Harpin Induces Local and Systemic Resistance Against Trichothecium Roseum in Harvested Hami Melons

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Postharvest Biology and Technology 38 (2005) 183–187

Research Note Harpin induces local and systemic resistance against Trichothecium roseum in harvested Hami melons

Bi Yang a,b, Tian Shiping b,∗, Zhao Jie c, Ge Yonghong c

a School of Life Science, Lanzhou University, Lanzhou 730000, PR China b Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, PR China c Department of Food Science, Gansu Agricultural University, Lanzhou 730070, PR China

Received 11 November 2004; accepted 27 May 2005

Abstract

Postharvest application of harpin to induce resistance was studied in two cultivars of Hami melon (Cucumis melo L. var. inodorus Jacq.) inoculated with Trichothecium roseum. Harpin signiﬁcantly reduced lesion diameter in inoculated fruit. A greater level of decay control was observed in long-term storage cultivars (cv. 8601). The treatment at 90 mg/L was the most effective concentration and higher concentrations over 90 mg/L failed to promote resistance and did not cause phytotoxicity to melons. Harpin did not demonstrate any fungicide effect in vitro, but suppressed lesion diameter in treated and untreated halves of the same fruit, suggesting induction of local and systemic resistance. Efﬁcacy of suppression lasted 8 and 6 days for ‘8601’ and 5 and 3 days for ‘New Queen’ cultivars in harpin-treated and untreated halves, respectively. The protection by harpin was associated with the activation of peroxidase (POD) and chitinase (CHT). © 2005 Elsevier B.V. All rights reserved.

Keywords: Harpin; Hami melons (Cucumis melo L. var. inodorus Jacq.); Trichothecium roseum; Induced resistance

1. Introduction and potential harmful effects on the environment and human health, new strategies for controlling posthar- Pink rot, caused by Trichothecium roseum,is vest diseases have been proposed. reported as one of principal postharvest diseases of Induction of natural disease resistance in harvested Hami melons (Bi et al., 2003) and the rot can be con- horticultural crops is a preferred strategy for disease trolled by iprodione and azoxystrobin (Ma et al., 2004). management (Terry and Joyce, 2004). Harpin, a bac- However, due to problems related to fungicide toxic- terial hypersensitive response (HR) elicitor, is a heat- ity, development of fungicide resistance by pathogens stable, glycine-rich protein that was ﬁrst described in Erwinia amylovora, which causes ﬁre blight of apple, ∗ Corresponding author. pear and other members of the Rosaceae (Wei et al., E-mail address: [email protected] (T. Shiping). 1992). Application of harpin induces systemic acquired

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184 B. Yang et al. / Postharvest Biology and Technology 38 (2005) 183–187

resistance (SAR) response in cucumber (Strobel et al., The effect of harpin on mycelial growth was assayed 1996). It has also been applied effectively as a posthar- using the method of Aharoni et al. (1993). Harpin at vest treatment to suppress the blue mould caused by 0, 30, 60, 90, 120 and 200 mg/L, was tested against Penicillium expansum in apples (de Capdeville et al., T. roseum. The chemical was added to PDA after 2003). It is commercially released in some countries as autoclaving for 15 min and cooling to 45–50 ◦C. A a plant health promoter under the name of Messenger®. 5 mm diameter disc of mycelial mat from a 7-day- The aim of this study was to test harpin for its ability old culture was placed in the center of each Petri to induce resistance in two cultivars of Hami melon fruit plate. The growth of the fungus was measured daily, against T. roseum and to analyze whether the induced over a 5-day incubation period at 25 ◦C and was fruit accumulate defense-related enzymes. determined as the average diameter of the mycelial mat following incubation. Each treatment was replicated three times and the experiment was repeated 2. Materials and methods twice. For in vivo assays, fruit treated with 0, 30, 60, 90, Hami melons (Cucumis melo L. var. inodorus Jacq. 120 and 200 mg/L of harpin were sampled after 2 days cvs. 8601 and New Queen) were obtained from Huang- of treatment. Fruit treated at 90 mg/L were removed dun State Farm in Dunhuang, Gansu. The fruit were after 2–6 days for ‘New Queen’ or 2, 4, 6, 8 and 10 days harvested at the beginning of physiological maturity for ‘8601’ of treatment, respectively. The melons were (42–43 days after full bloom for the short-term stor- disinfected with 70% ethanol and 9 wounds were made age cultivar ‘New Queen’ and 55–56 days after full with a sterile needle (3 mm deep) around the equator bloom for the long-term storage cultivar ‘8601’). They of treated and untreated halves of each melon. Twenty were sorted for uniform size and absence of obvious microlitres of spore suspensions (1 × 105 spores/mL) injuries, packed individually in 35 cm long net bags of T. ruseum were pipetted into each wound. After dry- of foam plastic (Fulihua Plastic Products Co. Ltd., ing in air, fruit were put in the boxes, covered with plas- Lanzhou), put in the standard melon shipping boxes tic film for high RH conditions (RH 85–90%) and kept (4 melons/box), transported to the laboratory arriving at 22 ± 2 ◦C. Fruit were evaluated for lesion diameter within 48 h of harvest and stored at room temperature 9 days after inoculation. Each treatment was applied (22 ± 2 ◦C) with a relative humidity (RH) of 55–60%. to three replicates of 12 melons. The experiment was T. roseum was isolated from infected Hami mel- repeated two times. ons and maintained on potato dextrose agar (PDA) For enzyme assays, fruit without inoculation were at 4 ◦C. Conidia were obtained from 10-day-old PDA used at the same degree of maturity (parameters tested cultures incubated at 25 ◦C. The spore concentration were firmness, color of the pulp and total soluble of the suspension was adjusted with distilled water to solids). Approximately 2 g fruit samples were taken 1 × 105 spores/mL with a hemacytometer. from 1.0–1.5 cm below the skin with a stainless steel Harpin (Messenger, Eden Bioscience Co., Bothell, cork borer around the equator of treated and untreated WA) was prepared at 30, 60, 90, 120 and 200 mg/L halves of each melon. Each sample was packed and with distilled water. One half of each melon (with both frozen in liquid nitrogen and kept at −80 ◦C until crude sun and ground sides) was dipped for 10 min in var- enzyme extraction. ious concentrations of harpin (treated half) and the Crude enzyme was extracted according to the remaining half (also with sun and ground sides) was not method of Venisse et al. (2001) with some modi- dipped (untreated half) and then the melons were air- fications. Two grams of frozen tissue was homoge- dried in the shade. Control fruit were similarly dipped nized with a mortar and pestle in 2 mL cold sodium half in water (water-treated and untreated halves). The phosphate buffer (50 mM, pH 7.5) containing 1 mM fruit were marked and packed individually in net bags polyethyleneglycol, 1 mM phenylmethylsulfonyl flu- of foam plastic, put in the standard melon shipping oride, 8% (w/v) polyvinypolypyrolidone and 0.01% boxes (4 melons/box) and stored at room temperature Triton X-100. Homogenates were centrifuged at (22 ± 2 ◦C, RH 55–60%) for inoculation and enzyme 16,000 × g for 20 min at 4 ◦C and supernatants were analysis. immediately assayed for enzyme activities. 中国科技论文在线 http://www.paper.edu.cn

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Peroxidase (POD) activity was determined accord- protein. All enzyme tests were performed with a min- ing to the methods of Venisse et al. (2001) using imum of three tissue samples replicated per treatment guaiacol as the substrate. Oxidation of guaiacol to and per time point. Each experiment was performed at tetraguaiacol was monitored spectrophotometrically at least three times. 470 nm for 1 min at 30 ◦C. Peroxidase activity was All statistical analyses were performed using SPSS. expressed as nmol tetraguaiacol produced per min and To test for the effect of the treatments at different con- per mg of proteins using a molar extinction coefficient centration and duration of protection, the data were of 26.6 mM/cm. analyzed by two- and one-way analysis of variance Chitinase (CHT) activity was assayed accord- (ANOVA). ing to the method of Venisse et al. (2001) using carboxymethyl-curdlan-remazol brilliant violet (CM- curdlan-RBV) as the substrate (Loewe Biochemica, 3. Results and discussion Otterfing, Germany). CHT activity was calculated from the difference of absorbance at 550 nm between the No significant differences were observed in the sample and reference. Data were expressed as the mycelial growth of T. roseum at different concentra- increase of absorbance per min and per mg of protein. tions of harpin from 30 to 200 mg/L when compared Protein content was determined according to the with the control. The average diameter of the mycelial method of Bradford (1976) using BSA as the standard mat was 55 mm after 5 days at 25 ◦C.

Fig. 1. Effect of harpin dips at 0, 30, 60, 90, 120 and 200 mg/L on lesion diameter in treated and untreated halves of inoculated fruit of both cultivars (A: 8601 and B: New Queen). Fruit were inoculated with 20 ␮L spore suspensions (1 × 105 spores/mL) of T. roseum 2 days after harpin treatment. Decay was evaluated after incubation at 22 ◦C for 9 days. The data were analyzed by two-way ANOVA. Bars with the same letters represent values that are not signiﬁcantly different (P = 0.05). 中国科技论文在线 http://www.paper.edu.cn

186 B. Yang et al. / Postharvest Biology and Technology 38 (2005) 183–187

Lesion diameter was significantly reduced by treat- temic. Moreover, no greater control was found at higher ment of harpin, depending upon the cultivars, concen- concentrations and phytotoxicity was not observed tration and treated or untreated half of the fruit. Harpin even at 200 mg/L of harpin in the two cultivars. provided greater control in ‘8601’ than in ‘New Queen’ The time between initial treatment with harpin fruit and the most effective treatment was 90 mg/L. In and subsequent inoculation with T. roseum signifi- ‘8601’ fruit, the treatment at 90 mg/L had the smallest cantly affected efficacy of the induction. In ‘8601’ lesion diameter, which was decreased by 28 and 18% in fruit, although treatment for a few hours early (0 treated and untreated halves, respectively (Fig. 1A). In day) or 8 days significantly reduced lesion diameter ‘New Queen’ fruit, the lesion diameter for the treatment in the harpin-treated half, the greatest reductions were at 90 mg/L was 21 and 13% less in treated and untreated observed with treatment periods of 2–6 days in harpin- halves, respectively (Fig. 1B). The treated and treated and untreated halves (Fig. 2A). In ‘New Queen’ untreated halves of the same fruit were protected, indi- fruit, the effect of harpin remained constant for 5 days cating that the effect of harpin was both local and sys- in the harpin-treated half. When the untreated half was

Fig. 2. Effect on lesion diameter of the time interval between application of harpin at 90 mg/L and wound inoculation with T. roseum in treated and untreated fruit of both cultivars (A: 8601 and B: New Queen). Fruit were inoculated with 20 ␮L spore suspensions (1 × 105 spores/mL) of T. roseum after harpin treatment. Decay was evaluated after inoculation for 9 days at 22 ◦C. The data were analyzed by one-way ANOVA. Bars with the same letters represent values that are not signiﬁcantly different (P = 0.05). 中国科技论文在线 http://www.paper.edu.cn

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inoculated 3 days after harpin treatment, the lesion (2001BA501A09), the National Natural Science Foun- diameter was significantly reduced (Fig. 2B). A time- dation of China (30430480) and Australia Cen- dependent response indicates that harpin-treated and ter of International Agricultural Research (ACIAR, untreated halves are able to resist the disease if they PHT/1998/140). We thank Dr. Stephen Morris (Syd- have enough time to develop higher levels of defense ney Postharvest Laboratory) for critical reading of the induction for slowing the ingress and advance of the manuscript. pathogen. Harpin induced a significant and progressively increasing activity of POD and CHT in treated and References untreated halves. In ‘8601’ fruit, POD and CHT activities increased in treated halves from a basal level Aharoni, Y., Copel, A., Fallik, E., 1993. Hinokitiol (␤-thujaplicin), of 130–426 nmol tetraguaiacol/min/mg of protein and for postharvest decay control on ‘Galia’ melons. New Zealand J. Crop Hort. Sci. 21, 165–169. from 3.38 to 8.67 OD550/min/mg of protein over 2 Bi, Y., Tian, S.P., Liu, H.X., Zhao, J., Cao, J.K., Li, Y.C., Zhang, days. In ‘New Queen’ fruit, POD and CHT activi- W.Y., 2003. Effect of temperature on chilling injury, decay and ties increased in treated halves from 132 to 274 nmol quality of Hami melon during storage. Postharvest Biol. Technol. tetraguaiacol/min/mg of protein and from 2.33 to 29, 229–232. Bradford, M.M., 1976. A rapid and sensitive method for quantita- 4.71 OD550/min/mg of protein in the same time period. tion of microgram quantities of protein utilizing the principle of POD activity reached a maximum after 6 and 4 days of protein–dye binding. Anal. Biochem. 72, 248–254. treatment in ‘8601’ and ‘New Queen’ fruit, being over Brisson, L.F., Tenhaken, R., Lamb, C.J., 1994. Function of oxida- 2.4 and 2.5 times higher in the treated than in the water- tive cross-linking of cell wall structural proteins in plant disease treated halves. The induced activation lasted at least 10 resistance. Plant Cell 6, 1703–1712. and 6 days in treated halves. The maximum of CHT de Capdeville, G., Beer, S.V., Watkins, C.B., Tedesehi, L.O., Aist, J.R., 2003. Pre- and postharvest harpin treatments of apples activity was found after 4 and 3 days of treatment in induced resistance to blue mold. Plant Dis. 89, 39–44. ‘8601’ and ‘New Queen’ fruit, being more than 1.7 and Ma, L.Y., Bi, Y., Zhang, Z.K., Zhao, L., An, Li., Ma, K.Q., 2004. 2 times higher in the treated halves than in the water- Control of pre- and postharvest main diseases on melon variety treated halves. The induced activation lasted at least 8 Yindi with preharvest azoxystrobin spraying. J. Gansu Agric. and 5 days in treated halves. POD and CHT activities Univ. 39, 14–17. Strobel, R.N., Gopalan, J.S., Kuc, J.A., He, S.Y., 1996. Induction were low and there was a detectable increase in water- of systemic acquired resistance in cucumber by Pseudomonas treated and untreated halves during the period. POD syringae pv. syringae 61 HrpZpss protein. Plant J. 9, 431–439. participates in cell wall reinforcement and is involved Terry, L.A., Joyce, D.C., 2004. Elicitors of induced disease resistance in the final steps of lignin biosynthesis and in the cross- in postharvest horticultural crops: a brief review. Postharvest linking of cell wall proteins (Brisson et al., 1994). CHT Biol. Technol. 32, 1–13. Van Loon, L.C., 1997. Induced resistance in plants and the role demonstrates mainly antifungal activity by hydrolyz- of pathogensis-related proteins. Eur. J. Plant Pathol. 103, 753– ing the cell walls of many phytopathogenic fungi (Van 765. Loon, 1997). Venisse, J.S., Gullner, G., Brisset, M.N., 2001. Evidence for the involvement of an oxidative stress in the initiation of infec- tion of pear by Erwinia amylovora. Plant Physiol. 125, 2164– 2172. Acknowledgements Wei, Z.M., Laby, R.J., Zumoff, C.H., Bauer, D.W., He, S.Y.,Collmer, A., Beer, S.V., 1992. Harpin, elicitor of the hypersensitive This research was supported by the grants from response produced by the plant pathogen Erwinia amylovora. Ministry of Science and Technology of China Science 257, 85–88.