| Drunken horse grass (Achnatherum inebrians) is a toxic perennial bunchgrass, which is so-named because it is associated with the narcosis of grazing animals on native grasslands in Northwest of China. This species is usually infected by the fungal endophyte Neotyphodium gansuense. The difference of the pest resistance of endophyte-infected (E+) and endophyte-free (E-) plants was investigated in this study. A series of experiments were conducted to study the effects of cadmium stress on seed germination, seedling growth and anti-oxidative systems of this grass. Drought and salt stresses can influence the concentrations of ergot alkaloids. The chemical composition and antifungal activity of the second metabolites (such as alkaloids and volatile oil) of E+and E-drunken horse grass were compared. The results were summarised as the follows:1. In the field experiment and laboratory tests, grasshopper species(Oedaleus decorus) and seed harvesting ant (Messor aciculatus) ate significantly (P<0.05) more E-than E+leaves and seeds, respectively.2. Germination and greenhouse experiments showed that the seed germination, seedling growth, antioxidative enzyme activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and the proline content of E+plants were significantly (P<0.05) higher than E-plants when the cadmium concentration was greater than or equal to100μmol/L. The malondialdehude (MDA) contents of E-seedlings were significantly (P<0.05) higher than E+seedlings. Endophyte infection was concluded to be of benefit to the growth and anti-oxidative mechanisms within A. inebrians under high concentrations of Cadmium chloride3. Greenhouse experiments showed that drought stress had a significant (P＜0.05) effect on the levels of ergonovine and ergine in both the Yuzhong (YZ) and Xiahe (XH) ecotypes. Levels of ergonovine were significantly (P＜0.05) higher than those of ergine and the difference between these two ecotypes was also significant (P＜0.05). Salt stress also had a significant (P＜0.05) effect on the levels of both ergonovine and ergine in the two ecotypes. Levels of ergonovine were also significantly (P＜0.05) higher than those of ergine and the difference between YZ and XH ecotypes was not significant (P>0.05).4. Compared with the method of ethanol immersion, ultrasound combined with hot alcohol reflux was a more effective method for extracting from drunken horse grass. Pure samples of the two ergot alkaloids from E+plants were obtained and the structures were identified as either ergonovinine and ergonovine, while these two alkaloids did not exist in the E-plants.5. Crude extractions from E+plants significantly (P<0.05) inhibited the fungi mycelia growth, spores germination and the lengths of germ tube of5pathogenic fungi (Alternaria alternata, Bipolaris sorokiniana, Curvularia lunata, Fusarium solani, Fusarium avenaceum) and Trichoderma viride. The fraction, which contained the two ergot alkaloids, ergonovine and ergonovinine, shown the strongest inhibiting effect. Effects of E+and E-petroleum ether extraction on17different fungi were different, and the effects on the same fungi were also different between E+and E-. Both of the two ergot alkaloids have the cytotoxicity on animal smooth muscle cells, the cell inhibition rate was increasing as the alkaloid concentrations increased and there exist a significantly (P＜0.05) positive correlation. The equation of the regression for these two alkaloids were Yergonovine=0.0041x+0.205(R2=0.945, P＜0.05) and Yergonovinine=0.0032x+0.2672(R2=0.9411, P＜0.05). The median lethal concentration (IC50) for ergonovine and ergonovinine was71.95μg/mL and72.75μg/mL, respectively.6. The volatile oils of E+/E-plants and3endophyte strains from the grass grown in Xiahe (XH), Yuzhong (YZ) and Sunan (SN) counties of Gansu province of China were obtained by hydro-distillation. The chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS). This resulted in the identification of61,59and34components for E+, E-and YZ strain, respectively. Heptadecane was the main component in both E+and E-volatile oils, and the mass fraction of this compound was up to9.06%and10.28%, respectively. There were31compounds identified that existed simultaneously in the E+and E-plants. There were7compounds identified that existed simultaneously in the E+/E-and YZ strain, and5compounds existed simultaneously in the E+and YZ strain. The presence of N. gansuense apparently affected some qualitative and quantitative differences in the volatile compounds production of this grass. The number of the compounds identified was36and42for the XH and SN strain. Trimethylsilyl methanol (63.31%) was the main component for XH, while3-Dioxolane (75.63%) and2-Methoxy-1,3-dioxolane (70.49%) was the main component for YZ and SN.7. Colonies of fungi were inhibited significantly (P＜0.05) by E+(vs. E-) volatiles when the oil’s concentration was greater than0.02mg/mL and fungi showed increased inhibition with increasing oil dose. It is clear that the effect of the E+oil on spore germination was stronger than that of E-oil. The maximum inhibitory effect of the E+(vs. E-) volatile oil were observed against Fusarium solani and Fusarium avenaceum, the IC50values being0.224mg/mL and0.417mg/L, respectively. The germination experiment showed that the seed germination, activities of POD, CAT and APX of perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinaced) and grassland bluegrass (Poe pratensis) under E-treatment were significantly (P＜0.05) higher than the E+treatment when the oil concentration was greater than or equal to300mg/mL. The experiment also showed that there was an increased inhibition with increasing oil dose, as did the contents of soluble sugar and proline. However, the activity of SOD, conductivity value and the MDA content of E+treatment were significantly (P<0.05) higher than E-treatment. |
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