Ergosterol (ERG) is a sterol found in fungal cell membranes and is not produced by most plants and animals. Thus, ERG is a potential biomarker of fungal invasion in grain. Previously, we developed an analytical method for ERG in grains by ref uxing samples with methanol-alkali, and performed a single laboratory validation of the method. However, the method is not suitable for screening purpose, which needs to be rapid and simple. Therefore a simplified method was developed for analysis of ERG in grains. In this simplified method, ERG was first extracted with methanol by horizontal shaking for 1 hr. After f ltration, ERG was partitioned into hexane, and then analyzed with reverse phase high performance liquid chromatography (HPLC). Recoveries of ERG obtained with this shaking method were compared with recoveries obtained with the refluxing method. In maize, recovery of ERG at 3 mg/kg using this new shaking method was 84 % of that of the ref uxing method and the relative standard deviation (RSD) of the shaking method and the ref uxing method were 1.5 % and 16 %, respectively. In wheat, recovery of ERG at 12 mg/kg by the shaking method was 108 % of that of the ref uxing method, and the RSD of the shaking method and the ref uxing method were 2.8 % and 5.9 %, respectively. This study demonstrates this simplif ed protocol for ERG analysis in grains to be a suitable method for screening purposes.
Epichloë festucae and the asexual derivative Neotyphodium lolii, are symbiotic fungi (Clavicipitaceae, Ascomycota) that systemically colonize the intercellular spaces of leaf primordia, leaf sheaths and leaf blades of vegetative tillers and the inf orescence tissues of reproductive tillers to form symbiotic associations with temperate grasses of the subfamily Pooideae. Previous studies have established that E. festucae, N. lolii and related endophytes confer bioprotective benefits to their host plants. Host benef ts from the symbiosis include enhancement of plant tolerance to a range of biotic and abiotic stresses, including drought, disease, and animal herbivory. Indole-diterpenes are an important class of bioprotective metabolites synthesized by the Epichloë endophyte in association with temperate grasses. The major indole-diterpene synthesized by E. festucae and N. lolii in association with perennial ryegrass is lolitrem B, a potent tremorgenic mycotoxin responsible for the mammalian syndrome, known as ‘ryegrass staggers’. Peramine, the only known natural occurring pyrrolopyrazine, is a potent insect feeding deterrent uniquely synthesized by Epichloë endophyte. Production of these bioprotective metabolites and expression of biosynthetic genes are specif cally up-regulated in planta, indicated that Epichloë endophyte strictly regulate the expression of bioprotective metabolite genes for effective production of metabolites principally for the protection of host plant.
Endophytic fungi of the Epichloë group systemically colonize the intercellular spaces of grass plants to establish a symbiotic association. Key requirements for microbes to initiate and establish mutualistic symbiotic interactions with plants are evasion of potential host defense responses and strict control of the growth of the fungal endophyte throughout the host plant. We have recently shown that reactive oxygen species (ROS) produced by a specif c fungal NADPH oxidase isoform NoxA, have a critical role in regulating hyphal growth in the mutualistic interaction between E. festucae and perennial ryegrass. Regulation of ROS production in the symbiosis requires two additional components, NoxR and RacA, homologues of the mammalian p 67phox and Rac 2. Perennial ryegrass host plants containing noxA, noxR or racA mutants lose apical dominance, become severely stunted, and undergo precocious senescence. Our working model proposes that hyphal tip growth and branching is controlled by localized production of ROS catalyzed by NoxA, following recruitment of NoxR and RacA from the cytosol to the membrane in response to signaling from the grass host.
Among endo-symbionts of plants (endophytes), fungi of the genus Neotyphodium (asexual derivatives of Epichloë species: epichloë endophytes) have been intensively studied for their protective effects to their hosts, grass species of the subfamily Pooideae, which includes major forage and turf grasses species. With their seed-borne (maternally transmitted) lifestyle, the endophytes can be maintained within the host plant population over generations to provide virtual genetic traits to the plant. Their use in agriculture began with ornamental/sports turf grasses, as some of them have toxicity not only to insects but also to grazing animals (mammals), but later, fungal strains not toxic to mammals were selected and their application was expanded to forage grasses. Their use may be extended to food crops in the future.