In vitro antifungal activity and mode of action of selected polyphenolic antioxidants on Botrytis cinerea

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In vitro antifungal activity and mode of action of selected polyphenolicantioxidants on Botrytis cinereaShutian Taoab; Shaoling Zhanga; Rong Tsaoc; Marie Thérèse Charlesb; Raymond Yangc; ShahrokhKhanizadehb

a College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu Province, P.R. China b

Horticultural Research and Development Centre, Agriculture and Agri-Food Canada, St-Jean-sur-Richelieu, Quebec, Canada c Food Research Program, Agriculture and Agri-Food Canada, Guelph,Ontario, Canada

Online publication date: 09 November 2010

To cite this Article Tao, Shutian , Zhang, Shaoling , Tsao, Rong , Charles, Marie Thérèse , Yang, Raymond andKhanizadeh, Shahrokh(2010) 'In vitro antifungal activity and mode of action of selected polyphenolic antioxidants onBotrytis cinerea', Archives Of Phytopathology And Plant Protection, 43: 16, 1564 — 1578To link to this Article: DOI: 10.1080/03235400802583834URL: http://dx.doi.org/10.1080/03235400802583834

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In vitro antifungal activity and mode of action of selected polyphenolic

antioxidants on Botrytis cinerea

Shutian Taoa,b, Shaoling Zhanga, Rong Tsaoc, Marie Therese Charlesb,Raymond Yangc and Shahrokh Khanizadehb*

aCollege of Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, JiangsuProvince, P.R. China; bHorticultural Research and Development Centre, Agriculture and Agri-FoodCanada, 430 Gouin Blvd., St-Jean-sur-Richelieu, J3B 3E6, Quebec, Canada; cFood Research Program,Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph N1G 5C9, Ontario, Canada

(Received 22 September 2008; final version received 22 October 2008)

Six selected antioxidants (catechin, quercetin-3-galactoside, cyanidin-3-glucoside,pelargonidin-3-glucoside, ellagic and gallic acids) were evaluated in vitro for theirantifungal activities and mode of action on Botrytis cinerea Pers., one of the mostimportant pathogens of strawberries. Inhibitory effects were found for all the testedantioxidants, but varied at different fungal developmental stages. Catechin andquercetin-3-galactoside showed linear inhibitory effects on germ tube elongation, withthe highest suppression ratios of 54.8% and 58.8% respectively. No significant effectwas found on spore germination between treatments and control. Gallic acid showedvery strong and linear inhibition on spore germination (r ¼ 70.95), but the effectdiminished after spore germination. Cyanidin-3-glucoside and pelargonidin-3-glucosideprovided effective control on the fungi as concentrations increased. The arresting effectof ellagic acid on development of B. cinerea was quadratic. Ellagic acid inhibited germtube elongation and mycelial growth at its highest and lowest concentrations, while noeffects were observed at its medium concentration used in this study.

Keywords: Botrytis cinerea; ellagic acid; gallic acid; anthocyanin; catechin; flavonol

Introduction

Strawberry (Fragaria x ananassa Duch) as an important cash crop in Canada is verynutritional but also delicate and perishable. Gray mold is the most destructive, commonand widely distributed fungal disease caused by Botrytis cinerea Pers., causing seriouslosses to strawberry during the growing season and storage. Disease control is difficult,because the pathogen can infect all plant parts at any stages of growth and also it is notpossible to use fungicide at harvesting. The effect of gray mold is more pronounced duringthe post-harvest storage due to environmental conditions and the declining resistance offruit with its ripening process which causes short shelf life and decay by as much as 55%(Martınez-Romero et al. 2007).

The sensitivity of Botrytis cinerea to plant phytochemicals such as phenolic compoundshave been studied by several research groups (Hebert et al. 2002; Khanizadeh et al.2002b; Daferera et al. 2003; Soylu 2006; Lee et al. 2007; Martınez-Romero et al. 2007;

*Corresponding author. Email: [email protected]

Archives of Phytopathology and Plant Protection

Vol. 43, No. 16, 1 November 2010, 1564–1578

ISSN 0323-5408 print/ISSN 1477-2906 online

� 2010 Taylor & Francis

DOI: 10.1080/03235400802583834

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Ehsani-Moghaddam et al. 2008; Khanizadeh et al. 2008a, 2008b); Chinese medicinal plantextracts, plant essential oil, rich in phenolics and carvacrol (a kind of phenolic compound)were found to be effective at inhibiting Botrytis cinerea growth in vitro (Daferera et al.2003; Lee et al. 2007; Martınez-Romero et al. 2007). Feucht et al. (1992) detected aboundary zone surrounding Botrytis-infected strawberry tissues enriched in catechin, themain constitutive unit of proanthocyanidin, which are oligomers of flavan-3-ols. In ourlab, we also established a positive correlation between the antioxidant capacity, shelf lifeand disease resistance (Khanizadeh et al. 2002b, 2007, 2008a, 2008b; Ehsani-Moghaddamet al. 2008).

Hebert et al. (2002) reported a higher degree of resistant to gray mold growth of sixstrawberry cultivars and concluded that ‘Seascape’ proanthocyanidins (PA) extract gavethe highest inhibition of mold radial growth (76.2%) and that its fruit was also the mostresistant to the appearance of mold during storage. However, the result did not indicatewhich phenolic composition or combinations are responsible for inhibiting growth of thegray mold in vitro.

Strawberries have been shown to be a rich source of phenolic compounds withantioxidant activity (Wang et al. 1996; Guo et al. 2003; Scalzo et al. 2005; Khanizadehet al. 2006, 2008a), but several factors influence the antioxidant capacity and thecomposition of bioactivity compounds including cultural practices, pre-harvest conditions,maturity, post-harvest handling and processing (Gil et al. 1997; Wang and Lin 2000, 2003;Wang and Zhang 2001; Ayala-Zavala et al. 2004; Kosar et al. 2004; Cordenunsi et al.2005; Vicente et al. 2006; Ferreyra et al. 2007; Hernanz et al. 2007; Wang et al. 2007) andgenetic variability is one of the most important factors.

In spite of many studies on the antioxidant and bioactive composition of strawberryfruits (Heinonen et al. 1998; Hebert et al. 2002; Wang et al. 2002; Terry et al. 2004; Heoand Lee 2005; Klopotek et al. 2005; Rekika et al. 2005; Khanizadeh et al. 2006, 2008a;Hernanz et al. 2007; Pinto et al. 2007), no study has been done on individual antioxidantsand their effect on gray mold growth and development.

The objective of this study is to simulate the effect of selected antioxidants on graymold growth and development including spore germination, germ tube elongation andradial growth of Botrytis cinerea, and if they can be of any use as biochemical markers toselect for B. cinerea resistance in a strawberry breeding program.

Materials and method

Chemicals

Catechin, cyaniding-3-glucoside, pelargonidin-3-glucoside and quercetin-3-galactosidewere purchased from Indofine Chemical Co. (Somerville, NJ, USA); ellagic acid, gallicacid, glycerol, NaOH and glucose were from Sigma-Aldrich (St. Louis, MO, USA); potatodextrose agar (PDA) was obtained from Difco Laboratories, Detroit (Sparks, MD, USA);KH2PO4 was from Fisher Scientific Company (Fair Lawn, NJ, USA); ethanol was fromCommercial alcohol Inc. (Brampton, ON, Canada).

Media preparation

Ellagic acid was dissolved in 0.2% NaOH aqueous solution; gallic acid, catechin andquercetin-3-galactoside were dissolved in 25% ethanol; cyaniding-3-glucoside andpelargonidin-3-glucoside were dissolved in double distilled water directly. All solutions

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were then sterilised with a 0.2 mm syringe filter (Nalgene, Rochester, NY, USA) and usedas stock solutions for making culture media.

The media were prepared by appropriate dilution of the stock solutions with PDA toevaluate mycelial growth. The following concentrations were made based on a preliminaryrange-finding study: ellagic acid, 18, 36, 54, 72, and 90 ppm; gallic acid, 50, 100, 150, 200and 250 ppm; catechin, 120, 240, 360, 480 and 600 ppm; cyaniding-3-glucoside, 10, 20,30,40 and 50 ppm; pelargonidin-3-glucoside, 10, 20, 30,40 and 50 ppm and quercetin-3-galactoside, 12, 24, 36, 48 and 60 ppm. PDA alone and PDA with NaOH or ethanol wereused together as controls when NaOH or ethanol was used to dissolve the antioxidants.

Mixtures of 0.3% KH2PO4, 1% glucose and 2.5% glycerol with a selectedconcentration of antioxidants were prepared for spore germination and germ tube growth(Li et al. 2003).

Gray mold isolate preparation

The isolation was performed based on Koch’s postulate and according to the method ofAgrios (1997) and Raposo et al. (1995) with some modification.

Strawberry fruits with typical symptoms of gray mold were collected and brought tothe lab from the green house of Horticultural Research and Development Centre(CRDH), Agriculture and Agri-Food Canada (AAFC-AAC) in St-Jean-Sur-Richelieu.Spores on the furthermost top surface of the fruit were picked out with inoculating needleunder a microscope, and transferred to PDA plates. The plates with spores were thenincubated in the dark at 18.58C for 7 d, and fungal culture was purified through sub-culturing. Finally, the pure isolates of Botrytis cinerea Pers. were maintained on PDAplates until required for the experiment.

The conidia of Botrytis cinerea Pers. were obtained by flooding dishes with sterile 5%glycerol aqueous solution. The suspensions were filtered through three lays of sterilecheesecloth in order to remove the adhering mycelia, and the concentration of conidia wasadjusted to 20,000 conidia/ml using a hemocytometer.

Inoculation and measurement

The PDAs enriched with different levels of antioxidants were inoculated with 4 mmdimeter mycelial plugs cut from the edge of seven-day-old actively growing cultures ofBotrytis cinerea Pers. (Tremblay et al. 2003). The cultures were then incubated in the darkat 18.58C for 72 h and the mycelia were measured at 24 h, 36 h, 48 h and 72 h intervalsafter incubation using a calliper. Spore germination and germ tubes were measured on asuspension of conidia adjusted to a final concentration of 105 conidia/ml after 24 hincubated in the dark at 18.58C (Li et al. 2003). An inverted microscope (Leica DMIL)connected with a cooled CCD camera (Lumenera, Infinty 3–1) was used to measure sporegermination and germ tube at 12 h, 18 h and 24 h intervals.

Experimental design and data analysis

A completely randomised design with three replicates was used to test the selected levels ofantioxidant at 24, 36, 48 and 72 h intervals for mycelial growth and 12, 18 and 24 hintervals for germ tube and spore germination. Three plates were used for each replicateand the experiment was repeated twice under the same conditions. Each experiment wasanalysed separately and the two set of data were pooled together after testing the

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homogeneity of the two experimental errors. Two controls (PDA and PDA plus solvent)were used to test the efficacy of the selected levels and a least significant test (LSD) wasused to separate the means. The effect of different levels of antioxidant for each timeinterval was tested using the orthogonal polynomial (Steel and Torrie 1980) contrast ofSAS using the GLM procedure (SAS Institute 1989).

Results

No significant difference was observed between the two controls used for testing ellagicand gallic acids, catechin and quercertin-3-galactoside, indicating that the addition ofeither 2% NaOH or 25% ethanol as a solvent during the media preparation had noeffect on spore germination rate, germ tube length and mycelial growth (Tables 1, 2, 5,and 6).

Ellagic acid

Ellagic acid had no effect on spore germination rate after inoculation (Table 1) while thegerm tube length and mycelial diameter were reduced significantly at all intervals afterinoculation. The effect of concentration of ellagic acid on germ tube and mycelial growthwas quadratic, which means the lower levels of ellagic acid had a negative effect on thegerm tube length and mycelial growth diameter, whereas higher concentrations elongatedthe germ tube and promoted mycelial growth. The optimum level to reduce the germ tubelength and mycelial growth diameter was estimated at about 36 ppm.

Gallic acid

Similar to ellagic acid, gallic acid did not have any effect on spore germination rate except24 h after inoculation when a significant negative linear relationship was observed betweengallic acid concentration and the spore germination rate (Table 2). Gallic acid had asignificant linear negative effect on germ tube length and it was very pronounced at 18 hafter inoculation (F ¼ 12.19) but its effect diminished after 24 h (Table 2). Mycelial growthdiameter was not affected by any level of gallic acid at any interval applied in this studyafter inoculation.

Cyanidin-3-glucoside

Cyanidin-3-glucoside showed significantly linear negative effects on spore germination,but its effect diminished after 18 h. Cyanidin-3-glucoside had a distinct effect on reducingthe germ tube length as well as mycelial growth diameters and this effect was carried overall examined periods. The most extreme effect was observed at 24 h (9.76***) and 72 h(14.90***) respectively for germ tube and mycelial growth (Table 3).

Pelargonidin-3-glucoside

Pelargonidin-3-glucoside, another anthocyanin, showed a significantly linear effect onspore germination over the examined period, however in germ tube growth, theeffect was quadratic until 18 h after inoculation and then linear at 24 h. Ana-logously, the linear effect did not appear in initial periods but was significant from 48 hon (Table 4).

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Table

1.

Spore

germination,germ

tubelength

andmycelialdiameter

ofBotrytiscinerea

intheculture

medium

enriched

withselected

levelsofellagic

acid.

Germinationrate

(%)

Germ

tubelength

(mm)

Mycelialgrowth

diameters(m

m)

Ellagic

acidlevels(ppm)

12h

18h

24h

12h

18h

24h

24h

36h

48h

72h

0(C

ontrol1)

52.93

54.79

53.81

118.10

181.21

244.72

11.69

27.59

42.06

70.86

0þ

0.004%

NaOH

(Control2)

55.56

53.33

53.38

99.88

175.56

225.27

10.34

26.04

40.22

69.31

18

50.00

56.61

54.58

85.38

147.06

193.49

9.46

22.47

36.54

66.61

36

48.61

49.45

50.81

78.31

117.23

165.77

8.52

20.94

35.33

65.05

54

52.40

49.55

50.71

92.00

119.55

179.97

8.64

21.71

35.90

65.14

72

46.94

48.55

50.06

114.50

139.50

199.83

9.48

27.00

37.21

66.23

90

49.15

50.28

49.98

111.93

152.81

214.60

10.39

27.86

38.77

68.11

Mean

50.80

51.79

51.90

100.01

147.56

203.38

9.79

24.80

38.00

67.33

LSD

0.05

9.07

7.06

7.25

32.62

40.79

50.39

1.68

3.94

3.09

3.38

Orthogonalcontrast

Control1vs.Control2

0.34ns

0.18ns

0.01ns

1.23ns

0.08ns

0.59ns

2.67ns

0.72ns

1.46ns

0.87ns

Controlsvs.all

3.33ns

3.88ns

1.25ns

1.67ns

12.58***

8.67***

12.31***

6.72**

23.80***

15.30***

Orthogonalpolynomialcontrast

Ns

ns

ns

Ns

Q***

Q***

Q***

Q***

Q***

Q***

Values

werethemeansofsixreplicates.

LSD

0.05:least

significantdifference

at0.05level.

ns,**and***:notsignificantorsignificantat0.05and0.01,respectively;orthogonalpolynomialcontrast:L¼

linear,Q¼

quadratic.

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Table2.

Spore

germination,germ

tubelength

andmycelialdiameter

ofBotrytiscinerea

intheculture

medium

enriched

withselected

levelsofgallicacid.

Germinationrate

(%)

Germ

tubelength

(mm)

Mycelialgrowth

diameters(m

m)

Gallic

acidlevels(ppm)

12h

18h

24h

12h

18h

24h

24h

36h

48h

72h

0(C

ontrol1)

47.48

48.69

48.46

148.51

167.36

211.26

10.72

31.58

41.03

71.20

0þ

0.5%

alcohol(C

ontrol2)

49.85

50.55

50.52

131.32

159.88

180.05

10.15

32.28

39.15

67.81

50

51.12

53.74

47.99

123.38

119.18

131.22

9.29

29.55

37.27

66.14

100

47.68

49.24

50.99

108.21

130.20

126.19

9.98

30.99

38.09

67.70

150

42.85

43.44

45.03

117.30

133.41

157.18

10.39

30.21

38.75

68.09

200

41.72

43.88

42.34

109.85

131.47

192.04

8.55

27.59

35.77

64.22

250

39.96

39.24

37.73

106.13

120.47

213.33

8.92

31.72

36.64

65.66

Mean

45.81

46.97

46.15

120.67

137.42

173.04

9.71

30.56

38.10

67.26

LSD

0.05

8.54

7.89

7.39

28.99

35.21

57.91

3.47

5.29

7.32

6.66

Orthogonalcontrast

Control1vs.Control2

0.32ns

0.23ns

0.32ns

1.38ns

0.18ns

1.14ns

0.11ns

0.08ns

0.27ns

1.07ns

Controlsvs.all

2.58ns

2.60ns

4.72**

9.70***

12.19***

3.36ns

0.99ns

1.72ns

1.71ns

2.62ns

Orthogonalpolynomialcontrast

ns

ns

L***

L***

L**

ns

ns

ns

ns

ns

Values

werethemeansofsixreplicates.

LSD

0.05:least

significantdifference

at0.05level.

ns,**and***:notsignificantorsignificantF

values

at0.05and0.01,respectively;orthogonalpolynomialcontrast:L¼

linear,Q¼

quadratic.

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Table3.

Spore

germination,germ

tubelength

andmycelialdiameter

ofBotrytiscinerea

intheculture

medium

enriched

withselected

levelsofcyanidin-3-

glucoside.

Germinationrate

(%)

Germ

tubelength

(mm)

Mycelialgrowth

diameters(m

m)

Cyanidin-3-glucosidelevels(ppm)

12h

18h

24h

12h

18h

24h

24h

36h

48h

72h

0(C

ontrol)

51.57

46.29

50.17

131.43

171.22

213.06

13.59

30.71

44.79

71.83

10

42.33

43.07

49.36

96.81

145.92

194.11

13.06

29.65

42.81

68.76

20

48.30

49.34

46.24

97.36

133.94

196.70

12.19

28.62

41.41

68.85

30

39.73

45.16

42.19

86.20

117.46

145.04

11.64

28.19

41.57

68.63

40

41.43

48.20

47.02

91.75

111.85

137.13

11.86

27.34

41.55

68.55

50

35.69

39.28

44.34

83.10

123.38

132.31

11.35

26.53

40.67

67.35

Mean

43.18

45.22

46.55

97.78

133.96

169.73

12.28

28.51

42.13

69.00

LSD

0.05

8.03

6.35

6.12

27.91

42.37

42.70

3.82

2.87

3.27

2.33

Orthogonalcontrast

Controlvs.all

10.92***

0.28ns

3.48ns

13.77***

7.33***

9.76***

1.17ns

5.94**

6.63**

14.90***

Orthogonalpolynomialcontrast

L***

ns

ns

L***

L***

L***

ns

L***

L**

L***

Values

werethemeansofsixreplicates.

LSD

0.05:least

significantdifference

at0.05level.

ns,**and***:notsignificantorsignificantF

values

at0.05and0.01,respectively;orthogonalpolynomialcontrast:L¼

linear,Q¼

quadratic.

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Table

4.

Spore

germination,germ

tubelength

and

mycelialdiameter

ofBotrytiscinerea

intheculture

medium

enriched

with

selected

levelsof

pelargonidin-3-glucoside.

Germinationrate

(%)

Germ

tubelength

(mm)

Mycelialgrowth

diameters(m

m)

Pelargonidin-3-glucosidelevels(ppm)

12h

18h

24h

12h

18h

24h

24h

36h

48h

72h

0(C

ontrol)

57.76

57.91

54.71

131.57

169.86

212.29

14.67

30.67

44.64

72.73

10

50.51

48.99

53.17

101.29

128.37

186.55

13.97

31.11

44.78

70.89

20

48.52

50.57

49.21

96.46

122.34

139.97

13.29

30.60

42.81

70.33

30

43.00

46.06

44.36

95.64

115.52

139.09

13.19

29.13

42.07

69.81

40

48.55

47.38

44.94

90.62

117.52

120.68

12.07

27.87

40.14

65.72

50

44.73

44.90

44.86

108.87

131.43

153.13

11.04

27.24

40.26

67.76

Mean

48.85

49.30

48.54

104.08

130.84

158.62

13.04

29.44

42.45

69.54

LSD

0.05

8.87

6.19

5.75

30.05

40.37

50.41

3.61

1.97

2.51

4.54

Orthogonalpolynomialcontrast

Controlvs.all

10.12***

19.34***

11.53***

7.93***

8.85***

10.74***

2.04ns

3.89ns

7.63***

4.93**

Orthogonalpolynomialcontrast

L***

L***

L***

Q**

Q**

L***

ns

ns

L***

L***

Values

werethemeansofsixreplicates.

LSD

0.05:least

significantdifference

at0.05level.

ns,**and***:notsignificantorsignificantFvalues

at0.05and0.01,respectively;orthogonalpolynomialcontrast:L¼

linear,Q¼

quadratic.

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Table5.

Spore

germination,germ

tubelength

andmycelialdiameter

ofBotrytiscinerea

intheculture

medium

enriched

withselected

levelsofcatechin.

Germinationrate

(%)

Germ

tubelength

(mm)

Mycelialgrowth

diameters(m

m)

Catechin

levels(ppm)

12h

18h

24h

12h

18h

24h

24h

36h

48h

72h

0(C

ontrol1)

50.49

51.06

50.66

136.98

180.39

224.22

13.42

33.93

43.96

71.69

0þ

0.5%

alcohol(C

ontrol2)

47.69

50.23

51.84

125.38

178.10

196.82

12.32

32.02

42.19

69.82

40

46.97

46.30

48.19

110.18

135.96

157.83

12.04

32.00

41.44

68.72

80

52.19

46.86

46.59

99.06

128.02

117.64

11.20

32.23

38.87

66.97

120

52.52

53.07

50.84

86.96

102.80

111.46

11.14

31.89

37.43

66.49

160

49.52

49.77

49.49

83.44

94.13

101.31

11.62

31.48

39.80

67.14

200

51.30

50.59

52.89

81.67

85.02

129.03

10.56

30.90

37.74

66.55

Mean

50.10

49.70

50.07

103.38

129.20

148.33

11.76

32.06

40.20

68.20

LSD

0.05

8.74

9.22

7.82

28.41

42.53

36.95

4.60

1.97

7.98

4.92

Orthogonalcontrast

Control1vs.Control2

0.42ns

0.03ns

0.10ns

0.65ns

0.01ns

2.16ns

0.24ns

4.31ns

0.20ns

0.60ns

Controlsvs.all

0.31ns

0.24ns

0.52ns

21.08***

30.49***

62.37***

1.34ns

5.50**

2.99ns

6.24**

Orthogonalpolynomialcontrast

ns

ns

ns

L***

L***

L***

ns

L**

ns

L**

Values

werethemeansofsixreplicates.

LSD

0.05:least

significantdifference

at0.05level.

ns,**and***:notsignificantorsignificantFvalues

at0.05and0.01,respectively;orthogonalpolynomialcontrast:L¼

linear,Q¼

quadratic.

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Table6.

Spore

germination,germ

tubelength

andmycelialdiameter

ofBotrytiscinerea

intheculture

medium

enriched

withselected

levelsofquercetin-

3-galactoside.

Germinationrate

(%)

Germ

tubelength

(mm)

Mycelialgrowth

diameters(m

m)

Quercetin-3-galactosidelevels(ppm)

12h

18h

24h

12h

18h

24h

24h

36h

48h

72h

0(C

ontrol1)

48.11

49.479

48.47

118.94

167.02

225.04

11.54

28.82

41.85

72.54

0þ

0.5%

alcohol(C

ontrol2)

47.22

49.102

49.44

108.24

158.30

209.84

11.74

28.74

41.41

69.41

12

41.87

44.47

44.40

101.59

132.06

193.54

9.80

27.06

39.21

67.80

24

42.99

45.24

48.22

87.61

119.35

140.63

9.40

26.36

38.79

67.26

36

47.99

48.42

50.69

77.30

107.42

103.58

9.49

26.07

39.12

68.13

48

49.17

48.98

46.61

73.22

101.01

93.12

8.43

26.15

39.02

67.44

60

42.31

45.61

51.37

69.12

88.51

92.64

8.68

25.49

38.43

66.35

Mean

45.67

47.33

48.46

90.86

124.81

151.20

9.87

26.96

39.69

68.42

LSD

0.05

7.51

8.72

7.68

22.05

44.94

58.55

2.29

3.77

4.65

3.93

Orthogonalcontrast

Control1vs.Control2

0.06ns

0.01ns

0.07ns

0.93ns

0.15ns

0.26ns

0.03ns

0.00ns

0.04ns

2.61ns

Controlsvs.all

1.64ns

1.17ns

0.10ns

23.40***

15.62***

28.18***

13.74***

5.40**

4.02ns

9.74***

Orthogonalpolynomialcontrast

ns

ns

ns

L***

L***

L***

L***

L**

ns

L***

Values

werethemeansofsixreplicates.

LSD

0.05:least

significantdifference

at0.05level.

ns,**and***:notsignificantorsignificantF

values

at0.05and0.01,respectively;orthogonalpolynomialcontrast:L¼

linear,Q¼

quadratic.

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Catechin

Catechin had no effect on spore germination rate but significantly reduced the germ tubelength and mycelial growth diameter and its effect was very pronounced especially after24 h (62.37***) and 72 h (6.24**), respectively.

Quercetin-3-galactoside

Quercetin-3-galactoside showed a similar effect to catechin. It had no effect on sporegermination rate, but significantly reduced the germ tube length and mycelial growthdiameter and its effect was very pronounced especially after 24 h (628.187***) and 72 h(9.74**), respectively.

Discussion

Gray mold development is one of the principal causes of premature termination of post-harvest shelf life of strawberry. Numerous studies have conclusively established a key rolefor constitutive and induced phenolic compounds with antioxidant properties in thedefence of plant tissues against pathogen attacks (Nicholson and Hammerschmidt 1992;Prusky 1996). Strawberry fruit, ellagic acid, gallic acid along with anthocyanins(cyaniding-3-glucoside, pelargonidin-3-glucoside), proanthocyanidins (catechin) andflavonols (quercetin-3-galactoside) are the main phenolic compounds with antioxidantproperties. These compounds are known to influence the disease resistance and shelf life ofthe fruit. A threefold increase in ellagic acid content was observed in raspberry leavesinfected by orange rust (Wang et al. 1994), and high level of catechins, the mainconstitutive unit of proanthocyanidins (PA), found in immature strawberry fruits wasassociated with resistance to gray mold (Feucht et al. 1992). In vitro studies revealed thatproanthocyanidins (PA) of some strawberry cultivars reduced hyphal growth of graymold, and fruit of such cultivars high in PA content were more resistant to storage rotdevelopment (Hebert et al. 2002). In the present study, antifungal activity was observed forall antioxidants, and dose dependent antifungal activity of the antioxidants was also foundat every developmental stage of B. cinerea, and almost every antioxidant showed asignificantly linear inhibitory effect on pathogen progress. For example, spore germinationwas strongly inhibited by increased concentrations of gallic acid (r ¼ 70.95); catechin andquercetin-3-galactoside show a negative relationship with germ tube elongation(r ¼ 70.96 and r ¼ 70.98, respectively), and cyanidin-3-glucoside and pelargonidin-3-glucoside also provided effective control of the fungi as concentrations increased. Theseindicate that strawberry cultivars with varied phenolic contents (Meyers et al. 2003; Aabyet al. 2005; Rekika et al. 2005; Khanizadeh et al. 2006, 2008a) should have different shelflives and susceptibility to pathogen infection. It has been reported that high antioxidantcapacity was found in Harmonie, Saint-Jean d’Orleans and Saint-Laurent d’Orleans,which exhibited better shelf life stability than Kent (Khanizadeh et al. 2005a, 2005b,2005c).

However, the rule is not always straightforward. For example, Saint Pierre which ischaracterised by very good shelf life (Khanizadeh et al. 2002a) displayed a similar level ofantioxidants (gallic acid, epicatechin, catechin and ellagic acid) to other cultivars with ashort shelf life. Rosemary oil had half the amount of eucalyptol compared with the sageoil, but it was shown to be more effective at fungal inhibition (Daferera et al. 2003). It isinteresting to note that the effects of almost all tested antioxidants were linear except for

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ellagic acid which was quadratic (Table 1). This indicates that ellagic acid has an effect ongerm tube and mycelial growth at low concentration (36 ppm), whereas the higher level ofellagic acid promotes germ tube elongation and mycelial growth similar to the effect ofgrowth hormone.

Several papers in the literature demonstrated that plants accumulate phenolic compoundsunder infection or elicited resistance to pathogen (Wang et al. 1994; Soylu 2006; Liu et al.2007), however, it is important to note that there might be an interaction between individualphenolic compounds which may cause a synergistic effect, and the presence of other enzymessuch as PPO, POD and H2O2 burst in the plant might also affect the susceptibility of acultivar (Khanam et al. 2005). It has been reported that grape pranthocyanidinscompetitively inhibit the activity of laccase produced by B. cinerea, but only in the presenceof tannins (Elad and Evensen 1995). Lee et al. (2007) reported that combinations of herbextracts had a higher inhibitory effect towards tested fungi than that of individual extract.Although the catabolic phloridzin derivatives did not inhibit fungal mycerial growth ofPhlyctaeea vagabunda, Lattanzio et al. (2001) reported that phloridzin and chlorogenic acidin combination with polyphenol oxidase activity could function to arrest P. vagabunda inquiescent infections associated with immature and ripening apple fruit.

In the present study, the effects of the six antioxidants were different at differentdevelopmental stages of the fungi progress, for example, catechin and quercetin-3-galactoside exhibited very significant linear inhibitory effects on germ tube elongation,with the highest suppression ratio of 54.8% and 58.8%, respectively, but no significanteffects were found on spore germination. Meanwhile, gallic acid inhibited sporegermination strongly but weakly on germ tube elongation and mycelial growth, andhigher concentrations may be needed (Hur et al. 2003). Similar results were found betweenchitosan and Penicillium expansum and B. cinerea (Liu et al. 2007). Furthermore, whenlettuce leaves were inoculated with B. cinerea, lettucenin A accumulation was associatedwith limited lesions restricting the spread of disease, but when leaves were inoculated withmycelial suspensions, lettucenin A accumulation appeared to be overcome by degradationprocesses, and the disease spread (Bennett et al. 1994). These indicate that differentcompounds have different antifungal mechanisms and/or the sensitivity of the fungi maychange with different stages of development. Some small phenolic compounds disruptfungal membranes and attach and polymerise within the wall of the fungal hyphae, thusreducing the plasticity of the fungus for growth (Wang et al. 1994). Gallic acid and methylgallate can act on a cAMP-related signalling pathway regulating appressorium formationin Magnaporthe grisea (Ahn et al. 2005).

In conclusion, almost every antioxidant used in the present study exerted significantinhibitory effects on B. cinerea progress, and their effects were significantly linear, exceptfor ellagic acid which was quadratic, however, different antioxidants have differentantifungal mechanisms and the sensitivity of the B. cinerea to the antioxidants may changeat different stages of development. The objective of future work is to test the combinationeffect of selected antioxidants and determine if there is an interaction between the selectedcompounds on gray mold growth and development.

The observed results will help to select lines in vitro in order to select lines resistant togray mold.

Acknowledgements

The authors would like to acknowledge the support received from ABIP-100 project entitled:Nutraceuticals Emerging from Agricultural Technologies Network (NEATNet) and ChinaScholarship Council (CSC) and International Scientific Cooperation Bureau (ISCB) of Agriculture

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and AgriFood Canada (AAFC). The authors also wish to thank Audrey Levasseur and MathieuTremblay from the Horticultural Research and Development Centre, Agriculture and Agri-FoodCanada for their technical assistance with this work.

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