2.9.20
OCCURRENCE OF THE TREMORGENIC ALKALOID LOLITREM B IN NEOTYPHODIUM LOLII-INFESTED PERENNIAL RYEGRASS (LOLIUM PERENNE)

J REINHOLZ1, VH PAUL1 and K KROHN2

1Universitaet-GH Paderborn, Fachbereich Agrarwirthschaft, Luebecker Ring 2, D-59494 Soest, Germany; 2Universitaet-GH Paderborn, Fachbereich Chernie und Chernietechnik, Warburger Strasse 100, D-33098 Paderborn, Germany

Background and objectives
The occurrence of endophytic fungi in grasses has been reported from almost all continents of the world. In our own study, the endophytic fungi of the Neotyphodium type have been detected in 10 out of 84 seed samples of Lolium perenne varieties from the German list of licensed L. ;perenne fodder grasses. The material examined was infested to between 1 and 20%. Little information is available 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.nbsp;lolii interaction is lolitrem B. Lolitrem B is the tremorgenic mycotoxin causing the nervous disorder of livestock known as ryegrass staggers (RGS). In addition, the fungus produces another alkaloid, peramine, which has beneficial effects for the host grass. The pyrrolopyrazine peramine deters a variety of insect pests from feeding on the grass [1].

Materials and methods
Because 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 Merklingsen (west Germany) and Asendorf (north Germany), sown in May 1994 as Neotyphodium-positive (infestation rate >80%) and as Neotyphodium-negative (infestation rate <10%) variant. In 1995 and 1996 the biomass of the variants was cut five times using a plot harvester. Representative samples of the Merklingsen harvest were freeze-dried, ground to a fineness of 1 ;mm using a hammer mill and analysed for their lolitrem B content according to [2]. At the Asendorf location. representative amounts were oven-dried at 65C and prepared as described for the Merklingsen samples.

A total of 10 Neotyphodium-positive plants from three European varieties and one New Zealand variety were cultivated in the greenhouse in 1997. The biomass was harvested eight times and prepared as described for the Merklingsen field samples. To investigate the influence of temperature on the content of lolitrem B in Neotyphodium-containing plants eight L. ;perenne varieties and ecotypes were cultivated under two different temperature conditions in climatic chambers: (1) 17C/10C and (11) 27C/19C, both with a daylength of 13 ;h (artificial light). The biomass of this samples was harvested three times and treated as described above.

Results and conclusions
At both locations lolitrem B was found only in endophyte-containing variants. At the Merklingsen location the amounts varied from 50 to 374 ;mg/kg dry matter (DM). In Asendorf, the values ranged from 20 to 267 ;mg/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 values determined were not sufficient to induce ryegrass staggers in livestock, but it is likely that this levels will aggravate the generation of the condition in grazing animals. 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 to 5000 ;mg/kg DM. This higher concentrations can be explained by the higher average temperature in the greenhouse and also the fact 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.<.p>

References
1. Bacon CW, 1895. Journal of Animal Science 73, 861-870.
2. Gallagher RT, Hawkes AD, Stewart JM, 1985. Journal of Chromatography 32, 217-226.