Study On The Response Of Active Oxygen Metabolism And Protective Enzyme System Of The Casuarina Equisetifolia L.roots Under Its Allelochemicals Stress

Continuous planting was an obstacle which has long been restricted the sustainable management of Casuarlna equisetifolia L.plantations,and Casuarina equisetifolia L.autotoxicity is one of important factor causing it.In the dissertation,clones of ’Huian No.T Casuarlna equisetifolia L.were studied,allelochemicals in its roots were isolated,purified and identified and Casuarina equisetifolia L.-endophytic fungus symbiont were built.Experiments were conducted to investigate the effects of allelochemical stress on some physiological processes of water-cultivated seedlings of Casuarina equisetifolia L.(Casuarina equisetifolia L.EF)and Casuarina equisetifolia L.-endophytic fungus synbiont(Casuarina equisetifolia L.El),including active oxygen metabolism,protective enzyme system and so on.The differential resistance of Casuarina equisetifolia L.and Casuarina equisetifolia L.-endophytic fungus symbiont was analyzed when subjected to allelochemical stress,which provide data accumulation and technical support to solve the problem.The main results are as follows:(1)The ethanol extract chemicals of ’Huian No.T Casuarina equisetifolia L.roots were isolated and identified by the techniques of high performance liquid chromatography(HPLC),column chromatography(CC),mass spectrum(MS),nuclear magnetic resonance(NMR).The result showed that the mainly chemical composition was phenolic acids.By spectrum analysis,the structure of the chemicals was identified as 12J3-dihydromicromeric acid(M-l),betulinic acid(M-2),3-O-coffee-acyl-lupeol(M-3),catechin(M-4)and epicatechin(M-5).The contents of M-l,M-2,M-3,M-4 and M-5 in roots were respectively 0.0547%,0.0612%,0.6095%,0.1623%and 0.0991%determined by HPLC.The chemicals extracted from the roots were biologically detected based on the seed germination experiments.In different concentrations of 12.5 mg·1-1,25 mg·L-1,50 mg·L-1,100 mg·L-1,200 mg·L-1 and 400mg·L-1,the restraining effects of extracts on seed germination were analysised by principal component analysis.Under various concentrations,the restraining effects of extracts on seed germination were M-3>M-1>M-2>M-4>M-5.The result showed that 5 kinds of extracts all restrained the seed germination and the restraint increased with higher concentrations.Therefore,allelopathy of the 5 extracts on Casuarina equisetifolia L.was assumed.(2)The mycorrhizal colonization rate and colonization intensity of Casuarina equisetifolia L.endophytic fungus(CGMCC No.6307)were studied in different colonization time(12,24,36,48,60,72,84,96 h).The results showed that mycorrhizal colonization rate was over 48%if seedlings were soaked for 60 h in endophytic fungus suspension culture and the differences of mycorrhizal colonization rate were not significant in 60-96 h(P>0.05),It suggested that 60 h was appropriate for endophytic fungus to colonize seeding roots.(3)Under the simulation of autotoxicity,Hydrogen peroxide(H2O2),malondlaldehyde(MDA)and superoxide radical(O2)in the root quantitatively increased with the concentration of allelochemicals over the time course.The activities of superoxide catalase(CAT),peroxidase(POD)and dismutase(SOD)in the root showed pulses,increasing at the early stage and then decreasing gradually to the level of the control.The occurrence of peak activities of CAT,POD and SOD,in the root was earlier with increased stress intensity.The effects of 5 allelochemicals on restraint intensity,o2 production rate,HZO2 and MDA of CAT,POD and SOD protective enzymes activity of Casuarina equisetifolia L,EF and Casuarina equisetifolia L.EI were M-3>M-2>M-1>M-4>M-5.The scavenging oxygen ability of CAT,POD and SOD was significant under different time and different strength stress.The protective enzymes in root of Casuarina equisetifolia L.are important for resisting oxidation damage induced by slight(12.5、25、50、100mg·L-1)allelochemical stress at the early stage(0-48h),but it would be restrained to the concentration of allelochemicals over the time course.The excess reactive oxygen produced inside the plant would likely lead to peroxidization of cytomembrane and therefore harms the plant.This may explain one of the intoxicating effects of allelochemicals on Casuarina equisetifolia L.The endophytic fungus colonization hindered the stress intensity to some degree and the effect was obvious in slight(12.5、25、50、100mg·L-1)allelochemical stress at the early stage(0-48h).The effective concentration and stress time was the same as those that Casuarina equisetifolia L.endured,which showed that the activities of Casuarina equisetifolia L.El protective enzyme improved by endophytic fungus shouid be built before protective enzyme system synthesis and degradation suffered from irreversible damage.