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Postharvest Biology and Technology 82 (2013) 51–58

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Postharvest Biology and Technology

jou rnal homepage: www.elsevier.com/locate/postharvbio

Impact of harvesting time and fruit ﬁrmness on the tolerance to fungal storage

diseases in an apple germplasm collection

a,∗ b a

Masoud Ahmadi-Afzadi , Ibrahim Tahir , Hilde Nybom

Balsgård, Department of Plant Breeding, Swedish University of Agricultural Sciences, Fjälkestadsvägen 459, SE-29194 Kristianstad, Sweden

Department of Plant Breeding, Swedish University of Agricultural Sciences, Box 101, SE-23053 Alnarp, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history: Blue mold and bitter rot, caused by Penicillium expansum and Colletotrichum gloeosporioides, respectively,

Received 16 October 2012

are two of the most devastating diseases during and after storage of apple. The present project was

Accepted 3 March 2013

conducted to evaluate the level of tolerance to these diseases in apple germplasm, and investigate possible

associations with other fruit characteristics such as harvest date, ﬁrmness at harvest, softening (loss

Keywords:

of ﬁrmness during storage) and sun-exposure. Apples were harvested at a maturation stage suitable

Malus × domestica

for storage, inoculated with spore suspensions of P. expansum (127 cultivars) or C. gloeosporioides (70

Blue mold

cultivars), and stored for 6 or 12 weeks for early- and late-maturing cultivars, respectively. Fruit ﬁrmness

Bitter rot

was measured after harvest and after storage, and the difference was used as a measure of fruit softening.

Fruit texture

PLS-DA Average lesion diameter varied signiﬁcantly among both early- and late-maturing cultivars. The amount

of damage caused by the two diseases was signiﬁcantly correlated across cultivars. Regression analyses

indicated that lesion diameter was positively affected by fruit softening and negatively affected by harvest

date and ﬁrmness at harvest. Impact of the independent variables was quantiﬁed with partial least squares

discriminant analysis; approximately 40% of the genetic variation could be explained by these variables

with harvest date being the most important. The effect of sun-exposure was analyzed on six bi-colored

cultivars but the results were not conclusive. Cultivars that showed relatively small symptoms in spite

of being early-maturing and/or only medium ﬁrm, may have other traits that are beneﬁcial for storage

and could therefore be especially useful in breeding programs.

1. Introduction after a few days at room temperature (Jones and Aldwinckle, 1990).

Presently, increasing concerns for environmental and health issues

Apple (Malus domestica Borkh.) is one of the economically limit the access to chemical compounds for efﬁcient plant pro-

most important fruit crops, and can have a strong impact on human tection. Moreover, prohibition of postharvest treatments in some

health due to its high accessibility, comparatively low price and countries has resulted in increasingly serious problems with stor-

high levels of antioxidant and phenolic compounds. Apples, in addi- age diseases (Jones and Aldwinckle, 1990; Gullino and Kuijpers,

tion to being processed into juice, sauce, slices, vinegar or cider, 1994; Janisiewicz and Korsten, 2002). These problems are even

are mainly consumed fresh or after storage (Folta and Gardiner, more pronounced in organic production; organically produced

2009). Presently, much of the commercially grown fruit is stored ‘Aroma’ fruit in Sweden suffered a 20-fold increase in fungal decay

for 4 months or longer before being sold, and very good storage compared to conventionally grown fruit (Jönssson et al., 2010).

potential is therefore an important requirement for apple cultivars Consequently, storage time must be substantially shortened for

(Ferguson and Boyd, 2002). organically grown fruit, resulting in lower availability for con-

Fungal diseases cause a wide array of postharvest damage on sumers and serious economic losses for growers.

fruit, thus restricting storage potential and constituting one of Blue mold caused by Penicillium expansum is one of the most

the main problems for commercial apple production. Attacks can common postharvest diseases of apple. This fungus produces

be initiated either during the growing season or at harvest and patulin, a carcinogenic mycotoxin which has attracted public con-

postharvest handling, but symptoms are seldom visible until after cern due to its potential impact on human health (Beretta et al.,

a certain storage period and, for some diseases, they appear only 2000; Pianzzola et al., 2004; Barreira et al., 2010). Bitter rot,

another important storage disease on fruit caused by Colletotrichum

gloeosporioides and Colletotrichum acutatum, is both a pre- and

∗ a postharvest disease, especially under hot and humid condi-

Corresponding author. Tel.: +46 445806; fax: +46 445830.

tions, and can result in 50–80% losses in apples harvested from

E-mail addresses: [email protected] (M. Ahmadi-Afzadi),

[email protected] (I. Tahir), [email protected] (H. Nybom). orchards without effective plant protectant spraying (Jones and

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52 M. Ahmadi-Afzadi et al. / Postharvest Biology and Technology 82 (2013) 51–58

Aldwinckle, 1990; Jurick et al., 2011). Concerns about posthar- 2.2. Analysis of level of resistance in apple germplasm

vest fungal diseases, in particular blue mold and bitter rot, have

increased signiﬁcantly during the last decades, possibly due to a On the day of harvest or the following day, fruit were washed

combination of global warming, increased organic production, pro- with distilled water in order to remove most of the naturally

hibition of several previously used fungicides and the increasing occurring fungi (as previously demonstrated by Tahir et al., 2009),

levels of resistance, e.g., P. expansum toward fungicides (Vinas˜ et al., wiped dry and then inoculated on opposite sides at a depth

5 −1

1991; Sholberg et al., 2005; Tahir and Jönsson, 2005; Weber, 2009). of 3 mm with 20 ␮L (1 × 10 conidia mL ) of P. expansum and

Unfortunately, most of the currently grown apple cultivars are C. gloeosporioides, respectively. The pathogens used were iso-

very sensitive to storage diseases. So far, no major genes provid- lated from naturally infected apples showing typical symptoms

ing resistance against the storage diseases have been identiﬁed but of the diseases, maintained on petri dishes with potato dex-

quantitatively inherited traits associated with chemical contents, trose agar augmented with penicillin G and streptomycin sulphate

−1

fruit texture, structure of the fruit epidermis and ripening behav- (each at 200 mg L agar), and stored separately as pure cul-

◦

ior may affect the ability of different cultivars to withstand fungal tures at 4 C. Pathogen virulence over time was conﬁrmed by

attacks (Prusky et al., 2004; Blazek et al., 2007; Nybom et al., 2008; periodic transfers through apples. Spores of the pathogens were

Johnston et al., 2009). Positive effects of increased ﬁrmness and removed from the surface of 10-day-old cultures and suspended

less softening due to calcium spraying, have been documented in a in 5 mL sterile distilled water containing 0.05% (v/v) Tween 80.

series of pre- and postharvest treatments of apple fruit, indicating The suspensions were ﬁltered, and spore concentrations were

5 −1

that fungal decay caused by P. expansum, Botrytis cinerea and Glom- adjusted to 1 10 conidia mL (Tahir et al., 2009; Weber and

erella cingulata can be signiﬁcantly reduced (Conway et al., 1991; Palm, 2010). Three replicates with 15 fruit in each were prepared

Sams et al., 1993; Conway et al., 2002). with each cultivar-fungus combination, as well as two controls

Variable levels of resistance to fungal decay have already been (fruit with no treatment, and wounded but not inoculated fruit,

reported in some studies. Thus, evaluation of the resistance to blue respectively).

mold in an apple germplasm collection from Kazakhstan, main- After inoculation, the fruit were stored in open-faced plastic

◦

tained in Geneva, NY, indicated the existence of greater genetic boxes at 2 C for either 6 weeks (2010: summer cultivars har-

diversity among the Kazak apples than among cultivated apples vested from August 10 to September 9; 2012: from August 9 to

(Janisiewicz et al., 2008). In another study, resistance to both blue September 7) or 12 weeks (2010: fall and winter cultivars har-

mold and bitter rot was identiﬁed in wild apple germplasm from vested from September 13 to October 26; 2012: from September

Kazakhstan (Jurick et al., 2011). 9 to October 9). Two alternatives for duration of storage were used

Genetic differences in tolerance to storage diseases have also since early-maturing cultivars could have deteriorated and become

been reported in some screenings of commercial cultivars (Spotts difﬁcult to evaluate if stored longer than 6 weeks whereas late

et al., 1999; Biggs and Miller, 2001) but data from large-scale cultivars might not have reached the climacteric stage if stored

screenings, especially of cultivars suitable for a cooler climate, are less than 12 weeks, thereby making it difﬁcult to observe dif-

lacking. The aims of this study were (1) to evaluate the level of tol- ferences in ﬁrmness and disease incidence. The chosen date for

erance to the postharvest diseases blue mold and bitter rot in an splitting the two groups reﬂects the division between ‘summer

apple germplasm collection, and (2) to investigate the association varieties’ which are consumed within a few weeks after harvest,

of fruit characteristics such as harvest date, ﬁrmness, softening and and ‘fall and winter varieties’ which are usually stored for several

skin color, with tolerance to postharvest diseases. months.

Lesion diameter (severity) was measured with a plastic ruler

on both inoculation sites of the infected apples at the end of the

2. Material and methods

storage period. Measurements were carried out immediately after

◦

storage for P. expansum, and after keeping the fruit at 20 C and 90%

2.1. Plant material

RH during 5 days for C. gloeosporioides.

This project comprised a total of 92 cultivars in 2010, with

2.3. Fruit ﬁrmness test

70 cultivars harvested in the apple germplasm orchard at Bals-

◦ ◦

gård in southern Sweden (N 56 6 23 , E 14 9 55 ) and 22 cultivars

Fruit ﬁrmness was measured with a penetrometer (model FT-

◦

harvested at the former research station in Kivik (N 55 40 57 ,

327, Efﬁgy, Alfonsine, Italy, plunger diameter 11.1 mm, depth

◦

E 14 13 18 ) about 60 km southwest of Balsgård (Figs. 1 and 2). In

7.9 mm), at opposite peeled sides and expressed as newtons.

2012, another experiment was carried out with 45 cultivars har-

Twenty fruit per cultivar were picked on the same day as harvest-

vested at Balsgård (Fig. 3), 10 of which were included also in the

ing for the inoculation tests. Firmness of 10 fruit was measured at

◦

2010 experiment (i.e. ‘Aroma’, ‘Discovery’, ‘Elise’, ‘Gloster’, ‘Graven-

harvest and the rest were stored in regular air (4 C and 90% RH) for

steiner’, ‘Ingrid Marie’, ‘Katja’, ‘Santana’, ‘Sylvia’ and ‘William’s

6 and 12 weeks of storage for early- and late-maturing cultivars,

Pride’). The orchards were fertilized, irrigated, pruned and sprayed

respectively. Fruit were removed after storage and left in room

as a commercial orchard, including 5–6 yearly administrations

temperature for 4 h before measuring the ﬁrmness. Average fruit

of standard fungicides [Candit (kresoxim-methyl), Delan WG

ﬁrmness at harvest and after storage, and fruit softening (differ-

(dithianon), Kumulus DF (sulphur), Scala (pyrimethanil), Signum

ence between ﬁrmness at harvest and ﬁrmness after storage) were

(boscalid and pyraclostrobin) and Topas 100EC (penconazole)] in

calculated for each cultivar.

spring and summer, with the last one at the end of June or in the

beginning of August.

2.4. Analysis of effects of sun-exposure

The iodine starch test (Smith et al., 1979) was applied up to 5

times for each cultivar in order to determine fruit maturation stage

In 2011, six cultivars (‘Delorina’, ‘Ingold’, ‘Jonathan’, ‘Red Baron’,

and select a harvest date suitable for long-term storage (starch con-

‘Winter Banana’ and ‘Winter Banana Spur’) with a pronounced

version value 4–5 on a 9-point scale, i.e. a pre-climacteric stage).

difference in color between the sun-exposed and the shaded sides

Harvest date was calculated by denoting the ﬁrst day of harvest

of the fruit, were harvested at Balsgård for analysis of possible

(August 10 in 2010, and August 9 in 2012) as ‘1 and then adding

effects on fungal tolerance due to sun-exposure. Freshly harvested

number of days to harvest for each cultivar.

fruit of each cultivar were inoculated on opposite sides with

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M. Ahmadi-Afzadi et al. / Postharvest Biology and Technology 82 (2013) 51–58 53

Mutsu Reglindis*

Prima*

Golden Delici ous Delor ina Redfree* Santana Jonathan

Gloster

Lobo*

Zarya Alatau Pinova Boskoop Sawa*

Rena* Gravensteiner gul* Queen Cox

Signe Tillisch*

Cox's Oran ge Pippin Gravensteiner röd* Cox's Pomona Boiken Kim Spässerud* Rubinola

Eir*

Laxton`s Su perb

Ingr id Marie

Summerred* Rödluva n* Cortland Dayton Agra* Samo* Ribston Zhigulevskoe Rajka

Goldrush

Dronnin g Louise James Gr ieve*

Karin Schneider

William´s Pride*

Hon eycrisp Charlamo vsky* Jaspi* Risäter* Nanna*

Idunn* Cultivars Holstein er Cox Frida Katja* Alice* Witos* Sultanat Quinte* Trogsta* Elise Prinsessäpple Richelieu

Kanka* Wor cesterpearmain

Julyred*

Mio*

Anana s Reinee Borgherre Heta* Guldborg* Aroma Amorosa Discovery* Joseﬁner* Siv*

Stenkyrke Vista Bella*

Mantet* Transparente Blanche* Filippa Fredrik Maglemer McIntosh Linda* K:1160 Stäringe Karin*

Ella Sävsta holm* Visjö* Sylvia* Eva-Loa Röd Astrakan*

Gyllenkroks Astrakan*

Close*

0 1 2 3 4 5 6 7 8 9 10

Lesion diameter.we ek-1 (mm)

Fig. 1. Average lesion diameter of fruits inoculated with P. expansum in 2010; symptoms for early- and late-maturing cultivars. Early cultivars are marked with “*” (mean ± SD,

n = 15).

P. expansum spores as described above. Three replicates with 10 and year separately, using average lesion diameter for the dif-

fruit in each were prepared, as well as three control replicates ferent cultivars as dependent variable and harvest date, ﬁrmness

(without any inoculation). Lesion diameter was evaluated after 6 and softening as independent variables. Data for the effects of

◦

weeks of storage at 4 C. sun-exposure were analyzed with paired t-tests (˛ = 0.05) for each

cultivar separately. All of the above analyses were conducted using

Minitab ver. 16 (Minitab Inc., State College, PA, USA).

2.5. Statistical analyses

Partial least squares discriminant analysis (PLS-DA) was applied

to investigate the ability of harvest date, ﬁrmness and soften-

Data for the fruit inoculations, i.e. average lesion diameter,

ing (the independent variables) to predict the response variable,

were subjected to one-way analysis of variance (ANOVA) for each

i.e. lesion diameter for each fungus separately. In this method, a

fungus separately to investigate inter-cultivar variation with the

multiple linear regression model is ﬁtted by projecting the inde-

general linear model (GLM). A possible co-occurrence of the two

pendent variables and the dependent response variable to a new

different fungal diseases across cultivars was investigated with a

variable space as deﬁned by the latent variables, maximizing the

Pearson correlation test. Association between 2010 and 2012 data

covariance between the dependent and independent variables.

for P. expansum inoculations was investigated with a Spearman

This model deﬁnes the multidimensional direction in the genotype

rank correlation test for 9 out of the 10 cultivars that had been

space that explains the maximum variance in the response variable.

inoculated in both years, excluding ‘Aroma’ which had been stored

PLS-DA was applied using The Unscrambler X.1 (CAMO Software,

as a late-maturing cultivar in 2010 but as an early-maturing in

2011).

2012. A set of regression analyses were conducted for each fungus

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54 M. Ahmadi-Afzadi et al. / Postharvest Biology and Technology 82 (2013) 51–58

Mutsu Stenkyrke

Reglindis* Redfre e*

Prima* Cox Oran ge Boskoop Pinova

Queen Cox

Rebel la

Ingr id Marie

Jonathan William s Pride*

Kanka* Laxton`s Su perb Kim Lobo* Elise

Boiken Zarya Al tau Idunn*

McIntosh

Cox Pomona

Hon eycrisp Ella Holsteiner Cox

Rena* Rödluva n* Prinsessäpple Guldborg*

Aroma Gravensteiner gul* Rajka Siv*

Spässerud* James Gr ieve*

Cultivars Fredrik Linda* Summerred* Dayton Heta*

Sävstaholm* Gravensteiner röd* Gloster

Filippa Karin Schn eider

K:1160 Wor cesterparmän Katja*

Sultanat

Borgherre

Anana s Reinee Goldrush Frida Quinte* Agra* Jaspi* Charlamovsky* Risäter* Alice* Trogsta* Mio* Samo*

Visjö* Stäringe Kar in* Discovery* Nanna*

Joseﬁner*

Transparente Blanche*

Eir*

0 2 4 6 8 10

-1

Lesion diameter.week (mm)

Fig. 2. Average lesion diameter of fruits inoculated with C. gloeosporioides; symptoms for early- and late-maturing cultivars. Early cultivars are marked with “*” (mean ± SD,

n = 15).

3. Results respectively, whereas it was 67.4 N and 68.5 N after storage,

respectively. A loss of 17% and 30% of the ﬁrmness at harvest was

3.1. Fruit ﬁrmness and softening thus observed for early- and late-maturing cultivars, respectively.

In 2010, mean ﬁrmness at harvest was 76.1 N for the early- 3.2. Storage disease tolerance

maturing cultivars and 88.1 N for the late-maturing, respectively,

while mean ﬁrmness after storage was 51.6 N and 47.1 N, respec- No infection was observed in either year in any of the control

tively. Mean fruit softening was 24.5 N (i.e. a loss of 32% of the fruit, i.e. fruit which had not received any treatment, and fruit that

ﬁrmness at harvest) for the early harvested cultivars that had had been wounded but not inoculated. Obviously the fungicide

been stored for 6 weeks, and 41.0 N (a loss of 47%) for the late sprayings in the orchards and the washing of harvested fruit in

harvested cultivars that had been stored for 12 weeks. Two culti- water had been sufﬁcient to protect against spontaneously occur-

vars ‘Antonovka Kamenitschka’ and ‘Antonovka Pamtorutka’, with ring decay, at least during the relatively short storage time used in

relatively high ﬁrmness at harvest (95.1 and 92.2 N, respectively), the present study.

had increased their ﬁrmness after 6 weeks of storage; 107.9 N and Inoculation with P. expansum, overall, produced easily inter-

101.0 N. These cultivars, however, had a rubbery fruit ﬂesh texture preted infections. By contrast, some failures were observed in fruit

after storage and would probably have yielded lower values with inoculated with C. gloeosporioides and therefore, only 70 cultivars

different technical equipment. In 2012, mean ﬁrmness at harvest were used for the statistical analyses. In addition, some secondary

was 81.4 N and 96.4 N for early- and late-maturing cultivars, infections with P. expansum were observed toward the end of

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M. Ahmadi-Afzadi et al. / Postharvest Biology and Technology 82 (2013) 51–58 55

Olga Kolinowskaya

Tönnes Severni j Sinap

Bersis

Pepin Scha franovij*

Ausma Birgit Bonni er*

Ontario

Nyck elby Björk a* Aroma* Katja* Arona Jedzenu Huvitus* Sandra*

Breaky*

Sörmlandsäppl e*

Gravenste iner* Gloster

Rörvik Slava Pet ersburg Vara

Cultivars Sinap Orlovski Kavlås*

Ingrid Marie

Barcha tnoje* Elise

Atlas

Göteborgs Flickäpple*

RigasRozab ele*

Kestr el William s Pride* Santana Luke

Raja Sylv ia* Alka Apelsinoe* Oranie*

Sariola Discov ery* Konsta

Juuso*

0 1 2 3 4 5 6 7

-1

Lesion size.week (mm)

Fig. 3. Average lesion diameter of fruits inoculated with P. expansum in 2012; symptoms for early- and late-maturing cultivars. Early cultivars are marked with “*” (mean ± SD,

n = 15).

the storage period for some of the late-maturing Colletotrichum- and 58.1 mm). Among the late-maturing cultivars, ‘Mutsu’ and

inoculated cultivars, especially ‘Gloster’ and ‘Kim’, thus making ‘Stenkyrke’ had the smallest lesions (21.4 and 24.4 mm) while ‘Gol-

proper estimations of lesion diameter very difﬁcult. drush’ and ‘Frida’ had the largest (73.8 and 79.1 mm). For this

In 2010, early-maturing cultivars inoculated with P. expansum, fungus, the range between the most tolerant and the most suscepti-

developed lesions that ranged from very small in ‘Reglindis’ and ble cultivars was smaller than for P. expansum (Fig. 2). Nevertheless,

‘Prima’ (average diameter 8.0 and 8.7 mm, respectively) to seven signiﬁcant variation was found among cultivars (P < 0.001 for both

times higher values in ‘Gyllenkroks Astrakan’ and ‘Close’ (55.2 early- and late-maturing cultivars).

and 56.3 mm, respectively). A similar range but overall somewhat The ability to tolerate one fungus seems to be extended to tol-

higher values were encountered in the late-maturing apples that erance of the other fungus. A positive correlation was thus found

had been stored for 12 weeks; with 14.0 and 19.4 mm, respectively, between Penicillium and Colletotrichum lesion diameter when cal-

in ‘Mutsu’ and ‘Golden Delicious’, and 88.4 and 98.3 mm in ‘Ella’ and culated across cultivars with data from 2010 for both inoculations;

‘Eva-Lotta’ (Fig. 1). Values for lesion diameter were less variable r = 0.622, P < 0.001 and r = 0.482, P = 0.005 for early- and late-

in 2012; ranging from 11.1 mm and 11.9 mm in ‘Pepin Schafra- maturing cultivars, respectively.

novij’ and ‘Birgit Bonnier’ respectively, to 34.2 mm and 37.2 mm

in ‘Discovery’ and ‘Juuso’, respectively, in early-maturing cultivars. 3.3. Association between disease tolerance, and harvest date and

In late-maturing cultivars, ‘Olga’ and ‘Kolinowskaya’ had the low- fruit texture

est values, 18.9 mm and 18.5 mm, while ‘Sariola’ and ‘Konsta’ had

the largest, 66.6 and 69.2 mm (Fig. 3). To facilitate comparisons In 2010, regression analyses using P. expansum lesion diameter

between early- and late-maturing cultivars, lesion diameter per as dependent variable and harvest date as independent vari-

week of storage was then calculated. In both years, analysis of variable, indicated that the tolerance to P. expansum increases with

ance demonstrated signiﬁcant variation among cultivars (P < 0.001 later harvest date in both early-maturing (R = 0.40, P < 0.001) and

for both early- and late-maturing cultivars). Data for 2010 and 2012 late-maturing (R = 0.21, P = 0.002) cultivars (Table 1). In 2012, P.

were signiﬁcantly correlated, r = 0.417, P = 0.02, suggesting that the expansum lesion diameter showed a signiﬁcant increase with har-

order of cultivars from least to most susceptible has a genetic com- vest date in late-maturing cultivars (R = 0.27, P = 0.007), but not

ponent. in early-maturing (Table 2). For C. gloeosporioides, harvest date

Among early-maturing cultivars inoculated with C. gloeospori- showed a negative, signiﬁcant effect on lesion diameter in the

oides, ‘Reglindis’ and ‘Redfree’ had the smallest lesions (12.5 and early-maturing cultivars (R = 0.24, P = 0.002) but not in the late-

15.3 mm) and ‘Transparente Blanche’ and ‘Eir’ the largest (56.9 maturing (Table 1).

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56 M. Ahmadi-Afzadi et al. / Postharvest Biology and Technology 82 (2013) 51–58

Table 1

Association of harvest date, fruit ﬁrmness, softening and level of fruit tolerance to P. expansum and C. gloeosporioides decay in early and late cultivars, in 2010.

P. expansum C. gloeosporioides

Early-ripening Late-ripening Early-ripening Late-ripening

2 2 2 2

Equation R Equation R Equation R Equation R

Firmness y = −0.46x + 8.5 0.05 y = −0.37 + 7.6 0.09 y = −0.36x + 8.9 0.04 y = −0.18x + 6.3 0.05

Softening y = 0.62 + 3.4 0.07 y = 0.46 + 2.4 0.16 y = 0.73x + 4.3 0.11 y = −0.009x + 4.7 0.0001

Harvest time y = −0.13x + 6.9 0.39 y = −0.13x + 10.5 0.21 y = −0.11x + 7.9 0.24 y = 0.006x + 4.3 0.003

Table 2

PLS-DA was calculated separately for each fungus on the 2010

Association of harvest date, fruit ﬁrmness, softening and level of fruit tolerance to

data, using lesion diameter divided by number of weeks in storage

P. expansum in early and late cultivars, in 2012.

as dependent variable, and harvest date, fruit ﬁrmness at har-

P. expansum

vest and fruit softening as independent variables. Since variation

Early-ripening Late-ripening due to the storage period could not be completely neutralized,

storage time (6 or 12 weeks) was included as an additional indepen- 2 2

Equation R Equation R

dent variable. For P. expansum, the explained variance reached 43%

Firmness y = −0.46x + 7.3 0.14 y = −0.33x + 6.3 0.15

of the observed variation in lesion size. The weighted regression

Softening y = −0.29x + 3.9 0.04 y = 0.43x + 1.9 0.17

coefﬁcients indicate the importance of the different independent

−

Harvest time y = 0.03x + 4.1 0.04 y = −0.06x + 6.03 0.27

variables in explaining lesion size variation; thus a strong positive

effect of softening and a still positive but weaker effect of storage

In 2010, regression analyses with P. expansum lesion diam- time was noted. Additionally, a strong negative effect of harvest

eter as dependent variable and fruit ﬁrmness at harvest as date and an almost similarly strong effect of ﬁrmness at harvest

independent variable, demonstrated a negative, signiﬁcant effect was found. These results indicate that later ripening cultivars with

in late-maturing cultivars (R = 0.09, P = 0.04) but not in early- initially ﬁrm fruit and relatively little softening have the highest

maturing. As expected, fruit softening instead had a positive effect tolerance to P. expansum (Fig. 4A).

on lesion diameter, which was again signiﬁcant in late-maturing For C. gloeosporioides, the explained variance reached 26% of

cultivars (R = 0.16, P = 0.006) but not in early-maturing. Data from the observed variation in lesion diameter when all 70 cultivars for

2012 produced similar regression equations as the 2010 Penicil- which we had data were analyzed. Harvest date and ﬁrmness at har-

lium data for both early- and late maturing cultivars (Table 2). vest were negatively associated with lesion diameter as expected,

Thus, lesion diameter decreased signiﬁcantly with higher ﬁrm- but also softening and storage time showed negative effects. Possi-

ness values (R = 0.15, P = 0.04) in late-maturing cultivars but not bly, secondary infections with P. expansum had distorted the results

in early-maturing. Similarly, softening had a positive effect on the for some of the late-maturing cultivars as suggested also by the

lesion diameter in late-maturing cultivars (R = 0.17, P = 0.04), but absence of signiﬁcant effects in the regression analyses (Table 1).

not in early-maturing. To conﬁrm this hypothesis, PLS-DA was conducted separately for

For C. gloeosporioides, fruit ﬁrmness at harvest had a nega- early- and late-maturing cultivars. Results indicated that a total

tive, but not signiﬁcant effect on lesion size for both early and of 41% of the variance is explained in the analysis for early har-

late-maturing cultivars. The positive effect of fruit softening was vested cultivars, with a positive association between lesion size and

signiﬁcant in early-maturing cultivars (R = 0.11, P = 0.049) but not softening while a negative association was found with ﬁrmness at

in late-maturing (Table 1). harvest and harvest date (Fig. 4B).

Fig. 4. Weighted regression coefﬁcients indicating the importance of independent variables (ﬁrmness at harvest, softening per week, maturation and storage time) in

explaining the dependent (lesion size) variation for (A) P. expansum and (B) C. gloeosporioides (only early-maturing cultivars).

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M. Ahmadi-Afzadi et al. / Postharvest Biology and Technology 82 (2013) 51–58 57

3.4. Effect of sun-exposure on disease tolerance was positively correlated with ﬁrmness at harvest and negatively

correlated with fruit softening rate but a PLS-DA revealed that ﬁrm-

Somewhat contradictory results were obtained concerning the ness at harvest was positively correlated with softening (ﬁrm fruit

effects of exposure versus shading of the fruit on lesion size after softens faster while already soft fruit continues to soften less fast).

inoculation with P. expansum. For three cultivars (‘Red Baron’, ‘Win- However, cultivars with a high tolerance to storage diseases thus

ter Banana’ and ‘Winter Banana Spur’), the t-tests did not ﬁnd appear to have not only above-average ﬁrmness at harvest but also

any signiﬁcant differences in the size of lesions on exposed and less softening during storage. The positive effect of higher ﬁrmness

shaded sides, respectively, on the same fruit. For ‘Delorina’, the (due to calcium inﬁltration) on fungal decay has already been well

sun-exposed side showed signiﬁcantly higher tolerance to P. expan- studied, indicating that improvement of fruit ﬁrmness can improve

sum (t = 6.04, P < 0.001), whereas the opposite was found in two storability in several ways (Conway et al., 1991; Sams et al., 1993).

other cultivars; ‘Ingold’ (t = 2.79, P = 0.009), and ‘Jonathan’ (t = 2.28, Similarly, a negative correlation was found between ﬁrmness

P = 0.03). and bitter rot severity in ﬁve apple cultivars where comparatively

more ripe (softer) fruit had larger lesions (Shi et al., 1995). The abil-

4. Discussion ity to tolerate the fungal attack is likely to be strongly inﬂuenced

by the fruit ripening stage and texture; a late harvest resulted

4.1. Tolerance to storage diseases in substantially higher fungal incidence compared to an earlier

harvest of the same cultivar (Bass and Birchler, 2012). By contrast,

Identiﬁcation of resistant or at least comparatively tolerant cul- severity of bitter rot after inoculation of wounded or non-wounded

tivars, and utilization of these in plant breeding programs, could fruit of 18 cultivars grown in USA, showed no associations with

improve postharvest quality and grower economy considerably. harvest date, ﬁrmness at harvest or content of soluble solids (Biggs

Assessment of relative levels of resistance must, however, be car- and Miller, 2001).

ried out on fruit in a similar physiological stage. A number of more Ethylene production has been described as one of the main fac-

or less labor-intensive methods for determination of maturation tors inﬂuencing fruit maturation and softening (Johnston et al.,

stage and storage potential in apple have been used, including 2009). Fruit of early-season cultivars apparently ripen quickly due

the quick and simple starch test (Fallik et al., 2003; Jemric et al., to a high climacteric respiration and higher levels of ethylene pro-

2006; Lafer, 2006; Villatoro et al., 2009; Llorente et al., 2012). In duction (Janisiewicz et al., 2008). Changes in ethylene production

a previous study of storage potential in Swedish apple cultivars, during ripening were shown to be highly correlated to maturation

correlations between starch value and internal ethylene content and harvest date in ‘Golden Delicious’ (Song and Bangerth, 1996).

(IEC) were signiﬁcant and of the same magnitude as the correla- Several ethylene production genes have been studied in apple like

tion between ﬁrmness and IEC, whereas both SSC and Streif index Md-ACS1 (1-aminocyclopropane-1-carboxylate synthase) and Md-

was less strongly correlated with IEC (Tahir and Nybom, 2013). ACO1 (1-aminocyclopropane-1- carboxylate oxidase). These genes

These results indicate that the starch test can be a valid replace- have been proposed to affect fruit ﬁrmness at harvest and/or fruit

ment for the more accurate but also more elaborate IEC test. Since softening during ripening (Costa et al., 2005, 2010; Zhu and Barritt,

all of our cultivars required repeated measurements to pinpoint the 2008). Fruit softening is also affected by cell wall degradation

desirable maturation stage, the quick starch test was used. To fur- which is more active in rapidly softening cultivars (Wakasa et al.,

ther minimize experimental artifacts in our study, fruit were picked 2006; Wei et al., 2010). Two genes that have been proposed to

when the starch value was quite low, i.e. 4–5, indicating an early have substantial effect on fruit softening in apple are Md-Exp7 (an

physiological and most likely pre-climacteric stage. In a previous expansin homologue) and Md-PG1 (endopolygalacturonase). In a

study of 8 Swedish-grown cultivars, a signiﬁcant increase in IEC large screening of 4 genes (Md-ACS1, Md-ACO1, Md-PG1 and Md-

−1 −1 −1 −1 −1 −1

␮ ␮

(from <1 L L kg h to >3 L L .kg h ) was not encoun- EXP7), alleles previously described as responsible for good texture

tered until the third or fourth harvesting week when starch values were associated with signiﬁcantly lower softening for Md-ACS1 and

had already passed 5 (Tahir and Nybom, 2013). Thus, starch val- Md-PG1, but the opposite was noted for Md-EXP7 while results were

ues of 4–5 are probably indicative of low and steady IEC values. In non-signiﬁcant for Md-ACO1 (Nybom et al., 2013). However, when

addition, fungal decay did not vary much between harvesting dates the predictive power was calculated with a partial least squares

when early to medium-early picked fruit of the same cultivar was discriminant analysis, these 4 genes accounted for only 15% of the

compared (Tahir and Nybom, 2013). Similarly, natural fungal decay observed variation in ﬁrmness at harvest and 18% of the variation in

remained stable from 2 weeks before optimal picking date (OPD) softening rate, suggesting that several unidentiﬁed genes are also

to OPD (Lafer, 2006). involved. Some of these unknown genes may also affect the ability

In the present study, signiﬁcant inter-cultivar variation was to tolerate storage diseases.

found in the amount of damage caused by two of the most impor-

tant storage diseases, blue mold and bitter rot, when evaluated in a 4.3. Additional factors and conclusions

total of 127 and 70 cultivars, respectively. The positive correlation

for blue mold lesion diameter in 9 control cultivars, i.e. inoculated In addition to ﬁrmness and softening, associations with skin

in both 2010 and 2012, indicates that there is a genetic component color, cuticular waxes and chemical contents have been investi-

to the estimated variability in disease susceptibility. gated as explanations for variation in resistance to fungal storage

diseases in apple but results have, on the whole been very incon-

4.2. Importance of harvest date, fruit ﬁrmness and softening clusive (Spotts et al., 1999; Biggs and Miller, 2001; Blazek et al.,

2007; Jurick et al., 2011). However, cultivars with comparatively

Harvest date had a pronounced inﬂuence on lesion diameter for high contents of anthocyanin and ascorbic acid showed less dam-

both diseases according to the regression analyses and the PLS- age to the fungal disease bull’s eye rot caused by Neofabraea spp.

DA. Fruit ﬁrmness at harvest and softening, i.e. the loss of ﬁrmness (Tahir and Gustavsson, 2009)

during storage, likewise proved to be closely associated with lesion A positive correlation was found between lesion diameters

diameter for both fungi in our study. Thus, cultivars with a higher caused by the two different fungi when calculated across cultivars

ﬁrmness at harvest and less softening are more tolerant. In a pre- in each of the two groups (early- and late-maturing, respectively)

vious study (Nybom et al., 2013), data on harvest date, ﬁrmness at in our study. In other reports, overlap in resistance has, however,

harvest and softening were collected for 127 cultivars; harvest date been very limited when these two fungi have been investigated

Author's personal copy

58 M. Ahmadi-Afzadi et al. / Postharvest Biology and Technology 82 (2013) 51–58

in multiple apple genotypes simultaneously (Spotts et al., 1999; Janisiewicz, W.J., Saftner, R.A., Conway, W.S., Forsline, P.L., 2008. Preliminary eval-

uation of apple germplasm from Kazakhstan for resistance to postharvest blue

Jurick et al., 2011), possibly due to the very different virulence

mold in fruit caused by Penicillium expansum. HortScience 43, 420–426.

mechanisms (Prusky et al., 2001, 2004). In cases where certain

Jemric, T., Lurie, S., Dumija, L., Pavicic, N., Hribar, J., 2006. Heat treatment and harvest

genotypes have shown similarity in levels of tolerance/resistance, date interact in their effect on superﬁcial scald of ‘Granny Smith’ apple. Sci.

Hortic.-Amsterdam 107, 155–163.

suggested explanations have involved an increased accumulation

Johnston, J.W., Gunaseelan, K., Pidakala, P., Wang, M., Schaffer, R.J., 2009. Co-

of compounds produced by the plant defence system (Jurick et al.,

ordination of early and late ripening events in apples is regulated through

2011) or the occurrence of protective characteristics in the fruit differential sensitivities to ethylene. J. Exp. Bot. 60, 2689–2699.

Jones, A.L., Aldwinckle, H.S., 1990. Compendium of apple and pear diseases. APS

epidermis and cortex tissue (Spotts et al., 1999).

Press, St. Paul, MN.

The photosynthetic capacity has been reported to be higher on

Jönssson, A., Nybom, H., Rumpunen, K., 2010. Fungal disease and fruit quality in an

the sun-exposed side than on the shaded side of an apple fruit (Chen apple orchard converted from integrated production to organic production. J.

and Cheng, 2007). Meanwhile, the wax is likely to be thicker on Sustain. Agr. 34, 15–37.

Jurick, W.M., Janisiewicz, W.J., Saftner, R.A., Vico, I., Gaskins, V.L., Park, E., Forsline,

the sun-exposed side in comparison with the shaded side, pos-

P.L., Fazio, G., Conway, W.S., 2011. Identiﬁcation of wild apple germplasm (Malus

sibly providing higher physical protection and improved defense

spp.) accessions with resistance to the postharvest decay pathogens Penicillium

against pathogens (Tahir et al., 2009). Since this could have effects expansum and Colletotrichum acutatum. Plant Breed 130, 481–486.

Lafer, G., 2006. Storability and fruit quality of ‘Golden Delicious’ as affected by har-

also on disease resistance, we analyzed ﬁve cultivars for possible

vest date, avg and 1-mcp treatments. J. Fruit Ornam. Plant Res. 14, 2.

differences in lesion diameter on the red (sun-exposed) and yellow

Llorente, D.D., Abrodo, P.A., González-Álvarez, J., de la Fuente, E.D., Alonso, J.J.M.,

(shaded) side, respectively. Results were inconclusive and further Álvarez, M.D.G., Gomis, D.B., 2012. A new analytical method to measure volatile

compounds in cider apples: application to evaluate the starch index. Food Bio-

studies are needed to conﬁrm to which extent fungal tolerance is

process Technol., http://dx.doi.org/10.1007/s11947-012-0879-5.

affected by intra-fruit differences in photosynthesis or other factors

Nybom, H., Ahmadi-Afzadi, M., Sehic, J., Hertog, M., 2013. DNA marker-assisted eval-

like fruit epidermis anatomy and physiology. uation of fruit ﬁrmness at harvest and post-harvest fruit softening in a diverse

apple germplasm. Tree Genet. Genom. 9, 279–290.

Present data on effects of harvest date, fruit ﬁrmness and soften-

Nybom, H., Sehic, J., Garkava-Gustavsson, L., 2008. Modern apple breeding is asso-

ing, however, suggest that the severity of these two diseases have

ciated with a signiﬁcant change in the allelic ratio of the ethylene production

a similar dependence on factors involved in fruit ripening and ﬂesh gene Md-ACS1. J. Hortic. Sci. Biotechnol. 83, 673–677.

Pianzzola, M.J., Moscatelli, M., Vero, S., 2004. Characterization of Penicillium isolates

texture. More research is thus needed on genetic inter-cultivar vari-

associated with blue mold on apple in Uruguay. Plant Dis. 88, 23–28.

ation in these respects, and in orchard management and storage

Prusky, D., McEvoy, J.L., Leverentz, B., Conway, W.S., 2001. Local modulation of

methods that can improve fruit texture and minimize softening. host pH by Colletotrichum species as a mechanism to increase virulence. Mol.

Plant–Microbe Interact. 14, 1105–1113.

Acknowledgement Prusky, D., McEvoy, J.L., Saftner, R., Conway, W.S., Jones, R., 2004. Relationship

between host acidiﬁcation and virulence of Penicillium spp. on apple and citrus

fruit. Phytopathology 94, 44–51.

This research was supported by grants to Hilde Nybom from the Sams, C.E., Conway, W.S., Abbott, J.A., Lewis, R.J., Benshalom, N., 1993. Firmness

and decay of apples following postharvest pressure inﬁltration of calcium and

Swedish Research Council for Environment, Agricultural Sciences

heat-treatment. J. Amer. Soc. Hortic. Sci. 118, 623–627.

and Spatial Planning, and from the Nordic Council of Ministers (Pub-

Shi, Y., Rom, C., Correll, J., 1995. Effect of fruit maturity on bitter rot of apple.

lic Private Partnership for Pre-breeding). The authors are grateful to HortScience 30, 762–762.

Sholberg, P.L., Harlton, C., Haag, P., Levesque, C.A., O’Gorman, D., Seifert, K., 2005.

Anna Zborowska, Jasna Sehic, Charlotte Håhus and Linnea Luthman

Benzimidazole and diphenylamine sensitivity and identity of Penicillium spp.

for technical assistance.

that cause postharvest blue mold of apples using beta-tubulin gene sequences.

Postharvest Biol. Technol. 36, 41–49.

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