| Taiwania flousiana Gaussen belongs to Taiwania of Taxodiaceae.The essential oil was obtained from the stem bark of T.flousiana by distillation.The chemical constituents of the essential oil were analyzed by gas chromatography-mass spectrometry?GC-MS?and its mechanism on Spirogyra communis?Hass.?Kutz was studied.The methanol extract was obtained from the stem bark T.flousiana,the isolation and identification of the active herbicidal ingredients were studied by bioassay guided methods.Moreover,the mechanism of action of the active ingredient was studied.The main results are as follows:GC-MS was used to analyze the chemical constituents of the essential oil of the stem bark of T.flousiana.A total of 36 compounds were isolated and identified,including alkanes,phenols,terpenes,and organic acids.The main chemical components were:9-methyl-nonadecane?17.97%?,nonacosane?15.42%?,dl-?-tocopherol?12.63%?,tetracosane?10.51%?,4-[?2-chloro-6-fluorobenzyl?thio]-1,3,5-triazin-2-amine?8.13%?,8-epimanoyl oxide?6.00%?,ferruginol?3.94%?,8 3-?3,4-dimethoxyphenyl?-7-hydroxy chromen-2-one?3.10%?,3-?3,4-dimethoxyphenyl?-7-hydroxybenzopyran-2-one?3.10%?,?-tocopherol?2.98%?,2,3,5,6-Tetrahydro-3,3,4,5,5,8-hexamethyl-s-indacene-1,7-dione?2.42%?,1,2,3,4,6,8-?-hexahydro-1-isopropyl-4,7-dimethylnaphthalene?1.68%?and dibutyl phthalate?1.43%?.The inhibitory effect of essential oils on S.communis was studied by determining the chlorophyll content.The inhibitory concentration(IC50)values of chlorophyll a of S.communis caused by the essential oil of T.flousiana at 24,48,72 and96 h were 90.10,47.13,41.89 and 52.29 mg·L-1,respectively.The treated S.communis cells were analyzed by transmission electron microscope?TEM?.It was found that essential oil at 200 mg·L-1 could damage the cell wall and the chloroplast,leaving the cells vacuolated,with some organelle remains in the cell.It is suggested that the main function site of the essential oil of T.flousiana be the cell wall and chloroplast.The herbicidal activities of the methanol extracts of T.flousiana were tested on Ageratum conyzoides L,Mikania micrantha Kunth,Bidens pilosa L,Eleusine indica?L.?Gaertn,Echinochloa crusgalli?L.?Beauv,Celosia argentea L,Digitaria sanguinalis?L.?Scop,Pistia stratiotes,Lemna minor,S.communis and Arabidopsis thaliala?L.?Heynh.The methanol extracts of T.flousiana showed obvious herbicidal activities on all the tested plants and the inhibition rates were positively correlated with the increase of the concentration.The IC50 values of the methanol extracts of T.flousiana on the inhibition rates of the root length of A.conyzoides,M.micrantha,D.sanguinalis,B.pilosa,E.indica and A.thaliala were 3.14,4.25,4.80,29.53,58.20 and 158.73 mg·L-1,respectively,7 d after the treatment.The IC50 values of the methanol extracts of T.flousiana on the inhibition rates of the fresh weight of A.conyzoides,M.micrantha,D.sanguinalis,B.pilosa,E.indica and A.thaliala were 3.74,4.33,61.66,41.16,45.67 and 174.81 mg·L-1,respectively,7 d after the treatment.The IC50 values of the methanol extracts of T.flousiana on the inhibition rates of the root length and the fresh weight of P.stratiotes were19.29 and 15.96 mg·L-1,respectively.Moreover,the IC50 values of the methanol extracts of T.flousiana on the corrected mortality of P.stratiotes were 510.04 mg·L-1?7 d?and 261.00mg·L-1?12 d?,respectively.The IC50 values of the methanol extracts of T.flousiana on the inhibition rates of chlorophyll a of L.minor and S.communis were 9.13 and 11.70 mg·L-1,respectively.The bioassay of the extracts of different solvents on the methanol crude extracts using the cup method showed that only the ethyl acetate extract had high herbicidal activity.After treatment for 7 days,the ethyl acetate extract significantly inhibited the increase of root length and fresh weight of A.conyzoides,M.micrantha,B.pilosa and E.indica and the IC500 values of the ethyl acetate extract on the inhibition rates of root length were 0.87,3.13,24.79 and 47.92 mg·L-1,respectively.The IC50 values of the ethyl acetate extract on the inhibition rates of fresh weight were 3.41,1.07,33.64 and 32.66 mg·L-1,respectively.Then the ethyl acetate extract was further studied for the isolation of the active ingredient.Fifteen compounds were isolated from the methanol extract of the stem bark of T.flousiana and were identified as the new compound TSC-3,helioxanthi,taiwanin E,taiwanin H,1,13,14-trihydroxy-8,11,13-podocarpatrien-7-one,diphyllin,justicidinA,7?-acetoxy-6?-hydroxyroyleanone,3,6-caryolanediol,8,11,13-Abietatriene-7,11,12,14-tetrol,6?,7a-diacetoxyroyleanone,taiwaninA,3?,14-dihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one,4-hydroxy-3-methoxy-3',4'-methylenedioxy-9,9'-lignanolide,4-cadinene-3,10-diol and4-muurolene-3,10-dil.After the treatment of TSC-3,glyphosate and berberine for 7 d,the IC50 values of the inhibition of the root length of B.pilosa was 0.83,1.35 and 4.56 mg·L-1,respectively,and the IC50 values of the inhibition of the fresh weight of B.pilosa were 1.14,11.57 and 7.69mg·L-1,respectively.After 14 d,the IC50 values of the inhibition of the root length of B.pilosa were 1.13,2.07,and 4.32 mg·L-1,respectively,and the IC50 values of the inhibition of the fresh weight of B.pilosa were 2.01,4.96,and 4.60 mg·L-1,respectively.After treatment of TSC-3,glyphosate,and berberine for 7 d,the IC50 values of the inhibition of the root length of f A.conyzoides were 0.29,0.38 and 2.07 mg·L-1,respectively,and the IC50 values of the inhibition of the fresh weight of A.conyzoides were 0.80,2.28 and 2.55mg·L-1,respectively.After treatment of TSC-3,glyphosate,and berberine for 7 d,the IC50values of the inhibition of the root length of M.micrantha were 0.18,0.87 and 1.68 mg·L-1,respectively,and the IC50 values of the inhibition of the fresh weight of M.micrantha were0.82 2.34 and 4.72 mg·L-1,respectively.After treatment of TSC-3,Taiwanin E,Taiwanin H,glyphosate,and berberine for 7 d,the IC50 values of the inhibition of the root length of A.thaliana were 0.63,1.41,16.36,0.11 and 0.21 mg·L-1,respectively.The IC50 values of the inhibition of the fresh weight of A.thaliana were 2.80,3.55,18.19,2.44 and 4.89 mg·L-1,respectively.It could be seen that the herbicidal activity of TSC-3 on A.conyzoides,M.micrantha,B.pilosa and A.thaliana was significantly higher than those of glyphosate and berberine.Since TSC-3 showed comparable,even better,herbicidal activities of glyphosate and berberine,therefore,the study of the absorption and transportation of TSC-3 was important for its future field application.Treatment of M.micrantha seedlings from 4 to 6 leaf stages with 10 mg·L-11 TSC-3 was carried out by hydroponic and leaf coating.The results showed that the plants were significantly inhibited,and the root length,root fresh weight,and whole length after 7 days were investigated.The fresh weight of the plant was significantly inhibited.However,the method of leaf coating did not have a significant effect.Water culture and leaf coating methods were used to study the transportation of TSC-3 in M.micrantha plants.HPLC analysis showed that after the M.micrantha seedlings were incubated with 10 mg·L-1 of TSC-3 for 0.5,1,2,3,5,and 7 days,the concentrations of TSC-3 in roots were 519.42,20.38,22.12,24.95,17.27 and 5.04?g·g-1,respectively,and the TSC-3 concentrations in the lower stems were 0,25.44,8.78,5.34,0.56 and 0.26?g·g-1,respectively.The concentrations of TSC-3 in the upper stem were 0,11.41,5.99,0,0 and 0?g·g-1,respectively,and the concentrations of TSC-3 in the lower leaves were 0,7.57,4.20,1.27,0 and 0?g·g-1,respectively.The concentrations of TSC-3 in the upper leaves were 0,7.48,3.79,0,0 and 0?g·g-1,respectively.TSC-3 could be detected in roots,stems and leaves with the extension of time;After 0.5,1,2,3,5,and 7 days of leaf treatment,the TSC-3 contents in the upper leaves were 0,9.88,10.13,0.94,0.40,and 0.14?g·g-1,respectively,and in the lower leaves,the contents of TSC-3 after 0.5,1,2,3,5 and 7 days were 0,4.16,4.43,0.51,0 and 0?g·g-1,respectively.In the upper stem,the contents of TSC-3 after 0.5,1,2,3,5 and 7 days were 0,2.00,0.20,0.14,0 and 0?g·g-1,respectively,and in the lower stem,the contents of TSC-3 after 0.5,1,2,3,5 and 7 days were 0,0.17,1.31,2.67,0.98 and 0?g·g-1.In the root,the contents of TSC-3 after 0.5,1,2,3,5 and 7days were 0,2.13,5.92 4.58,1.15 and 0?g·g-1,respectively.These results indicated that TSC-3 could be absorbed by root and leaf and was a systemic herbicidal chemical.Further,pot experiment showed that TSC-3 at 10 mg·L-1 caused a totally fresh weight inhibition to A.conyzoides and M.micrantha 7 d after treatment.Mechanism studies revealed that TSC-3 affected the distribution of AUX1 at the root tip.As the increase of time,auxin gradually diffused to the stele and cortex,and the gradient distribution patterns of auxin at root tip was destroyed.The distribution of PIN1 in the root tip was also changed,as evidenced by the diffusion of PIN1 to the cortex and then the whole root tip.The distribution of PIN2 at the apex was changed too.After the treatment,PIN2 gradually diffused from the cortex to the stele and eventually spreaded to the entire root tip.Further studies demonstrated that TSC-3 also disrupted the polymerization and stability of microtubules,reduced the redox potential in the quiescent center and the meristem,and increased redox potential in the elongation zone. |
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