Tall Fescue Endophyte Detection: Commercial Immunoblot Test Kit Compared with Microscopic Analysis

of N. coenophialum mycelial proteins was first proposed by Gwinn et al. (1991) using polyclonal antiserum proA reliable, efficient, and accurate detection method for presence duced in rabbits. This technique worked equally well of the endophytic fungus Neotyphodium coenophialum (MorganJones and Gams) Glenn, Bacon, and Hanlin comb. nov. in tall fescue with either seed or plant tissue but seed analysis was (Festuca arundinacea Schreb.) seed and plant tissue would be benefimore tedious and time consuming. Seed had to be scaricial for tall fescue breeding and seed lot analysis. This experiment was fied, allowed to imbibe water overnight, and split longiconducted to determine the accuracy, reliability, and reproducibility of tudinally prior to immunoblot analysis. Assays using the Phytoscreen Neotyphodium immunoblot detection kit (Agrinosthis technique produced comparable results to ELISA tics Ltd. Co., Watkinsville, GA). Tissue immunoblot was compared and did not require specialized equipment to conduct with histological staining followed by microscopic analysis on tall the analysis. fescue tillers from a greenhouse grow-out test, field grown spaced Polyclonal antibodies are found in the immunoglobuplants, established field paddocks, and different tall fescue seed lots. lin fraction of serum, which is composed of an almost Endophyte-infected and endophyte-free tall fescue populations were infinite array of molecules of varying affinities and quanevaluated by both methods. Results obtained by both methods were similar regardless of the infection level of the population, type of tissue tities. Over 90% of the Ig molecules present in the serum assayed, or the technician that conducted the assay. The immunoblot have little or no affinity for the target antigen (Roitt, detection kit was accurate and reliable and readily accommodated 1994). In contrast, monoclonal antibodies are produced large numbers of samples. by a single hybridoma and all have the same structure, affinity, and specificity to a given epitope. Hence, a monoclonal antibody-based immunoblot technique has the advantages over a polyclonal antibody-based techT presence of ergot alkaloid-producing endophyte nique of greater specificity and defined affinity (Hiatt (N. coenophialum) in tall fescue may be viewed as et al., 1997). a positive or negative attribute, depending upon Private seed testing laboratories and commercial tall whether the fescue is used for turf or forage. Endophytefescue breeders currently use histological staining folinfected forage germplasms that are non-toxic (produclowed by microscopic analysis to determine endophyte ing little or no ergot alkaloids) are being produced (Adinfection status (S. Davidson, 1996, personal communicock et al., 1997; J.H. Bouton, 1998, personal communication). Neotyphodium-specific polyclonal antisera is cation). Knowledge of endophyte infection status of not available to the private sector, therefore ELISA seed lots and breeding populations of tall fescue is imtechniques and tissue immunoblot techniques are also portant. Seed testing services currently use histologically not available. A monoclonal antibody-based tissue imstained plant tissue for microscopic analysis (Clark et munoblot technique requiring little immunological al., 1983). Histological staining procedures are tedious, training is now available to public and private scientists time consuming, and difficult to use for large numbers and seed analysts. It is marketed in a kit format and can of samples. A rapid, inexpensive, and reproducible techbe used on seed, greenhouse-grown grow-out seedlings, nique is needed to evaluate tall fescue for the presence and field-grown plants. or absence of the endophyte. The purpose of this study was to compare the reliabilEnzyme linked immunosorbent assays (ELISA) have ity, reproducibility, and accuracy of a commercially been used to detect or quantitate endophyte in tall fesavailable, monoclonal antibody-based immunoblot decue seed and leaf sheath tissue (Johnson et al., 1982; tection assay with microscopic analysis for presence of Musgrave et al., 1986; Reddick, 1988; Reddick and Colendophyte in (i) greenhouse-grown tall fescue seedling lins, 1988; Hiatt et al., 1997; Hiatt and Hill, 1997). The plants, (ii) field-grown tall fescue plants, and (iii) tall ELISA methods are sensitive, specific, consistent, and fescue seed. capable of performing analyses on large numbers of samples. The ELISA procedures, however, require exMATERIALS AND METHODS pensive laboratory equipment and specialized preparaDetection of Endophyte in Greenhouse-Grown tion to perform the analysis. Seedling Tall Fescue Plants A tissue print immunoblot technique for the detection Seeds of endophyte-infected and endophyte-free ‘Jesup Improved’ tall fescue were planted into a commercial potting E.E. Hiatt, III, N.S. Hill, and J.H. Bouton, Dep. of Crop and Soil soil in flats containing 72 cells (each cell 5 35 by 35 by 60 Science, Univ. of Georgia, Athens GA 30602; J.A. Stuedemann, mm; Landmark Plastic Corporation, Akron, OH). One hunUSDA-ARS, J. Phil Campbell, Sr. Natural Resource Conservation dred plants were harvested each week after emergence, for Center, Watkinsville, GA 30677. Received 29 June 1998. *Correspond8 wk (50% of seed emergence 5 Day 0). Plants were sampled ing author ([email protected]). Abbreviations: ELISA, enzyme linked immunosorbent assay. Published in Crop Sci. 39:796–799 (1999).