Neotyphodium hyphae are found only in the intercellular spaces of plant tissue, never outside the plant. Endophyte infection begins with an infected seed. From the seed, the mycelium spreads to the shoot apical meristem (i.e., growing point) of the young seedling. In mature plants, the spread of endophyte from the meristem into the leaves is not fully understood, but is thought to be a rather passive process with the fungus being carried along by an emerging leaf as it originates from the meristems. This topic is discussed and illustrated in Chapter 14. Hyphae are thought to not actually grow into leaves, but that growth is an intricately coordinated process where cell division of cells at the hyphal tip occurs at a rate identical to the elongation of leaf cells (Schmid and Christensen, 1999). As leaves elongate, especially in spring when rapid growth occurs, they may exceed the ability of hyphae to grow as rapidly. The consequence is a tendency for a pattern of decreasing endophyte abundance toward the leaf tip, with fungal mass being more concentrated near the plant base. Any measurement of endophyte mass or ergovaline concentration must state exactly which plant part was measured, because the height of sampling above ground level greatly affects the results. Ultimately, endophytic hyphae are carried in the developing seedhead as it emerges and matures. The hyphae proliferate inside the ripening seed, thus completing the infection cycle.
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Fig. 15-4. Concentration ( μg/kg) of ergovaline in leaves (laminae), leaf sheaths (pseudostems), and seedheads of tall fescue in spring and summer at Columbia, MO. (Data adapted from Rottinghaus et al., 1991.)
In tall fescue at a vegetative stage, endophytic mycelia (Fig. 15-4) are most abundant in the grass sheath (i.e., the pseudostem). Although hyphae originate from the meristem, the meristem is so small and difficult to sample that it is impractical to measure it. The standard test for endophyte is to sample near the base of a fescue tiller, since sampling at the meristem is difficult, especially when the plant crown is at or below ground level. In practice, most endophyte tests recommend sampling as near to the ground as possible, usually within 2.5 cm of the apical meristem.
The distributions of endophyte and alkaloids are related closely, since the alkaloids are produced by the fungi, using plant precursors. Ergovaline has low water solubility; hence, its distribution is linked closely to that of the endophyte. This is not the case for water-soluble alkaloids, such as peramine in perennial ryegrass.
Endophytic hyphae of N. coenophialum are sparse in tall fescue leaves (Fig. 15-4) and are not found in roots. Variation in the occurrence of endophyte in leaves is likely the result of plant genotype-race interactions, with vigorous genotype-race associations having endophyte in their leaves, whereas weak genotype-race associations have no endophyte in their leaves (Christensen et al., 1997).
Alkaloids from the Neotyphodium endophyte are concentrated in tall fescue seedheads; therefore, grazing during flowering in late spring and after formation of seedheads in early summer can be extremely toxic to livestock (Fig. 15-4). Good grazing management (see Chapter 6 and Chapter 7) aims to minimize the number of seedheads in the pasture. In some circumstances, however, usually as a result of understocking or mismanagement, livestock may consume many seedheads. Grazing under such circumstances may have more severe effects on livestock than might be predicted from simple endophyte infestation rates; accurate prediction of livestock performance may be possible only by measurement of the alkaloid concentration.
The Neotyphodium endophyte is a seed-borne fungus. Infected seed is the most prevalent method for endophyte infection into new grass stands (see Chapter 5). It is imperative that seed for new pasture be tested before planting. Most reputable seed companies sell seed with a certified endophyte test (see Chapter 19 and Chapter 25). If test results are not provided with the seed, producers are advised to conduct a test before planting. The test is inexpensive compared with the potential cost of dealing with an endophyte contamination. Now that nontoxic endophyte tall fescue seed is sold commercially, it is equally important to test for the presence and viability of the nontoxic endophyte.
Endophyte viability in seed is short-lived, and appreciable losses in viability occur after 1 to 2 yr (Rolston et al., 1986). For this reason it is important that any endophyte test of seed be conducted within the last 2 to 3 mo before use. One option for producing endophyte free (E-) seed is simply to store the seed for several years, especially at the high humidity and temperatures prevailing in common storage at ambient temperature. However, this practice also would compromise seed viability dramatically, resulting in low germination percentages and poor seed vigor.
In the case of E- seed, there is no mechanism by which seed becomes infected during storage. Thus, a negative test will be valid forever. There have been several cases where infected stands have been attributed to the purchase of contaminated seed. While such cases are rare, it is recommended that a sample of any seed sown be retained. Freezing seed at -18°C (0°F) may reduce the deterioration of endophyte viability with time. However, seed that is going to be used should not be frozen, but rather stored at 2 to 4°C (36-40°F).
One consequence of the short viability of endophyte in seed is that some tests will show the endophyte present in seed, even after the endophyte has died. To be able to distinguish live from dead fungus in seed, grow-out tests have to be used, whereby a seed sample is planted in the greenhouse and the endophyte infection of seedlings is tested about 6 wk after emergence. Results from grow-out tests can range from 0% infection, indicating that the endophyte is dead, all the way up to the level of infection present in the seed sample, meaning that the endophyte present is all viable. Endophyte viabilities of 50 to 75% are not uncommon.
Endophyte remains in stored forages even after harvest and drying for hay. While the endophyte might not remain viable, it can be detected when an antibody method is used. Of greater significance than the viability of the endophyte is the concentration of alkaloids present in these forages. In silage, alkaloids remain relatively stable and toxic during storage. There is evidence that alkaloid concentrations may decrease during haymaking (Norman et al., 2007), especially if the hay is ammoniated (Roberts et al., 2002). Endophyte cannot be detected easily in stored forages using microscopy or antibodies, and its potential toxicity usually is tested by measuring the concentration of ergovaline using HPLC.
Usually, it has been assumed that endophyte is uniformly or randomly distributed throughout a tall fescue field. Thompson et al. (1989) found evidence of random distribution of the fungal endophyte. More recently, Brown et al. (2007) found evidence that endophyte may have a patchy distribution, with closely grazed patches having lower endophyte occurrence and ungrazed patches having higher endophyte occurrence.
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