Effect of harpin on physiological characteristics of walnut under low temperature stress

Abstract：【Objective】‘Xiangling'is an early fruiting walnut cultivar widely cultivated in north China, but it is susceptible to cold and often damaged by late frost, which results in significant decline in yield or even total crop failure. Harpin is a stable polypeptide isolated from Erwinia amylovory, and has been used as a pure biopesticide that induce resistance in plants. In this study, the effect of harpin on physiological characteristics of walnut under low temperature stress was studied in order to explore the mechanism of harpin improving cold resistance of walnut and to provide theoretical basis for the application of harpin in walnut production.【Methods】1.5 g·L^-1 of harpin was sprayed to 6-year-old trees of walnut cultivar‘Xiangling'prior to female flower stage with distilled water spray as the control. The experiment was conducted with a random block design, with 5 trees in each of 3 blocks. 7 d after spraying, shoots of the same diameter and length were sampled from the same direction of the canopy of each treated walnut tree and placed into a phytotron (RXZ-0288) and exposed to different low temperatures (2, 0, -2, -4 and-6 ℃) . The temperature in the phytotron was initially set at 10 ℃ and programmed to decrease at a rate of 4 ℃·h-1and maintained at each target low temperature for 8 h, and then the temperature was set to 10 ℃ again. Thethird leaf from the top was taken from the young sprout after 5 h at 10 ℃. The samples were frozen in liquid nitrogen and stored under-70 ℃. The relative electricity conductivity was determined using an electricity conductivity meter. Soluble protein was determined with coomassie brilliant blue. Soluble sugars were determined with anthrone colorimetric method, proline content with acidic-ninhydrin reaction method, and malondialdehyde (MDA) with thiobarbituric acid method. The superoxide dismutase (SOD) activity was determined with the method of Xue Guoxi, peroxidase (POD) with the method of guaiacol, and catalase (CAT) with the method of ultraviolet absorption.【Results】With the decrease of temperature, the relative electricity conductivity and malondialdehyde (MDA) content in walnut leaves increased in both the treatment (sprayed with harpin) and the control (spraying distilled water) , but they were lower in the former than in the latter. The relative electricity conductivity in the treatment and the control was 27.71%and 32.13% at -6 ℃, respectively, and their content of MDA was 24.51 μmol·g^-1and 18.25 μmol·g^-1, respectively. The relative electricity conductivity in the treatment and the control had significant difference under various low temperatures and the MDA content had significant differences under the low temperature of-2, -4 and-6 ℃. The activities of SOD, POD and CAT increased with the decrease in temperature and were higher in the treatment. The activity of SOD in the treatment (119.23 U·g^-1) was 1.22 times that of the control (97.71 U·g-1) at-6 ℃; POD activity in the treatment (0.032 μmol·g^-1·min^-1) was 1.39 times that of the control (0.023 μmol·g^-1·min^-1) ; and CAT activity in the treatment (0.124 μmol·g^-1·min^-1) was1.36 times that of the control (0.091 μmol·g^-1·min^-1) . The activities of SOD, POD and CAT were significantly different between the treatment and the control under the temperatures of 0, -2, -4 and -6 ℃. The contents of proline and soluble proteins increased with the decrease in temperature, and they were higher in the treatment than in control. Proline content in the treatment (98.85 μg·g^-1) was 1.112 times that of the control (88.93 μg·g^-1) under the-6 ℃. Soluble protein content in the treatment (12.17 mg·g^-1) was1.166 times that of the control (10.44 mg·g^-1) . Under the low temperatures of 0, -2, -4 and-6 ℃, the contents of proline and soluble proteins were significantly different between the treatment and the control.With the decrease in temperature, the content of soluble sugars in the treatment and the control decreased, but the content of soluble sugars in the treatment was higher than in the control. When the temperature decreased from 2 to-6 ℃, the percentage of decrease in soluble sugar content in the treatment (11.8%) was lower than in the control (15.2%) . But the difference in soluble sugar content between the treatment and the control was not significant.【Conclusion】Spraying harpin increased the contents of proline and soluble proteins and the activities of antioxidant enzymes in the leaves of walnut but reduced the relative electricity conductivity and MDA content under low temperature stress. The treatment helped the walnut to maintain normal physiological status of cells, preventing membrane lipid peroxidation, maintaining the integrity of membrane structure, and enhancing the cold resistance.