Seed testing for endophytes by microscopic and immunoblot procedures

The presence of alkaloid-producing endophytes in forage grasses are generally considered detrimental, but are beneficial to turf grasses. Endophyte technology is changing to where they are now viewed positively for forages as well. Therefore, endophyte testing is likely to occur more frequently in the future. To do so will require a less laborious method than current histological/microscopic techniques. The objective of this project was to compare an immunological test kit with the histological/microscopic procedure for endophyte (Neotyphodium coenophialum and N. lolii) detection among independent laboratories. Three tall fescue and perennial ryegrass seed lots testing low, medium and high for endophyte were sent in three blind replicates to laboratories in the United States, Netherlands, United Kingdom and Argentina. Commercial immuno-chemical test kits were sent to each laboratory, as well as the International Seed Testing Association/Association of Official Seed Analysts histochemical protocol for endophyte detection. All laboratories analyzed fifty seeds with both procedures. Within and between laboratory variability were compared and data from the two procedures regressed to determine specificity across the range of data. Endophyte values from one laboratory were outliers compared to values from the other three laboratories for both methods. Data from that lab was omitted from the test. Intra-laboratory variability ranged from 0.0 to 9.6 percentage units while inter-laboratory variability ranged from 0.0 to 7.7 percentage units. Intra- and inter-laboratory variability was similar for both endophyte detection methods. When microscopic and immunoblot data were regressed with each other the intercepts of the regression lines were not different from 0.0, slopes were not different from 1.0 and regression coefficients were 0.88 or higher. The immunoblot procedure is an acceptable alternative to microscopic analysis of tall fescue and perennial ryegrass seed for endophyte.