| Perennial ryegrass (Lolium perenne), as an important forage and turf grass in China and world, plays an important role in grassland livestock production and landscape turf establishment. It is well known that Epichloe festucae var. lolii endophyte enhances the biotic and abiotic stress tolerance of host plants. Previous studies on perennial ryegrass and endophyte symbiont focused on the effects of endophyte on their hosts under abiotic stress. The study of interactions between perennial ryegrass and endophyte symbiont stressed by pathogenic fungi was only less reported. In this research, perennial ryegrass varieties (Fairway) from the United States were used to determine the interactions of perennial ryegrass, endophyte and pathogenic fungi under the laboratory, greenhouse and field conditions. Firstly, we established endophyte-infection (E+) and endophyte-free (E-) populations in the field, and harvested seeds from E+ and E- populations in the next year. A series of experiments were undertaken to evaluate the disease resistance of grass-endophyte symbiont under various conditions. Mechanisms of the symbiont resistance to Bipolaris sorokiniana was further determined by microscopic observation and detection of resistance-related enzymes, osmotic substance, and ergot alkaloids. The ethanol extracts of perennial ryegrass, ergot alkaloids against pathogenic fungus, nitrogen and phosphorus contents of perennial ryegrass were also examined. The results were summarized as the follows:1. Seedborne fungi and their pathogenicity to perennial ryegrass seedlings. Fungi isolated from ryegrass seeds, regardless of E+ or E-, including Alternaria alternata, Bipolaris sorokiniana, Curvularia lunata, Fusarium avenaceum and Ascochyta leptospora. The total fungi isolation rate of unsterilized seeds was higher than that of sterilized ones. The fungi isolation rate of E+ seeds was lower than that of E- seeds by 7.1% in the sterilized treatment, but no significant difference was found.2. Disease resistance of E+ and E- seedlings. Germination percentage, germination index, shoot length, fresh and dry weight of E+ seeds or seedlings were significantly (P<0.05) higher than those of E- seeds or seedlings after inoculated with the seedborne fungi mentioned previously, The proline content, POD activity and SOD activity of E+ seedlings was significantly (P<0.05) higher than that of E- seedlings, respectively, while MDA content of E+ seedlings was significantly (P<0.05) lower than that of E-seedlings.3. Resistance of pot plant to Bipolaris sorokiniana. The disease incidence, lesion numbers and disease index of E+ plants were lower than those of E- plants by 34.7%～37.1%, 50.9%～61.6% and 24.8%～34.0%, respectively, measured within 15 days after the pathogenic fungus inoculation. The tiller number, plant height and chlorophyll content of E+ plants were higher than those of E- plants in 10 weeks after inoculation.4. Resistance of field growing plant to rust. The disease incidence of E+ and E- plants in the field was 21.9% and 65.0%, respectively. In less severe and severe diseased plants, the loss diseased leaf of E+ plants was fewer than those of E- plants by 49.4% and 18.3%, respectively. The net photosynthetic rate, transpiration rate and stomatal conduction of E+ plants were higher than those of E- plants by 71.7% and 78.0%,64.5% and 55.5%,26.1% and 45.5%, respectively. The relative water content, chlorophyll content and soluble sugar content of E+ plants were higher than those of E- plants by 8.2% and 29.4%,22.1% and 64.5%,17.1% and 28.2%, respectively. The proline content, SOD and POD activity of E+ plants were higher than those E-plants by 25.7% and 22.6%,16.9% and 24.5%,39.5% and 21.5%, respectively. The MDA content of E+ plants were lower than that of E- plants by 49.6% and 62.3%.5. Mechanism of perennial ryegrass-endophyte symbiont resistant to Bipolaris sorokiniana: The spore germination rate, germ tube length and number of infection sites at E+ leaves were significantly (P<0.05) lower than those at E- ones. There were significant (P<0.05) differences in PPO activity, chitinase activity, and β-1,3-glucanase activity between E+ and E- plants in 10 weeks after inoculation, which E+ plants were increased by 25.1%,36.3% and 26.1% (P<0.05), respectively, compared with those of E- plants. On the other hand, the total amino acid content, proline content and the soluble sugar content in E+ plants were 53.4%,49.0% and 37.7% more than the E-plants, respectively, which were significantly different between the two kind of plants (P<0.05).6. The ergine and ergonovine content increased before they were reduced in E+ treatment. Maximum of ergine and ergonovine content appeared in 4 weeks and 2 weeks, respectively, after inoculation. The ergine content in blade, sheath and root was 22.1～82.3 mg kg-1DW,16.8～47.2 mg kg-1DW and 11.4～35.0 mg kg-1DW, respectively, from 1 to 10 weeks after inoculation. The ergonovine content in blade, sheath and root was 5.4～20.8 mg kg-1DW,5.0～16.0 mg kg-1DW and 3.5～8.9 mg kg-1DW, respectively, from 1 to 10 weeks after inoculation. Ergot alkaloids in E+ treatments could inhibit colony growth, spore germination and germ tube length of Bipolaris sorokiniana, compared with ergot alkaloids in E+ control groups. Inhibition of ergot alkaloids from blade> inhibition of ergot alkaloids from sheath> inhibition of ergot alkaloids from root. The inhibition of ergot alkaloids from each part in E+ plants decreased following the aggravation of treatment plants. The ethonal extracts from E+ blades could inhibit colony growth, spore germination and germ tube length of Bipolaris sorokiniana compared with those from E- blades.7. The nitrogen content of E+ plants decreased and that of E- plants increased from 5 to 10 weeks after inoculation. There were significant (P<0.05) differences in nitrogen contents between E+ and E- plants from 7 to 10 weeks after inoculation. The phosphorus content of E+ and E- plants decreased from 7 week and 4 week, respectively, after inoculation. There were significant (P<0.05) differences in phosphorus content between E+ and E- plants from 5 to 10 weeks after inoculation. |
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