The development of new inoculation techniques and viability tests for Neotyphodium endophytes

• Main Author: Gillanders, Timothy James
• Language: en
• Published: University of Canterbury. Biological Sciences 2010
• Subjects: Neotyphodium; Festuca; RT-PCR; Fungi; Lolium
• Online Access: http://hdl.handle.net/10092/3677

Neotyphodium endophytes (Claviceptaceae) are asexual filamentous fungi found living between the cells of many cool season forage grasses including tall fescue, meadow fescue and perennial ryegrass. They produce a range of alkaloids, including ergovaline and lolitrem B, which have been shown to be directly associated with the livestock disorders fescue toxicosis and ryegrass staggers syndrome, while others, including peramine and the lolines, have been linked to increased insect and drought resistance of the grass host. In the past decade, the Neotyphodium strains AR1, MaxQ and MaxP were selected because they did not produce the alkaloids associated with livestock disorders. Subsequently, artificial associations were established between them and commercial forage grass cultivars. The slow growth rate of Neotyphodium endophytes in vitro and the low success rate of the present methods for establishing artificial associations between endophytes and grass hosts are limiting the rate at which new novel endophytes can be incorporated into plant breeding programs and eventually commercialised. In this thesis, the type and concentration of the growth medium was shown to affect radial growth rate, colony appearance and mycelial morphology of three strains of Neotyphodium endophytes. The floret inoculation of meadow fescue with the U2 strain of N. uncinatum using several techniques involving liquid culture was attempted but was unsuccessful in creating any artificial associations. Neotyphodium endophytes are unstable in stored seed. In New Zealand, it is critical that pastures are infected with protective Neotyphodium endophytes to ensure that they will not be destroyed by exotic pests. The present methods for determining the percentage of viable endophyte infection of a seed lot are too slow for efficient use in the commercial seed industry. In this thesis, primers specific to the â-tubulin gene of N. coenophialum, N. lolii and N. uncinatum were designed and successfully used to detect these species in planta. However, using these primers to develop a method to accurately determine the viable endophyte infection rate of a seed lot using RT-qPCR was unsuccessful.