| The effects of chitinooligosaccharide on the germination vigor and capacity of tobacco seeds, the inhibitive activity of chitinooligosaccharide to two important tobacco pathogens---Phytophthora nicotianae Breda de Hean var. nicotianae Waterh and Alternaria alternata (Fr.) Keissler, the control effectiveness of chitinooligosaccharide to tobacco black shank and brown spot based on the preparation and refine of Trichodema chitinase and chitinooligosaccharide were systematically studied in this paper respectively. And the dynamic activity of resistant enzymes such as superoxide dismutase(SOD) in the tobacco plants treated by chitinooligosaccharide with artificial inoculation were measured to explore the bio-control effects and mechanism of chitinooligosaccharide to tobacco black shank and brown spot. The main results were as follows. 1. The chitinase activities at different time and different temperature (20℃,30℃,35 and 40℃) were determined with Trichodema isolate YHS-1 respectively. The results showed that the optimum temperature for the production of chitinase was 35℃, the optimum time was the forth day and the enzyme activity was 55U on this condition. Chromatographically pure chitinase could be obtained by (NH4)2SO4 fractional precipitation, anion-exchange chromatography, SDS-PAGE, and a singular and high chromatography peak could be found and two electrophoretic bands could be validated from gel electrophoresis. The two bands are exochitinase and endochitinase. Pure chitinooligosaccharide could be obtained by the purification of the crude extract with protein precipitation and refrigerated centrifuge. 2. The germination vigor and rate of K326 tobacco seeds socked with different concentrations chitinooligosaccharide solution were tested respectively. The results indicated that chitinooligosaccharide could improve germination vigor and rate of tobacco seeds, and that the optimum concentration for treatment was 0.04mg/ml. The germination vigor and rate of seeds treated by the optimum concentration were 40%, 6% higher than those of the contrast, respectively. The activities of polyphenol oxidase (PPO),SOD and phenylalanine deaminase (PAL ) in tobacco germination seeds treated by chitooligodaccharide were higher than those of the contrast. The activity of SOD was nearly 2 times higher than that of the contrast, while there were no activities of POD and PAL in the contrast, and the activities in the germination seeds treated by chitooligodaccharide could reach to 42U and 20U respectively. The results mentioned above suggested that germination seeds socked by chitooligodaccharide could induce activities of PPO,SOD and PAL. 3. Inoculating the tested isolate AL-1 A. alternata on the tested varieties, hongda and K326 was conducted, with mycelium block inoculation, mycelium suspension inoculation, spore hanging drop inoculation, spore spraying inoculation and spore suspension in cotton ball inoculation respectively. The results suggested that mycelium block inoculation and spore suspension in cotton ball inoculation could reflect the resistance difference of tobacco to the tested pathogen, and the high rate of success and repeatability of the pathogenic organism suggested that the two methods were suitable to the inoculation of the pathogenic orgamism. 4. In vitro, the inhibitive activities of chitiooligose to A. alternata and P .nicotianae. var. nicontianae were tested with mycelium block inoculation. The results showed that chitooligodaccharide could inhibit the growth of A. alternata .efficiently but couldn't restraint the mycelium growth of P. nicotianae. var .nicotianae. 5. The control effectiveness of the five treatments such as chitooligodaccharide, chitooligodaccharide +Trichodema +chitin were investigated on the condition of artificial inoculation and pot cultivation in greenroom. Corresponding, the experiment results indicated that the black shank lesion area of tobacco stems was 42% of those in contrast and the control effectiveness was 58% in the treatment with chitooligodaccharide solel... |
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