| Allelopathy refers to the direct or indirect positive or negative effects exerted on plant itself or other plants,microbes or insects by plant's release of chemicals to the environment,including intraspecific autotoxicity and interspecific allelopathy.Domestic researchers have proved that the accumulation of allelochemicals will cause the decline of soil fertility,productivity and shelter effect in Casuarina equisetifolia L.stand,which leads to its regeneration difficulty.In the dissertation,clones of 'Huian No.1'Casuarina equisetifolia L.were studied,allelochemicals in its branchlets 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 symbiont(Casuarina equisetifolia L.EI),including active oxygen metabolism,protective enzyme system,root activity,root endogenous hormone and chlorophyll fluorescence parameters.The differential proteome of water-cultivated seedling branchlets of Casuarina equisetifolia L.and Casuarina equisetifolia L.-endophytic fungus symbiont was analyzed when subjected to allelochemical stress.The main results are as follows:1.Identification of isolated Casuarina equisetifolia L.allelochemicals and building of Casuarina equisetifolia L.-endophytic fungus symbiontThe ethanol extract chemicals of 'Huian No.1' Casuarina equisetifolia L.branchlets were isolated and identified by the techniques of column chromatography(CC),high performance liquid chromatography(HPLC),mass spectrum(MS),nuclear magnetic resonance(NMR).The result showed that the chemicals were composed of flavonoid and gallogen.By spectrum analysis,the structure of the chemicals was identified as quercetin-3-O-?-D-galactopyranoside(M-1),kaempferol-3-O-a-L-rhamnop-yranoside(M-2),Ellagic acid(M-3),3-O-Methylellagicacid-4'-O-?-D-xylopyranoside(M-4)and kaem-pferol-3-O-?-D-glucopyranoside(M-5).The contents of M-1,M-2,M-3,M-4 and M-5 in branchlets were respectively 0.0651%,0.5540%,0.1506%,0.0871%and 0.0596%determined by HPLC.The chemicals extracted from the branchlets were biologically detected based on the seed germination experiments and the result showed that 5 kinds of extracts all restrained the seed germination and the restraint increased with higher concentrations.In different concentrations of 12.5 mg · L-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 M-2>M-1>M5>M3>M4 by principal component analysis.Therefore,allelopathy of the extracts on Casuarina equisetifolia L.was assumed and flavonoid allelochemicals had higher restraining effects on seed germination than gallogen allelochemicals.The mycorrhizal colonization rate and colonization intensity of Casuarina equisetifolia L.endophytic fungus(CGMCC No.6309)were studied in different colonization time(12,24,36,48,60,72,84,96 h).The results showed that mycorrhizal colonization rate was over 40%if seedlings were soaked for 48 h in endophytic fungus suspension culture and 60 h was appropriate for endophytic fungus to colonize seeding roots.2.Effects of allelochemicals on reactive oxygen metabolism and its scavenging system of Casuarina equisetifoia L.seedlings and Casuarina equisetifolia L.-endophytic fungus symbiontHydrogen peroxide(H2O2),malondlaldehyde(MDA)and superoxide radical(O2)in the branchlet quantitatively increased with the concentration of allelochemicals over the time course.The activities of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)in the branchlet showed pulses,increasing at the early stage and then decreasing gradually to the level of the control.The occurrence of peak activities of SOD,CAT and POD in the branchlet was earlier with increased stress intensity.The effects of 5 allelochemicals on restraint intensity,O2 production rate,H2O2 and MDA of 3 protective enzymes' activity of Casuarina equisetifolia L.EF and Casuarina equisetifolia L.El were M-2>M-1>M-5>M-3>M-4.The scavenging of reactive oxygen like H2O2 and O2 by SOD,CAT and POD in Casuarina equisetifolia L.EF branchlets was effected by stress time and intensity.The protective enzymes in banchlet of Casuarina equisetifolia L.are important for resisting oxidation damage induced by slight(25?12.5 mg · L-1)and medium(100?50 mg ·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(25?12.5 mg · L-1)and medium(100?50 mg ·L-1)allelochemical stress at the early stage(0-48h).The effective concentration and stress time was in accordance with those that Casuarina equisetifolia L.endured,which showed that Casuarina equisetifolia L.El protective enzyme system improved by endophytic fungus needed to be built.before protective enzyme system synthesis and degradation suffered from lethal harms.According to the reactive oxygen metabolism and protective enzyme activity variation of Casuarina equisetifolia L.EF and Casuarina equisetifolia L.El by the stress of 5 allelochemicals,M-3 and M-4 differed with M-1,M-2 and M-5 in effecting reactive oxygen metabolism and protective enzyme activity,due to the fact that M-3 and M-4 were gallogen while M-1,M-2 and M-5 were flavonoid.3.Effect of tow allelochemicals of Casuarina equisetifolia on dynamic change of root activity and endogenous hormone of its seedling under hydroponic cultivationWith increased concentration of allelochemicals over the time course,the decreasing trend was shown except the root activity stressed by 12.5 mg · L-1 and 25 mg · L-1 of M-3 and M-4 which showed its decreasing following its increasing.Stressed by M-1,M-2 and M-5,the root activity and the content of Gibberellin(GAs)and heteroauxin(IAA)were significantly decreased and the content of ABA was significantly increased.By slight(12.5 mg ·-1)stress of M-3 and M-4 at the early stage(5d),the contents of GAs and IAA increased,followed by decreased trend and by slight(12.5 mg · L-1)stress of M-4 at the early stage(5d),the contents of ABA decreased,followed by increased trend and the variation of GAs,IAA and ABA in other concentrations showed similar trend with those stressed by M-1,M-2 and M-5.Though the endophytic fungus colonization differently increased the root activity,GAS and IAA of Casuarina equisetifolia L.El and decreased the accumulation of ABA,the effect was slowly imposed.Over the time course,GAs and IAA contents of Casuarina equisetifolia L.El increased more compared with those of Casuarina equisetifolia L.EF by the same treatment,however,with increased concentration and stress time of allelochemicals,the GAs and IAA contents in seedlings suffered greater restraint,and the GAs and IAA contents increased by endophytic fungus was based on the extremely low GAs and IAA contents,therefore the physiological metabolism processes of Casuarina equisetifolia L by the stress of allelochemicals were only partly adjusted.The effects of different allelochemicals on restraint intensity of root activity,GAs and IAA and increase intensity of ABA of Casuarina equisetifolia L.EF and Casuarina equisetifolia L.El were M-2>M-1>M-5>M-3>M-4.Casuarina equisetifolia L showed different hormonal balance when stressed by gallogen(M-3 and M-4)and flavonoid(M-1,M-2 and M-5).Casuarina equisetifolia L.EF increased ABA contents against stresses by M-1,M-2 and M-5 while it increased GAs and IAA contents against stressed by M-3 and M-4.4.Effect of allelochemicals and endophytic fungus of Casuarina equisetifolia L.on chlorophyll fluorescence parameters in branchlet of its seedlingLow concentration stress of M-3 and M-4(12.5 mg·L-1)caused the decrease of F0 and increase of Fm,Fv/F0 and Fv/Fm in branchlets of Casuarina equisetifolia L EF and Casuarina equisetifolia L El while with the increase of concentration.the above parameters showed reverse trends.By the stress of M-1,M-2 and M-5,the Fo values in branchlets increased while Fm,Fv/F0 and Fv/Fm decreased,showing that allelochemical stress lowed the efficiency of primary conversion of light energy,restrained the activity of PS II reaction center,affected linear electron transfer,reduced the transfer of chloroplast excitation energy from LHC II to PS II.decreased photochemistry reaction rate(Prate),boosted non-photochemical quenching(qN),photosynthetic function relative limit(LPAR)and antenna pigment dissipation rate(Drate).reduced PS II actual photochemical efficiency(?PSII)and photochemical quenching(qP),to the extent of inactivation of PS II reaction center.The self protection mechanism of Casuarina equisetifolia L.was more effective by the stress of M-3 and M-4.Based on the analysis of FO,Fm,Fv/F0,Fv/Fm,qP,qN,?PS ?,Prate,Drate and LPAR,50 mg· L-1.was assumed to be the threshold for Casuarina equisetifolia L.seedlings to resist M-3 and M-4 stresses.The concentration lower than it had a certain unobvious effects on chlorophyll fluorescence parameters,showing a good self protection mechanism of seedlings against slight M-3 and M-4 stresses while M-1,M-2 and M-5 had more harm to photosynthetic apparatus.Endophytic fungus slowered the increase of FO and decrease of Fm,Fv/F0 and Fv/Fm in branchlets stressed by slight and medium allelochemical stresses but the effect varied according to allelochemicals.The membership function values of 10 chlorophyll fluorescence parameters in branchlets of Casuarina equisetifolia L.EF and Casuarina equisetifolia L.El were calculated by membership function of fuzzy mathematics and the stress extents by allelochemicals were M-2>M-1>M-5>M-4>M-3.The difference between flavonoid(M-1,M-2 and M-5)and gallogen(M-3 and M-4)allelochemicals comprehensive stress values of Casuarina equisetifolia L.El was higher than that of Casuarina equisetifolia L EF,indicating a better resistance against allelochemical stress on photosynthetic apparatus by endophytic fungus of Casuarina equisetifolia L.-5.The co-effects of Casuarina equisetifolia L.allelochemical and endophytic fungus on proteome differential expression of seedlings50mg · L-1 M-2 was used to stress Casuarina equisetifolia L.EF and Casuarina equisetifolia L.El and total proteins in branchlets was extracted at 0 h,12 h,24 h,36 h and 48 h.IEF/SDSPAGE gel electrophoresis was applied to compare the proteome differential expression.The results showed that the protein dots of protein expression profile of 0 h,12 h,24 h,36 h and 48 h were respectively 1592,1627,1631,1611 and 1624 in Casuarina equisetifolia L.EF while 1612,1634,1617,1640 and 1631 in Casuarina equisetifolia L.EI.The matching discrepancy between Casuarina equisetifolia L EF and Casuarina equisetifolia L.El showed that there were 12 downregulated proteins and 21 upregulated proteins at 0 h,53 downregulated proteins and 51 upregulated proteins at 12 h,15 downregulated proteins and 6 upregulated proteins at 24 h,53 downregulated proteins and 20 upregulated proteins at 36 h and 36 downregulated proteins and 47 upregulated proteins at 48 h.The matching discrepancy within Casuarina equisetifolia L.EF indicated that differential proteins turned to 18,54,73 and 99 over time,among which downregulated proteins were 11,32,46 and 72 while upregulated proteins were 7,22,27 and 2.The matching discrepancy within Casuarina euisetifolia L El showed that differential proteins turned to 65,48,76 and 62 over time,among which downregulated proteins were 31,33,54 and 38 while upregulated proteins were 34,15,22 and 24.The regulation of gene expression of Casuarina equisetifolia L.by M-2 stress varied and the proteins in Casuarina equisetifolia L EF and Casuarina equisetifolia L El had different expressing responses to M-2 stress.The upregulated and downregulated proteins of Casuarina equisetifolia L.El differential proteins outnumbered those of Casuarina equisetifolia L.EF indicated the slow molecular response of Casuarina equisetifolia L EF and possible improvement of Casuarina equisetifolia L.El resistance by endophytic fungus.20 protein dots were identified when 61 differentially expressed proteins were analyzed by MALDI-TOF/MS and the identification success rate was 32.78%.By bioinformatics software analysis,some proteins related to allelopathy stress were identified,such as spermidine synthetase,phosphoglyceric acid kinase,ATP-dependent Clp protease congener CD4B,heat shock 70 kDa protein,copper-zinc oxide SOD etc.The protein differential expression in branchlets showed apparent regulations and variations between Casuarina equisetifolia L.EF and Casuarina equisetifolia L.EL Compared with Casuarina equisetifolia L.EF,protein differential expression of Casuarina equisetifolia L.El showed better regulation metabolism,photosynthesis,respiration,reactive oxygen scavenging,gene regulation,stress signal transduction and stress resistance,which was proved by the results of physiological experiments.Further researches will be conducted to study the relation and influencing mechanism between endophytic fungus colonization and improved physiological metabolism of Casuarina equisetifolia L.. |
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