中国科技论文在线 http://www.paper.edu.cn 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 0925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2005.05.012 转载 中国科技论文在线 http://www.paper.edu.cn 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 repli- cated 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 B. Yang et al. / Postharvest Biology and Technology 38 (2005) 183–187 185 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.