The greatest problem in detecting N. coenophialum in tall fescue is the lack of morphological characteristics of infected tillers or seeds that point clearly to endophyte presence (see Chapter 14). Factors such as poor utilization by grazing livestock, greenness during drought, and absence of insect damage are associated with endophyte presence, but are not definitive characteristics of endophyte presence or absence. Several techniques have been developed to detect the endophyte.
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Fig. 15-2. Neotyphodium coenophialum hyphae stained with aniline blue in an epidermal strip from tall fescue (photo: N.S. Hill).
Fig. 15-3. Qualitative analysis of ergot alkaloids in Neotyphodium endophyte infected seed. The top four rows, with yellow stain, are analyses of individual seeds containing a nontoxic endophyte. The bottom four rows are analyses of individual seeds from a lot that was 90% infected with a toxic endophyte-the cells F4, F11, G6, and G9 are stained yellow, and the clear color indicates presence of fescue alkaloids. (Photo by N. Hill, University of Georgia.)
Endophyte has been identified most commonly in tall fescue using a microscope (Clark et al., 1983). The hyphae are present in the intercellular spaces of grass pseudostems and to a lesser extent in leaves, and can be identified by aniline blue staining of a leaf epidermal peel (Shelby and Dalrymple, 1987; Hiatt et al., 1999; Hill et al., 2002a) (Fig. 15-2). Hyphae can be seen with a light microscope or with a transmission electron microscope for more detailed studies (Christensen et al., 2002).
Monoclonal antibody methods increasingly are becoming the preferred method for detecting endophyte (Hiatt et al., 1997). During 2000, Agrinostics Ltd. (Watkinsville, GA) commercialized an antibody kit for testing the presence or absence of Neotyphodium endophyte (Fig. 15-1). This kit is as reliable as microscope testing, faster, and less subject to operator bias than microscope testing (Hiatt et al., 1999; Hill et al., 2002a). One expert technician in one of the authors' laboratory completed the antibody test on 2400 tillers in 2 d (D.J. Barker, unpublished data, 2003). Kits are available for testing endophyte in seed and fresh tillers. Monoclonal antibodies specific to fungal races, such as AR542, also have been produced (Hill et al., 2002b).
Perhaps more important than the identification of the endophyte in the grass host is the recognition of the presence and the quantification of toxic alkaloids that have been produced by that endophyte. This question is even more important because nontoxic novel endophytes, such as AR542, that do not produce ergovaline, could be present. Ergovaline is measured quantitatively by high performance liquid chromatography (HPLC) (Hill et al., 1993; Rottinghaus et al., 1991; Spiering et al., 2002; Kallenbach et al., 2003). An ergovaline concentration exceeding 150 μg/kg may be toxic to livestock (Stamm et al., 1994). Alkaloids from tall fescue have been identified also using mass spectrometry (Yates et al., 1985).
Another method for measuring toxic alkaloids in tall fescue is enzyme-linked immunoassay (ELISA) (Reddick, 1988; Gwinn et al., 1991). The ELISA method has some distinct advantages over HPLC because it can test for total ergot alkaloids, not just ergovaline. All ergot alkaloids are toxic to mammalian systems, and the question of identifying the specific alkaloid(s) responsible for tall fescue toxicosis is still under investigation. Thus, the detection of total ergot alkaloids provides a good indication of the potential toxicity in the sample.
In addition to plant measurement, it has been shown that urinary excretion of ergot alkaloids measured by ELISA is inversely proportional to animal performance (Hill et al., 2000). The test can be formatted for a qualitative analysis for quality control in plant tissues infected with nontoxic endophytes (Fig. 15-3) or it can be used with dilutions of lysergic acid standards to establish quantitative measures. Eventually, there may be merit in diagnosing livestock symptoms using urinary testing. This method is available commercially.
A study by Christensen et al. (1993) used isozymes of 11 different metabolic enzymes to differentiate among endophytes of perennial ryegrass (L. perenne L.), meadow fescue [Lolium pratense (Huds.) Darbysh.], and tall fescue and also to differentiate among races of endophytes within a species. A similar approach used isozymes of phosphoglucose isomerase to differentiate between two races of the ryegrass endophyte N. lolii Latch, M.J. Christensen & Samuels (Barker et al., 1997).
The Neotyphodium endophyte in tall fescue has been identified in the laboratory by allowing the fungus to grow out from the plant tissue into a culture medium (Clark et al., 1983). Different endophytes and endophyte races have distinctive growth patterns in culture that include distinctive patterns of hyphal growth and distinct sexual phases (Christensen et al., 1993, 2002)
Endophytic fungi also have DNA unique from their host plant (Al-Samarrai and Schmid, 2000). Diagnostic probes (i.e., short and specific sequences of nucleotides) can be used to identify endophytes in grasses. This method can be used in research to differentiate among endophyte fungal species and races because the DNA sequences are unique for each species and race. This research method is not used commercially.
Endophyte also has been quantified in plant tissue using nuclear magnetic resonance (NMR) (Green et al., 1997) and near-infrared spectroscopy (NIR) (Roberts et al., 1997, 2005). Other endophytes than N. coenophialum can be identified in tall fescue, for example, p-endophytes Phialophora spp. (Christensen et al., 2002); however, no effect adverse to livestock by these endophytes has been reported. Usually, these endophytes are identified by electron microscopy.
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