flow-through system. After the low O
2
stress, the fruit were stored in dynamic slowly ventilated CA under 3 or 1.5 O
2
at 1°C, using a flow rate of 0.18 void volume exchanges per h.
Some fruit so treated were again subjected to the same low O
2
stress treatments after 2 months in CA. Other groups of fruit were stored in air or in
3 or 1.5 O
2
CA without prior low O
2
stress.
2
.
1
.
2
. Scald index measurement Examination of fruit for scald index was made
after 5 and 8 months of storage plus 1 week post-storage in air at 20°C. Scald data are ex-
pressed as a scald index based on the percentage
of fruit surface area affected, where no scald = 0, B
25 = 1, 25 – 50 = 2, and \ 50 = 3. The scald index was normalized to 100 by multiplying
the values by 1003.
2
.
1
.
3
. Analysis of 6olatiles To determine the effects of ILOS on ripening,
scald development, and production of a-farnesene and its oxidation product MHO, fruit were re-
moved from storage at appropriate intervals and transferred to 20°C in air. Solid phase microex-
tractiongas chromatographymass spectrometry SPMEGCMS was employed to identify and
quantify the volatiles including a-farnesene and MHO as described by Song et al. 1997. Five to
eight fruit from each treatment were enclosed in 4-l glass jars at 20°C for 1.5 h. The headspace was
sampled by SPME equilibrating for 4 min to absorb volatiles and analyzed as described previ-
ously. At the same time intervals, samples 14 mg of epicuticular wax were removed from the
‘Granny Smith’ fruit and placed in glass vials 2 ml for 3 h at 20°C to determine the amount of
a -farnesene and MHO partitioned in the epicutic-
ular wax
by SPMEGCMS
as described
previously.
2
.
2
. Continuous low O
2
stress ‘Granny Smith’ apples as used for the ILOS
studies were employed. Fruit were placed in 10-l glass desiccators at 1°C and ventilated with 0.13,
0.25, 0.4, 0.5, 0.75, 1.0, 1.25 or 1.5 O
2
or with air 20.7 O
2
. The low O
2
atmospheres were made and distributed as described above. The
ventilation rate through the chambers was 54 ml min
− 1
approximately 0.6 void volume exchanges per h. Volatiles analyses commenced 1 week after
the low O
2
levels were established and continued at weekly intervals throughout the 8-month stor-
age period. The volatiles profile of more than 60 compounds produced by intact fruit in a flow
through system at 1°C and while in various stor- age atmospheres was obtained. Fruit were exam-
ined for scald and other physiological disorders after removal from storage and again after 7 days
at 20°C.
2
.
3
. Initial low O
2
stress trials under commercial conditions
Apples were obtained in 1997 from six Michi- gan orchards. Fruit were stored in four 42 m
3
CA storage rooms at CHES. ‘Red Delicious’ strains
were ‘Red Chief’, ‘Starkrimson’, ‘NuRed’, ‘Ace’ and ‘Super Spur’. ‘Rome’ strains were ‘Law
Rome’ and ‘Galia Beauty’. ‘Winesap’ strains were ‘Stayman’ and ‘Turley’. All fruit were harvested
preclimacteric and loaded into the rooms between 6 and 13 October. Each of the four rooms con-
tained 126 bins about 0.63 m
3
per bin. The rooms were loaded with an identical stacking
pattern and sealed on 13 October and brought to 2.3 O
2
by 18 October. Dry lime was employed to scrub carbon dioxide. A nitrogen generator
Permea™ experimental prototype, 1987 was used for initial O
2
pull-down and as needed to maintain O
2
levels. CA room c 1 was maintained at 0.7 O
2
with 0.5 carbon dioxide throughout the storage period at 0.1°C. CA room c 2 was
maintained at 1.5 O
2
with 0.5 carbon dioxide throughout the storage period at 0.1°C. Fruit in
CA room c 3 had ILOS at 0.55 9 0.19 for 18 days from 20 October to 6 November and be-
tween 24 October and 4 November 12 days the O
2
level was 0.48 9 0.11. Fruit in CA room c 4 had ILOS at 0.61 9 0.21 for 18 days beginning
on 20 October and between 27 October and 7 November 12 days the O
2
level was 0.51 9 0.13. For CA rooms 3 and 4, the carbon dioxide
level was 0.5. At the end of the ILOS, the temperature was raised in CA rooms 3 and 4 from
0.1 to 2°C for 3 days then returned to 0.1°C and the O
2
level was raised to 1.5 for the duration of the storage period. The rooms were opened after
about 8 months in CA. Three 40-fruit samples were obtained from each lot and examined for
scald, internal disorders and flavor immediately after storage and again after a week at 20°C.
3. Results
