Biotechnology of Endophytic Fungi

Plants in arid and semi-arid countries like Iran require specific mechanisms in order to survive. The Iran-native endophytic fungi previously studied for the first time by Khayam Nekouei et al. are considered as a promising source with unique agricultural applications. The endophytic fungi enhance the plant strength in order to fight against biotic and abiotic stresses. Endophytic fungus Neotyphodium belonging to the family Clavicipitaceae lives symbiotically with cool-season grasses. The fungus modifies biological, morphological, physiological and ecological characteristics of plant making it more tolerant to biotic and abiotic threats. Endophytic fungi also help their hosts by producing some metabolites. Having considered the significance of this symbiotic relationship between the fungus and plants, six projects have been conducted to study the different aspects of the relationship. To investigate the genetic diversity of grass endophytes, SSR markers were used in 19 endophyt isolates obtained from tall fescus and perennial rye grasses. These grasses belonged to eight different geographical zones in Iran. Cluster analysis of the results obtained indicated five distinct groups in which Epichloe and Neotyphodium species had the highest polymorphism. A correlation was also found between genetic distances of endophytic fungi and geographical zones. According to the results, SSR markers are identified as suitable tools for classification and genetic distance estimation of endophytic fungi. To examine the effect of endophytic fungi on increasing the plant tolerance to environmental stresses a factorial design was used in a hydroponic system. The first factor was two classes of clones (with and without endophytes) which were isolated from fescus plants using a fungicide. Moreover, five levels of salt-stress conditions were considered. Mean analysis of data showed that the plants including endophytes were superior to those lacking them. Therefore, endophytic fungi are introduced as an effective biological factor for enhancing the production efficiency of grasses during stress conditions. To identify and select the best artificial inoculation methods and fungus transfer techniques from one genotype to another, two plant genotypes were selected and their symbiotic endophytes were extracted by three various methods including Latch and Christensen approach, Johanson- Cicales approach, and insertion of mycelium into the seeds. In this study, the existence of endophytes in new hosts were investigated by using microscopic methods, AFLP molecular markers and Koch's postulates. The results obtained revealed that the Latch and Christensen approach was the best method resulting in production of inoculated genotypes. While microscopic methods were found appropriate for recognition of endophyte existence in inoculated hosts, AFLP molecular markers were not efficient at all. Applying Koch's postulates indicated that endophytic fungi in new inoculated hosts did not show any morphological changes. To assess symbiotic fungal endophytes in the native tall fescus of Iran and in order to determine the ergoline alkaloids contents at three different growth stages of plants, HPLC system was used. Based on the results, it could be concluded that the alkaloids were at their highest level at the pollination stage and ergoline was at its lowest level at tilling time. To evaluate the effect of symbiotic fungal endophytes on grass plants tolerance during abiotic stress conditions such as hydrocarbon polluted soils, soil samples from two regions located in the Southeast of the Persian Gulf were obtained and dried at room temperature. Then, each sample was treated with three different petroleum compounds i.e. Naphthalene, Chrysene, and Naphthaline-chrysene. Each treatment included 150 mg of the compound per 1 kg of soil with three replicates. Subsequently, plants including and excluding endophytes were planted in the prepared soil samples. The results obtained showed that Naphthalene and Kryzen concentrations were reduced significantly in two soil samples in which endophytic inoculated plants were grown. Petroleum-polluted soil remediation by plants including endophytes could be ascribed to increased root volume and its consequent secretions in the surrounding soil. Therefore, by using endophyte-infected plants, hydrocarbon contaminated soil remediation could be efficiently achieved.