In the reinfection strategy, only a limited number of plants (200) from the recipient cultivar have been infected initially with a novel endophyte (Bouton et al., 2002). Therefore, the first seed increase requires pollinating these newly infected plants with a large number of plants from the recipient cultivar to ensure maximum preservation of all the positive traits of the cultivar. However, the authors reported that, in some combinations, the initial transmission rate was only 43%, indicating that certain combinations did not exhibit good strain-host compatibility. In addition, Paul et al. (2000) estimated there could be up to 5% infection loss during this generative seed increase phase, even where compatibility was good. Thus, it is normal to lose endophyte infection rate with each generation of seed production, simply due to the biology of the infection process itself. Although the most recent information indicates that the endophyte colonizes the ovaries and then the embryos early in their development (see Chapter 14, particularly Fig. 14-7), tall fescue has an indeterminate flowering habit, and the endophyte may not colonize some of the late-maturing seeds to a viable extent before seed harvest. Therefore, late-maturing seed may end up with viable embryos but not enough viable infection; hence, the total seed crop harvested is less than 100% infected. Therefore, the number of early generation seed increases must be kept to a minimum, and strict monitoring of infection and contamination rates must be maintained to ensure the highest level of infection in the seed labeled as breeder's seed (Rolston and Agee, 2007).
Since the main basis for any endophyte commercialization will be through a plant cultivar, the testing phase before actual release into the commercial seed trade is the process that consumes the most time and resources. For commercial tall fescue cultivars with novel endophytes, there is the added essential requirement of animal safety and performance trials that must be added to the standard agronomic testing protocols.
Animal responses to tall fescue endophytic toxins can be grouped into four categories (Stuedemann and Thompson, 1993): (i) decreased productivity, as expressed by weight gain, milk production, and/or pregnancy rate; (ii) changes in behavior, such as decreased feed consumption but increased water intake; (iii) physiological responses, such as increased respiration rates, and elevated rectal and core body temperatures; and (iv) changes in levels of serum or plasma constituents, such as decreased serum prolactin and cholesterol. One or more of these responses usually is measured when assessing fescue toxicosis (Bouton and Easton, 2005; Stuedemann and Thompson, 1993). Safety trials to measure these responses are therefore expensive and resource consuming, but were conducted before commercialization of MaxQ (Bouton et al., 2002) and should be conducted for any new product if it is to be sold with the claim of being nontoxic to livestock.
Agronomic testing for new cultivar-reinfected strain combinations must be rigorous. It must examine the main biotic and abiotic environmental stresses for the region of interest (see Chapter 3 and Chapter 4), contain the best checks (i.e., both E+ and E- entries) and be realistically applied and proven (Bouton and Easton, 2005). In addition to measuring yield and persistence across broadly based environments, there is a need to conduct trials that assess response to specific stresses, such as grazing, grass competition, insects, diseases, and nematodes, that can individually or in combination lead to poor plant survival and performance. Although the overall trends are for E+ tall fescue to be more persistent that E- fescue, especially in producer fields, this is not observed routinely, consistently, and rapidly for E+ and E- check cultivars in most university performance trials. The standards for any persistence trial should be no less stringent than those expected for pest resistance assays, where entries are scored by comparison with known resistant and susceptible differential cultivars that differ in a consistent manner. This type of assay then allows tests to be conducted through time with consistent scoring because the check cultivars perform differently but consistently. In this regard, Bouton et al. (2001) reported that exposing tall fescue cultivars and germplasms to increasing levels of stress created by grazing and association with bermudagrass [Cynodon dactylon (L.) Pers.] resulted in the most consistent and rapid separation of the E+ and E- check cultivars. On the basis of these findings, consideration needs to be given to designing a tall fescue persistence assay that employs planting all entries and check cultivars into bermudagrass sods and grazing intensely with livestock. Stands of the E+ and E- entries could then be monitored until they differed in a predicted manner. Each entry would then be scored for its statistical comparison to the known checks.
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