Hyphae throughout the host remain metabolically active for the life of the tissue. Sustained metabolic activity in endophytes after hyphal growth has ceased may be a consequence of the continued supply of nutrients to the hyphae, both from the apoplast and possibly also from the cells of the host to which hyphae are attached (Tan et al., 2001). Once leaves and hyphae stop growing and extending, hyphal filaments remain in situ, and evidence of ongoing metabolic activity becomes apparent. These changes are visible using light and transmission electron microscopy. A predominant cytoplasmic feature of hyphae in young plant tissue is the presence of mitochondria among numerous much smaller ribosomes (Fig. 14-26). As the age of the leaf, and thus of hyphae, increases so does the cytoplasmic complexity (Fig. 14-27). Lipid droplets accumulate, and in some Neotyphodium species (but not N. coenophialum), crystalloid inclusions and microtubule bodies (Fig. 14-28 and 14-29) also become visible (Christensen et al., 2002; Fineran et al., 1983; Siegel et al., 1987). The function of these structures, which are not formed when the endophytes are grown in culture, is unknown. Hyphae expressing GFP remain fluorescent for the life of the host, and even when leaves begin to senesce and die, the endophytes do not alter visibly in morphology or recommence growth in the manner of a saprotroph (Fig. 14-30) (Christensen and Voisey, 2007). These characteristics of regulated growth are unique features of the symbioses between Neotyphodium and Epichloë (and probably Balansia) endophytes with grasses.

 

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Fig. 14-26. A. Neotyphodium lolii hypha in a young ryegrass leaf blade that is typical in being thin-walled with a simple ultrastructure, the most conspicuous organelles being mitochondria (M).

      

Fig. 14-27. Neotyphodium coenophialum hypha (in mature tall fescue leaf blade tissue) with complex cytoplasmic contents including lipid droplets (L).

 

 

                  

Fig. 14-28. Neotyphodium lolii hyphae in mature ryegrass leaves typically contain crystalloid inclusions (C) and microtubule bodies (T).

 

 
                             
                 

Fig. 14-29. Closer view of crystalloid inclusions (C) from Fig. 14-28.

      

Fig. 14-30. Hyphae of Epichloë festucae expressing GFP in a senescent ryegrass leaf sheath.

        
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