| Ginseng (Panax ginseng C.A. Meyer) is a perennial herbaceous plant in the family Araliaceae which has been grown in northeast China for centuries, is known as the king of medicine. Cylindrocarpon root rot of ginseng caused by Cylindrocarpon destructans is the main disease which led to serious crop yield losses, and the average loss is up to20%-30%. The soilborne disease is very difficult to control. The use of high toxic pesticide is forbidden because of GAP cultivation. It maybe provide a new way to control Cylindrocarpon root rot of ginseng by using the principle of plant induced disease resistance to improve the resistance of ginseng plant and using molecular detection method to monitor pathogen population. The inducing mechanism of Cylindrocarpon root rot was studied and the pathogen molecular detection system was established based on plant induced disease resistance from the physiological and biochemical reaction, proteomics, and molecular detection. The results were as follows:1. The effects of salicylic acid, cinnamic acid, ferulic acid, benzoic acid, p-hydroxybenzoic acid and methyl jasmonate on C. destructans were studied. These chemicals were added into media to incubate C. destructans to investigate their effects on the pathogen. The results indicated that the growth and spore germination of C. destructans were suppressed by cinnamic acid, ferulic acid, benzoic acid, while p-hydroxybenzoic acid had no influence on the pathogen. At low concentration of salicylic acid and methyl jasmonate, the growth of C. destructans was hardly affected, while at higher concentrations (>200μg mL-1) the growth of pathogen was prohibited.2. The resistance of ginseng to C. destructans could be induced by low concentrations of SA and MeJA. Of these6kinds of chemicals, the induced effects of SA and MeJA were better than other chemicals. It showed that ginseng treated with low concentration of salicylic acid and jasmonic acid methyl jasmonate could enhance the resistance of ginseng plant itself, the control effects were up to67.9%and56.6%, respectively, and the two kinds of chemical factors in low concentration had promoting effects on the growth of ginseng. However, the other chemicals had harmful effects on ginseng growth. Therefore, SA and MeJA were chosen for next study.3. The resistance of ginseng induced by SA and MeJA was firstly demonstrated. The physiological and biochemical mechanisms of ginseng resistant to Cylindrocarpon root rot induced by exogenous salicylic acid and methyl jasmonate were studied. SA and MeJA treatment could effectively decrease the contents of MDA and relative electricity conductivity, but increase the contents of proline, soluble sugar and total phenols. The activities of PAL, CAT, PPO, POD,(β-1,3-glucanase and chitinase could be increased significantly in the ginseng roots induced by SA and MeJA than those in control. The incidence and disease severity were reduced with ginseng plant resistance increased obviously due to exogenous applied SA and MeJA, and the incidences were reduced by39.1%and34.8%, respectively. It demonstrated that SA and MeJA could induce the system resistance of ginseng to C. destructans. In addition, the impact of SA was better than MeJA. Thus, SA is chosen as the chemical factor for further study.4. A stable and vigorous growth of ginseng suspension cell lines was established using plant tissue culture technology. The establishment of the system provides a stable and uniform test material in ginseng proteomics research. MS culture medium was used in callus induction, supplement with3mg/L2,4-D and0.2mg/L KT,0.8%sucrose, pH5.8to6.0. Successive transfer culture added2mg/L2,4-D and0.5mg/L BA. The hormone of suspension cell liquid medium was at the same levels of successive transfer culture. The culture condition of callus is25℃, dark culture. The suspension cell masses were grown in the flasks on a rotary shaker (110rpm)at24±2℃.5. The proteome of salicylic acid inducing ginseng suspension cell was firstly studied by two-dimensional gel electrophoresis technology. A high-throughput and high-resolution two-dimensional gel electrophoresis system was established for suspension-cultured ginseng cells proteins. The proteins were extracted by using the phenol extraction, the elements of lysis buffer were composed of9M urea,2M Thiourea,2%IPG buffer,4%CHAPS,65mM DTT and1%TBP. The pH5-8IPG strip was used. SDS-PAGE was performed in12%polyacrylamide gels. The proteins related to resistance were identified.2-DE was adopted to separate the differentially expressed proteins from suspension-cultured ginseng cells induced by SA. About800protein spots were detected on the2-DE gels. Twenty-four differentially expressed proteins that changes in protein abundance in more than2times and good repeatability were analyzed by MALDI-TOF-MS, of23proteins were successfully identified. In the absence of pathogen,12proteins had increased levels, two proteins had decreased levels and one protein was newly induced in suspension cultures treated directly with SA, in comparison to the control. Two proteins were up-regulated and one protein was down-regulated after SA treatment in the presence of C. destructans. Five proteins were up-regulated and only one protein was down-regulated in suspension cultures were differentially expressed after both SA treatment and pathogen infection. These identified proteins were predicted to be involved in defense and stress responses, energy and metabolism, signal transduction/transcription, protein synthesis and metabolism, photosynthesis and others. The proteins related to defense responses, such as Chaperonin60, monodehydroascorbate reductase, CAD and orcinol O-methyltransferase, were more abundant in suspension cultures of ginseng roots after application of S A.6. The molecular detection of Cylindrocarpon root rot of ginseng was established. A pair of primers specific for C. destructans was designed from comparisons of the internal transcribed spacer (ITS) regions1and2of isolates of diverse origins with sequences from commonly encountered fungi in soil. Under stringent PCR conditions, primers CD-F/CD-R amplify a450bp fragment from C. destructans DNA but not from other species or negative control. And C. destructans could be specifically detected with CD-F/CD-R from ginseng tissues and soil samples inoculated artificially. |
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