Effect of Plum pox virus on Chemical Composition and Fruit Quality of Plum

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Article

The Effect of Plum pox virus on ChemicalComposition and Fruit Quality of Plum

Valentina Usenik, Damijana Kastelec, Franci Stampar, and Mojca Virscek MarnJ. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505330t • Publication Date (Web): 15 Dec 2014

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The Effect of Plum pox virus on Chemical Composition and Fruit Quality of Plum

Valentina Usenik,*† Damijana Kastelec,‡ Franci Stampar,† and Mojca Virscek Marn◊

†University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Chair for Fruit, Wine and

Vegetable Growing, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

‡University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Chair for Genetics,

Biotechnology, Statistics and Plant Breeding, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

◊Agricultural Institute of Slovenia, Plant Protection Department, Hacquetova 17, SI-1000 Ljubljana,

Slovenia

*Corresponding author, Tel: +386 1 320 31 43; Fax: +386 1 423 10 88; E-mail:

[email protected]

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ABSTRACT: The quality of fruit from PPV (Plum pox virus)-infected plum (Prunus domestica L.) 1

trees, was investigated during the last 3 weeks of ripening using healthy tree (T3), tree with short-2

term infection (T2) and tree with long-term infection (T1). The pomological variables (presence of 3

necrosis on fruit, colour of fruit, colour of flesh, firmness, soluble solids content, fruit weight), 4

composition of nutritive compounds (sugars, organic acids) and bioactive compounds (phenolics: 5

anthocyanins, flavonols and hydroxycinnamic acids) were evaluated. The results indicated that the 6

PPV infection modified the ripening process (the most in T1) and the composition of nutritive and 7

bioactive compounds in the healthy-looking part of the fruit. Long-term infected tree (T1) yielded 8

fruit with the poorest pomological traits and with the most modified composition of nutritive and 9

phenolic compounds. The short-term PPV infected tree also produced fruit with significantly 10

altered phytochemicals composition, although the pomological traits had not changed significantly. 11

12

KEYWORDS: Plum pox virus, Prunus domestica, pomological characteristics, phytochemicals, 13

nutritive compounds, phenolics, composition data analysis 14

15

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INTRODUCTION 16

Sharka (plum pox), caused by the Plum pox virus (PPV), is the most serious viral disease 17

threatening the stone fruit industry, particularly because it causes severe losses in susceptible 18

cultivars and is spread efficiently by aphids.1 Over an approximately 80-year period since the 19

description of the sharka disease, PPV has been thoroughly studied at various stages of the infection 20

cycle.2 On unripe fruit, shallow depressions of darker colour appear. During ripening, these develop 21

into deep rings and irregular lines, which cause deformation of the fruit. The epidermal tissue and 22

underlying cells become necrotic, and the flesh turns brownish red. The necrotic tissue often 23

extends down to the seed. These fruit are tasteless and fibrous.3 The diseased fruit contain more 24

organic acids, but less sugar and, especially, less sucrose. The fruit of infected susceptible varieties 25

are also smaller in size.4 26

The influence of viruses on trees depends on the pathogen and its strain, as well as the fruit 27

species, cultivar, rootstock, nutrition supply, time and mode of infection, vector species and the age 28

of the tree when infected.5 Trees newly infected with PPV show minor symptoms, usually only on 29

one part of the tree, but with passing years infection of the tree increases.6 The 'Požegača' cultivar is 30

known to be very susceptible to PPV. This cultivar is widely distributed in Europe and has many 31

synonyms, like ‘Vânăt Românesc’, ‘Vinete Româneşti’ or ‘Bistriţene’ in Romania; ‘Kiustendilska’ 32

in Bulgaria; ‘Vengerka’ in Moldova; ‘Quetsche’ in France; ‘Hauszwetsche’ in Germany’; 33

‘Madjarica’ in Croatia; ‘Domača češplja’ in Slovenia etc.7 Severe premature fruit drop (from 80 to 34

100%) has been reported for this cultivar. The dropping of the fruit begins 30-40 days before 35

ripening.4 36

A number of physiological, biochemical, and structural changes occur during the ripening of 37

plum,8 resulting in modifications to pomological traits as well as to phytochemicals, which are 38

dependent on the species,9,10 cultivar,9,11-13, ripening stage,11-15 part of fruit,13,16 and rootstock.17,18 39

Phytochemicals, particularly phenolics are also good indicators of changed plant metabolism, owing 40

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to stress caused by infection; however, there has been no report about the effect of the PPV 41

infection on phytochemicals in plum fruit. 42

In this study the effect of PPV-Rec strain on external and internal quality of 'Brkinska češpa' was 43

studied during the last 3 weeks of ripening. The quality of fruit from a tree that has been infected 44

with PPV for at least 5 years and from a recently infected tree was compared to that of fruit from 45

healthy tree grown at the same location. To our knowledge this is the first report about the effect of 46

PPV infection on composition of sugars, organic acids and phenolics (anthocyanins, flavonols and 47

hydroxycinnamic acids) in plum fruit during the last stages of ripening. The results will improve the 48

knowledge regarding the impact of PPV infection on ripening process, pomological traits and 49

especially the composition of phytochemicals in the healthy-looking part of the susceptible Prunus 50

domestica plum fruit. 51

52

MATERIALS AND METHODS 53

Sample collection. Fruit of 'Brkinska češpa’, the local biotype of ‘Domača češplja’ (Prunus 54

domestica L.), were collected from 20 August to 19 September in 2013 from plum orchard in the 55

south western part of Slovenia (45°32’ latitude, 14°15’ longitude; 600 m altitude), with 1500 mm of 56

average annual precipitation. Three trees growing on its own roots in the close vicinity were 57

selected based on the abundance of symptoms. 58

All the trees were over 30 years old and propagated through root suckers. The rate of infection 59

on leaves and fruits was estimated visually on each sampling time. One tree showed severe 60

symptoms of PPV infection on almost all fruit and severe premature dropping of fruit and it was 61

infected by PPV for at least 5 years (T1) (long-term infection), since the owner discovered 62

symptoms on the leaves in spring 2009. The sharka symptoms on the recently infected tree were 63

less severe. Mild symptoms were observed on few fruits. The first symptoms of PPV infections on 64

leaves of this tree were observed by the owner in 2012 (T2) (short-term infection). On the third tree 65

symptoms of PPV infection were absent (T3) (control). 66

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The visual observations of PPV symptoms were confirmed by laboratory analyses. The 67

presence/absence of PPV and other viruses that might cause similar symptoms or aggrevate the 68

symptoms of PPV was checked on 12th of March, 2014 in buds collected throughout the canopy 69

using DAS-ELISA. Antibodies (Bioreba AG, Reinach, Switzerland) for detection of Plum pox virus 70

(PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic virus 71

(ACLSV) and Apple mosaic virus (ApMV) were used. Total RNA isolated from the two PPV 72

positive samples was used in RT-PCR using PPV3 MODIF/P3M and PPV3 MODIF /P3D primer 73

pairs.19,20 Obtained amplicons were directly sequenced. Strain designations of all sequences were 74

determined based on the 99% nucleotide identity to sequences in the NCBI database using blastn 75

tool. 76

Fruit were picked at 8 to 13-day intervals through the last 3 weeks of ripening: 20 August (d0, in 77

Figures: 0), 29 August (d9, in Figures: 9), 11 September (d22, in Figures: 22) and 19 September 78

(d30, in Figures: 30 - only T3) and immediately transferred to laboratory for further analytical 79

determinations. For each sampling date and each tree 30 fruit were selected randomly. The sample 80

of 30 fruit was randomly divided into five repetitions (subsamples). The percentage of premature 81

dropping of fruit was estimated for each tree and each sampling date. 82

Pomological variables. Pomological variables (colour of fruit, colour of flesh, firmness, fruit 83

weight, and soluble solids content (SSC) were measured and the presence of necrosis on plum fruit 84

was assessed visually on each fruit using the following scale: 0 = without symptoms, 1 = symptoms 85

covering less than 10% of the fruit skin, 2 = symptoms covering from 10 to 30% of the skin, 3 = 86

symptoms covering more than 30% of the skin. Using a Konica Minolta Portable Colour Reader 87

CR-10 (Konica Minolta, Inc., Osaka, Japan; measuring diameter 8 mm), hue (h*) was measured in 88

fruit with an uninjured blush on two sides of each fruit, the values averaged, (hpeel) and in flesh 89

(hflesh). With a penetrometer (Chatillon penetrometer model DFG 50, John Chatillion & Sons, New 90

York, U.S.A.; insertion depth 5 mm), equipped with a round jagged stainless steel probe (diameter 6 91

mm), firmness was measured on two sides of each fruit immediately after the measurement of hpeel, 92

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and the values averaged. SSC was measured in fruit juice with a digital refractometer (WM-7, 93

Atago Co. Ltd., Tokyo, Japan). 94

Fruit weight was measured only on 11 September and 19 September. The water content of edible 95

part was calculated as a difference from the fresh and dry weight of each subsample. 96

Samples (peel + flesh) for further determination of sugars, organic acids and phenolic 97

compounds were taken on four sides of each fruit with a round stainless steel probe (diameter 6 98

mm). In fruit with necrosis healthy-looking part of fruit was taken for analyses. Fruit tissue of each 99

subsample (4 parts of 6 fruit) was frozen and kept at -20 ºC until the extraction. The extraction was 100

made for each subsample at once: from one part of fruit tissue for determination of sugars and 101

organic acids and from the other part for determination of phenolic compounds. Determination of 102

sugars, organic acids and phenolic compounds was therefore made on the same plant material. 103

Sugars and organic acids. Extraction: 1 gram of plum tissue was homogenized in 5 mL of 104

twice-distilled water and left for 30 min at room temperature with frequent stirring. The extracts 105

were centrifuged at 12800g for 7 min at 10 ºC (Eppendorf Centrifuge 5810R, Hamburg Germany). 106

The supernatants were filtered through a 0.20 µm cellulose ester filter (Macherey-Nagel; Düren, 107

Germany), transferred into a vial and 20 µL of the sample used for the analysis. 108

The analysis of sugars was carried out using a Surveyor HPLC system with a refractive index 109

(RI) detector (Thermo Finnigan). Separation of sugars was carried out using Rezex-RCM-110

monosaccharide column (300 x 7.8 mm; Phenomenex, Torrance, CA) operated at 65 °C. The 111

mobile phase was double distilled water. The flow rate was 0.6 mL/min. The run time was 30 min. 112

Organic acids were analysed with the same HPLC system, equipped with a UV detector set at 113

210 nm using a Rezex ROA column from Phenomenex HPLC (Thermo Scientific, Finnigan Spectra 114

system, Waltham, MA, USA) operated at 65 °C. The elution solvent was 4 mM sulfuric acid at a 115

flow rate of 0.6 mL min−1. The duration of the analysis was 30 min. 116

The concentration of an individual metabolite was calculated according to a calibration curve of 117

corresponding standard solution. The concentrations of sugars and organic acids were expressed as 118

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grams per kilogram fresh weight (g kg-1 FW). From the data of individual sugars and individual 119

organic acids, sum of sugars (SUG) and sum of acids (ACID) and their ratio SUG/ACID were 120

calculated. 121

Phenolics. Extraction: plum tissue was ground to a fine paste in a mortar chilled with liquid 122

nitrogen. One gram was extracted with 2.4 mL of methanol containing 3% (v/v) formic acid and 1% 123

(w/v) 2.6-di-tert-butyl-4-methylphenol (BHT) in a cooled ultrasonic bath for 1 h. BHT was added to 124

the samples to prevent oxidation during the extraction and formic acid for anthocyanin stabilization. 125

After extraction, samples were centrifuged at 12800g for 5 min at 4 °C and filtered through 126

Chromafil A0-20/25 polyamide filters (Macherey-Nagel, Duren; Germany). 127

The analysis was performed immediately after extraction on a Surveyor HPLC system with a 128

PDA detector (Thermo Scientific, San Jose, CA). The column was a Gemini C18 (150 x 4.6 mm; 129

3µm; Phenomenex, Torrance, CA, USA) protected with a Phenomenex security guard column and 130

operated at 25 ºC. The elution solvents were aqueous 0.1% formic acid in double-distilled water (A) 131

and 0.1% formic acid in acetonitrile (B). Samples were eluted using a linear gradient from 5 to 20% 132

B in the first 15 min, followed by a linear gradient from 20 to 30% B for 5 min, then an isocratic 133

mixture for 5 min, followed by a linear gradient from 30 to 90% B for 5 min, and then an isocratic 134

mixture for 15 min before returning to the initial conditions21 with some modifications, with an 135

injection amount of 20 µL and a flow rate of 0.6 mL min−1. Hydroxycinnamic acids were detected 136

and quantified at 280 nm, flavonol derivatives at 350 nm, and anthocyanins at 530 nm. 137

Phenolic compounds were identified using a mass spectrometer (Thermo Scientific, LCQ Deca 138

XP MAX) with an electrospray ionization (ESI) operating in positive (anthocyanins) and negative 139

(all the other phenolics) ionization mode. Analyses were carried out using full scan, data-dependent 140

MSn scanning from m/z 100 to 1000. The MS data are presented in Table 1. The capillary 141

temperature was 250 °C, the sheath gas and auxiliary gas were 60 and 15 units, respectively, and 142

the source voltage was 3 kV for negative and 4 kV for positive ionization. 143

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The identification of compounds was confirmed by comparing retention times and their spectra 144

as well as by adding the standard solution to the sample and by fragmentation. The concentrations 145

of phenolics were calculated from peak areas of samples and corresponding standards. For 146

compounds lacking standards, quantification was carried out using similar compounds as standards 147

(Table 1). Concentrations were expressed in milligrams per kilogram fresh weight (mg kg-1 FW). 148

For the general presentation of the results the sum of four hydroxycinnamic acids (HCA), the 149

sum of five flavonols (FLAVO), and the sum of four anthocyanins (ANTH) were calculated. 150

Chemicals. The following standards were used for the quantification of sugars, organic acids, 151

and phenolic compounds: sucrose, fructose, glucose, sorbitol, citric acid, malic acid, fumaric acid, 152

p-coumaric acid, quercetin-3-O-galactoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, 153

cyanidin-3-O-galactoside, peonidin-3-O-glucoside from Fluka Chemie GmBH (Buchs, 154

Switzerland); shikimic acid, neochlorogenic (3-caffeoylquinic) acid, chlorogenic (5-caffeoylquinic) 155

acid and rutin (quercetin-3-O-rutinoside) from Sigma–Aldrich Chemical (St. Louis, MO, USA), and 156

isorhamnetin-3-O-glucoside from Extrasynthese (Genay Cedex, France). Methanol for the 157

extraction of phenolics was acquired from Sigma–Aldrich Chemical. The chemicals for the mobile 158

phases were HPLC–MS grade acetonitrile and formic acid from Fluka Chemical GmBH. Water for 159

the mobile phase was twice distilled and purified with the Milli-Q system (Millipore, Bedford, MA, 160

USA). 161

Statistical analysis. The data were analysed as a two-factor experiment design, the first factor 162

expressing the level of PPV infection with three levels: T1, T2 and T3; the second factor expresses 163

the time during ripening and also has three levels (d0, d9 and d22). In the case of pomological 164

variables, there were 30 replications, and one plum represents an experimental unit. In the case of 165

sugars, organic acids and phenolic compounds, there were 5 replications; samples of flesh + peel 166

were prepared from 6 plums. The impact of two fixed factors on pomological and chemical 167

variables was examined on the basis of statistical analysis, performing linear models with one 168

categorical explanatory variable (PPV infection) and one numeric explanatory variable (time). In 169

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most cases the interaction between PPV infection and time was statistically significant, indicating 170

differing time trends for variables at different levels of PPV infection. The influence of PPV 171

infection during the last stage of ripening on sugars, organic acids and phenolic composition 172

(relative values) was examined on the basis of compositional data analysis.22 Three out of the four 173

components in the four-part sub-compositions of sugars, acids and groups of phenolic components 174

were first transformed using isometric log-ratio transformation (ilr) based on sequential binary 175

partition. Multivariate and further univariate analysis of variance (MANOVA and ANOVA) was 176

performed on ilr coordinates. Statistical analysis was done with the R program.23 For compositional 177

data analysis, the “compositions” package was used.24,25 178

179

RESULTS / DISCUSSION 180

DAS-ELISA testing showed the absence of all tested viruses with the exception of PPV. The 181

presence of PPV was confirmed in both trees showing sharka symptoms (T1 and T2). The 182

symptomless tree (T3) proved to be PPV free. Owing to the absence of other viruses that can affect 183

stone fruit, all the differences found can be ascribed to the effect of PPV. The 1226 nucleotide long 184

sequences from both PPV positive trees obtained with the primers PPV3 MODIFIED19 and P3M20 185

were found to be identical and to belong to the PPV-Rec strain. Strain identification is important, 186

since PPV isolates are diverse and have been assigned to 9 strains,26 which differ in their 187

pathogenicity, host range, aphid transmissibility, and geographic distribution. 188

The results show that premature fruit drop is higher in infected trees, and that it increases with 189

the duration of infection (Table 2). 190

Pomological variables. Our results show the effect of PPV infection on the pomological 191

characteristics during the final phases of ripening (Table 2). The analysis of variance for all 192

pomological characteristics show that the interaction between infection and ripening stage (time) 193

was statistically significant. This means that the level of PPV infection does influence the process of 194

ripening. 195

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The medians of the necrosis score evaluated for 30 plums of each tree/time combination differed 196

from 0 only for T1 (Table 2). At the first sampling date, no necrosis was found on T2 and T3 fruit, 197

but all fruit from T1 showed necrotic symptoms; score 1: 12 plums, score 2: 12 plums, and score 3: 198

6 plums. At the second sampling (d9), 12 plums from T1 got a score of 2 and 18 plums a score of 3. 199

Two fruit from T2 got a score of 1, all others 0. At the last sampling (d22), 10 plums from T1 got a 200

score of 1, 14 plums a score of 2 and 5 plums a score of 3; 5 plums from T2 got a score of 1, 1 a 201

score of 2 and 1 a score of 3. This shows that the severity of symptoms increases with the duration 202

of infection. These results also confirmed our estimation of infection rate done in the field. It is 203

known that the infection of trees and the symptom expression increases with years,6 thus the relative 204

differences in the rate of infection among trees of the same cultivar and rootstock on the same 205

location can be estimated based on the abundance of symptoms on the leaves and on the fruit. 206

Significantly the lowest average values of hpeel were observed in T1 at first measurement (d0) 207

(Table 2). The colour of T1 at d0 was magenta-blue and did not change significantly over the next 208

22 days. Ripening of T2 fruit resulted in changed colour of fruit peel from magenta (d0) to 209

magenta-blue (d22). The colour of T3 changed to magenta-blue at d30. When plums become more 210

highly coloured, the hpeel decreases.15 These results show the faster process of fruit ripening in fruit 211

from infected trees (T1 and T2) and confirm the observations of Atanassov (cited in Kamenova and 212

Milusheva)4, that plums from PPV infected trees ripen from 10 to 15 days or more ahead of fruit 213

from healthy trees. 214

Colour intensity of flesh (hflesh) significantly decreased during ripening (the colour changed from 215

green to yellow), more evidently in T2 and T3 than in T1 fruit. The fruit of T1 on d22 had 216

significantly higher values of hflesh than T2 and T3, showing a more greenish flesh. The hflesh of T3 217

decreased the most from d22 to d30. Assessment of ripeness using flesh colour is consistent with 218

the results of other pomological characteristics. 219

Our results show variation in reduction of firmness among fruit with various levels of infection. 220

At the first measurement (d0), fruit from the PPV-free (T3) and from the tree infected for only a 221

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short time (T2) showed equal levels of firmness. Fruit from the long-term infected tree (T1) were 222

statistically softer. After 9 days, differences in firmness among the fruit almost disappeared, but 223

after 22 days the firmness of T1 and T2 fruit decreased to almost the same level, while T3 fruit 224

remained firmer. The results for firmness also show that a high level of PPV infection causes earlier 225

ripening of fruit, since during the final ripening phases a change in firmness is the only useful 226

indicator of plum maturity.15 227

Ripening did not change soluble solids content (SSC) in T1, but increased it significantly in T2 228

between d9 and d22 and in T3 between d22 and d30. Given that ripening increase SSC11 it can be 229

assumed that PPV infection caused earlier ripening of T1 and T2 fruit. SSC is an essential quality 230

variable in stone fruit,27,28 which has been proposed as the most reliable maturation parameter for 231

plums.29,30 The average SSC of 10 selections of 'Brkinska češpa’ varied from 16.2 to 19.8.31 232

Infection had no impact on the water content of the edible part of plum fruit (Table 2). 233

The level of PPV infection did influence average fruit weight, which was measured only on d22 234

and d30. The fruit of T1 and T2 on d22 had a similar fruit weight (10.8 g and 11.1 g), which was 235

much lower compared to T3 (15.9 g). By the last sampling day (d30), the average fruit weight of T3 236

had increased to 16.5 g. Ripening process increase fruit weight.11 The average fruit weight of 10 237

selections of 'Brkinska češpa’ varied from 17.4 g to 21.4 g. 31 238

Nutritive compounds. Our results show the effect of PPV infection on the composition of 239

nutritive compounds in the plum fruit of ‘Brkinska češpa’ during the final ripening phases. Two 240

groups of nutritive compounds were identified: sugars and organic acids. In general, the relative 241

content of sugars increased and the relative content of acids decreased during ripening (Figure 1, 242

Table 3 and 4). T1 fruit had significantly less SUG than T2 and T3 fruit (Table 3). 243

The average content of individual sugars is shown in Table 3 and their average composition in 244

Figure 2. Glucose, fructose, sorbitol and sucrose were detected.13 Relative content of sucrose during 245

the final phases of ripening generally increased (30.0% to 47.5%), but decreased in the case of 246

glucose (39.5% to 27.8%), fructose (17.4% to 10.5%), and sorbitol (19.4% to 8.0%). 247

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The interaction between infection and time was significant for sucrose, the increase of sucrose 248

during ripening was the slowest for T3. The average glucose content significantly linearly decreased 249

during ripening, with the same rate for all levels of infection. At first measurement (d0), there was a 250

significant difference in average glucose between T1 and the other two infection rates; T3 had on 251

average 10.4 g/kg more, and T2 on average 7.3 g/kg more glucose than T1. The average content of 252

fructose increased during ripening in T2 and T3 and decreased in T1. Only in T1 and T2 did 253

ripening significantly decrease sorbitol. 254

In general, the results for sugars show similarities between T2 and T3, but in case of sucrose at 255

d22 similarities between T1 and T2. Fruit of T1 had less total sugars, glucose and sorbitol than T2 256

and T3. Fruit of T1 and T2 had at d22 more sucrose than T3 (Table 3). 257

To examine relative changes in the composition of sugars, the analysis was also done on the ilr 258

(isometric log-ratio transformation) transformed four-component sugar composition (Figure 2). The 259

MANOVA for the ilr coordinates shows that the infection/time interaction was statistically 260

significant. This means that sugar composition changes during ripening are not the same for all 261

levels of PPV infection. The three ilr coordinates were calculated: ilr1 represents the balance 262

between glucose and sucrose ilr1= c1*ln(glucose/sucrose); ilr2 represents the balance between 263

fructose and the group of sucrose and glucose) ilr2 = c2*ln(fructose/g(sucrose*glucose)), and ilr3 264

represents balance between sorbitol and the other three components 265

ilr3 = c3*ln(sorbitol/g(sucrose*glucose*fructose)). The constants c1, c2 and c3 are determined on the 266

basis of sequential binary partition and play a normalization role. The function g(.) represents the 267

geometric mean of composition components. The univariate ANOVA for ilr1 showed a significant 268

influence of infection and time; the highest occurred in T3 and the lowest in T1, and it decreased 269

with time, owing to increased sucrose content relative to glucose. The infection/time interaction was 270

statistically significant for ilr2. The ilr2, which for T1 at d0 was significantly higher than T2 and T3 271

during ripening, decreased significantly in T1, but in T2 and T3 increased slightly. The ilr3 differed 272

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among levels of infection (the highest was in T3) and decreased over time – the most in T1 and T2, 273

owing to a reduction in sorbitol concentration relative to the other sugars. 274

The ilr1 and ilr2 coordinates show more similarities between T2 and T3, but ilr3 coordinate 275

similarity between T1 and T2. 276

The average content of individual organic acids and the sum of acids (ACID) are shown in 277

Table 4 and their average composition in Figure 2. In general, the relative concentration of acids 278

during ripening decreased:11,13 from 98.4% to 97.5% malic acid, from 2.0% to 1.2% citric acid, 279

from 0.3% to 0.2% shikimic acid, and from 0.2% to 0.1% fumaric acid. 280

The infection/time interaction was significant for ACID (Table 4). Fruit of T2 and T3 had 281

significantly higher average ACID (20.0 and 15.4 g/kg, respectively) than T1 (9.3 g/kg) at first 282

measurement (d0). Ripening decreased organic acids in plum, the most in T1 and T2. Fruit of T3 283

had significantly higher average ACID than T1 and T2 at d22. There is evident decrease of ACID in 284

T3 between d22 and d30. 285

The ANOVA for each acid separately (Table 4) showed that the citric acid content of T3 was 286

significantly higher compared to T1. During ripening the content significantly decreased, the most 287

in T2. The ANOVAs for average levels of malic acid, shikimic acid and fumaric acid show that the 288

infection/time interaction was statistically significant. The content of malic acid and shikimic acid 289

of T2 and T3 was significantly higher than that of T1. The negative time trend (from d0 to d22) was 290

significant for all acids. There was an evident decrease in the main organic acid, malic acid, and 291

ACID in T3 between d22 and d30. 292

In general, also the results for organic acids show that PPV infection causes earlier ripening of 293

fruit. More similarities were between T1 and T2. 294

Four-component organic acid composition (Figure 2) was transformed into three ilr coordinates: 295

ilr1 = c1*ln(malic/citric), ilr2 = c2*ln(fumaric/shikimic) and 296

ilr3 = c3*ln(g(citric*malic)/g(shikimic*fumaric)). MANOVA showed that the infection/time 297

interaction was statistically significant for all ilr coordinates. The ilr1 was, at first measurement, 298

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lowest in T3 and highest in T2, but at d22 it was highest in T3, owing to the higher relative 299

concentration of malic acid against citric acid, and it was lowest in T2. There were significant 300

differences among the levels of infection in ilr2; it was higher in T1 and T2, thus explaining the 301

higher relative concentration of fumaric acid against shikimic acid. Ripening decreased the ilr3 of 302

T2 and T3 due to decreased relative concentrations of malic acid and citric acid. 303

The coordinates ilr1 and ilr2 show more similarities between T1 and T2, but ilr3 similarity 304

between T2 and T3. 305

Additionally, the ratio SUG/ACID (Table 4) shows that the level of PPV infection influences the 306

process of ripening. To consider the assumptions of ANOVA, the log-ratio was analysed. The 307

average SUG/ACID ratio of T1 increased significantly between d9 and d22 to 19.1 (95% CI is 15.5 308

to 21.7); the average SUG/ACID ratio of T2 increased linearly from d0 to d22, and the average 309

SUG/ACID ratio of T3 increased slowly between d0 and d22 and then jumped to 22.1 (95% CI is 310

18.4 to 25.8) by d30, owing to the increase SUG and the decrease in ACID between d22 and d30. 311

Phenolics. Three main groups of phenolics were detected in the edible part of ‘Brkinska češpa’ 312

fruit: anthocyanins, flavonols and the most abundant hydroxycinnamic acids. The average 313

composition according to these groups (sum of anthocyanins (ANTH), sum of flavonols (FLAVO) 314

and sum of hydroxycinnamic acids (HCA)) is shown in Figure 1 (right). Average concentrations, 315

along with standard errors for individual phenolics inside groups are shown in Tables 5, 6 and 7, 316

and the average composition within the phenolic group, in Figure 3. 317

Two ilr coordinates were calculated: ilr1 = c1*ln(FLAVO/HCA) and 318

ilr2 = c2*ln(ANTH/g(HCA*FLAVO)). The MANOVA for the ilr coordinates showed that the 319

infection/time interaction was statistically significant. The phenolic composition of T3 was 320

significantly different from that of T1 and T2. Ripening significantly increased the ilr1 of T1 and 321

T2, owing to the decreased relative content of HCA and increased FLAVO. This resulted in 322

significantly higher ilr1 in T1 and T2 than in T3 at d22. The ilr2 was significantly increased during 323

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ripening, owing to increased ANTH and decreased HCA, evidently more in T1 and T2 than in T3 324

(Figure 1). 325

The four hydroxycinnamic acid derivatives were identified in ‘Brkinska češpa’ fruit: the main 326

neochlorogenic acid,13,31 p-coumaroylquinic acid, chlorogenic acid, and cryptochlorogenic acid, in 327

accordance with the results of Slimestad et al.10 328

The ANOVA for each hydroxycinnamic acid separately shows that infection/time interaction 329

was statistically significant for the average HCA and neochlorogenic acid prevailed: T2 and T3 had 330

significantly more HCA than T1 (T2 more than T3) at d0. Ripening significantly decrease HCA and 331

neochlorogenic acid only in T2. At the last sampling (d22), T3 had a significantly higher content of 332

neochlorogenic acid and HCA than T2 and especially T1 (Table 5). 333

The PPV infection significantly influenced the average content of p-coumaroylquinic acid. Fruit 334

of T1 had a significantly higher average content of p-coumaroylquinic acid than T2 and T3. There 335

was no time-trend. 336

Infection and time significantly influenced average content of chlorogenic acid. Fruit of T2 and 337

T3 had significantly more chlorogenic acid than T1. Analysis shows a positive time trend. 338

There were no significant differences among the levels of infection in content of 339

cryptochlorogenic acid; the content significantly decreased during ripening (the most in T3). 340

In the case of HCA (Figure 3), three ilr coordinates were calculated: ilr1 = c1*ln(p-341

coumaroylquinic/neochlorogenic), ilr2 = c2*ln(cryptochlorogenic/chlorogenic) and 342

ilr3 = c3*ln(g(neochlorogenic*p-coumaroylquinic)/g(chlorogenic*cryptochlorogenic)). The 343

MANOVA for ilr coordinates showed that the infection/time interaction was statistically 344

significant. The coordinate ilr1 was significantly influenced by infection – the greatest in T1 and the 345

lowest in T3, owing to the higher relative content of neochlorogenic acid. Ripening had no 346

significant influence on ilr1.13 The coordinate ilr2 was significantly influenced by infection (the 347

greatest in T1 and the lowest in T2) and time (ripening decreased the ilr2). Our results show a 348

negative time trend for the ilr3 coordinate, with no significant effect from infection. 349

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The coordinates ilr1 and ilr3 show similarities between T2 and T3, but ilr2 similarity between T1 350

and T3 on d0 and d9 and similarity between T2 and T3 on d22. 351

Four anthocyanins, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, peonidin-3-O-glucoside, 352

and peonidin-3-O-rutinoside were identified in ‘Brkinska češpa’ fruit, which is in accordance with 353

previous reports.13,32 The relative concentration of cyanidin-3-O-rutinoside ranged from 34.8% to 354

49.4%, peonidin-3-O-rutinoside 11.1% to 44.4%, cyanidin-3-O-glucoside 7.0% to 33.8%, and 355

peonidin-3-O-glucoside 5.6% to 11.5% (Figure 3). Cyanidin-3-O-rutinoside was ascertained as the 356

main anthocyanin in Prunus domestica plum.10,12,13 357

Ripening significantly increased ANTH,11-13 in T1 and T2 more than in T3. At the beginning of 358

measurements, the highest content of ANTH was measured in T1. A higher content of ANTH in 359

infected fruit shows a stress-protective role of anthocyanins in plant tissue given that they are acting 360

as general antioxidants in various stress situations. The measurement of hpeel show similar results; 361

T1 fruit was coloured more than T2 and T3 at d0. 362

The ANOVA for each anthocyanin separately shows that infection/time interaction was 363

statistically significant for the average cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside. The 364

highest content of both compounds was measured in T1 and T2. Ripening from d0 to d22 increased 365

the content of both compounds significantly only in T1 and T2. In T3 the content of both cyanidins 366

increased between d22 and d30. The content of peonidin-3-O-glucoside which increased with time 367

was significantly higher in T1 and T2 than in T3. The infection/time interaction was statistically 368

significant for peonidin-3-O-rutinoside; the content increased with time, the most in T2 and T3. 369

In the case of ANTH (Figure 3), three ilr coordinates were calculated: ilr1 = c1*ln(cyanidin-3-O-370

rutinoside/cyanidin-3-O-glucoside), ilr2 = c2*ln(peonidin-3-O-rutinoside/peonidin-3-O-glucoside) 371

and ilr3 = c3*ln(g(cyanidin-3-O-glucoside*cyanidin-3-O-rutinoside)/g(peonidin-3-O-372

glucoside*peonidin-3-O-rutinoside)). The infection/time interaction was statistically significant for 373

the ilr1; the log-ratio of T1 and T2 during ripening decreased because of an relative increase in 374

cyanidin-3-O-glucoside, but that of T3 increased slightly, owing to a relative increase in cyanidin-3-375

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O-rutinoside. The ilr2 was significantly affected by infection; the least effect was in T1 and the 376

greatest in T3, owing to the higher relative content of peonidin-3-O-rutinoside. The infection/time 377

interaction was statistically significant for the coordinate ilr3; ripening increased the log-ratio and 378

resulted in a higher relative content of cyanidins in T1 and T2, but not in T3. 379

The ilr1, ilr2 and ilr3 coordinates show significant differences in the composition of anthocyanins 380

between T1 and T3. T2 shows more similarities with T1. 381

The following five flavonols were identified in ‘Brkinska češpa’ fruit: rutin, quercetin-3-O-382

galactoside, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside. 383

Rutin was the main flavonol compound (Figure 3).13,32 Quercetin-3-O-galactoside and quercetin-3-384

O-glucoside have been previously identified in plums of Prunus domestica32 and Prunus salicina33. 385

Two novel compounds in plum fruit were identified: kaempferol-3-O-rutinoside and isorhamnetin-386

3-O-rutinoside (Table 1). 387

Infection and time significantly influenced average FLAVO (Table 7). T1 had a significantly 388

higher content of FLAVO than T2 and T3. The increase in FLAVO during ripening was highest in 389

T1 and lowest in T3. The levels of PPV infection which represent various levels of stress do 390

influence the content of FLAVO. A higher content of FLAVO in infected fruit shows a stress-391

protective role of flavonols. 392

The ANOVA for each individual flavonol shows that infection and time significantly influenced 393

the average content of rutin and quercetin-3-O-galactoside. T1 had a significantly higher content of 394

rutin and quercetin-3-O-galactoside than T3. The time trend for both compounds was positive. 395

The infection/time interaction was statistically significant for quercetin-3-O-glucoside. The 396

content was significantly the highest in T1. Ripening increased quercetin-3-O-glucoside – the most 397

in T1 and the least in T3. 398

Infection had no significant effect on kaempferol-3-O-rutinoside and isorhamnetin-3-O-399

rutinoside. A positive time trend was significant for both compounds. The content of kaempferol-3-400

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O-rutinoside increased the most in T2 and the least in T3. The content of isorhamnetin-3-O-401

rutinoside increased the most in T1 and the least in T3. 402

In the case of FLAVO (Figure 3), four ilr coordinates were calculated: ilr1 = c1*ln(quercetin-3-403

O-galactoside/rutin), ilr2 = c2*ln(kaempferol-3-O-rutinoside/quercetin-3-O-glucoside), 404

ilr3 = c3*ln(g(quercetin-3-O-glucoside*kaempferol-3-O-rutinoside)/isorhamnetin-3-O-rutinoside) 405

and ilr4 = c4*ln(g(quercetin-3-O-glucoside * kaempferol-3-O-rutinoside*isorhamnetin-3-O-406

rutinoside)/g(rutin*quercetin-3-O-galactoside)). There was no statistically significant effect of 407

infection on the coordinate ilr1 which from d0 to d22 decreased due to decreased relative content of 408

rutin (Figure 3). The infection/time interaction was statistically significant for the ilr2, ilr3 and ilr4 409

coordinates. The ilr2 coordinate decreased during ripening in T1 and T2 owing to a higher increase 410

in quercetin-3-O-glucoside. In T1 and T2, the ilr3 and ilr4 coordinates decreased significantly 411

during ripening due to increased relative content of quercetin-3-O-glucoside and decreased relative 412

content of rutin. Evident changes in the ilr3 and ilr4 of T3 occurred only between d22 and d30, when 413

the relative content of rutin decreased but quercetin-3-O-galactoside and quercetin-3-O-glucoside 414

increased. These results confirm the other data; T3 fruit was ripe by d30, but the infection forced 415

the process of ripening – T1 and T2 fruit ripened earlier. 416

Our results show that the level of PPV infection did modify the ripening process, especially fruit 417

coloration, fruit softening and fruit weight. The ripening process was the most forced in the long-418

term infected tree (T1), but resulted in inferior pomological traits. The fruit from the short-term 419

infected tree (T2) were softer, smaller and riper than fruit from the healthy tree (T3), but the 420

percentage of fruit showing serious symptoms on fruit peel and in the flesh was lower than for T1. 421

PPV infection significantly modified the composition of nutritive and phenolic compounds, the 422

most in fruit from the long-term PPV-infected tree (T1). The long-term infected tree (T1) yielded 423

fruit with less glucose, malic acid, shikimic acid, sum of organic acids, neochlorogenic acid and 424

sum of hydroxycinnamic acids, but with more anthocyanins and flavonols than both T2 and T3. The 425

healthy tree yielded fruit with more neochlorogenic acid and sum of hydroxycinnamic acids and 426

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less cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, peonidin-3-O-glucoside and sum of 427

anthocyanins than the short-term infected tree (T2). 428

Additionally, the short-term PPV infected tree produces fruit with a significantly changed 429

phytochemicals composition. The visual appearance of such fruit has not changed significantly, but 430

the composition of phytochemicals has, and this does determine the taste of plum, the quality of 431

products processed from fruit and ultimately, human health. 432

433

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potyvirus and a major disease. Mol. Plant Pathol. 2014, 15, 226-241. 436

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(Prunus domestica) from Bosnia and Herzegovina. Acta Hort. 2012, 968, 81-83. 449

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maturation parameters to define the harvest maturity of plums (Prunus domestica L.): A case study 451

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Serrano, M. Changes in physicochemical and nutritive parameters and bioactive compounds during 454

development and on tree ripening of eight plum cultivars: a comparative study. J. Sci. Food Agric. 455

2008, 88, 2499-2507. 456

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plum cultivars (Prunus domestica L.). J. Agric. Food Chem. 2009, 57, 11370-11375. 458

(11) Usenik, V.; Kastelec, D.; Veberič, R.; Štampar, F. Quality changes during ripening of plums 459

(Prunus domestica L.). Food Chem. 2008, 111, 830-836. 460

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domestica L.) during ripening. Food Chem. 2009, 114, 529-534. 462

(13) Usenik, V.; Štampar, F.; Kastelec, D. Phytochemicals in fruits of two Prunus domestica L. 463

plum cultivars during ripening. J. Sci. Food Agric. 2013, 93, 681-692. 464

(14) Tomás-Barberán, F. A.; Gil, M. I.; Cremin, P.; Waterhouse, A. L.; Hess-Pierce, B.; Kader, 465

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Agric. Food Chem. 2001, 49, 4748-4760. 467

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composition and antioxidant capacity of various botanical parts of the fruits of Prunus x domestica 471

L. from the Lorraine region of Europe. Food Chem. 2012, 133, 697-706. 472

(17) Rato, A. E.; Agulheiro, A. C.; Barroso, J. M.; Riquelme, F. Soil and rootstock influence on 473

fruit quality of plums (Prunus domestica L.). Sci. Hortic.-Amsterdam, 2008, 118, 218-222. 474

(18) Milošević, T.; Milošević, N. The physical and chemical attributes of plum influenced by 475

rootstock. Acta Aliment. Hung 2012, 41, 293-303. 476

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virus isolates in Montenegro. J. Plant Pathol. 2012, 94, 201-204. 478

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(20) Candresse, T.; Cambra, M.; Dallot, S.; Lanneau, M.; Asensio, M.; Gorris, M. T.; Revers, F.; 479

Macquaire, G.; Olmos, A.; Boscia, D.; Quiot, J. B.; Dunez, J. Comparison of monoclonal antibodies 480

and PCR assays for the typing of isolates belonging to the D and M serotypes of Plum Pox 481

Potyvirus. Phytopathology 1998, 88, 198-204. 482

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foundation for statistical computing, Vienna, Austria. 2013. URL http://www.R-project.org/. 488

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Compositional data analysis: From theory and practise; Buccianti, A., Mateu-Figueras, G., 490

Pawlowsky-Glahn, V. Eds.; Special Publication of the Geological Society of London, 2006, 119-491

127. 492

(25) Van den Boogart, K. G.; Tolosana-Delgado, R. »Compositions«: A unified R package to 493

analyse compositional data. Computers and Geosciences, 2008, 34, 320-33. 494

(26) James, D.; Varga, A.; Sanderson, D. Genetic diversity of Plum pox virus: strains, disease and 495

related challenges for control. Can. J. Plant Pathol. 2013, 35, 431-441. 496

(27) Di Miceli, C.; Infante, R.; Inglese, P. Instrumental and sensory evaluation of eating quality 497

of peaches and nectarines. Eur. J. Hort. Sci. 2010, 75, 97-102. 498

(28) Infante, R.; Contador, L.; Rubio, P.; Mesa, K.; Meneses, C. Non-destructive monitoring of 499

flesh softening in the black-skinned Japanese plums 'Angeleno' and 'Autumn beaut' on-tree and 500

postharvest. Postharvest Biol. Technol. 2011, 61, 35-40. 501

(29) Prasanna, V.; Prabha, T. N.; Tharanathan, R. N. Fruit ripening phenomena – An overview. 502

Crit. Rev. Food Sci. Nutr. 2007, 47, 1-19. 503

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(30) Casquero, P. A.; Guerra, M. Harvest parameters to optimize storage life of European plum 504

‘Qullins Gage’. Int. J. Food Sci. 2009, 44, 2049-2054. 505

(31) Usenik,V.; Štampar, F.; Fajt N. Pomological and phenological characteristics of some 506

autochthonous Slovenian plum cultivars. Acta Horti. 2007, 734, 53-59. 507

(32) Treutter, D.; Wang, D. W.; Farag, M. A.; Baires, G. D. A.; Ruhmann, S.; Neumuller, M. 508

Diversity of phenolic profiles in the fruit skin of Prunus domestica plums and related species. J. 509

Agric. Food Chem. 2012, 60, 12011−12019. 510

(33) Venter, A.; Joubert, E.; de Beer, D. Characterisation of phenolic compounds in South 511

African plum fruits (Prunus salicina Lindl.) using HPLC coupled with diode-array, fluorescence, 512

mass spectrometry and on-line antioxidant detection. Molecules 2013, 18, 5072-5090. 513

514

Funding 515

This work is part of programme Horticulture No. P4-0013-0481 funded by the Slovenian Research 516

Agency. The work was financially supported also by the FP7 Project CropSustain, grant agreement 517

FP7-REGPOT-CT2012-316205. 518

519

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Figure captions 520

Figure 1. The effect of PPV infection (T1: long-term infection; T2: short-term infection; T3: 521

control) on the average composition of nutritive compounds (left) and phenolics (right) of plum 522

'Brkinska češpa’ during last stages of fruit ripening (day) (0; 9; 22; 30 - only T3) 523

524


control) on the average composition of sugars (left) and organic acids (right) of plum 'Brkinska 526

češpa’ during last stages of fruit ripening (day) (0; 9; 22; 30 - only T3) 527

528


control) on the average composition of hydroxycinnamic acids (top left), flavonols (top right) and 530

anthocyanins (bottom right) of plum 'Brkinska češpa’ during last stages of fruit ripening (day) (0; 9; 531

22; 30 - only T3) 532

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Table 1. Identification of Phenolic Compounds in Plum Fruit in Negative and Positive Ionization with HPLC-MS, MS2 and MS3

phenolic group M+ or

[M-H]-

(m/z)b

MS2

(m/z) MS3 (m/z)

tentative identification expressed in

anthocyanins 449 287 cyanidin-3-O-glucoside cyanidin-3-O-galactoside

595 449 287 cyanidin-3-O-rutinoside cyanidin-3-O-galactoside

463 301 peonidin-3-O-glucoside peonidin-3-O-glucoside

609 463 301 peonidin-3-O-rutinoside peonidin-3-O-glucoside

hydroxycinnamic acids 353 191, 179 3-O-caffeoylquinic acid (neochlorogenic acid) 3-O-caffeoylquinic acid

337 173 4-O-p-coumaroylquinic acid p-coumaric acid

353 191, 179 5-O-caffeoylquinic acid (chlorogenic acid) chlorogenic acid

353 173, 179 4-O-caffeoylquinic acid (cryptochlorogenic acid ) chlorogenic acid

flavonols 609 301 quercetin-3-O-rutinoside (rutin) quercetin-3-O-rutinoside

463 301 quercetin-3-O-galactoside quercetin-3-O-galactoside

463 301 quercetin-3-O-glucoside quercetin-3-O-glucoside

593 285 kaempferol-3-O-rutinoside kaempferol-3-O-glucoside

623 315 isorhamnetin-3-O-rutinoside isorhamnetin-3-O-glucoside bM+ (m/z) anthocyanins were obtained in the positive ion mode, other phenolics in the negative ion mode

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Table 2. Average Score of Premature Dropping of Fruit (Fruit drop), Median of Necrosis (Nec) and

Averages with Standard Errors of hpeel, Firmness, hflesh, Soluble Solids Content (SSC), Water Content

of Edible Part (%), and Fruit Weight (g) for Three Levels of PPV Infection (T1: Long-Term

Infection; T2: Short-Term Infection; T3: Control) during Last Stages of Fruit Ripening (Day); n=30

fruit

hpeel firmness

hflesh SSC

water content

fruit weight

day drop nec SE

SE

SE

SE SE

SE T1 0 20 2 277.4 cd 8.8 1.0 b 0.4 91.4 b 7.5 18.8 cd 1.3 81.2 1.3 9 45 3 270.5 cd 6.5 0.9 bcd 0.3 87.0 c 9.2 19.0 cd 0.8 81.0 0.8 22 60 2 268.7 d 9.0 0.5 e 0.1 85.2 cd 5.0 19.4 bc 0.2 80.6 0.2 10.8 2.2 T2 0 2 0 310.3 a 24.7 1.6 a 0.4 96.4 a 6.4 18.4 de 0.5 81.7 0.5 9 10 0 278.2 c 9.5 0.9 cd 0.3 82.8 de 5.5 18.2 de 0.3 81.8 0.2 22 30 0 270.9 cd 12.8 0.6 e 0.2 78.0 f 6.3 20.4 a 0.8 79.6 0.8 11.1 1.8 T3 0 2 0 303.5 a 20.1 1.6 a 0.5 94.5 ab 3.3 18.6 cd 0.2 81.4 0.2 9 5 0 295.0 b 11.6 1.0 bc 0.2 80.0 ef 5.7 17.6 e 0.4 82.4 0.4 22 5 0 306.9 a 28.5 0.8 d 0.2 76.8 f 6.5 18.1 de 0.4 81.9 0.4 15.9 2.0 30 8 0 272.3 cd 4.0 0.9 cd 0.2 70.0 g 3.5 19.9 ab 0.4 80.1 0.4 16.5 1.5

a,b,c,d – No statistically significant differences between averages denoted with the same letter.

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Table 3. Average Contents with Standard Errors (g/kg FW) of Individual Sugars and Sum of Sugars

(SUG) for Three Levels of PPV Infection (T1: Long-Term Infection; T2: Short-Term Infection; T3:

Control) during Last Stages of Fruit Ripening (Day); n=5

sucrose glucose fructose sorbitol SUG

day

SE

SE

SE

SE

SE T1 0 30.5 d 4.9 34.4 bA 2.6 17.6 a 1.5 19.0 a 3.2 101.4 bB 7.7 9 44.6 bc 4.5 28.8 bB 0.9 15.7 ab 1.6 15.0 b 3.1 104.1 bAB 6.6 22 49.7 ab 5.1 31.3 bB 1.4 13.0 cd 1.8 10.6 c 1.1 104.7 bA 7.4 T2 0 31.5 d 4.4 40.3 aA 8.3 11.5 d 2.4 19.4 a 2.1 102.6 aB 15.0 9 43.7 c 2.2 37.6 aB 2.2 13.7 bcd 1.2 14.4 b 2.1 109.4 aAB 2.4 22 54.8 a 3.5 36.1 aB 4.1 17.7 a 1.9 9.8 c 2.9 118.3 aA 9.2 T3 0 33.5 d 5.1 43.2 aA 4.7 11.6 d 2.2 21.2 a 2.0 109.5 aB 10.4 9 41.3 c 2.9 39.2 aB 2.8 12.2 cd 1.1 19.3 a 2.5 112.0 aAB 8.6 22 43.8 c 2.0 34.0 aB 3.0 14.5 bc 0.8 19.0 a 2.0 111.2 aA 4.0 30 45.5 2.7 38.4 1.0 17.3 0.5 18.8 0.9 119.9 2.3

a,b,c,d – No statistically significant differences between averages denoted with the same letter. For the variables with statistically significant interaction infection/time only small letters are used for indication of differences between treatments; if the interaction was not statistically significant small letters indicate differences between rates of infection and capital letters the differences between different time points.

Page 26 of 34



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Table 4. Average Contents with Standard Errors (g/kg FW) of Individual Organic Acids, Sum of

Organic Acids (ACID) and SUG/ACID Ratio with 95% Confidence Intervals (CI) for Three Levels of

PPV Infection (T1: Long-Term Infection; T2: Short-Term Infection; T3: Control) during Last Stages

of Fruit Ripening (Day); n=5

citric acid malic acid shikimic acid fumaric acid ACID SUG/ACID ratio

day

SE

SE

SE

SE

SE 95% CI T1 0 0.16 bA 0.03 9.1 de 0.9 0.02 d 0.01 0.014 bcd 0.001 9.3 de 0.9 10.9 9.2. 12.9 9 0.21 bB 0.04 11.0 cd 2.9 0.02 d 0.01 0.016 bc 0.003 11.2 cd 3.0 9.8 8.1, 11.3 22 0.08 bC 0.02 5.8 fg 1.9 0.01 e 0.00 0.008 f 0.002 5.9 fg 2.0 19.1 15.5, 21.7 T2 0 0.29 abA 0.05 19.6 a 3.0 0.05 a 0.01 0.023 a 0.002 20.0 a 3.0 5.2 4.3, 6.1 9 0.11 abB 0.02 7.3 ef 1.6 0.02 d 0.00 0.011 def 0.001 7.4 ef 1.7 15.3 12.7, 17.8 22 0.09 abC 0.01 4.4 g 0.5 0.01 e 0.00 0.011 ef 0.002 4.5 g 0.5 26.4 22.2, 31.1 T3 0 0.32 aA 0.11 15.0 b 1.0 0.04 a 0.00 0.017 b 0.002 15.4 b 1.1 7.2 6.0, 8.4 9 0.16 aB 0.04 12.9 bc 0.9 0.04 b 0.00 0.013 cde 0.001 13.1 bc 0.9 8.6 7.2, 10.1 22 0.14 aC 0.02 11.2 cd 1.2 0.03 c 0.00 0.017 b 0.003 11.4 cd 1.2 9.9 8.3, 11.6 30 0.14 0.03 5.4 1.1 0.01 0.00 0.011 0.001 5.6 1.1 22.1 18.4, 25.8


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Table 5. Average Contents with Standard Errors (mg/kg FW) of Individual Hydroxycinnamic Acids

and Sum of Hydroxycinnamic Acids (HCA) for Three Levels of PPV Infection (T1: Long-Term

Infection; T2: Short-Term Infection; T3: Control) during Last Stages of Fruit Ripening (Day); n=5

neochlorogenic acid p-

coumaroylquinic acid

chlorogenic acid cryptochlorogenic acid

HCA

day

SE

SE

SE

SE

SE T1 0 222.6 de 32.6 13.8 aA 1.5 43.5 bB 5.3 9.9 aA 2.3 289.7 de 34.6 9 166.9 e 66.5 14.3 aA 3.5 59.3 bB 21.6 8.1 aB 3.6 248.5 e 91.6 22 220.4 de 46.7 14.5 aA 0.9 67.6 bA 7.8 8.3 aB 0.9 310.8 de 52.9 T2 0 383.5 a 44.9 14.3 bA 1.6 78.0 aB 23.8 9.4 aA 2.4 485.1 a 61.8 9 270.1 cd 24.1 11.3 bA 1.4 67.6 aB 14.4 6.0 aB 0.3 354.9 cd 37.6 22 241.7 d 22.2 11.5 bA 2.0 83.4 aA 13.1 5.6 aB 0.5 342.1 cd 24.9 T3 0 320.8 bc 55.5 12.1 bA 2.1 53.0 aB 15.0 11.5 aA 5.2 397.4 bc 75.1 9 331.3 ab 30.8 10.6 bA 1.0 66.8 aB 12.2 8.2 aB 0.6 416.8 abc 32.2 22 335.4 ab 34.5 11.0 bA 0.8 83.5 aA 7.9 6.5 aB 1.1 436.4 ab 40.0 30 321.6 24.9 8.6 0.8 64.1 8.8 6.3 0.8 400.6 32.8


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Table 6. Average Contents with Standard Errors (mg/kg FW) of Individual Anthocyanins and Sum of

Anthocyanins (ANTH) for Three Levels of PPV Infection (T1: Long-Term Infection; T2: Short-Term

Infection; T3: Control) during Last stages of Fruit Ripening (Day); n=5

cyanidin-3-glucoside

cyanidin-3-rutinoside

peonidin-3-glucoside

peonidin-3-rutinoside

ANTH

day

SE

SE

SE

SE

SE T1 0 26.8 c 6.1 52.3 cd 18.0 8.6 aC 2.4 29.9 d 6.0 117.6 cd 27.6 9 65.7 b 14.5 111.6 b 31.3 16.6 aB 2.7 34.0 bc 8.5 227.9 b 50.9 22 113.8 a 20.9 167.9 a 39.9 19.6 aA 6.4 37.5 ab 6.9 338.8 a 57.8 T2 0 4.5 d 2.6 19.4 e 4.1 4.4 aC 0.9 19.3 d 3.0 47.5 e 6.4 9 26.4 c 10.7 69.9 c 16.2 14.4 aB 2.3 45.3 c 15.0 156.0 c 39.7 22 99.6 a 33.1 151.1 a 32.8 19.6 aA 4.3 69.6 a 13.1 339.9 a 76.6 T3 0 4.3 d 1.7 14.0 e 5.5 4.0 bC 1.1 16.8 d 2.6 39.0 e 10.2 9 6.4 d 1.9 26.3 de 2.9 8.7 bB 4.2 31.4 c 15.1 72.8 de 19.9 22 14.6 cd 4.2 63.3 c 8.6 11.8 bA 4.9 53.8 c 4.8 143.4 c 13.5 30 29.5 11.1 98.0 28.6 17.1 4.4 56.7 8.4 201.3 49.1


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Table 7. Average Contents with Standard Errors (mg/kg FW) of Individual Flavonols and Sum of

Flavonols (FLAVO) for Three Levels of PPV Infection (T1: Long-Term Infection; T2: Short-Term

Infection; T3: Control) during Last Stages of Fruit Ripening (Day); n=5

rutin

quercetin-3-galactoside

quercetin-3-glucoside

kaempferol-3-rutinoside

isorhamnetin-3-rutinoside

FLAVO

day

SE

SE

SE SE

SE

SE T1 0 33.6 aB 7.0 1.7 aB 0.3 2.2 c 0.8 1.6 aB 0.6 2.9 aB 0.8 42.0 aB 8.9

9 66.5 aA 24.5 2.6 aAB 0.9 8.8 a 2.2 2.5 aAB 0.8 5.6 aA 1.8 86.0 aA 29.2 22 59.9 aA 11.5 2.6 aA 0.4 10.9 a 3.6 2.3 aA 0.7 5.3 aA 1.5 81.1 aA 15.7

T2 0 34.4 abB 19.2 2.0 abB 1.0 1.8 c 1.3 1.7 aB 1.2 3.4 aB 2.0 43.3 bB 24.5 9 36.7 abA 11.7 1.6 abAB 0.4 2.4 c 0.7 1.7 aAB 0.5 3.5 aA 1.0 45.9 bA 14.2 22 56.7 abA 18.6 2.7 abA 0.9 6.4 b 2.3 3.1 aA 1.4 5.0 aA 1.2 73.9 bA 23.8 T3 0 31.0 bB 5.4 1.7 bB 0.4 1.6 c 0.5 1.7 aB 0.4 3.2 aB 0.6 39.2 bB 7.1 9 35.9 bA 16.6 1.7 bAB 0.6 1.8 c 1.0 1.6 aAB 0.9 3.6 aA 1.6 44.5 bA 20.6 22 36.0 bA 10.5 1.7 bA 0.4 1.8 c 0.6 1.6 aA 0.6 3.8 aA 0.8 44.9 bA 12.9 30 43.1 8.8 4.2 1.5 3.7 1.3 2.3 0.6 4.9 1.1 58.2 12.7 a,b,c,d – No statistically significant differences between averages denoted with the same letter. For the variables with statistically significant interaction infection/time only small letters are used for indication of differences between treatments; if the interaction was not statistically significant small letters indicate differences between rates of infection and capital letters the differences between different time points.

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Effect of Plum pox virus on Chemical Composition and Fruit Quality of Plum