Cross inoculation of tall fescue genotypes with endophytic fungi and its detection through DNA markers

Mutual relationship between pasture grasses and fungal endophytes genus Neotyphodium are one of the most important symbiossa in nature. These fungi are members of Clavicipitaceae family and symbiosist to grasses of subfamily Pooideae. Endophytes grow intercellulary and asymtomtically within grasses. Fungal hyphae are found in all parts of the plant except the root, with highest concentration found in the leaf sheath. These fungi exhibit no sexual phase of reproduction but as a way of asexually reproduction, they grow inside ovules and become incorporated into seeds and transmit to next generation. Endophytes have many advantageous effects such as pest and disease resistance, and resistance to environmental stresses including drought, soil acidity, mineral imbalance, salinity and pH fluctuation, for their host plants. Endophyts have interactive relations with their host plants. Precise investigation of interaction between tall fescue and its corresponding endophytic fungus requires application of inoculation methods. The purpose of this research was to evaluate and compare capabilities of different cross inoculation method and to determine the best methods for transferring endophyte fungus from carrying plants to other plant genotypes. Two genotypes of tall fescue known as 75 and 83 were selected and their endophytes were isolated. Six methods of cross inoculation were used in this research consisting Latch and Christensen approach, placing fungus mycelium on coleoptile without making a split, insertion of mycelium into the seeds, Johanson- Cicales approach, cross inoculation of endophyte fungus to seedling in sand pot and finally, injection of mycelium fragments into the stem and crown tissue using hypodermic needle. Endophytes were detected in new host using microscopy examination of stained tissues, AFLP marker and Kokh's postulates. Results showed that Latch and Christensen approach was the only technique by which 75 and 83 endophytes isolates were successfully inoculated into both original host genotypes and other studied tall fescue genotypes. With the other investigated cross inoculation methods, no infected plant was obtained. Success in cross inoculation seems to be dependent on splitting procedure and youth of seedlings. Furthermore, rate of contamination to saprophytic fungi in Latch and Christensen approach was less than that of resulted from other methods. Detection methods used to confirm the presence of endophyte fungi in plants, indicated that microscopy examination method was the most qualified for detection. AFLP marker confirmed endophyte fungus in some plants, but not in all. That is possibly because of owing low concentrations of fungal hyphae in new host. Kokh's postulates showed that endophytic fungi had no morphological alterations after being inoculated into new host. Considering potent disadvantages resulted from introduction of endophytes into new grasses, it must be carried out with great cautions and complete considerations.