High-affinity iron uptake is required for optimal Epichloë festucae colonization of Lolium perenne and seed transmission.

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte-grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.

Effect of Sodium Selenite on the Metabolite Profile of Epichloë sp. Mycelia from Festuca sinensis in Solid Culture.

Selenium (Se) is an essential micronutrient with many beneficial effects for humans and other living organisms. Numerous microorganisms in culture systems enrich and convert inorganic selenium to organic selenium. In this study, Epichloë sp. from Festuca sinensis was exposed to increasing Na2SeO3 concentrations (0, 0.1, 0.2, 0.3, and 0.4 mmol/L) in Petri dishes with potato dextrose agar (PDA) for 8 weeks. Epichloë sp. mycelia were immediately collected after mycelial diameters were measured at 4, 5, 6, 7, and 8 weeks of cultivation, respectively. Gas chromatography-mass spectrometer (GC-MS) analysis was performed on different groups of Epichloë sp. mycelia. Different changes were observed as Epichloë sp. was exposed to different selenite conditions and cultivation time. The colony diameter of Epichloë sp. decreased in response to increased selenite concentrations, whereas the inhibitory effects diminished over time. Seventy-two of the 203 identified metabolites did not differ significantly across selenite treatments within the same time point, while 82 compounds did not differ significantly between multiple time points of the same Se concentration. However, the relative levels of 122 metabolites increased the most under selenite conditions. Specifically, between the 4th and 8th weeks, there were increases in 2-keto-isovaleric acid, uridine, and maltose in selenite treatments compared to controls. Selenium increased glutathione levels and exhibited antioxidant properties in weeks 4, 5, and 7. Additionally, we observed that different doses of selenite could promote the production of carbohydrates such as isomaltose, cellobiose, and sucrose; fatty acids such as palmitoleic acid, palmitic acid, and stearic acid; and amino acids such as lysine and tyrosine in Epichloë sp. mycelia. Therefore, Epichloë sp. exposed to selenite stress may benefit from increased levels of some metabolite compounds.