Biochemical Mechanism Of Festuca Sinensis-Epichloe Symbiosis Exposure To Cold Stress Based On Metabolomics

Many factors such as extreme temperatures, drought, salt, pathogens, strongly affect plant productivity. Festuca sinensis is a perennial forage species symbiotic with Epichloe fungal endophyte, has many advantages e.g.:high nutrition, palatability, cold, drought and disease tolerance. In this study, three F. sinensis ecotypes, i.e.:Xiahe (XH) of Gansu, Yushu (YS) and Haibei (HB) of Qinghai, China, were used as test materials, and the effects of Epichloe and/or exogenous calcium on endophyte alkaloids, metabolites, and sugars at low temperature stress were discussed, and the antifungal activity of Epichloe sp. XH03 was assayed by many methods. The main findings were as follows:1. The concentrations of the ergot alkaloids, ergine and ergonovine differed largely in the three endophyte-infected (E+) ecotypes exposed to different temperatures (5℃,10℃,22℃) (P< 0.05) based on high performance liquid chromatography (HPLC), and were not detected in endophyte-free (E-) plants. The concentrations of ergine, ergonovine, and total ergot alkaloids were significantly higher at 5℃ than at 22℃ (P<0.05).Twenty-four marker metabolites, including nitrogen compounds, acids, sugars, alcohols, and some unknown compounds could be considered by load diagram of the principal component analysis (PCA) scores based on gas chromatography-mass spectrometry (GC-MS) metabonomics, which were commonly higher in E+ plants than those in E- ones. These marker metabolites were influenced by ecotypes. The hierarchical cluster analysis revealed three distinct clusters among different materials, including plants under normal temperature, E+ plants under cold stress, and E-plants under cold stress. Moreover, there were two distinct groups of metabolites, one consisted of eight compounds, whereas the other was of 109 compounds divided into three clusters. These metabolites may exert their effectiveness on host cold tolerance.2. Many targeted compounds showed dynamic changes when F. sinensis responsed to low temperature (1±1℃). Levels of nitrogen compounds, glutamate, glutamine, valine, aspartate, alanine, phenylalanine, tryptophan, and GABA were increased under cold stress in all three ecotypes. In the two ecotypes, a small decrease in a large number of organic acids was also evident. All ecotypes had a different response both under cold stress and normal conditions. Epichloe endophyte increased metabolic compounds which relate to biosynthetic pathway of EMP, TCA, and amino acids. Here, the results indicated that Epichloe endophyte has the ability to enhance F. sinensis to cold stress.3. The results of principal component analysis and hierarchical cluster analysis revealed differences in the metabolic profiles of Epichloe XHO3 grown under low and normal temperature. The marker compounds were nitrogen compounds, sugars and organic acids. Contents of nitrogen compounds, most notably cadaverine, ethanolamine, leucine, serine, pyroglutamate, glutamate, aspartate, asparagine, aminomalonic acid, and phenylalanine were increased under cold stress. Levels of sugars, fructose and glucose were accumulated, however sucrose contents was decreased under cold stress. A small decrease of partial organic acids was aslo confirmed under cold stress.4. The effects of exogenous calcium and/or Epichloe endophyte on leaf relative water content (RWC), chlorophyll content, root activity and sugars contents were varied among three ecotypes. In all ecotypes, the levels of fructose increased on the 14 day and decreased on the 28 day, and the levels of glucose in root increased continuously under field cold conditions. In ecotype XH, Epichloe endophyte promoted RWC on the 14 and 28 day, chlorophyll contents on the 0 day, root activity on the 0 and 14 day, soluble sugar and glucose contents in shoot, and sucrose contents in root on the 28 day. Exogenous calcium in ecotype XH, promoted chlorophyll contents, root activity on the 28 day, soluble sugar contents in shoot or root, fructose content in root or of E+ plants in shoot, and sucrose contents of E+ in shoot higher than E- plants, and decreased RWC of E+. In ecotype HB, Epichloe endophyte promoted RWC on the 0 and 14 day, fructose and glucose contents in shoot, and negatively affected on carotenoid contents on the 14 and 28 day. Moreover, exogenous calcium promoted carotenoid levels, chlorophyll contents on the 28 day, root activity, soluble sugar contents in shoot or root, fructose contents in shoot or root, glucose contents in root, glucose contents of E- plants in shoot, and sucrose contents of E+ plants in shoot, decreased RWC of E+ plants, and the levels of sucrose in root were promoted by both Epichloe endophyte and exogenous calcium. In ecotyoe YS, Epichloe endophyte promoted RWC on the 28 day, root activity, soluble sugar contents in shoot or root, and fructose contents in root, and play negative role in carotenoid contents. Exogenous calcium in ecotype YS, promoted RWC on the 28 day, total chlorophyll and carotenoids contents of E- on the 28 day, carotenoids contents of E+ on the 14 day, root activity, soluble sugar contents in shoot or root, fructose and glucose contents of E+ in shoot, and sucrose content of E- in root or shoot on the 28 day. These results indicated that exogenous calcium contents, endophyte, and ecotypes, reduced variation in the levels of sugars, soluble sugar, fructose, sucrose, and exogenous calcium and/ or enhanced the host cold tolerance.5. In dual culture assays, the growth of Alternaria alternata, Bipolaris sorokiniana, andCurvularia lunata was inhibited by Epichloe fungal extracts at least 50%. The Extracts from mycelial supernatant broth of the strain produced inhibitory effects against the fungi, with ethyl acetate extracts giving the strongest inhibitory effects. Biotrophic regulation was shown to induce inhibitory effects against the pathogenic fungi of A. alternata, B. sorokiniana, C. lunata and Fusarium acuminatum, compared with E- leaves, the disease incidence and size of lesions on E+ detached leaves significantly reduced. When inoculating conidial supernatant of fungi, and/or ethyl acetate extracts to leaves, the lesion sizes were significantly different. A wide range of products were identified by GC-MS in the ethyl acetate extracts of the strain.