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Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Control of postharvest grey mould decay of nectarine by tea polyphenol combined with tea saponin X.P. Yang, X.D. Jiang, J.J. Chen and S.S. Zhang Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, China

Significance and Impact of the Study: This study demonstrates that the combination of TP and TS has exhibited synergistic antifungal interactions against Botrytis cinerea, and it suggests that their combination may be useful and effective agents for the control of nectarine grey mould decay. Such natural products therefore represent a promising alternative to synthetic fungicides in the control of nectarine postharvest diseases.

Keywords antifungal activity, Botrytis cinerea, nectarine, tea polyphenol, tea saponin. Correspondence Xiao-Ping Yang, Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China. E-mail: [email protected] 2013/1119: received 7 June 2013, revised 17 July 2013 and accepted 29 July 2013 doi:10.1111/lam.12139

Abstract The control efficacy of tea polyphenol (TP) in combination with tea saponin (TS) against nectarine grey mould decay caused by Botrytis cinerea and the underlying mechanism were investigated. The in vitro experiments showed that both TP and TS inhibited the mycelial growth in a dose-dependent manner, and their combinations exhibited synergistic antifungal interactions with the synergistic ratios (SR) exceeding 15. The in vivo experiments showed that disease incidence and lesion diameter of grey mould of inoculated fruit were significantly lowered after being treated with the combination of TP and TS; furthermore, the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO), chitinase and b-1,3-glucanase of inoculated fruit as well as the contents of total phenolic and lignin were significantly induced, the respiration rate of inoculated fruit was significantly decreased and therefore the quality decrease was accordingly retarded. These results revealed that TP in combination with TS could control grey mould of inoculated nectarines and their mechanism of action might be attributed to their active components, the induction of defensive system and the regulation of respiration.

Introduction Nectarines arise as peach mutants. Similarly to peaches, nectarines are highly perishable at ambient temperature due to rapid ripening and high susceptibility to pathogens (Navarro et al. 2011). Grey mould decay, caused by Botrytis cinerea Pers.: Fr., is one of the major postharvest diseases of nectarines, and it causes huge losses during the postharvest storage (Zhang et al. 2008; Navarro et al. 2011). Currently, the disease is mainly controlled by the application of synthetic fungicides. However, the application often results in pesticide residues on fruit that may affect human health, environmental contamination and the development of pathogen resistance to many currently used pesticides (Ragsdale and Sisler 1994). These 502

problems have led to search for promising alternatives to synthetic fungicides in the control of B. cinerea. Some plant extracts, for their fungistatic activities, have shown a great potential as an effective alternative to synthetic fungicides to control postharvest diseases of fruit. It has been reported that several natural compounds from plants, such as Citrus lemon L. (Viuda-Martos et al. 2008), Cistus villosus (Talibi et al. 2012) and Inula viscosa (Askarne et al. 2012), can effectively control postharvest fruit fungi. The mechanisms underlying their antifungal effects are still obscure, but may be attributed, at least in part, to the induction of defensive system. For example, it was reported that the treatment with jasmonic acid and its derivatives suppressed fungal decay through inducing disease resistance of fruit (Pe~ na-Cortes et al. 2005).

Letters in Applied Microbiology 57, 502--509 © 2013 The Society for Applied Microbiology

X.P. Yang et al.

Control of nectarine grey mould

Tea (Camellia sinensis) and its extract have recently attracted much attention for good antimicrobial activity (Almajano et al. 2008). Several studies have reported that tea polyphenol (TP) has antifungal activity against postharvest pathogens of fruit, such as Diplodia natalensis (Liu et al. 2010a) and B. cinerea (Liu et al. 2010b), as well as tea saponin (TS) against Penicillium italicum, Penicillium digitatum and Geotrichum candidum (Hao et al. 2010). Moreover, both TP and TS enhance the antimicrobial activity of fungicides and reduce the necessary amounts of these fungicides (Hirasawa and Takada 2004; Hao et al. 2010; Yang and Zhang 2012). In a recent study, Chen et al. (2013) have found that TP and TS possess a potent antifungal activity against Monilinia fructicola. However, to our knowledge, few data exist regarding the control mechanism of fruit postharvest diseases using TP and TS as well as their effect on postharvest fruit quality, which affects their application in fruit preservation. The present study aims to evaluate the following: (i) the in vitro and in vivo antifungal activities of the combination of TP and TS against B. cinerea on nectarine fruit, (ii) the effect of their combination on physiological and biochemical responses of inoculated fruit and (iii) the mechanism underlying the control. Results and discussion In vitro antifungal activity The in vitro antifungal activities of TP and TS individually or their combination are shown in Table 1. The results indicated that both TP and TS inhibited the mycelial growth of B. cinerea in a dose-dependent manner and exhibited good antifungal activities. Moreover, each combination of TP and TS showed synergistic antifungal Table 1 In vitro synergistic interaction between tea polyphenol (TP) and tea saponin (TS) against the mycelial growth of Botrytis cinerea

TP : TS

Toxic regression equation

R2

EC50 (obs) (mg ml 1)

EC50 (th) (mg ml 1)

SR

10 : 0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0 : 10

Y Y Y Y Y Y Y Y Y Y Y

0976 0983 0991 0992 0994 0983 0979 0993 0986 0965 0985

870 450 433 419 385 383 367 338 359 402 710

– 851 832 815 808 798 782 766 751 737 –

– 189 192 195 210 208 213 227 209 183 –

= = = = = = = = = = =

1057X 1158X 1065X 1295X 1242X 1474X 3572X 3609X 2657X 4464X 1567X

+ + + + + + + + + + +

4006 4244 4322 4195 4272 4140 2982 3090 3526 2304 3663

R2, the coefficient of determination; EC50, the concentration that inhibits mycelial growth by 50%; SR, synergistic ratio.

interaction with SR value exceeding 150. The combination of TP and TS at the ratio of 3 : 7 among all combinations exhibited the highest SR value of 227. Therefore, this ratio was used in the subsequent experiments. Tea polyphenol (TP) and tea saponin (TS), two main active ingredients of tea, have been reported to have antifungal activity against postharvest pathogens of fruit (Hao et al. 2010; Liu et al. 2010a,b). Moreover, it has been reported that both TP and TS could enhance the antifungal activity of fungicides and reduce their necessary amounts. For example, Hirasawa and Takada (2004) reported that TP enhanced the antifungal effect of amphotericin B or fluconazole against Candida albicans; Hao et al. (2010) reported that TS improved the antifungal activity of prochloraz or imazalil against P. italicum, P. digitatum as well as G. candidum; we previously demonstrated that TS enhanced the antifungal activity of mancozeb against Pestalotiopsis theae (Yang and Zhang 2012) and TP combined with TS exhibited a synergistic antifungal interaction against M. fructicola (Chen et al. 2013). These findings provided encouraging evidences for the application of the combination of TP and TS. However, the mechanism of their synergic effect is unknown. The synergistic mechanism may be as follows: first, both TP and TS have good antifungal activities against B. cinerea and their antifungal activities are related to their antifungal effects; second, TP acts on and damages fungal membranes (Hirasawa and Takada 2004) and TS can result in better retention of TP on B. cinerea (Hao et al. 2010), which can improve the efficacy of TP. Effect of the combination treatment of TP and TS on the control of grey mould On the first day after inoculation, all fruits in the control treatment showed typical lesions of grey mould decay. On the fourth day after inoculation, average diameter of rotten area in the control treatment was over 46 mm and about 10% of fruits even rotted away (rotten area was 100%). Therefore, we only provided the control effect of the combination treatment against grey mould of inoculated fruit for 4 days in Fig. 1. As shown, the combination significantly reduced grey mould incidence from 933% on the first day to 50% on the fourth day and lesion diameter from 467 cm on the first day to 3162 cm on the fourth day compared with the control, which showed that the combination significantly reduced the growth of grey mould on nectarine fruit. This study suggested that the combination of TP and TS could effectively control grey mould decay of postharvest fruit, which is in agreement with the report of Soylu et al. (2010) that several plant extractions could be used as biofungicide in the protection of tomato against B. cinerea.


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Disease incidence (%)

120

a

(a)

a

a

a

100 80

b

60

b

40

b b

20

a a

0 0

1

2

3

4

Inoculation time (days)

Lesion diameter (mm)

50

a (b)

40 Figure 1 Effect of the combination of tea polyphenol (TP) and tea saponin (TS) on disease incidence (a) and decay severity (b) against grey mould of nectarines inoculated with Botrytis cinerea. ( ) CK, the treatment with sterile distilled water and (h) TP + TS. Significant differences between means were indicated by different letters above histogram bars.

a

30 20

a

10

b b

a

b

b

a a 0

0

1

2 Inoculation time (d)

3

4

Table 2 Effect of the combination treatment of tea polyphenol (TP) and tea saponin (TS) on lignin, total phenolic and MDA contents of nectarines Day (d)

Treatment

Lignin (SAE 100 g 1)

0 1

CK CK TP + CK TP + CK TP + CK TP +

082 086 095 092 103 096 133 103 140

2 3 4

TS TS TS TS

002 002b 003a 002b 001a 001b 011a 008b 011a

Total phenolic (GAE 100 g 1) 6757 8254 9600 9764 13431 11124 15148 13276 15574

317 227b 271a 206b 547a 686b 1022a 665b 821a

MDA (lmol g 1) 1035 2053 1547 2742 1954 3170 2284 3917 2764

121 154a 090b 145a 170b 193a 140b 129a 135b

SAE, sinapyl alcohol equivalent; GAE, gallic acid equivalent; MDA, malondialdehyde; CK, the treatment with sterile distilled water. Values within a column followed by different lowercase letters are significantly different at P < 005 according to Tukey’s HSD tests.

Effect of the combination treatment of TP and TS on the defensive enzyme and substance Endogenous defence mechanisms of plants can be induced in response to attack by pathogens. In the current study, we found that the inoculation of B. cinerea significantly induced the accumulations of total phenolic and lignin (Table 2) as well as the increase in the enzymatic activities such as PPO, PAL, POD, b-1,3-glucanase and chitinase (Fig. 2) in nectarines. Phenolic compounds are toxic to fungi in nature and are produced or released by the host plant during fungal infection. Lignin renders cell walls highly resistant to pathogen invasion through 504

forming covalent cross-links with carbohydrates and proteins (Whitmore 1978). PAL, POD and PPO have been reported to strengthen the defence system in plants by biosynthesizing metabolites such as phenols to form lignin or toxic quinines (Milosevic and Slusarenko 1996). b-1,3-glucanase and chitinase have been considered as key enzymes having direct activity against pathogens in plant disease resistance systems (Ji and Kuc 1996). Therefore, the accumulation of phenolic compounds and lignin as well as the increase in PPO, PAL, POD, b-1,3-glucanase and chitinase activities improved nectarine resistance to B. cinerea and lowered the incidence and severity of grey mould.


X.P. Yang et al.


PPO (U g–1 FW)

800

a

(a) a

b 600

a

b a

b

a

a b

400 200 0 0

1

2

3

4

PAL (U g–1 FW )

Inoculation time (days) 700 600 500 400 300 200 100

b a

0

POD (U g–1 FW)

500

Chitinase (U g–1 FW)

a b

a b

1

(c)

2 3 Inoculation time (days)

4

a

b

400 b

300

a

200 a

a

0

1

(d)

a

0·2

a

4

a

a

b

a

a

b

2 3 Inoculation time (days)

b a

a

b

100

0·25

a

0·15

b

0·1 0·05 0

β-1,3- glucanase (U g–1 FW)

a b

a

0

0

Figure 2 Effect of the combination of tea polyphenol (TP) and tea saponin (TS) on the activities of polyphenol oxidase (a), phenylalanine ammonia-lyase (b), peroxidase (c), chitinase (d) and b -1,3-glucanase (e) of nectarines. ( ) CK, the treatment with sterile distilled water and (h) TP + TS. Significant differences between means were indicated by different letters above histogram bars.

a

(b)

0·3

0

1

2 Inoculation time (days)

(e)

b

a

a

a

b

0·2

3

4

a b

a b

a

0·1

0

0


1

2 Inoculation time (days)

3

4

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In present study, we observed that the treatment of TP and TS alone could induce the accumulations of total phenolic and lignin as well as the increase in the above enzymatic activities in inoculated nectarines (data not shown), and the treatment of the combination of TP and TS significantly enhanced the induction (except chitinase activity on the first day) compared with the control, thus significantly improving nectarine resistance to B. cinerea and reducing the growth of grey mould on inoculated fruit. It is consistent with the above results that the combination significantly lowered the incidence and lesion diameter of grey mould. To our knowledge, it is the first report of the combination of TP and TS controlling grey mould decay on nectarine by inducing resistance to B. cinerea. Yu et al. (2009) reported that methyl jasmonate lowered grey mould decay incidence by inducing resistance to B. cinerea in tomato. Chen et al. (2013) reported that the combination of TP and TS reduced the growth of M. fructicola in inoculated fruit through increasing the defensive enzymatic activities. Liu et al. (2005) also reported that BTH treatment induced the resistance of peach fruit to the infection of P. expansum through enhancing PAL, PPO and POD activities as well as total phenolic content. These are in agreement with the current results. Therefore, the induction of the defence system might be one of the mechanisms of controlling grey mould of postharvest nectarine fruit by the combination treatment of TP and TS. Effect of the combination treatment of TP and TS on the respiration and quality of nectarines

first day. Respiration is a major factor contributing to postharvest losses of fruit, which converts stored sugar to energy in the presence of an oxygen substrate and advances fruit senescence, thus significantly shortening shelf life and decreasing quality of fruit (McLaughlin and O’Berne 1999). The present study showed that the inoculation led to the continuous decrease in fruit firmness, total soluble solids (TSS) content, titratable acidity (TA) content and ascorbic acid (Vc) content as well as the continuous increase in weight loss (Table 3) and malondialdehyde (MDA) content (Table 2) during the 4 day period of observation. Moreover, fruit senescence conversely contributes to pathogen infection and decay (Ju and Curry 2001). We also found that the rotten area in the control group increased rapidly and some fruits even rotted away 4 days after inoculation. Therefore, it is very important to control the respiration rate of fruit. In the present study, we observed that the combination of TP and TS significantly decreased the respiration rate of inoculated fruit and significantly lowered firmness loss, weight loss as well as the contents of TSS, MDA and Vc. Thus, the senescence and pathogen infection of fruit were retarded. Current results are consistent with those of Gonzalez-Ure~ na et al. (2003) who reported that the exogenous application of resveratrol maintained the postharvest quality of grapes and apples against B. cinerea. Therefore, the regulation of respiration might also be one of the mechanisms of controlling grey mould of postharvest nectarine fruit by the combination treatment of TP and TS. Materials and methods

Nectarine is climacteric fruit. When the fruit is attacked by pathogens during storage, its respiration rate increases (Navarro et al. 2011). As shown in Table 3, the respiration rate of nectarines after inoculation increased significantly and the first respiration peak was observed on the

Fungal pathogen Botrytis cinerea was originally isolated from infected nectarines with typical grey mould symptoms, purified and identified. Botrytis cinerea conidia were collected and

Table 3 Effect of the combination of tea polyphenol (TP) and tea saponin (TS) on the respiration and quality of nectarines Day (d)

Treatment

Respiration (mg kg

0 1

CK CK TP + CK TP + CK TP + CK TP +

4803 9790 8376 7546 5727 7004 5496 8405 7371

2 3 4

TS TS TS TS

102 127a 503b 370a 488b 391a 625b 312a 380b

1

h 1)

TSS (%) 1088 940 1008 732 912 639 810 532 720

Vc (lg g 1)

TA (%) 088 038b 059a 045b 056a 042b 067a 065b 049a

087 084 087 079 086 077 083 075 080

005 004 003 001 005 002 004 002 003

9002 8500 9137 6947 8140 4537 6007 2677 3917

155 029b 068a 035b 177a 130b 145a 083b 182a

Firmness (N) 5125 3927 4506 2548 3637 2173 3266 1411 2781

125 212b 114a 222b 175a 151b 118a 167b 118a

Weight loss (%) 0 291 234 346 291 400 341 453 392

021a 020b 018a 015b 026a 016b 024a 025b

TSS, total soluble solids; TA, titratable acidity; Vc, ascorbic acid; CK, the treatment with sterile distilled water. Values within a column followed by different lowercase letters are significantly different at P < 005 according to Tukey’s HSD tests.

506


X.P. Yang et al.

suspended in sterile water and filtered through a gradient of cheesecloth. The conidial suspension was adjusted to 5 9 106 conidia ml 1 using a haematocytometer (HX034; VEDENG, Nanjing, China). The conidial suspension was distributed onto water agar amended with 100 lg ml 1 streptomycin sulfate in Petri dishes. A hyphal tip from a germinating conidium was transferred aseptically to potato dextrose agar (PDA), and single-spore cultures were used for further experimentation. Chemicals and fruits Tea polyphenol (purity of 98%) was purchased from Nanjing Qingze Medical Technological Development Co. Ltd (China). TS (purity of 98%) was purchased from Hunan Geneham Biomedical Technology Ltd. All other chemicals and reagents were of analytical grade. Nectarine fruits (Prunus persica var. nectarina cv. Shuguang) were harvested at commercial ripening stage from Wuhan, China. Fruits were free of damage and visual infection. They were selected for uniformity of size and ripeness without chemical postharvest treatments. In vitro antifungal assay Antifungal assays were performed by mycelial growth assay. Serial concentrations of TP, TS and their combination (025, 05, 10, 20, 40, 80 and 160 mg ml 1) were tested for their action against mycelial growth of B. cinerea, using sterile distilled water as a control. The solutions were respectively added to sterilized PDA at 40–50°C and poured into Petri dishes (9 cm in diameter). A mycelial disc of approximately 10 mm in diameter was cut from the edge of 2-day-old culture of B. cinerea and was placed at the centre of each Petri dish. The inoculated plates were incubated at 25°C for 2 days until the growth in the control plates reached the edge of the Petri plates. The EC50 value (the concentration of each chemical that inhibited 50% of mycelial growth) was calculated by probit analysis with the help of probit package of SPSS 115 software (SPSS Inc., Chicago, IL, USA). Each test was replicated three times and each treatment within a replication was repeated five times. The synergistic interaction between TP and TS was evaluated according to Horsfall (1945). The synergistic ratio (SR) was calculated as SR = EC50 (th)/EC50 (obs), where EC50 (obs) is the observed EC50 value of a twocomponent mixture by mycelial growth assay and EC50 (th) is the theoretical EC50 value of the specific mixture. EC50 (th) was calculated as EC50 (th) = (a + b)/[(a/ EC50A) + (b/EC50B)], where A and B represent the two components, and a and b represent the ratio of the components in the mixture. Synergism was considered


significant if SR ≥ 15, antagonism if SR ≤ 05 and additive interactions at 05 < SR < 15. Inoculation and treatment procedure Nectarine fruits used in this study were washed with running tap water. Their surfaces were sanitized with 2% sodium hypochlorite for 2 min, rinsed twice in sterile distilled water and dried in ambient air. A single puncture was made on the equator of the fruit with a stainless steel rod with a 2-mm-wide and 4-mm-long tip. Each puncture was inoculated with 25 ll of inoculant containing 5 9 104 conidia ml 1 of B. cinerea and held at 25°C for 2 h. Then, the fruits were treated with 50 ll of the combination solution (60 mg ml 1) of TP and TS at the same puncture. Control fruits (CK) were treated with sterile distilled water. The treated fruits were stored at 25°C with 90% RH for 4 days. There were 20 fruits in each treatment and each treatment was replicated three times. The experiment was repeated three times. Decay evaluation Disease incidence (the percentage of fruit with visible disease development) and lesion diameter (including median diameter of inoculation wound) on each fruit were recorded daily. When the visible rot zone beyond the wounded area on each fruit was more than 1 mm wide, it was considered as decayed fruit. Disease incidence was assessed by counting infected fruit, and disease severity was assessed by measuring diameter of rotten area using a calliper. Enzyme analysis All enzyme extraction procedures were conducted at 4°C. The pulp (100 g) was respectively sampled from the junction between the healthy and rotten part on 0, 1, 2, 3 and 4 days after the inoculation. The pulp was homogenized on dry ice in 20 ml of 01 mol l 1 borate buffer (pH 88) for PAL, 02 mol l 1 phosphate buffer (pH 68) for PPO and POD, 01 mol l 1 sodium acetate buffer (pH 52) for b-1,3-glucanase and chitinase. The homogenate was centrifuged at 12 000 g for 20 min at 4°C, and the supernatant was collected for enzyme assay. PAL, PPO and POD activities were assayed according to Liu et al. (2005). b-1,3-glucanase and chitinase activities were assayed according to Yang and Zhang (2012). PAL, PPO and POD activities were expressed as U, where one U was respectively defined as change in OD290 nm (PAL), OD420 nm (PPO) and OD470 nm (POD) per minute per gram fresh weight (FW) of pulp. Chitinase activity was expressed as U, where one unit was defined as the


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amount of liberated N-acetyl-D-glucosamine per hour per gram FW of pulp. b-1,3-glucanase activity was expressed as U, where one unit was defined as the amount of enzyme that produced one micromole of glucose per minute per gram FW of pulp. Determination of the content of total phenolic, lignin and MDA Total phenolic content was measured according to the methods of Pirie and Mullins (1976). One gram of flesh tissue was homogenized with 5 ml ice-cold 1% HCl– methanol solution and then centrifuged at 4°C for 10 min at 12 000 g. The supernatant was collected and the absorbance was measured at 280 nm. The results are expressed as grams of gallic acid equivalent (GAE) per 100 g FW of pulp. Lignin content was determined as previously described (Morrison 1972). The result is expressed as grams of sinapyl alcohol equivalent (SAE) per 100 g FW of pulp. Ten grams of flesh tissue was homogenized with 20 ml of 02 mol l 1 phosphate buffer (pH 64) and then centrifuged at 12 000 g for 20 min at 4°C. The supernatant was used to determine MDA content according to the method of Yang et al. (2009). Respiration assay Ten fruits from each treatment were individually weighed and placed into 40 l glass jars equipped with septa and sealed for 2 h at 20°C. One-ml gas samples were withdrawn from the headspace and injected into a gas chromatograph equipped with a thermal conductivity detector (Shimadzu, Tokyo, Japan) for the detection of CO2 levels. Each data point consists of 10 replicate containers. Respiration rate as CO2 production is expressed in mg kg 1 h 1. Determinations of quality parameters Fruit firmness was determined using a hand-held fruit firmness tester (GY-1: China) equipped with a 5-mm diameter probe. The probe was inserted to a depth of 05 cm and the force recorded in newtons. The fruits were weighed regularly to determine weight loss, which was expressed as percentage. Pulp (25 g) from 10 fruits of each treatment was homogenized using a grinder and then centrifuged at 4800 g for 10 min. The supernatant was collected and analysed for the amount of TSS using a hand refractometer (model N1; ATAGO, Tokyo, Japan); TA content were determined by titration with 01 mol l 1 NaOH, and Vc content was determined by 2,6-dichlorophenolindorhenol titration (AOAC 1984). 508

Statistical analysis The data were analysed by one-way analysis of variance (ANOVA) using SPSS 115 (SPSS Inc.) for Windows. All results were expressed as the means SD. ANOVA was carried out using Tukey’s HSD tests at P < 005 to examine significant differences between groups. Acknowledgements This work was financially supported by the Fundamental Research Funds for the Central Universities, China (grant no. 2009QC029) and natural science foundation of Hubei province, China (grant no. 2011CDB136). Conflict of interest No conflict of interest declared. References Almajano, M.P., Carb o, R., Jimenez, J.A.L. and Gordon, M.H. (2008) Antioxidant and antimicrobial activities of tea infusions. Food Chem 108, 55–63. AOAC. (1984) Official Methods of Analysis of the Association of Official Analytical Chemists, 14th edn. Washington, DC: AOAC. Askarne, L., Talibi, I., Boubaker, H., Boudyach, E.H., Msanda, F., Saadi, B. and Ait Ben Aoumar, A. (2012) Use of Moroccan medicinal plant extracts as botanical fungicide against citrus blue mould. Lett Appl Microbiol 56, 37–43. Chen, J.J., Zhang, S.S. and Yang, X.P. (2013) Control of brown rot on nectarines by tea polyphenol combined with tea saponin. Crop Prot 45, 29–35. Gonzalez-Ure~ na, A., Orea, J.M., Montero, C. and Jimenez, J.B. (2003) Improving postharvest resistance in fruits by external application of trans-resveratrol. J Agr Food Chem 51, 82–89. Hao, W.N., Zhong, G.H., Hu, M.Y., Luo, J.J., Weng, Q.F. and Rizwan-ul-Haq, M. (2010) Control of citrus postharvest green and blue mould and sour rot by tea saponin combined with imazalil and prochloraz. Postharvest Biol Technol 56, 39–43. Hirasawa, M. and Takada, K. (2004) Multiple effects of green tea catechin on the antifungal activity of antimycotics against Candida albicans. J Antimicrob Chemoth 53, 225–229. Horsfall, J.G. (1945) Fungicides and Their Action. Waltham: Chronica Bot. Co. Ji, C. and Kuc, J. (1996) Antifungal activity of cucumber b-1,3-glucanase and chitinase. Physiol Mol Plant Pathol 49, 257–265. Ju, Z.G. and Curry, E.A. (2001) Plant oil emulsions prevent senescent scald and core breakdown and reduce fungal decay in ‘Bartlett’ pears. J Am Soc Hortic Sci 126, 358–363.


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