- Email: [email protected]
Accumulation of phenolic compounds in apple in response to infection by the scab pathogen, Venturia inaequalis
Physiological and Molecular Plant Pathology 74 (2009) 60–67
Contents lists available at ScienceDirect
Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
Accumulation of phenolic compounds in apple in response to infection by the scab pathogen, Venturia inaequalis M. Mikulicˇ Petkovsˇek*, F. Sˇtampar, R. Vebericˇ University of Ljubljana, Biotechnical Faculty, Agronomy Department, Chair for Fruit Growing, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 3 September 2009
The research dealt with phenolics in healthy versus scab infected apple leaves and fruits. The leaf samples were picked in the period from May to September and the fruit samples at technological maturity. Infection with the Venturia inaequalis fungus enhanced the metabolism of phenolics at the infected sites, especially in the boundary tissue. Tissue infected with Venturia inaequalis showed in comparison to the healthy tissue up to 7.6 times more hydroxycinnamic acids, up to 2.6 times more flavan-3-ols and up to 2.9 times higher values of flavanols. The content level of total phenolics in the infected tissue was 1.3–2.4 times higher than in the healthy leaves and fruit. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Malus domestica Apple scab Hydroxycinnamic acids Flavonoids Dihydrochalcones Hydroxybenzoic acids
1. Introduction Apple scab, which is caused by the ascomycete Venturia inaequalis (Cke.) Wint., is the most important disease in all applegrowing areas with high spring and summer rainfall. The disease manifests as lesions on the surface of leaves, buds or fruit. Severe leaf spotting causes a reduction in the assimilation surface and leads to defoliation. If infection on fruit occurs early, the fruit do not expand properly at the infected portions and become undersized and gnarled. Such fruit often crack, allowing fruit-rotting organisms to invade into the apple. The disease reduces fruit yields and fruit quality. Affected fruit are not marketable, owing to the presence of the black fungal lesions. Disease control in commercial orchards can require up to 15 fungicide treatments per year. An alternative approach is the use of resistant cultivars [1] or treatment with chemical agents (i.e. prohexadione-Ca), which cause higher resistance to scab disease [2]. The secondary compounds in plants are strongly involved in the interaction between the pathogen and the plant. Phenolic compounds may contribute to the resistance of apple to Venturia inaequalis. Phenolics are toxic to pathogens. Examples of such phenolic compounds are flavanols and hydroxycinnamic acids [3,4]. In apple, flavanols play an important role in resistance to the V. inaequalis fungus [5,6]. Resistant apple cultivars have a higher content of hydroxycinnamic acids and flavanols [7,8], although susceptible cultivars are also able to accumulate flavanols in the
* Corresponding author. Tel.: þ386 1 423 11 61; fax: þ386 1 423 10 88. E-mail address: [email protected] (M. Mikulicˇ Petkovsˇek). 0885-5765/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2009.09.003
tissue surrounding infection sites [6]. Sierotzki and Gessler [9] reach the contradictory conclusion that there is no positive correlation between resistance and pre-formed flavan-3-ols in the relationship between Malus domestica and V. inaequalis. It has been reported that phloridzin also plays a role in host resistance to pathogens. It has been suggested that phloridzin can be hydrolysed in vivo by various fungi such as V. inaequalis to give phloretin, which in turn, is degraded to phloroglucinol, phloretic acid and p-hydroxybenzoic acid, which inhibits the development of the fungus [10]. Phenol-oxidizing enzymes oxidize phenolics to quinones, which are often more toxic to pathogens than the original phenol [3]. Thus Hrazdina et al. [11] reported that malusfuran produced in response to invasion by V. inaequalis inhibited spore germination and growth of V. inaequalis. The aglycone of malusfuran showed higher toxicity than malusfuran and acts as the actual phytoalexin. The production and accumulation of phenolics occurs in healthy plant cells surrounding wounded or infected cells, and they are stimulated by alarm substances produced and released by the damaged cells and diffusing into the adjacent healthy cells. Therefore, the activity of many phenol-oxidizing enzymes is generally higher in the infected tissue than in the uninfected tissue of healthy plants [3]. Changes at the phenolic level can play a role in the protection of the plant [12–15]. The phenolic derivatives can oxidize and react with proteins, thus causing a loss of enzyme function, and restricting the viability of aggressors, or they can be deposited inside the cell wall as an important first line in plant defence against infection [16]. Previous studies [17,18] revealed that, for successful protection, rapid biosynthesis of flavanols, starting from phenylalanine, were necessary.
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
The inhibition of the enzyme phenylalanine-ammonia-lyase (PAL) resulted in severe sporulation symptoms in a resistant cultivar. The biosynthesis and accumulation of secondary compounds are dependent on growth and environmental conditions. Previous investigations have shown that high nitrogen supply reduced flavonoid accumulation in leaves [19,20]. This situation is positively related to increased susceptibility to scab [19,21]. Veberic et al. [22] reported that growing technology also has an influence on the content of phenolic substances. Organically grown apples exhibit higher phenolics because of different stress factors in the orchard, such as disease, and pests, lack of mineral nutrients – all of which induce accumulation of phenolic compounds. The relationship between phenolics and apple scab has been the topic of several papers. The researchers mainly focused on how the infection alters phenolic accumulation in apple leaves. In contrast, the aim of our study was to investigate how the infection changed the synthesis of phenolic compounds in healthy and scab infected fruit. To monitor the plant response, three zones on the fruit were chosen: healthy peel, peel with the scab lesion, and the boundary zone of the scab lesion with a 1–2 mm narrow strip of healthy peel. This was done on fruit at their technological maturity in the ’Jonagold’ and ’Golden Delicious’, which are important cultivars in Europe. During the 2006 growing season the phenolic profile in healthy and scab – infected apple leaves was also monitored. Based on previous studies, we hypothesized that the infected tissue would have a higher content of certain phenolic compounds compared with healthy tissue. 2. Materials and methods 2.1. Materials 2.1.1. Plant material and growing conditions The experiment was carried out in the growing seasons 2005 and 2006. For the experiment samples were taken from apple trees grafted on M9 rootstock growing in two locations: Ljubljana (central Slovenia) and Maribor (north-eastern Slovenia). In these orchards the trees were cultivated according to the guidelines of integrated production. Despite the use of fungicides, some weak symptoms of apple scab on leaves and fruit were present. Weather parameters (temperature and rainfall) in the years 2005 and 2006 were favorable to the development of apple scab. Leaves of cultivars were sampled in year 2006 on both locations. Fruit samples were taken in both years on location Maribor. Ten fully developed healthy or infected leaves were collected from each tree. The leaves were sampled from five trees on different dates. The 3rd or 4th fully developed leaf from annual shoots was taken for analysis. From the infected leaves, only infected tissue with a surrounding narrow zone of healthy cells (1–2 mm) was taken. From each tree, five healthy or scab – infected apple fruit in technological maturity were also collected. On the fruit peel three zones were defined: healthy peel, scab lesion on peel, and a marginal section of the scab lesion with a 1–2 mm narrow strip of healthy peel. Before extraction, the leaves and fruit peel were frozen in liquid nitrogen and stored at 20 C. The leaf sampling was made on several occasions during growing season. Sampling at Ljubljana began on May 25th (34 days after full bloom) and at Maribor on May 25th (36 days after full bloom). Sampling of leaves was completed on September 2nd. 2.1.2. Chemicals The following standards were used for quantification of single phenolic compounds: protocatechuic acid was obtained from Merck (Darmstadt, Germany), chlorogenic acid (5-caffeoylquinic acid) and rutin (quercetin-3-O-rutinoside) from Sigma (St. Louis, MO, USA),
61
caffeic acid, ferulic acid, ()-epicatechin, quercitrin (quercetin-3-Orhamnoside), p-coumaric acid and phloridzin dihydrate from Fluka (Buchs, Switzerland) and (þ)-catechin from Roth (Karlsruhe, Germany). Methanol was acquired from Sigma. Water was bidistilled and purified with the Milli-Q system (Millipore, Bedford, MA, USA). For the total phenolic content, Folin-Ciocalteu reagent (Fluka), sodium carbonate (Merck), gallic acid (Sigma) and ethanol (Sigma) were used. For the antioxidant capacity 1,1-Diphenyl-2-picrylhydrazyl (DPPH), trolox and methanol were purchased from Sigma. 2.2. Extraction and determination of phenolic compounds The frozen leaves were lyophilized and ground in a mortar. Extraction with some modification was made as described by Colaric et al. [23]. The fine powder (50 mg) was extracted with methanol (20 ml) containing 1% 2.6-di-tert-butyl-4-methylphenol (BHT) for 30 min in a cooled water bath, using sonification. BHT was added to the samples to prevent oxidation during the extraction. It did not interfere with the extracted phenols during the subsequent HPLC analysis, because it was eluted at the end of the gradient or in the equilibration delay between the two analyses. The apples were peeled with a mechanical peeler, and the peel was separated from the pulp. The extraction of peel samples was done as described by Escarpa and Gonzalez [24], with some modification. The samples of 2 g peel were extracted with methanol containing 1% 2.6-di-tert-butyl-4-methylphenol (BHT) in a cooled water bath using sonification. BHT was added to the samples to prevent oxidation. The samples were extracted with 5 ml of extraction solution for 1 h, 5 ml for 30 min, and then 2.5 ml for 30 min. The extracts were then combined to a final volume of 12.5 ml. The leaf or peel extracts were centrifuged at 10.000 rpm for 10 min at 4 C, and the supernatant was filtered through a 0.45 mm membrane filter (Macherey–Nagel, Du¨ren, Germany) prior to the injection to HPLC. The phenolic compounds were analyzed on a Thermo Finnigan Surveyor HPLC system with a diode array detector, at 280 and 350 nm. The hydroxycinnamic acids (chlorogenic, p-coumaric, ferulic and caffeic acid), the hydroxybenzoic acid (protocatechuic acid) and the monomeric flavan-3-ols (catechin, epicatechin) were detected at 280 nm, whereas rutin, phloridzin and quercetin-3rhamnoside (quercitrin) were estimated at 350 nm. Spectra of the compounds were recorded between 200 and 400 nm. The column used was a Phenomenex Gemini C18 (150 4.60 mm, 3 micron), operated at 25 C. The elution solvents were A (aqueous 0.01 M phosphoric acid) and B (100% methanol). The samples were eluted according to a linear gradient described by Escarpa and Gonzales [24], with slight changes: 5% B initially; 50% B for 10 min; 70% B for 5 min; 80% B for 5 min and, finally, 100% B for 10 min. The injection amount was 10 ml for the leaf and 20 ml for the peel extract; the flow-rate was 1 ml/min. The identification of compounds was achieved by comparing the retention times and spectra as well as by the addition of the standard solution to the sample. Concentrations of phenolic compounds were calculated from the peak areas of the sample and the corresponding standards. The concentrations were expressed as mg/100 g dry weight or mg/g dry weight (DW) for leaves and as mg/kg fresh weight (FW) for peel. 2.3. Determination of total phenolic content The extraction of leaf or peel samples for the determination of total phenolics was made according to the same protocol as for phenolics, with the difference that no BHT was added. The total phenolic content (TPC) of extracts was assessed by using the FolinCiocalteu phenol reagent method [25]. To 100 ml of the sample extracts (diluted 1: 5 (v/v) with MeOH), 6 ml of bidistilled water
62
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
and 500 ml of Folin-Ciocalteu reagent were added; after resting between 8 s and 8 min at room temperature, 1.5 ml of sodium carbonate (20% w/v) was added. The extracts were mixed and allowed to stand for 30 min at 40 C before measuring the absorbance on a spectrophotometer (Perkin Elmer, UV/VIS Lambada Bio 20) at 765 nm. A mixture of water and reagents was used as a blank. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g dry weight of leaf tissue and in mg/kg fresh weight of peel tissue. Absorption was measured in three replications. 2.4. Determination of antioxidant activity with the DPPH radical scavenging method The extraction of peel samples for the determination of antioxidant activity was made according to the same protocol as for total phenolics. The free radical scavenging activity of apple extracts was measured according to the DPPH (1.1-diphenyl-2picrylhydrazyl) method reported by Brand-Williams et al. [26], with some modifications. A methanolic solution (50 ml) of peel extract (diluted 1:6) was placed in 96-well microplates, and 200 ml of a 0.1 mM methanolic solution of DPPH was added and allowed to react in the dark at room temperature. The decrease in absorbance of DPPH at 520 nm was measured at 5 min intervals by the Spectrophotometer MRX Dynex Technologies (USA), until absorbance stabilized (30 min). Methanol was used as a blank solution, and a DPPH solution without test samples served as the control. The radical stock solution was prepared fresh daily. All samples were performed in triplicate. The DPPH radical scavenging activity of peel methanolic extracts was expressed as mM of trolox per 100 g of fresh peel in 30 min of reaction time. Determination of antioxidant capacities of the samples at various concentrations was made using the standard curves of trolox. 2.5. Statistical analysis The data were analyzed by using the Statgraphics Plus 4.0 program (Manugistics. Inc.; Rockville, Maryland, USA). The significance of the infection with V. inaequalis on individual and total phenolic content was tested using the one-way analysis of variance (ANOVA). Differences between treatments were tested with the LSD test at a significance level of 0.05 Also a two-way analysis of variance was carried out to determine the significance of cultivar and year in the apple peel as well as a three-way analysis of variance for testing differences in phenolics in the apple leaves for year, cultivar and location. 3. Results and discussion In both apple cultivars we have identified eleven phenolics belonging to different groups (Tables 3 and 4, Figs. 1 and 2). In apple skin at technological maturity 3 different tissues were analyzed: healthy tissue, scab lesion and the marginal tissue around the scab lesion. In Figs. 1 and 2 the response of the plant with synthesis of phenolic compounds in these three different tissues can be seen. For individual phenolic compounds, total phenolics and antioxidant capacity the influence of the year, cultivar and their interaction was also statistically evaluated and is presented in Table 1. The interaction between year and cultivar was significant for all types of tissue in all phenolics. Since the year had a greater influence on the amount of phenolics than the cultivar, we assume that the interaction is mainly the consequence of that factor. As can be seen from Figs. 1 and 2, in the year 2006 the amounts of most of the synthesised phenolics were higher. This is probably because of influence of the environmental factors. The influence of the cultivar on the amount of phenolics is much lower and significant in only a few
cases. The reason is probably that the two cultivars in our study are closely related and the response to infection is similar. In the case of leaves, the year and cultivar were also taken into account as well as the location, since we obtained the data from two different locations (Table 2). Also in this case the interaction of all three factors was significant on the amount of phenolics in healthy and scab infected leaves. The only exception was in the infected leaves in the case of ferulic acid. All three factors contributed to the different outcome with the strongest influence again being the year. From these results we concluded that the accumulation of phenolic compounds in apple tissues is, apart from apple scab infection, also influenced by the growing season as well as variety and location. Similar results were also reported by Veberic et al. [27], who noticed that in the cultivar ’Fuji’ the amounts of analyzed phenolics were strongly dependent upon the growing season. Especially if there is a short time of stress condition this can influence the results. That cultivars can also have an influence on the amount of phenolics was reported by many papers [8,22]. In our case it was however noticed that the amounts were quite similar when observing the phenolics in the fruit peel (Table 1). The location can influence the amounts of phenolics in different ways: either is the influence of pedoclimatic factors or the influence of technological measures applied in the orchard [22,28,29]. 3.1. Phenolic acids Among the hydroxybenzoic acids, we have identified protocatechuic acid in the fruit skin. Healthy skin of all the varieties under investigation contained statistically the lowest amount of protocatechuic acid, while the lesion and the tissue around it contained a statistically higher quantity of this acid (Fig. 1A). On the lesion, up to 2.5 times more protocatechuic acid was synthesised in comparison with the healthy skin. Of the hydroxycinnamic acids, the amount of chlorogenic acid is the highest. In different tissue, healthy apple skin contained statistically the least amount of chlorogenic acid, i.e. from 96.32 to 158.26 mg/kg FW. Significantly the highest amount was observed in the tissue around the lesion and from the lesion itself. If the content of chlorogenic acid in healthy skin and in lesions is compared, its content in the latter was up to 4 times higher (Fig. 1B). The content of chlorogenic acid in healthy leaves was from 4.2 to 85.3 mg/100 g. In infected leaves, its content increased by 1.1–7.6 times in comparison with healthy leaves (Tables 3 and 4). The content of the other hydroxycinnamic acids analyzed was lower than the chlorogenic acid content. Scab infection caused increased synthesis of caffeic, p-coumaric and ferulic acid in fruit (Fig. 1C–E). In comparison with healthy skin, lesions contained 1.1–3.2 times more p-coumaric acid, 1.2–4.2 times more ferulic and 1.2–3.7 times more caffeic acid. In apple leaves, the content of p-coumaric acid, ferulic and caffeic acid changed minimally over the growth period. Generally, it may be concluded that the content of hydroxycinnamic acids increased by 1.2–3 times in comparison with healthy leaves. Our previous investigation reported that infection with scab caused an increase in their content [30]. Moreover, the findings of previous publications have shown the response of apple-tree to fungus infection as indicated by the accumulation of phenols. Back in 1969, Williams and Kuc´ [31] found that hydroxycinnamic acids inhibited the growth and sporulation of the fungus V. inaequalis. Michalek et al. [32] reported on the accumulation mainly of derivatives of caffeic acid, which is a precursor of flavonols in apple tissue. Rat-Morris et al. [33] found significant differences in the content of chlorogenic acid in appletree leaves between the scab-resistant variety ‘Florina’ and the susceptible variety ‘Melrose’. Resistant varieties contained more chlorogenic acid in comparison with susceptible ones [8]. Similar
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
60
2005
B
2006
Chlorogenic acid (mg/kg FW)
Protocatechuic acid (mg/kg FW)
A
b
50
b b
40
b 30
c b
20 10
b
a
a
b a
a
800 700
JG
GD
healthy
spot
300
b
b a
200
a
a
a
100
JG
GD
healthy
spot
JG
boundary
D 21 c
20
c
c 15
b c
b b b
10
a
a
a
a
p-Coumaric acid (mg/kg FW)
Caffeic acid (mg/kg FW)
b
b
400
boundary
0
18
c
2006
2005
b
15
c
12
b
9
a
6 3
a
b
c
c
a
healthy
spot
b a
0 GD
JG
GD
healthy
spot
JG
GD
F
21
c
2005
2006
b 15
b
b
b
b
b
9
a a
a
GD
JG
boundary
600
2006
2005
c
12
a
JG
boundary
Catechin (mg/kg FW)
Ferulic acid (mg/kg FW)
b
b
GD
2006
2005
6
c
500
JG
C 25
18
2006
c
0 GD
E
2005
600
0
5
63
c
500
b
c 400
b
300
c c
200
b
a
b
a
a
a 100
3 0 GD
JG
healthy
GD
spot
JG
0 GD
boundary
JG
healthy
GD
spot
JG
boundary
Fig. 1. The content of single phenolics in healthy and in infected apple peel (healthy tissue, scab spot, boundary zone between scab spot and healthy tissue) at cultivars ‘Golden Delicious’ and ‘Jonagold’ at location Maribor in years 2005 and 2006. Different letters denoted statistically significant differences between treatments within one cultivar in particular year.
results were obtained by Picinelli et al. [34], who found a higher content of p-coumaric acid in resistant varieties. Mayr et al. [12] reported that it was impossible to find out exactly how much a particular stress factor influenced the content of phenolic compounds in a plant, since it is an effect resulting from all the stress and other environmental factors. 3.2. Flavan-3-ols Healthy apple skin contained from 111.7 to 203.9 mg/kg of catechin, which is 1.2–2.6 times less than in lesions (Fig. 1F), and 148.4– 198.4 mg/kg of epicatechin, which is 1.4–1.9 times less in comparison with the lesions themselves (Fig. 2A). The tissue around the lesion contained significantly the highest amounts of catechin and epicatechin in comparison with the lesion and healthy skin tissue (Figs. 1F, 2A). For all dates at both locations there were significant differences in the content of flavan-3-ols between healthy and infected leaf tissue (Tables 3 and 4). Healthy leaves contained from 7.3 to 81.2 mg/100 g DW catechin and 22.3–137.4 mg/100 g DW
epicatechin. The catechin and epicatechin content of infected leaves increased approximately 1–3 times in comparison with healthy leaves. Some researchers [5,35] also observed a dramatic increase in catechins and their polymers in the boundary zones around the infection of V. inaequalis in apple tissue. Flavan-3-ols may interact with proteins and inhibit the enzymes secreted by diverse pathogenic fungi, which is probably the reason for the presence of catechins in defence mechanisms of plants [5]. Mayr et al. [17] reported that a rapid accumulation of catechins and oligomeric proanthocyanidins at the infection site restricted fungal spread. Feucht et al. [36] indicate that epicatechin is the main flavanol synthesised during damage to fruit by the V. inaequalis fungus. 3.3. Flavonols Among flavonols, we determined the content of rutin and quercetin-3-rhamnoside (quercitrin). The content of quercetin-3-Orutinoside (rutin), quercetin-3-O-galactoside (hyperin) and quercetin-3-O-glucoside (isoquercitrin) was expressed as an equivalent of
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
64
B
500
Epicatechin (mg/kg FW)
2005
2006
400
c
2005
b
b
c
b 200
a
a
a
a
100
JG
GD
healthy
D
a
c b
a
450
c
b
300
b
a
a
a
JG
b
c
b
GD
spot
JG
boundary
300
2005
2006
b
2006
b
250
b
b c
200
a
b c
b
150
a b
100 a
a
50
GD
JG
GD
healthy
spot
2006 c
3000 2500
b
F
b
b
b
b a
a a
a
JG
GD
healthy
b
c
2000
GD
boundary
4000
2005
0
JG
AC mM trolox/100g FW
Total phenolic content (mg GAE/kg FW)
a
a
healthy
a
1500
a
boundary
Phloridzin (mg/kg FW)
Quercetin-3-rhamnoside (mg/kg FW)
spot
c
2005
600
3500
400
a
c
b
GD
0
E
b
JG
900
150
600
0 GD
750
2006
200
0
C
c
800
b
b
300
1000
c Rutin (mg/kg FW)
A
1000
1,4
spot
boundary
2006
2005
1,2
b
1 b
0,8
JG
b b
b
b
b b
a
a a
a
0,6 0,4 0,2
500 0
0 GD
JG
healthy
GD
spot
JG
GD
JG
boundary
GD
healthy
spot
JG
boundary
Fig. 2. The content of single phenolics, total phenolics and antioxidant capacity in healthy and in infected apple peel (healthy tissue, scab spot, boundary zone between scab spot and healthy tissue) at cultivars ‘Golden Delicious’ and ‘Jonagold’ at location Maribor in years 2005 and 2006. Different letters denoted statistically significant differences between treatments within one cultivar in particular year.
Table 1 Two-way ANOVA for single phenolic compounds, total phenolics (TPC) and antioxidant capacity in apple peel of two years, two cultivars and the interaction year cultivar. Phenolic compound
Protocatechuic acid Chlorogenic acid Caffeic acid p-Coumaric acid Ferulic acid Catechin Epicatechin Rutin Quercetin-3-rhamnoside Phloridzin TPC Antioxidant capacity
Year
Cultivar
healt.
spot
boun.
healt.
*
***
***
*
***
***
**
***
*
**
***
***
***
*
*
NS
***
***
***
***
NS
***
***
***
NS
NS
NS NS
***
***
***
*
***
**
NS
***
***
Year cultivar spot
boun.
NS
NS NS
***
*
NS NS NS NS NS
NS NS NS NS
NS NS NS NS NS NS NS NS
**
***
***
NS NS NS
NS NS NS
NS NS NS
*
healt., healthy peel; spot, scab spot on peel; boun., boundary zone between scab spot and healthy tissue. NS, not significant. *Statistically significant differences at P-value below 0.05. **Statistically significant differences at P-value below 0.01. *** Statistically significant differences at P-value below 0.001.
healt.
spot
boun.
**
***
***
***
***
***
***
**
***
***
***
***
**
**
*
***
***
***
*
***
***
**
***
*
*
***
***
***
***
***
**
***
***
**
***
***
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
65
Table 2 Three-way ANOVA for single phenolic compounds and total phenolics (TPC) in apple leaves of two years, two cultivars, two locations and the interaction year cultivar location (Y C L). Phenolic compound
Year
Cultivar
healthy Chlorogenic acid Caffeic acid p-Coumaric acid Ferulic acid Catechin Epicatechin Rutin Quercetin-3-rhamnoside Phloridzin Phloretin TPC
infected
Location
healthy
YCL
infected
healthy
infected
healthy
infected
***
***
***
NS
***
*** ***
NS
***
*
**
NS
***
***
***
*
***
***
***
*
NS
NS NS
***
***
**
NS NS
***
***
***
**
**
NS
***
***
***
***
**
NS
***
***
***
***
***
***
***
***
NS
NS
***
***
***
***
**
***
*
***
***
***
**
***
*
***
**
NS
***
***
***
***
***
***
NS
**
***
***
*
***
*
***
***
NS
***
NS
***
***
***
***
NS, not significant. *Statistically significant differences at P-value below 0.05. **Statistically significant differences at P-value below 0.01. ***Statistically significant differences at P-value below 0.001.
rutin. The boundary area between the lesion and the healthy peel contained from 1.2 to 2.9 times more rutin than healthy skin (Fig. 2B). In apple leaves rutin was the main flavonol. The content of rutin in healthy leaves was from 5.1 to 10.3 mg/g DW (Tables 3 and 4). In leaves infected with apple scab, the content of rutin increased by 1.2–2.4 times. The content of quercetin-3-rhamnoside (quercitrin) in healthy apple skin was from 121.3 to 463.1 mg/kg. Its content in scab infected skin increased significantly, so that the lesion contained from 1.1 to 1.7 times more quercitrin in comparison with healthy skin (Fig. 2C). In the marginal zone between the lesion and healthy skin tissue, the values of quercitrin were statistically the highest, 1.3–2.4 times more than in healthy peel. Healthy apple-tree leaves contain from 3.9 to 8.9 mg/g DW quercetin-3-rhamnoside. Its content changed considerably during the months observed (Tables 3 and 4). The infected leaves contained 1.2–1.9 times more quercitrin in comparison with
the healthy ones. The scab infection caused increased synthesis of flavonols of rutin as well as those of quercetin-3-rhamnoside. According to Treutter [28], leaves contained higher values of quercitrin than apple peel. Feucht [37] gave the same conclusion, that leaves infected with the V. inaequalis fungus accumulated flavonols. In contrast, Picinelli et al. [34] found no relation between flavonol levels and scab resistance in apples. 3.4. Dihydrochalcones The dihydrochalcones phloridzin and phloretin are often related to resistance to numerous diseases. Healthy apple skin tissue had significantly lower values of phloridzin in comparison with infected tissue (Fig. 2D). The values of phloridzin in healthy skin were 1.3–2.1 times lower than in the lesion and even to 2.9 times lower than in the marginal tissue around the lesion. In apple leaves, phloridzin was
Table 3 The content of single phenolic compounds [mean standard error in mg/100 g or in mg/g dry weight (phloretin, rutin, phloridzin, quercetin-3-rhamnoside)] and total phenolics (mean standard error expressed as mg GAE/g DW) in healthy and infected leaves of the ‘Golden Delicious’ cultivar on different dates at the Ljubljana (LJ) and Maribor (MB) location in year 2006. Date
Loc. Treat. Catechin
Epicatechin
May 25
LJ
13.8 22.4 81.2 120.8
1.6 1.6 3.1 1.8
39.6 70.4 131.3 170.6
1.2 5.6 7.3 3.2
67.3 78.5 79.6 83.5
2.9 4.2 0.2 3.9 11.9 1.2 1.4 34.4 1.2 1.6 45.7 0.8
11.3 13.2 26.3 37.9
0.3 0.4 1.7 1.5
8.6 10.5 18.4 30.0
0.2 0.8 1.2 1.9
2.9 11.3 17.5 21.7
0.1 7.3 0.2 42.5 1.2 10.6 0.3 86.3 1.3 8.0 0.2 53.7 0.8 15.8 0.3 151.7
0.9 5.3 0.1 70.0 3.1 10.4 0.5 92.6 2.1 8.9 0.2 65.5 5.5 14.5 0.3 133.4
2.1 2.4 2.0 3.8
June 19
LJ
H I MB H I
11.9 28.8 59.2 84.2
1.2 1.1 2.5 0.7
79.2 145.6 125.2 133.1
0.9 2.3 2.9 5.9
50.0 115.5 39.0 66.3
3.2 3.1 3.2 3.8
16.7 28.7 24.2 29.1
0.3 0.6 0.7 0.7
8.0 14.6 22.4 26.5
0.3 0.7 0.4 0.6
9.9 21.3 15.2 63.8
0.2 0.5 1.6 2.5
4.0 16.8 8.8 42.7
0.2 8.4 0.2 44.6 0.7 12.4 0.4 67.3 1.3 5.6 0.2 55.5 1.2 10.3 0.4 112.9
1.8 8.2 0.2 67.5 3.3 10.2 0.2 91.6 1.9 5.9 0.2 51.7 2.9 9.5 0.2 115.5
2.2 2.9 1.0 2.2
July 14
LJ
H I MB H I
11.7 16.4 7.9 22.4
1.2 1.2 1.5 2.2
80.2 135.2 41.7 127.1
2.6 3.1 3.6 13.1
28.7 81.0 7.6 35.4
2.9 1.8 1.1 6.1
16.5 23.6 10.4 16.7
0.6 0.6 1.3 1.9
9.4 12.2 11.0 16.5
0.5 0.2 1.4 1.2
10.7 24.5 8.4 12.9
0.7 0.7 1.0 1.4
11.4 17.3 11.3 17.2
0.2 10.3 0.2 0.5 14.7 0.3 0.2 8.2 0.3 2.4 10.9 0.4
43.4 67.9 69.6 91.9
1.9 3.3 3.2 3.8
1.7 1.6 1.7 3.3
August 8
LJ
15.0 31.6 22.8 31.5
1.1 1.7 2.0 5.4
74.2 148.8 43.3 83.1
8.6 4.0 5.1 2.5
28.0 138.3 12.6 45.9
2.7 12.8 2.4 8.3 32.9 1.4 1.4 5.1 0.9 1.8 8.3 0.9
7.0 22.4 10.0 14.7
1.4 0.7 0.8 1.7
7.3 30.9 5.8 11.4
2.7 1.1 0.9 1.8
11.4 28.9 11.0 59.3
0.6 9.1 0.2 0.8 19.1 0.5 0.3 8.1 0.3 1.2 11.3 0.2
46.1 91.8 59.2 88.9
2.0 6.5 0.2 65.6 1.8 5.2 10.0 0.3 123.4 2.8 1.9 5.7 0.2 69.1 2.1 2.9 8.1 0.4 123.3 2.7
September 2 LJ
9.2 21.3 55.8 125.2
10.7 20.0 12.5 19.8
1.2 0.5 0.4 0.3
14.8 35.5 24.5 39.5
1.6 1.4 0.3 1.4
8.1 15.4 21.0 25.2
0.6 7.4 0.2 57.8 2.5 5.9 0.4 13.2 0.4 102.8 3.5 7.8 0.5 7.9 0.3 51.7 1.7 6.9 1.6 11.8 0.3 88.9 3.6 10.2
H I MB H I
H I MB H H I H MB I H
Chlorogenic Caffeic acid p-Coumaric Ferulic acid Phloretin acid acid
2.1 70.9 4.4 39.7 2.4 1.0 153.3 2.9 161.6 5.1 1.7 137.4 3.0 25.3 0.6 11.1 232.6 15.5 98.9 5.9
14.3 28.6 23.2 40.3
1.3 1.1 1.0 2.5
Rutin
Phloridzin
Q-3-ram
6.2 8.7 7.3 8.5
0.6 0.2 0.3 0.4
TPC
72.2 82.4 55.9 92.9
0.2 54.6 1.1 0.2 102.9 4.2 0.2 57.4 2.7 0.1 99.5 3.2
*Statistically significant differences between healthy and infected leaves were noted in all dates (LSD test. p < 0.05). Comparison was made between healthy and infected leaves on the same date at one location. Loc., location; Treat., treatment; Q-3-ram, quercetin-3-rhamnoside; TPC, total phenolic content; GAE, gallic acid equivalents; H, healthy; I, infected.
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
66
Table 4 The content of single phenolic compounds [mean standard error in mg/100 g or in mg/g dry weight (phloretin, rutin, phloridzin, quercetin-3-rhamnoside)] and total phenolics (mean standard error expressed as mg GAE/g DW) in healthy and infected leaves of the ‘Jonagold’ cultivar on different dates at the Ljubljana (LJ) and Maribor (MB) location in year 2006. Date
Loc. Treat. Catechin
May 25
LJ MB
June 19
LJ MB
July 14
LJ MB
August 8
LJ MB
September 2 LJ MB
Epicatechin Chlorogenic Caffeic acid p-Coumaric Ferulic acid Phloretin acid acid 59.9 77.1 85.3 96.7
Phloridzin
Q-3-ram
TPC
H I H I
23.1 36.7 76.2 117.8
1.4 55.1 2.2 74.9 1.9 71.6 1.5 152.2
3.9 4.9 1.8 1.4
10.7 16.2 20.9 30.1
0.7 2.2 2.5 0.5
7.8 11.7 22.4 25.5
0.4 1.2 0.6 0.6
7.4 9.5 18.5 21.4
0.4 1.1 0.6 0.4
69.7 107.5 13.4 15.5
5.6 8.2 0.3 59.3 9.5 10.2 0.2 70.5 0.7 5.6 0.2 46.0 0.9 13.2 0.2 127.9
3.0 3.5 1.7 2.9
7.8 9.1 4.5 9.9
0.2 61.9 0.5 95.5 0.1 57.3 0.3 125.4
1.2 2.3 0.9 2.9
H I H I
29.1 62.9 40.1 55.4
2.1 70.1 2.4 28.8 3.0 1.4 158.6 2.3 135.4 2.7 3.1 88.4 6.3 32.7 3.3 4.0 153.0 5.1 82.4 3.4
18.5 32.6 18.7 32.5
0.6 0.7 0.9 3.1
14.3 23.7 22.9 28.6
1.4 0.8 1.0 0.4
12.2 24.4 10.7 18.6
0.5 0.7 1.7 0.9
18.9 78.4 32.5 59.7
0.9 7.3 0.3 42.3 5.8 10.7 0.3 60.9 0.5 5.1 0.1 51.5 1.7 10.9 0.9 103.3
2.2 3.4 1.4 6.4
4.9 6.3 5.5 8.8
0.2 66.1 0.2 98.5 0.2 55.1 0.3 133.8
1.8 2.0 0.8 4.5
H I H I
14.7 47.9 22.2 51.9
9.0 0.7 0.5 70.5 1.2 21.9 0.3 1.2 164.7 1.9 105.2 7.2 33.4 0.6 1.0 59.3 6.9 21.5 2.1 11.7 1.4 2.3 118.4 2.5 68.5 4.9 25.7 1.9
8.0 16.6 16.8 28.0
0.2 1.0 1.4 0.8
15.4 26.2 10.1 22.6
0.5 0.5 1.3 0.9
13.1 19.7 7.5 11.9
0.2 7.3 0.2 37.7 0.3 11.1 0.2 63.2 0.3 6.6 0.1 66.1 1.6 11.8 0.4 127.5
1.4 2.3 2.8 2.9
4.5 5.4 5.3 9.2
0.1 59.4 0.1 86.6 0.2 55.7 0.2 110.8
3.5 1.4 1.5 4.2
H I H H
13.3 22.6 7.3 21.4
0.9 69.7 2.0 22.9 1.4 13.2 0.5 2.1 176.8 3.1 127.2 5.9 31.7 0.5 0.6 22.3 1.8 4.2 0.4 2.7 0.4 2.6 60.2 7.1 32.2 4.3 7.9 1.1
8.4 20.2 5.8 13.2
0.6 0.8 0.3 2.2
15.0 28.7 7.3 13.6
0.5 0.7 0.6 1.5
8.9 19.7 6.8 12.3
0.2 7.9 0.2 43.1 0.2 12.7 0.3 79.1 0.2 7.6 0.2 64.7 0.3 12.2 0.4 103.2
1.6 3.0 2.1 2.6
4.6 6.4 4.5 6.1
0.1 64.9 0.1 93.0 0.2 68.1 0.2 133.6
1.0 2.1 1.4 4.8
I H I H
21.3 39.5 15.8 25.6
1.3 89.4 1.9 36.4 0.6 17.5 1.3 2.8 179.3 3.2 141.1 3.4 37.4 0.5 1.5 63.0 2.3 11.4 0.8 8.2 1.1 1.3 116.4 2.9 65.8 1.9 20.4 0.9
12.3 19.9 6.7 11.0
0.8 1.3 0.2 0.9
20.1 31.8 12.4 29.9
0.6 0.7 0.6 3.1
12.6 21.0 8.1 16.4
0.4 7.4 0.2 47.4 0.2 13.1 0.2 73.2 0.2 7.3 0.2 63.3 0.6 12.4 0.4 104.4
1.7 2.8 2.0 6.8
4.4 6.8 3.9 5.6
0.1 59.1 0.1 87.7 0.1 70.8 0.4 113.1
1.4 0.7 2.5 4.5
3.5 7.2 1.6 2.0
Rutin
*Statistically significant differences between healthy and infected leaves were noted in all dates (LSD test. p < 0.05). Comparison was made between healthy and infected leaves on the same date at one location. Loc., location; Treat., treatment; Q-3-ram, quercetin-3-rhamnoside; TPC, total phenolic content; GAE, gallic acid equivalents; H, healthy; I, infected.
the main phenol according to quantity. The content of phloridzin in healthy leaves ranged from 37.7 to 69.6 mg/g DW (Tables 3 and 4). The infected leaves contained statistically more phloridzin in comparison with the healthy ones – i.e. from 1.2 to 2.8 times more. It is evident that infection with the V. inaequalis fungus increased higher phloridzin synthesis. This effect was also confirmed by Leser and Treutter [21]. Based on our results, it may be suggested that plants have the capacity to induce a specific environment which could limit the growth of the fungus. Obwald and Elstner [38] reported that leaves of apple-trees resistant to scab contained high concentrations of phloridzin. The proportion of flavan-3-ols and phloridzin in a plant is crucial for resistance to scab [34]. The aglycone phloretin changed considerably between dates. Its content in healthy leaves ranged from 2.9 to 69.7 mg/100 g of leaves. Differences in the content of phloretin between healthy leaves and those infected by scab were statistically significant for all dates studied (Tables 3 and 4). The infected leaves had 1.1–5.4 times more phloretin in comparison with the healthy ones. Additionally, Leser and Treutter [21] reported that the contents of aglycone phloretin fluctuated very much in apple leaves. 3.5. Total phenolic content and antioxidative activity Owing to infection with the V. inaequalis fungus, an increase in total phenols in the infected spots of apple peel occurred (Fig. 2E). The lesion had 1.3–2.1 times higher values of total phenols compared to the healthy skin, while the boundary zone between healthy tissue and lesion contained as much as 2.5 times more total phenols than healthy peel. Similar to the case of phenols, values for the antioxidative capacity of skin increase when it is scab infected (Fig. 2F); scab lesions had 1.2–1.9 times higher antioxidative values than healthy apple peel. The values of antioxidative activity of healthy skin ranged from 0.54 to 0.68 mM trolox/100g, and the values for the lesion were 1.2–1.9 times higher. Also, the scab infected leaves contained significantly higher values of total
phenols than healthy leaves (Tables 3 and 4). From these results, it is evident that the V. inaequalis fungus caused a content of total phenols that was 1.4–2.4 times higher in infected tissue in comparison with healthy leaf tissue. Our previous publication also reported that healthy tissue has significantly less total phenolic content than the scab-infected leaves [30]. If we consider the content of phenolic substances in healthy and infected tissue of either leaves or fruit peel, we can see a similar response to infection. The proliferation of phenolic compounds in the tissue in and around a symptomatic spot confirms the important role of phenolics in the plant response to infection. This was underlined by the fact that the content of phenolic compounds in the boundary area between healthy and infected tissue is the highest. In some cases this was also significantly higher when compared to tissue from the symptomatic spot itself. However it should be considered that a high accumulation of phenolics could also be part of mechanisms of general response to a stress situation, in this case caused by V. inaequalis. Further studies of the velocity of synthesis and abundance of phenolic compounds as a response to scab infection should be carried out. This could also yield clues to the difference between the responses of scab-susceptible and scabresistant cultivars. Acknowledgements This work is part of the programme Horticulture No P4-00130481 funded by the Slovenian Research Agency. References [1] Brun L, Didelot F, Parisi L. Effects of apple cultivar susceptibility to Venturia inaequalis on scab epidemics in apple orchards. Crop Prot 2008;27:1009–19. [2] Bazzi C, Messina C, Tortoreto L, Stefani E, Bini F, Brunelli A, et al. Control of pathogen incidence in pome fruits and other horticultural crop plants with prohexadione-Ca. Europ J Hort Sci 2003;68(3):108–14.
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67 [3] Agrios NG. How plants defend themselves against pathogens. In: Plant pathology. 4th ed. San Diego: Academic Press; 1997. p. 93–114. [4] Gessler C, Patocchi A, Sansavini S, Tartarini S, Gianfranceschi L. Venturia inaequalis resistance in apple. Crit Rev Plant Sci 2006;25:473–503. [5] Treutter D, Feucht W. The pattern of flavan-3-ols in relation to scab resistance of apple cultivars. J Hortic Sci 1990;65:511–7. [6] Treutter D, Feucht W. Accumulation of flavan-3-ols in fungus-infected leaves of Rosaceae. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 1990;97: 634–41. [7] Picinelli A, Suarez B, Moreno J, Dapena E, Mangas J. HPLC analysis of polyphenols in apple tree leaves. In: Brouillard R, Jay M, Scalbert A, editors. Polyphenols 94. International conference on polyphenol; 1994; Palma de Malorca, Spain. Paris: INRA; 1994. p. 315–6. 1994. [8] Mikulic Petkovsˇek M, Stampar F, Veberic R. Parameters of inner quality of the scab resistant and susceptible apple in organic and integrated production. Sci Hortic 2007;114:37–44. [9] Sierotzki H, Gessler C. Flavan-3-ols content and the resistance of Malus x domestica to Venturia inaequalis (Cooke.) Wind. Physiol Mol Plant Pathol 1993;42:291–7. [10] Hamauzu Y. Role and evolution of fruit phenolic compounds during ripening and storage. Stewart Posthar Rev 2006;2(2):1–7. [11] Hrazdina G, Borejsza-Wysocki W, Lester C. Phytoalexin production in apple cultivar resistant to Venturia inaequalis. Phytopathology 1997;87:868–76. [12] Mayr U, Batzdorfer R, Treutter D, Feucht W. Surfactant-induced changes in phenol content of apple leaves and fruit skins. Acta Hortic 1994;381: 479–87. [13] Mikulic Petkovsˇek M, Usenik V, Stampar F. The role of chlorogenic acid in the resistance of apples to apple scab (Venturia inaequalis (Cooke) G. Wind. Aderh.). Res Rep Biotech Fac Univ Ljubljana 2003;81(2):233–42. [14] Usenik V, Mikulic Petkovsˇek M, Solar A, Stampar F. Flavonols of leaves in relation to apple scab resistance. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 2004;111:137–44. [15] Treutter D. Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biol 2005;7:581–91. [16] Schwalb P, Feucht W. Changes in the concentration of phenolic substances in the bark during the annual development of the cherry tree (Prunus avium L.). Adv Hortic Sci 1999;13:71–5. [17] Mayr U, Michalek S, Treutter D, Feucht W. Phenolic compounds of apple and their relationship to scab resistance. J Phytopathol 1997;145:69–75. [18] Michalek S, Mayr U, Treutter D, Lux-Endrich A, Gutmann M, Feucht W, et al. Role of flavan-3-ols in resistance of apple trees to Venturia inaequalis. Acta Hortic 1999;484:535–9. [19] Ru¨hmann S, Leser C, Bannert M, Treutter D. Relationship between growth, secondary metabolism and resistance of apple. Plant Biol 2002;4:137–43. [20] Strissel T, Halbwirth H, Hoyer U, Zistler C, Stich K, Treutter D. Growthpromoting nitrogen nutrition affects flavonoid biosynthesis in young apple (Malus domestica Borkh.) leaves. Plant Biol 2005;7:677–85. [21] Leser C, Treutter D. Effect of nitrogen supply on growth content of phenolic compounds and pathogen (scab) resistance of apple trees. Physiol Plant 2005;123:49–56.
67
[22] Veberic R, Trobec M, Herbinger K, Hofer M, Grill D, Stampar F. Phenolic compounds in some apple (Malus domestica Borkh) cultivars of organic and integrated production. J Sci Food Agric 2005;85:1687–94. [23] Colaric M, Stampar F, Hudina M. Changes in sugars and phenolics concentrations of Williams pear leaves during growing season. Can J Plant Sci 2005;86:1203–8. [24] Escarpa A, Gonzalez MC. Optimization strategy and validation of one chromatographic method as approach to determine the phenolic compounds from different sources. J Chromatogr A 2000;897:161–70. [25] Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdicphospholungistic acid reagents. Am J Enol Vitic 1965;16:144–58. [26] Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 1995;28:25–30. [27] Veberic R, Zadravec P, Stampar F. Fruit quality of ‘Fuji’ apple (Malus domestica Borkh.) strains. J Sci Food Agric 2007;87:593–9. [28] Treutter D. Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regul 2001;34:71–89. [29] Asami DK, Hong Y-T, Barret DM, Mitchell AE. Comparison of the total phenolics and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn using conventional, organic, and sustainable agricultural practices. J Agric Food Chem 2003;5:1237–41. [30] Mikulic Petkovsˇek M, Stampar F, Veberic R. Increased phenolic content in apple leaves infected with the apple scab pathogen. J Plant Pathol 2008;90:49–55. [31] Williams EB, Kuc´ J. Resistance in Malus to Venturia inaequalis. Annu Rev Phytopathol 1969;7:223–46. [32] Michalek S, Treutter D, Mayr U, Lux-Endrich A, Gutmann M, Feucht W. Role of flavan-3-ols in resistance of apple trees to Venturia inaequalis. In: Vercauteren J, Cheze C, Dumon M, Weber J, editors. Polyphenols communications 96. 18th International conference on polyphenols; 1996 Jul 15–18; Bordeaux, France. Bordeaux: Secretariat du Groupe Polyphenols; 1996. p. 26–7. [33] Rat-Morris E, Amiot MJ, Tacchini M, Lespinasse Y. Phenolic composition of apple leaves, prior to infestation, associated with the resistance of the apple cv. Florina-Querina to the apple scab Venturia inaequalis and to the rosy apple aphid Pysaphis plantaginea. In: Vercauteren J, Cheze C, Dumon M, Weber J, editors. Polyphenols communications 96. 18th International conference on polyphenols; 1996 Jul 15–18; Bordeaux, France. Bordeaux: Secretariat du Groupe Polyphenols; 1996. p. 355–6. [34] Picinelli A, Dapena E, Mangas JJ. Polyphenolic pattern in apple tree leaves in relation to scab resistance. A preliminary study. J Agric Food Chem 1995;43:2273–8. [35] Feucht W, Treutter D, Christ E. The precise localization of catechins and proantocyanidins in protective layers around fungal-infections. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 1992;99(4):404–13. [36] Feucht W, Treutter D, Schwalb P. Principles of barrier formation of scab infected fruits. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 1998;105(4):394–403. [37] Feucht W. The localization of phenols at the cellular and tissue level. Acta Hortic 1994;381:803–15. [38] Obwald WF, Elstner EF. Die Wirt-Parasit-Interaktionen: Bakterien und Pilze als Parasiten. In: Hock B, Elstner EF, editors. Schadwirkungen auf Pflanzen. Wien: Bibliographisches Institut; 1988. p. 241–82.
Contents lists available at ScienceDirect
Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
Accumulation of phenolic compounds in apple in response to infection by the scab pathogen, Venturia inaequalis M. Mikulicˇ Petkovsˇek*, F. Sˇtampar, R. Vebericˇ University of Ljubljana, Biotechnical Faculty, Agronomy Department, Chair for Fruit Growing, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 3 September 2009
The research dealt with phenolics in healthy versus scab infected apple leaves and fruits. The leaf samples were picked in the period from May to September and the fruit samples at technological maturity. Infection with the Venturia inaequalis fungus enhanced the metabolism of phenolics at the infected sites, especially in the boundary tissue. Tissue infected with Venturia inaequalis showed in comparison to the healthy tissue up to 7.6 times more hydroxycinnamic acids, up to 2.6 times more flavan-3-ols and up to 2.9 times higher values of flavanols. The content level of total phenolics in the infected tissue was 1.3–2.4 times higher than in the healthy leaves and fruit. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Malus domestica Apple scab Hydroxycinnamic acids Flavonoids Dihydrochalcones Hydroxybenzoic acids
1. Introduction Apple scab, which is caused by the ascomycete Venturia inaequalis (Cke.) Wint., is the most important disease in all applegrowing areas with high spring and summer rainfall. The disease manifests as lesions on the surface of leaves, buds or fruit. Severe leaf spotting causes a reduction in the assimilation surface and leads to defoliation. If infection on fruit occurs early, the fruit do not expand properly at the infected portions and become undersized and gnarled. Such fruit often crack, allowing fruit-rotting organisms to invade into the apple. The disease reduces fruit yields and fruit quality. Affected fruit are not marketable, owing to the presence of the black fungal lesions. Disease control in commercial orchards can require up to 15 fungicide treatments per year. An alternative approach is the use of resistant cultivars [1] or treatment with chemical agents (i.e. prohexadione-Ca), which cause higher resistance to scab disease [2]. The secondary compounds in plants are strongly involved in the interaction between the pathogen and the plant. Phenolic compounds may contribute to the resistance of apple to Venturia inaequalis. Phenolics are toxic to pathogens. Examples of such phenolic compounds are flavanols and hydroxycinnamic acids [3,4]. In apple, flavanols play an important role in resistance to the V. inaequalis fungus [5,6]. Resistant apple cultivars have a higher content of hydroxycinnamic acids and flavanols [7,8], although susceptible cultivars are also able to accumulate flavanols in the
* Corresponding author. Tel.: þ386 1 423 11 61; fax: þ386 1 423 10 88. E-mail address: [email protected] (M. Mikulicˇ Petkovsˇek). 0885-5765/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2009.09.003
tissue surrounding infection sites [6]. Sierotzki and Gessler [9] reach the contradictory conclusion that there is no positive correlation between resistance and pre-formed flavan-3-ols in the relationship between Malus domestica and V. inaequalis. It has been reported that phloridzin also plays a role in host resistance to pathogens. It has been suggested that phloridzin can be hydrolysed in vivo by various fungi such as V. inaequalis to give phloretin, which in turn, is degraded to phloroglucinol, phloretic acid and p-hydroxybenzoic acid, which inhibits the development of the fungus [10]. Phenol-oxidizing enzymes oxidize phenolics to quinones, which are often more toxic to pathogens than the original phenol [3]. Thus Hrazdina et al. [11] reported that malusfuran produced in response to invasion by V. inaequalis inhibited spore germination and growth of V. inaequalis. The aglycone of malusfuran showed higher toxicity than malusfuran and acts as the actual phytoalexin. The production and accumulation of phenolics occurs in healthy plant cells surrounding wounded or infected cells, and they are stimulated by alarm substances produced and released by the damaged cells and diffusing into the adjacent healthy cells. Therefore, the activity of many phenol-oxidizing enzymes is generally higher in the infected tissue than in the uninfected tissue of healthy plants [3]. Changes at the phenolic level can play a role in the protection of the plant [12–15]. The phenolic derivatives can oxidize and react with proteins, thus causing a loss of enzyme function, and restricting the viability of aggressors, or they can be deposited inside the cell wall as an important first line in plant defence against infection [16]. Previous studies [17,18] revealed that, for successful protection, rapid biosynthesis of flavanols, starting from phenylalanine, were necessary.
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
The inhibition of the enzyme phenylalanine-ammonia-lyase (PAL) resulted in severe sporulation symptoms in a resistant cultivar. The biosynthesis and accumulation of secondary compounds are dependent on growth and environmental conditions. Previous investigations have shown that high nitrogen supply reduced flavonoid accumulation in leaves [19,20]. This situation is positively related to increased susceptibility to scab [19,21]. Veberic et al. [22] reported that growing technology also has an influence on the content of phenolic substances. Organically grown apples exhibit higher phenolics because of different stress factors in the orchard, such as disease, and pests, lack of mineral nutrients – all of which induce accumulation of phenolic compounds. The relationship between phenolics and apple scab has been the topic of several papers. The researchers mainly focused on how the infection alters phenolic accumulation in apple leaves. In contrast, the aim of our study was to investigate how the infection changed the synthesis of phenolic compounds in healthy and scab infected fruit. To monitor the plant response, three zones on the fruit were chosen: healthy peel, peel with the scab lesion, and the boundary zone of the scab lesion with a 1–2 mm narrow strip of healthy peel. This was done on fruit at their technological maturity in the ’Jonagold’ and ’Golden Delicious’, which are important cultivars in Europe. During the 2006 growing season the phenolic profile in healthy and scab – infected apple leaves was also monitored. Based on previous studies, we hypothesized that the infected tissue would have a higher content of certain phenolic compounds compared with healthy tissue. 2. Materials and methods 2.1. Materials 2.1.1. Plant material and growing conditions The experiment was carried out in the growing seasons 2005 and 2006. For the experiment samples were taken from apple trees grafted on M9 rootstock growing in two locations: Ljubljana (central Slovenia) and Maribor (north-eastern Slovenia). In these orchards the trees were cultivated according to the guidelines of integrated production. Despite the use of fungicides, some weak symptoms of apple scab on leaves and fruit were present. Weather parameters (temperature and rainfall) in the years 2005 and 2006 were favorable to the development of apple scab. Leaves of cultivars were sampled in year 2006 on both locations. Fruit samples were taken in both years on location Maribor. Ten fully developed healthy or infected leaves were collected from each tree. The leaves were sampled from five trees on different dates. The 3rd or 4th fully developed leaf from annual shoots was taken for analysis. From the infected leaves, only infected tissue with a surrounding narrow zone of healthy cells (1–2 mm) was taken. From each tree, five healthy or scab – infected apple fruit in technological maturity were also collected. On the fruit peel three zones were defined: healthy peel, scab lesion on peel, and a marginal section of the scab lesion with a 1–2 mm narrow strip of healthy peel. Before extraction, the leaves and fruit peel were frozen in liquid nitrogen and stored at 20 C. The leaf sampling was made on several occasions during growing season. Sampling at Ljubljana began on May 25th (34 days after full bloom) and at Maribor on May 25th (36 days after full bloom). Sampling of leaves was completed on September 2nd. 2.1.2. Chemicals The following standards were used for quantification of single phenolic compounds: protocatechuic acid was obtained from Merck (Darmstadt, Germany), chlorogenic acid (5-caffeoylquinic acid) and rutin (quercetin-3-O-rutinoside) from Sigma (St. Louis, MO, USA),
61
caffeic acid, ferulic acid, ()-epicatechin, quercitrin (quercetin-3-Orhamnoside), p-coumaric acid and phloridzin dihydrate from Fluka (Buchs, Switzerland) and (þ)-catechin from Roth (Karlsruhe, Germany). Methanol was acquired from Sigma. Water was bidistilled and purified with the Milli-Q system (Millipore, Bedford, MA, USA). For the total phenolic content, Folin-Ciocalteu reagent (Fluka), sodium carbonate (Merck), gallic acid (Sigma) and ethanol (Sigma) were used. For the antioxidant capacity 1,1-Diphenyl-2-picrylhydrazyl (DPPH), trolox and methanol were purchased from Sigma. 2.2. Extraction and determination of phenolic compounds The frozen leaves were lyophilized and ground in a mortar. Extraction with some modification was made as described by Colaric et al. [23]. The fine powder (50 mg) was extracted with methanol (20 ml) containing 1% 2.6-di-tert-butyl-4-methylphenol (BHT) for 30 min in a cooled water bath, using sonification. BHT was added to the samples to prevent oxidation during the extraction. It did not interfere with the extracted phenols during the subsequent HPLC analysis, because it was eluted at the end of the gradient or in the equilibration delay between the two analyses. The apples were peeled with a mechanical peeler, and the peel was separated from the pulp. The extraction of peel samples was done as described by Escarpa and Gonzalez [24], with some modification. The samples of 2 g peel were extracted with methanol containing 1% 2.6-di-tert-butyl-4-methylphenol (BHT) in a cooled water bath using sonification. BHT was added to the samples to prevent oxidation. The samples were extracted with 5 ml of extraction solution for 1 h, 5 ml for 30 min, and then 2.5 ml for 30 min. The extracts were then combined to a final volume of 12.5 ml. The leaf or peel extracts were centrifuged at 10.000 rpm for 10 min at 4 C, and the supernatant was filtered through a 0.45 mm membrane filter (Macherey–Nagel, Du¨ren, Germany) prior to the injection to HPLC. The phenolic compounds were analyzed on a Thermo Finnigan Surveyor HPLC system with a diode array detector, at 280 and 350 nm. The hydroxycinnamic acids (chlorogenic, p-coumaric, ferulic and caffeic acid), the hydroxybenzoic acid (protocatechuic acid) and the monomeric flavan-3-ols (catechin, epicatechin) were detected at 280 nm, whereas rutin, phloridzin and quercetin-3rhamnoside (quercitrin) were estimated at 350 nm. Spectra of the compounds were recorded between 200 and 400 nm. The column used was a Phenomenex Gemini C18 (150 4.60 mm, 3 micron), operated at 25 C. The elution solvents were A (aqueous 0.01 M phosphoric acid) and B (100% methanol). The samples were eluted according to a linear gradient described by Escarpa and Gonzales [24], with slight changes: 5% B initially; 50% B for 10 min; 70% B for 5 min; 80% B for 5 min and, finally, 100% B for 10 min. The injection amount was 10 ml for the leaf and 20 ml for the peel extract; the flow-rate was 1 ml/min. The identification of compounds was achieved by comparing the retention times and spectra as well as by the addition of the standard solution to the sample. Concentrations of phenolic compounds were calculated from the peak areas of the sample and the corresponding standards. The concentrations were expressed as mg/100 g dry weight or mg/g dry weight (DW) for leaves and as mg/kg fresh weight (FW) for peel. 2.3. Determination of total phenolic content The extraction of leaf or peel samples for the determination of total phenolics was made according to the same protocol as for phenolics, with the difference that no BHT was added. The total phenolic content (TPC) of extracts was assessed by using the FolinCiocalteu phenol reagent method [25]. To 100 ml of the sample extracts (diluted 1: 5 (v/v) with MeOH), 6 ml of bidistilled water
62
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
and 500 ml of Folin-Ciocalteu reagent were added; after resting between 8 s and 8 min at room temperature, 1.5 ml of sodium carbonate (20% w/v) was added. The extracts were mixed and allowed to stand for 30 min at 40 C before measuring the absorbance on a spectrophotometer (Perkin Elmer, UV/VIS Lambada Bio 20) at 765 nm. A mixture of water and reagents was used as a blank. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g dry weight of leaf tissue and in mg/kg fresh weight of peel tissue. Absorption was measured in three replications. 2.4. Determination of antioxidant activity with the DPPH radical scavenging method The extraction of peel samples for the determination of antioxidant activity was made according to the same protocol as for total phenolics. The free radical scavenging activity of apple extracts was measured according to the DPPH (1.1-diphenyl-2picrylhydrazyl) method reported by Brand-Williams et al. [26], with some modifications. A methanolic solution (50 ml) of peel extract (diluted 1:6) was placed in 96-well microplates, and 200 ml of a 0.1 mM methanolic solution of DPPH was added and allowed to react in the dark at room temperature. The decrease in absorbance of DPPH at 520 nm was measured at 5 min intervals by the Spectrophotometer MRX Dynex Technologies (USA), until absorbance stabilized (30 min). Methanol was used as a blank solution, and a DPPH solution without test samples served as the control. The radical stock solution was prepared fresh daily. All samples were performed in triplicate. The DPPH radical scavenging activity of peel methanolic extracts was expressed as mM of trolox per 100 g of fresh peel in 30 min of reaction time. Determination of antioxidant capacities of the samples at various concentrations was made using the standard curves of trolox. 2.5. Statistical analysis The data were analyzed by using the Statgraphics Plus 4.0 program (Manugistics. Inc.; Rockville, Maryland, USA). The significance of the infection with V. inaequalis on individual and total phenolic content was tested using the one-way analysis of variance (ANOVA). Differences between treatments were tested with the LSD test at a significance level of 0.05 Also a two-way analysis of variance was carried out to determine the significance of cultivar and year in the apple peel as well as a three-way analysis of variance for testing differences in phenolics in the apple leaves for year, cultivar and location. 3. Results and discussion In both apple cultivars we have identified eleven phenolics belonging to different groups (Tables 3 and 4, Figs. 1 and 2). In apple skin at technological maturity 3 different tissues were analyzed: healthy tissue, scab lesion and the marginal tissue around the scab lesion. In Figs. 1 and 2 the response of the plant with synthesis of phenolic compounds in these three different tissues can be seen. For individual phenolic compounds, total phenolics and antioxidant capacity the influence of the year, cultivar and their interaction was also statistically evaluated and is presented in Table 1. The interaction between year and cultivar was significant for all types of tissue in all phenolics. Since the year had a greater influence on the amount of phenolics than the cultivar, we assume that the interaction is mainly the consequence of that factor. As can be seen from Figs. 1 and 2, in the year 2006 the amounts of most of the synthesised phenolics were higher. This is probably because of influence of the environmental factors. The influence of the cultivar on the amount of phenolics is much lower and significant in only a few
cases. The reason is probably that the two cultivars in our study are closely related and the response to infection is similar. In the case of leaves, the year and cultivar were also taken into account as well as the location, since we obtained the data from two different locations (Table 2). Also in this case the interaction of all three factors was significant on the amount of phenolics in healthy and scab infected leaves. The only exception was in the infected leaves in the case of ferulic acid. All three factors contributed to the different outcome with the strongest influence again being the year. From these results we concluded that the accumulation of phenolic compounds in apple tissues is, apart from apple scab infection, also influenced by the growing season as well as variety and location. Similar results were also reported by Veberic et al. [27], who noticed that in the cultivar ’Fuji’ the amounts of analyzed phenolics were strongly dependent upon the growing season. Especially if there is a short time of stress condition this can influence the results. That cultivars can also have an influence on the amount of phenolics was reported by many papers [8,22]. In our case it was however noticed that the amounts were quite similar when observing the phenolics in the fruit peel (Table 1). The location can influence the amounts of phenolics in different ways: either is the influence of pedoclimatic factors or the influence of technological measures applied in the orchard [22,28,29]. 3.1. Phenolic acids Among the hydroxybenzoic acids, we have identified protocatechuic acid in the fruit skin. Healthy skin of all the varieties under investigation contained statistically the lowest amount of protocatechuic acid, while the lesion and the tissue around it contained a statistically higher quantity of this acid (Fig. 1A). On the lesion, up to 2.5 times more protocatechuic acid was synthesised in comparison with the healthy skin. Of the hydroxycinnamic acids, the amount of chlorogenic acid is the highest. In different tissue, healthy apple skin contained statistically the least amount of chlorogenic acid, i.e. from 96.32 to 158.26 mg/kg FW. Significantly the highest amount was observed in the tissue around the lesion and from the lesion itself. If the content of chlorogenic acid in healthy skin and in lesions is compared, its content in the latter was up to 4 times higher (Fig. 1B). The content of chlorogenic acid in healthy leaves was from 4.2 to 85.3 mg/100 g. In infected leaves, its content increased by 1.1–7.6 times in comparison with healthy leaves (Tables 3 and 4). The content of the other hydroxycinnamic acids analyzed was lower than the chlorogenic acid content. Scab infection caused increased synthesis of caffeic, p-coumaric and ferulic acid in fruit (Fig. 1C–E). In comparison with healthy skin, lesions contained 1.1–3.2 times more p-coumaric acid, 1.2–4.2 times more ferulic and 1.2–3.7 times more caffeic acid. In apple leaves, the content of p-coumaric acid, ferulic and caffeic acid changed minimally over the growth period. Generally, it may be concluded that the content of hydroxycinnamic acids increased by 1.2–3 times in comparison with healthy leaves. Our previous investigation reported that infection with scab caused an increase in their content [30]. Moreover, the findings of previous publications have shown the response of apple-tree to fungus infection as indicated by the accumulation of phenols. Back in 1969, Williams and Kuc´ [31] found that hydroxycinnamic acids inhibited the growth and sporulation of the fungus V. inaequalis. Michalek et al. [32] reported on the accumulation mainly of derivatives of caffeic acid, which is a precursor of flavonols in apple tissue. Rat-Morris et al. [33] found significant differences in the content of chlorogenic acid in appletree leaves between the scab-resistant variety ‘Florina’ and the susceptible variety ‘Melrose’. Resistant varieties contained more chlorogenic acid in comparison with susceptible ones [8]. Similar
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
60
2005
B
2006
Chlorogenic acid (mg/kg FW)
Protocatechuic acid (mg/kg FW)
A
b
50
b b
40
b 30
c b
20 10
b
a
a
b a
a
800 700
JG
GD
healthy
spot
300
b
b a
200
a
a
a
100
JG
GD
healthy
spot
JG
boundary
D 21 c
20
c
c 15
b c
b b b
10
a
a
a
a
p-Coumaric acid (mg/kg FW)
Caffeic acid (mg/kg FW)
b
b
400
boundary
0
18
c
2006
2005
b
15
c
12
b
9
a
6 3
a
b
c
c
a
healthy
spot
b a
0 GD
JG
GD
healthy
spot
JG
GD
F
21
c
2005
2006
b 15
b
b
b
b
b
9
a a
a
GD
JG
boundary
600
2006
2005
c
12
a
JG
boundary
Catechin (mg/kg FW)
Ferulic acid (mg/kg FW)
b
b
GD
2006
2005
6
c
500
JG
C 25
18
2006
c
0 GD
E
2005
600
0
5
63
c
500
b
c 400
b
300
c c
200
b
a
b
a
a
a 100
3 0 GD
JG
healthy
GD
spot
JG
0 GD
boundary
JG
healthy
GD
spot
JG
boundary
Fig. 1. The content of single phenolics in healthy and in infected apple peel (healthy tissue, scab spot, boundary zone between scab spot and healthy tissue) at cultivars ‘Golden Delicious’ and ‘Jonagold’ at location Maribor in years 2005 and 2006. Different letters denoted statistically significant differences between treatments within one cultivar in particular year.
results were obtained by Picinelli et al. [34], who found a higher content of p-coumaric acid in resistant varieties. Mayr et al. [12] reported that it was impossible to find out exactly how much a particular stress factor influenced the content of phenolic compounds in a plant, since it is an effect resulting from all the stress and other environmental factors. 3.2. Flavan-3-ols Healthy apple skin contained from 111.7 to 203.9 mg/kg of catechin, which is 1.2–2.6 times less than in lesions (Fig. 1F), and 148.4– 198.4 mg/kg of epicatechin, which is 1.4–1.9 times less in comparison with the lesions themselves (Fig. 2A). The tissue around the lesion contained significantly the highest amounts of catechin and epicatechin in comparison with the lesion and healthy skin tissue (Figs. 1F, 2A). For all dates at both locations there were significant differences in the content of flavan-3-ols between healthy and infected leaf tissue (Tables 3 and 4). Healthy leaves contained from 7.3 to 81.2 mg/100 g DW catechin and 22.3–137.4 mg/100 g DW
epicatechin. The catechin and epicatechin content of infected leaves increased approximately 1–3 times in comparison with healthy leaves. Some researchers [5,35] also observed a dramatic increase in catechins and their polymers in the boundary zones around the infection of V. inaequalis in apple tissue. Flavan-3-ols may interact with proteins and inhibit the enzymes secreted by diverse pathogenic fungi, which is probably the reason for the presence of catechins in defence mechanisms of plants [5]. Mayr et al. [17] reported that a rapid accumulation of catechins and oligomeric proanthocyanidins at the infection site restricted fungal spread. Feucht et al. [36] indicate that epicatechin is the main flavanol synthesised during damage to fruit by the V. inaequalis fungus. 3.3. Flavonols Among flavonols, we determined the content of rutin and quercetin-3-rhamnoside (quercitrin). The content of quercetin-3-Orutinoside (rutin), quercetin-3-O-galactoside (hyperin) and quercetin-3-O-glucoside (isoquercitrin) was expressed as an equivalent of
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
64
B
500
Epicatechin (mg/kg FW)
2005
2006
400
c
2005
b
b
c
b 200
a
a
a
a
100
JG
GD
healthy
D
a
c b
a
450
c
b
300
b
a
a
a
JG
b
c
b
GD
spot
JG
boundary
300
2005
2006
b
2006
b
250
b
b c
200
a
b c
b
150
a b
100 a
a
50
GD
JG
GD
healthy
spot
2006 c
3000 2500
b
F
b
b
b
b a
a a
a
JG
GD
healthy
b
c
2000
GD
boundary
4000
2005
0
JG
AC mM trolox/100g FW
Total phenolic content (mg GAE/kg FW)
a
a
healthy
a
1500
a
boundary
Phloridzin (mg/kg FW)
Quercetin-3-rhamnoside (mg/kg FW)
spot
c
2005
600
3500
400
a
c
b
GD
0
E
b
JG
900
150
600
0 GD
750
2006
200
0
C
c
800
b
b
300
1000
c Rutin (mg/kg FW)
A
1000
1,4
spot
boundary
2006
2005
1,2
b
1 b
0,8
JG
b b
b
b
b b
a
a a
a
0,6 0,4 0,2
500 0
0 GD
JG
healthy
GD
spot
JG
GD
JG
boundary
GD
healthy
spot
JG
boundary
Fig. 2. The content of single phenolics, total phenolics and antioxidant capacity in healthy and in infected apple peel (healthy tissue, scab spot, boundary zone between scab spot and healthy tissue) at cultivars ‘Golden Delicious’ and ‘Jonagold’ at location Maribor in years 2005 and 2006. Different letters denoted statistically significant differences between treatments within one cultivar in particular year.
Table 1 Two-way ANOVA for single phenolic compounds, total phenolics (TPC) and antioxidant capacity in apple peel of two years, two cultivars and the interaction year cultivar. Phenolic compound
Protocatechuic acid Chlorogenic acid Caffeic acid p-Coumaric acid Ferulic acid Catechin Epicatechin Rutin Quercetin-3-rhamnoside Phloridzin TPC Antioxidant capacity
Year
Cultivar
healt.
spot
boun.
healt.
*
***
***
*
***
***
**
***
*
**
***
***
***
*
*
NS
***
***
***
***
NS
***
***
***
NS
NS
NS NS
***
***
***
*
***
**
NS
***
***
Year cultivar spot
boun.
NS
NS NS
***
*
NS NS NS NS NS
NS NS NS NS
NS NS NS NS NS NS NS NS
**
***
***
NS NS NS
NS NS NS
NS NS NS
*
healt., healthy peel; spot, scab spot on peel; boun., boundary zone between scab spot and healthy tissue. NS, not significant. *Statistically significant differences at P-value below 0.05. **Statistically significant differences at P-value below 0.01. *** Statistically significant differences at P-value below 0.001.
healt.
spot
boun.
**
***
***
***
***
***
***
**
***
***
***
***
**
**
*
***
***
***
*
***
***
**
***
*
*
***
***
***
***
***
**
***
***
**
***
***
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
65
Table 2 Three-way ANOVA for single phenolic compounds and total phenolics (TPC) in apple leaves of two years, two cultivars, two locations and the interaction year cultivar location (Y C L). Phenolic compound
Year
Cultivar
healthy Chlorogenic acid Caffeic acid p-Coumaric acid Ferulic acid Catechin Epicatechin Rutin Quercetin-3-rhamnoside Phloridzin Phloretin TPC
infected
Location
healthy
YCL
infected
healthy
infected
healthy
infected
***
***
***
NS
***
*** ***
NS
***
*
**
NS
***
***
***
*
***
***
***
*
NS
NS NS
***
***
**
NS NS
***
***
***
**
**
NS
***
***
***
***
**
NS
***
***
***
***
***
***
***
***
NS
NS
***
***
***
***
**
***
*
***
***
***
**
***
*
***
**
NS
***
***
***
***
***
***
NS
**
***
***
*
***
*
***
***
NS
***
NS
***
***
***
***
NS, not significant. *Statistically significant differences at P-value below 0.05. **Statistically significant differences at P-value below 0.01. ***Statistically significant differences at P-value below 0.001.
rutin. The boundary area between the lesion and the healthy peel contained from 1.2 to 2.9 times more rutin than healthy skin (Fig. 2B). In apple leaves rutin was the main flavonol. The content of rutin in healthy leaves was from 5.1 to 10.3 mg/g DW (Tables 3 and 4). In leaves infected with apple scab, the content of rutin increased by 1.2–2.4 times. The content of quercetin-3-rhamnoside (quercitrin) in healthy apple skin was from 121.3 to 463.1 mg/kg. Its content in scab infected skin increased significantly, so that the lesion contained from 1.1 to 1.7 times more quercitrin in comparison with healthy skin (Fig. 2C). In the marginal zone between the lesion and healthy skin tissue, the values of quercitrin were statistically the highest, 1.3–2.4 times more than in healthy peel. Healthy apple-tree leaves contain from 3.9 to 8.9 mg/g DW quercetin-3-rhamnoside. Its content changed considerably during the months observed (Tables 3 and 4). The infected leaves contained 1.2–1.9 times more quercitrin in comparison with
the healthy ones. The scab infection caused increased synthesis of flavonols of rutin as well as those of quercetin-3-rhamnoside. According to Treutter [28], leaves contained higher values of quercitrin than apple peel. Feucht [37] gave the same conclusion, that leaves infected with the V. inaequalis fungus accumulated flavonols. In contrast, Picinelli et al. [34] found no relation between flavonol levels and scab resistance in apples. 3.4. Dihydrochalcones The dihydrochalcones phloridzin and phloretin are often related to resistance to numerous diseases. Healthy apple skin tissue had significantly lower values of phloridzin in comparison with infected tissue (Fig. 2D). The values of phloridzin in healthy skin were 1.3–2.1 times lower than in the lesion and even to 2.9 times lower than in the marginal tissue around the lesion. In apple leaves, phloridzin was
Table 3 The content of single phenolic compounds [mean standard error in mg/100 g or in mg/g dry weight (phloretin, rutin, phloridzin, quercetin-3-rhamnoside)] and total phenolics (mean standard error expressed as mg GAE/g DW) in healthy and infected leaves of the ‘Golden Delicious’ cultivar on different dates at the Ljubljana (LJ) and Maribor (MB) location in year 2006. Date
Loc. Treat. Catechin
Epicatechin
May 25
LJ
13.8 22.4 81.2 120.8
1.6 1.6 3.1 1.8
39.6 70.4 131.3 170.6
1.2 5.6 7.3 3.2
67.3 78.5 79.6 83.5
2.9 4.2 0.2 3.9 11.9 1.2 1.4 34.4 1.2 1.6 45.7 0.8
11.3 13.2 26.3 37.9
0.3 0.4 1.7 1.5
8.6 10.5 18.4 30.0
0.2 0.8 1.2 1.9
2.9 11.3 17.5 21.7
0.1 7.3 0.2 42.5 1.2 10.6 0.3 86.3 1.3 8.0 0.2 53.7 0.8 15.8 0.3 151.7
0.9 5.3 0.1 70.0 3.1 10.4 0.5 92.6 2.1 8.9 0.2 65.5 5.5 14.5 0.3 133.4
2.1 2.4 2.0 3.8
June 19
LJ
H I MB H I
11.9 28.8 59.2 84.2
1.2 1.1 2.5 0.7
79.2 145.6 125.2 133.1
0.9 2.3 2.9 5.9
50.0 115.5 39.0 66.3
3.2 3.1 3.2 3.8
16.7 28.7 24.2 29.1
0.3 0.6 0.7 0.7
8.0 14.6 22.4 26.5
0.3 0.7 0.4 0.6
9.9 21.3 15.2 63.8
0.2 0.5 1.6 2.5
4.0 16.8 8.8 42.7
0.2 8.4 0.2 44.6 0.7 12.4 0.4 67.3 1.3 5.6 0.2 55.5 1.2 10.3 0.4 112.9
1.8 8.2 0.2 67.5 3.3 10.2 0.2 91.6 1.9 5.9 0.2 51.7 2.9 9.5 0.2 115.5
2.2 2.9 1.0 2.2
July 14
LJ
H I MB H I
11.7 16.4 7.9 22.4
1.2 1.2 1.5 2.2
80.2 135.2 41.7 127.1
2.6 3.1 3.6 13.1
28.7 81.0 7.6 35.4
2.9 1.8 1.1 6.1
16.5 23.6 10.4 16.7
0.6 0.6 1.3 1.9
9.4 12.2 11.0 16.5
0.5 0.2 1.4 1.2
10.7 24.5 8.4 12.9
0.7 0.7 1.0 1.4
11.4 17.3 11.3 17.2
0.2 10.3 0.2 0.5 14.7 0.3 0.2 8.2 0.3 2.4 10.9 0.4
43.4 67.9 69.6 91.9
1.9 3.3 3.2 3.8
1.7 1.6 1.7 3.3
August 8
LJ
15.0 31.6 22.8 31.5
1.1 1.7 2.0 5.4
74.2 148.8 43.3 83.1
8.6 4.0 5.1 2.5
28.0 138.3 12.6 45.9
2.7 12.8 2.4 8.3 32.9 1.4 1.4 5.1 0.9 1.8 8.3 0.9
7.0 22.4 10.0 14.7
1.4 0.7 0.8 1.7
7.3 30.9 5.8 11.4
2.7 1.1 0.9 1.8
11.4 28.9 11.0 59.3
0.6 9.1 0.2 0.8 19.1 0.5 0.3 8.1 0.3 1.2 11.3 0.2
46.1 91.8 59.2 88.9
2.0 6.5 0.2 65.6 1.8 5.2 10.0 0.3 123.4 2.8 1.9 5.7 0.2 69.1 2.1 2.9 8.1 0.4 123.3 2.7
September 2 LJ
9.2 21.3 55.8 125.2
10.7 20.0 12.5 19.8
1.2 0.5 0.4 0.3
14.8 35.5 24.5 39.5
1.6 1.4 0.3 1.4
8.1 15.4 21.0 25.2
0.6 7.4 0.2 57.8 2.5 5.9 0.4 13.2 0.4 102.8 3.5 7.8 0.5 7.9 0.3 51.7 1.7 6.9 1.6 11.8 0.3 88.9 3.6 10.2
H I MB H I
H I MB H H I H MB I H
Chlorogenic Caffeic acid p-Coumaric Ferulic acid Phloretin acid acid
2.1 70.9 4.4 39.7 2.4 1.0 153.3 2.9 161.6 5.1 1.7 137.4 3.0 25.3 0.6 11.1 232.6 15.5 98.9 5.9
14.3 28.6 23.2 40.3
1.3 1.1 1.0 2.5
Rutin
Phloridzin
Q-3-ram
6.2 8.7 7.3 8.5
0.6 0.2 0.3 0.4
TPC
72.2 82.4 55.9 92.9
0.2 54.6 1.1 0.2 102.9 4.2 0.2 57.4 2.7 0.1 99.5 3.2
*Statistically significant differences between healthy and infected leaves were noted in all dates (LSD test. p < 0.05). Comparison was made between healthy and infected leaves on the same date at one location. Loc., location; Treat., treatment; Q-3-ram, quercetin-3-rhamnoside; TPC, total phenolic content; GAE, gallic acid equivalents; H, healthy; I, infected.
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67
66
Table 4 The content of single phenolic compounds [mean standard error in mg/100 g or in mg/g dry weight (phloretin, rutin, phloridzin, quercetin-3-rhamnoside)] and total phenolics (mean standard error expressed as mg GAE/g DW) in healthy and infected leaves of the ‘Jonagold’ cultivar on different dates at the Ljubljana (LJ) and Maribor (MB) location in year 2006. Date
Loc. Treat. Catechin
May 25
LJ MB
June 19
LJ MB
July 14
LJ MB
August 8
LJ MB
September 2 LJ MB
Epicatechin Chlorogenic Caffeic acid p-Coumaric Ferulic acid Phloretin acid acid 59.9 77.1 85.3 96.7
Phloridzin
Q-3-ram
TPC
H I H I
23.1 36.7 76.2 117.8
1.4 55.1 2.2 74.9 1.9 71.6 1.5 152.2
3.9 4.9 1.8 1.4
10.7 16.2 20.9 30.1
0.7 2.2 2.5 0.5
7.8 11.7 22.4 25.5
0.4 1.2 0.6 0.6
7.4 9.5 18.5 21.4
0.4 1.1 0.6 0.4
69.7 107.5 13.4 15.5
5.6 8.2 0.3 59.3 9.5 10.2 0.2 70.5 0.7 5.6 0.2 46.0 0.9 13.2 0.2 127.9
3.0 3.5 1.7 2.9
7.8 9.1 4.5 9.9
0.2 61.9 0.5 95.5 0.1 57.3 0.3 125.4
1.2 2.3 0.9 2.9
H I H I
29.1 62.9 40.1 55.4
2.1 70.1 2.4 28.8 3.0 1.4 158.6 2.3 135.4 2.7 3.1 88.4 6.3 32.7 3.3 4.0 153.0 5.1 82.4 3.4
18.5 32.6 18.7 32.5
0.6 0.7 0.9 3.1
14.3 23.7 22.9 28.6
1.4 0.8 1.0 0.4
12.2 24.4 10.7 18.6
0.5 0.7 1.7 0.9
18.9 78.4 32.5 59.7
0.9 7.3 0.3 42.3 5.8 10.7 0.3 60.9 0.5 5.1 0.1 51.5 1.7 10.9 0.9 103.3
2.2 3.4 1.4 6.4
4.9 6.3 5.5 8.8
0.2 66.1 0.2 98.5 0.2 55.1 0.3 133.8
1.8 2.0 0.8 4.5
H I H I
14.7 47.9 22.2 51.9
9.0 0.7 0.5 70.5 1.2 21.9 0.3 1.2 164.7 1.9 105.2 7.2 33.4 0.6 1.0 59.3 6.9 21.5 2.1 11.7 1.4 2.3 118.4 2.5 68.5 4.9 25.7 1.9
8.0 16.6 16.8 28.0
0.2 1.0 1.4 0.8
15.4 26.2 10.1 22.6
0.5 0.5 1.3 0.9
13.1 19.7 7.5 11.9
0.2 7.3 0.2 37.7 0.3 11.1 0.2 63.2 0.3 6.6 0.1 66.1 1.6 11.8 0.4 127.5
1.4 2.3 2.8 2.9
4.5 5.4 5.3 9.2
0.1 59.4 0.1 86.6 0.2 55.7 0.2 110.8
3.5 1.4 1.5 4.2
H I H H
13.3 22.6 7.3 21.4
0.9 69.7 2.0 22.9 1.4 13.2 0.5 2.1 176.8 3.1 127.2 5.9 31.7 0.5 0.6 22.3 1.8 4.2 0.4 2.7 0.4 2.6 60.2 7.1 32.2 4.3 7.9 1.1
8.4 20.2 5.8 13.2
0.6 0.8 0.3 2.2
15.0 28.7 7.3 13.6
0.5 0.7 0.6 1.5
8.9 19.7 6.8 12.3
0.2 7.9 0.2 43.1 0.2 12.7 0.3 79.1 0.2 7.6 0.2 64.7 0.3 12.2 0.4 103.2
1.6 3.0 2.1 2.6
4.6 6.4 4.5 6.1
0.1 64.9 0.1 93.0 0.2 68.1 0.2 133.6
1.0 2.1 1.4 4.8
I H I H
21.3 39.5 15.8 25.6
1.3 89.4 1.9 36.4 0.6 17.5 1.3 2.8 179.3 3.2 141.1 3.4 37.4 0.5 1.5 63.0 2.3 11.4 0.8 8.2 1.1 1.3 116.4 2.9 65.8 1.9 20.4 0.9
12.3 19.9 6.7 11.0
0.8 1.3 0.2 0.9
20.1 31.8 12.4 29.9
0.6 0.7 0.6 3.1
12.6 21.0 8.1 16.4
0.4 7.4 0.2 47.4 0.2 13.1 0.2 73.2 0.2 7.3 0.2 63.3 0.6 12.4 0.4 104.4
1.7 2.8 2.0 6.8
4.4 6.8 3.9 5.6
0.1 59.1 0.1 87.7 0.1 70.8 0.4 113.1
1.4 0.7 2.5 4.5
3.5 7.2 1.6 2.0
Rutin
*Statistically significant differences between healthy and infected leaves were noted in all dates (LSD test. p < 0.05). Comparison was made between healthy and infected leaves on the same date at one location. Loc., location; Treat., treatment; Q-3-ram, quercetin-3-rhamnoside; TPC, total phenolic content; GAE, gallic acid equivalents; H, healthy; I, infected.
the main phenol according to quantity. The content of phloridzin in healthy leaves ranged from 37.7 to 69.6 mg/g DW (Tables 3 and 4). The infected leaves contained statistically more phloridzin in comparison with the healthy ones – i.e. from 1.2 to 2.8 times more. It is evident that infection with the V. inaequalis fungus increased higher phloridzin synthesis. This effect was also confirmed by Leser and Treutter [21]. Based on our results, it may be suggested that plants have the capacity to induce a specific environment which could limit the growth of the fungus. Obwald and Elstner [38] reported that leaves of apple-trees resistant to scab contained high concentrations of phloridzin. The proportion of flavan-3-ols and phloridzin in a plant is crucial for resistance to scab [34]. The aglycone phloretin changed considerably between dates. Its content in healthy leaves ranged from 2.9 to 69.7 mg/100 g of leaves. Differences in the content of phloretin between healthy leaves and those infected by scab were statistically significant for all dates studied (Tables 3 and 4). The infected leaves had 1.1–5.4 times more phloretin in comparison with the healthy ones. Additionally, Leser and Treutter [21] reported that the contents of aglycone phloretin fluctuated very much in apple leaves. 3.5. Total phenolic content and antioxidative activity Owing to infection with the V. inaequalis fungus, an increase in total phenols in the infected spots of apple peel occurred (Fig. 2E). The lesion had 1.3–2.1 times higher values of total phenols compared to the healthy skin, while the boundary zone between healthy tissue and lesion contained as much as 2.5 times more total phenols than healthy peel. Similar to the case of phenols, values for the antioxidative capacity of skin increase when it is scab infected (Fig. 2F); scab lesions had 1.2–1.9 times higher antioxidative values than healthy apple peel. The values of antioxidative activity of healthy skin ranged from 0.54 to 0.68 mM trolox/100g, and the values for the lesion were 1.2–1.9 times higher. Also, the scab infected leaves contained significantly higher values of total
phenols than healthy leaves (Tables 3 and 4). From these results, it is evident that the V. inaequalis fungus caused a content of total phenols that was 1.4–2.4 times higher in infected tissue in comparison with healthy leaf tissue. Our previous publication also reported that healthy tissue has significantly less total phenolic content than the scab-infected leaves [30]. If we consider the content of phenolic substances in healthy and infected tissue of either leaves or fruit peel, we can see a similar response to infection. The proliferation of phenolic compounds in the tissue in and around a symptomatic spot confirms the important role of phenolics in the plant response to infection. This was underlined by the fact that the content of phenolic compounds in the boundary area between healthy and infected tissue is the highest. In some cases this was also significantly higher when compared to tissue from the symptomatic spot itself. However it should be considered that a high accumulation of phenolics could also be part of mechanisms of general response to a stress situation, in this case caused by V. inaequalis. Further studies of the velocity of synthesis and abundance of phenolic compounds as a response to scab infection should be carried out. This could also yield clues to the difference between the responses of scab-susceptible and scabresistant cultivars. Acknowledgements This work is part of the programme Horticulture No P4-00130481 funded by the Slovenian Research Agency. References [1] Brun L, Didelot F, Parisi L. Effects of apple cultivar susceptibility to Venturia inaequalis on scab epidemics in apple orchards. Crop Prot 2008;27:1009–19. [2] Bazzi C, Messina C, Tortoreto L, Stefani E, Bini F, Brunelli A, et al. Control of pathogen incidence in pome fruits and other horticultural crop plants with prohexadione-Ca. Europ J Hort Sci 2003;68(3):108–14.
M. Mikulicˇ Petkovsˇek et al. / Physiological and Molecular Plant Pathology 74 (2009) 60–67 [3] Agrios NG. How plants defend themselves against pathogens. In: Plant pathology. 4th ed. San Diego: Academic Press; 1997. p. 93–114. [4] Gessler C, Patocchi A, Sansavini S, Tartarini S, Gianfranceschi L. Venturia inaequalis resistance in apple. Crit Rev Plant Sci 2006;25:473–503. [5] Treutter D, Feucht W. The pattern of flavan-3-ols in relation to scab resistance of apple cultivars. J Hortic Sci 1990;65:511–7. [6] Treutter D, Feucht W. Accumulation of flavan-3-ols in fungus-infected leaves of Rosaceae. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 1990;97: 634–41. [7] Picinelli A, Suarez B, Moreno J, Dapena E, Mangas J. HPLC analysis of polyphenols in apple tree leaves. In: Brouillard R, Jay M, Scalbert A, editors. Polyphenols 94. International conference on polyphenol; 1994; Palma de Malorca, Spain. Paris: INRA; 1994. p. 315–6. 1994. [8] Mikulic Petkovsˇek M, Stampar F, Veberic R. Parameters of inner quality of the scab resistant and susceptible apple in organic and integrated production. Sci Hortic 2007;114:37–44. [9] Sierotzki H, Gessler C. Flavan-3-ols content and the resistance of Malus x domestica to Venturia inaequalis (Cooke.) Wind. Physiol Mol Plant Pathol 1993;42:291–7. [10] Hamauzu Y. Role and evolution of fruit phenolic compounds during ripening and storage. Stewart Posthar Rev 2006;2(2):1–7. [11] Hrazdina G, Borejsza-Wysocki W, Lester C. Phytoalexin production in apple cultivar resistant to Venturia inaequalis. Phytopathology 1997;87:868–76. [12] Mayr U, Batzdorfer R, Treutter D, Feucht W. Surfactant-induced changes in phenol content of apple leaves and fruit skins. Acta Hortic 1994;381: 479–87. [13] Mikulic Petkovsˇek M, Usenik V, Stampar F. The role of chlorogenic acid in the resistance of apples to apple scab (Venturia inaequalis (Cooke) G. Wind. Aderh.). Res Rep Biotech Fac Univ Ljubljana 2003;81(2):233–42. [14] Usenik V, Mikulic Petkovsˇek M, Solar A, Stampar F. Flavonols of leaves in relation to apple scab resistance. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 2004;111:137–44. [15] Treutter D. Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biol 2005;7:581–91. [16] Schwalb P, Feucht W. Changes in the concentration of phenolic substances in the bark during the annual development of the cherry tree (Prunus avium L.). Adv Hortic Sci 1999;13:71–5. [17] Mayr U, Michalek S, Treutter D, Feucht W. Phenolic compounds of apple and their relationship to scab resistance. J Phytopathol 1997;145:69–75. [18] Michalek S, Mayr U, Treutter D, Lux-Endrich A, Gutmann M, Feucht W, et al. Role of flavan-3-ols in resistance of apple trees to Venturia inaequalis. Acta Hortic 1999;484:535–9. [19] Ru¨hmann S, Leser C, Bannert M, Treutter D. Relationship between growth, secondary metabolism and resistance of apple. Plant Biol 2002;4:137–43. [20] Strissel T, Halbwirth H, Hoyer U, Zistler C, Stich K, Treutter D. Growthpromoting nitrogen nutrition affects flavonoid biosynthesis in young apple (Malus domestica Borkh.) leaves. Plant Biol 2005;7:677–85. [21] Leser C, Treutter D. Effect of nitrogen supply on growth content of phenolic compounds and pathogen (scab) resistance of apple trees. Physiol Plant 2005;123:49–56.
67
[22] Veberic R, Trobec M, Herbinger K, Hofer M, Grill D, Stampar F. Phenolic compounds in some apple (Malus domestica Borkh) cultivars of organic and integrated production. J Sci Food Agric 2005;85:1687–94. [23] Colaric M, Stampar F, Hudina M. Changes in sugars and phenolics concentrations of Williams pear leaves during growing season. Can J Plant Sci 2005;86:1203–8. [24] Escarpa A, Gonzalez MC. Optimization strategy and validation of one chromatographic method as approach to determine the phenolic compounds from different sources. J Chromatogr A 2000;897:161–70. [25] Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdicphospholungistic acid reagents. Am J Enol Vitic 1965;16:144–58. [26] Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 1995;28:25–30. [27] Veberic R, Zadravec P, Stampar F. Fruit quality of ‘Fuji’ apple (Malus domestica Borkh.) strains. J Sci Food Agric 2007;87:593–9. [28] Treutter D. Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regul 2001;34:71–89. [29] Asami DK, Hong Y-T, Barret DM, Mitchell AE. Comparison of the total phenolics and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn using conventional, organic, and sustainable agricultural practices. J Agric Food Chem 2003;5:1237–41. [30] Mikulic Petkovsˇek M, Stampar F, Veberic R. Increased phenolic content in apple leaves infected with the apple scab pathogen. J Plant Pathol 2008;90:49–55. [31] Williams EB, Kuc´ J. Resistance in Malus to Venturia inaequalis. Annu Rev Phytopathol 1969;7:223–46. [32] Michalek S, Treutter D, Mayr U, Lux-Endrich A, Gutmann M, Feucht W. Role of flavan-3-ols in resistance of apple trees to Venturia inaequalis. In: Vercauteren J, Cheze C, Dumon M, Weber J, editors. Polyphenols communications 96. 18th International conference on polyphenols; 1996 Jul 15–18; Bordeaux, France. Bordeaux: Secretariat du Groupe Polyphenols; 1996. p. 26–7. [33] Rat-Morris E, Amiot MJ, Tacchini M, Lespinasse Y. Phenolic composition of apple leaves, prior to infestation, associated with the resistance of the apple cv. Florina-Querina to the apple scab Venturia inaequalis and to the rosy apple aphid Pysaphis plantaginea. In: Vercauteren J, Cheze C, Dumon M, Weber J, editors. Polyphenols communications 96. 18th International conference on polyphenols; 1996 Jul 15–18; Bordeaux, France. Bordeaux: Secretariat du Groupe Polyphenols; 1996. p. 355–6. [34] Picinelli A, Dapena E, Mangas JJ. Polyphenolic pattern in apple tree leaves in relation to scab resistance. A preliminary study. J Agric Food Chem 1995;43:2273–8. [35] Feucht W, Treutter D, Christ E. The precise localization of catechins and proantocyanidins in protective layers around fungal-infections. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 1992;99(4):404–13. [36] Feucht W, Treutter D, Schwalb P. Principles of barrier formation of scab infected fruits. Z Pflanzenkr Pflanzenpathol Pflanzenschutz 1998;105(4):394–403. [37] Feucht W. The localization of phenols at the cellular and tissue level. Acta Hortic 1994;381:803–15. [38] Obwald WF, Elstner EF. Die Wirt-Parasit-Interaktionen: Bakterien und Pilze als Parasiten. In: Hock B, Elstner EF, editors. Schadwirkungen auf Pflanzen. Wien: Bibliographisches Institut; 1988. p. 241–82.