6.109
OCCURRENCE OF THE TREMORGENIC ALKALOID LOLITREM B IN NEOTYPHODIUM LOLII INFESTED PRENNIAL RYEGRASS (LOLIUM PERENNE)
J REINHOLZ l,VH PAUL l and K KROHN2

lUniversitaet-GH Paderbom, Fachbereich AgrarMrtschaft, Luebecker Ring 2, D-59494 Soest, Germany; 2Universitaet-GH Paderbom, Fachbereich Chemie und Chemietechnik, Warburger Strasse 100, D-33098 Paderborn, Germany


Background and objectives
The occurrence of endophytic fungi in grasses has been reported from virtually all continents of the world. In our own study the endophytic fungi of the Neotyphodium-type have been detected in 10 of 84 seed samples of Lolium perenne varieties of the German list of licensed L. perenne fodder grasses. The examined material was infested between 1-20%. There is only little information available so far on the occurrence of the alkaloids produced in these infested grasses under the climatic conditions of Germany. In reports from New Zealand, Australia and the USA the production of specific alkaloids in endophyte containing perennial ryegrass and tall fescue is well documented. The most important alkaloid of the perennial ryegrass N. lolii interaction is lolitrem B. Lolitrem B is the tremorgenic mycotoxin causing the nervous disorder of livestock known as ryegrass staggers (RGS). In addition to this the fungus produces an other alkaloid, peramine, which has beneficial effects to the host grass. The pyrrolopyrazine peramine deters a variety of insect pests from feeding on the grass [1].


Materials and methods
Field trials
On account of the low infestation levels of the European varieties of perennial ryegrass we used the cultivar Ellett (origin: New Zealand) for our field trials. The field trials were located in Meridingsen (West Germany) and Asendorf (North Germany), sown in May 1994 as Neotyphodiumpositive (infestation rate > 80%) and as Neotyphodium-negative (infestation rate < 10 %) variant. In 1995 and 1996 the biomass of the variants was cut 5 times in total using a plot harvester. Representative samples of the Merklingsen harvest were freeze-dhed, ground to a fineness of 1 mm using a hammer mill and analysed for their folitrem B content according to [2]. At the location Asendorf representive amounts were oven-dried at 65 OC and prepared as described for the Merklingsen samples. Greenhouse and climatic chamber experiments 1 0 Neotyphodium-positive plants of 3 European and 1 New Zealand variety were cultivated in the greenhouse in 1997. The biomass was harvested 8 times and prepared as described for the Merklingsen field samples.
To investigate the influence of temperature on the content of lolitrem B in Neotyphodiumcontaining plants 8 L. perenne varieties and ecotypes were cultivated under two different temperature conditions in climatic chambers ( 1) 17 OC/10 OC and 11) 27 OC/19 OC; day (13 h artificial light). The biomass of this samples was harvested 3 times and was treated as described above.


Results and conclusions
At both locations lolitrem B was found only in endophyte containing variants. At the location Merklingsen the amounts varied from 50-374 pg/kg DM. In Asendorf the values ranged from 20-267 pg/kg DM. This study showed that production of lolitrem B can occur in Neotyphodium infested perennial ryegrass when grown under the climatic conditions of Germany. The determined values were not sufficient to induce ryegrass staggers in livestock, but it is likely that this levels vviii aggravate the general condition of grazing animal.
In contrast to the results of the field trial the lolitrem B concentrations found in the greenhouse experiments were much higher. The values of lolitrem B for the four varieties ranged from 200 pglkg DM up to 5000 pglkg DM. This higher concentrations can be explained first through the higher average temperature in the greenhouse and second that only Neotyphodium-positive plants were harvested.


The experiments in the climatic chambers with two different temperature conditions showed that the lolitrem B production is temperature dependent.

References
1. Bacon CW. 1995 Journal of Animal Science 73, pp 861-870. 2. Gailagher RT, Hawkes AD, Stewart JM. 1985 Journal of Chromatography 32, pp 217-226.