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Fig. 14-31. Hyphae of Epichloë festucae expressing GFP in the shoot apex of a ‘Resolute' tall fescue tiller. The arrows indicate a network of dividing hyphae in the meristematic tissue at the base of leaves.

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Fig. 14-32. Diagrammatic representation of intercalary extension in hyphae among enlarging plant cells of the leaf expansion zone. The plant cells increase in size and the attached hyphal strand increases in length accordingly to accommodate the increased distance between the two lateral hyphal branches that penetrate between files of expanding host cells. New compartments are formed along the length of the elongating hyphal strand. Intercalary hyphal growth enables the hyphal branches to remain intact rather than being sheared off the primary filament by upward movement of the host cells.


The understanding of the association between Neotyphodium and Epichloë endophytes and host grasses as outlined in this review has provided a model of growth for these fungi. In grasses, endophyte growth and elongation is limited to tissues where host cells are either dividing or elongating. Hyphae grow, most probably by apical extension, to form a heavily branched mycelium among dividing plant cells (Fig. 14-31), which in vegetative plants is largely in the shoot apex. When plant cells enter the leaf expansion zone, individual cells increase in size and the leaf undergoes intercalary extension; likewise the hyphae extend by intercalary growth (Fig. 14-32). Epichloë and Neotyphodium endophytes stop branching and elongating when among plant cells that are not dividing or extending.

The exception to this model of growth comes with the onset of development of inflorescences when Epichloë can continue to grow, resulting in the conspicuous mass of hyphae (stromata) in which the sexual stage can form, located above the top visible node of reproductive tillers.

Features of the Endophyte-Grass Association Explained by this Growth Model

Intercalary growth explains why hyphae in leaves are nearly all orientated parallel with the longitudinal leaf axis. Hyphae in the expansion zone of the leaf are subjected to stretching forces when the plant cells to which they are attached expand. Hyphal morphology thus reflects the direction of growth of the host tissue. It also explains why hyphae grow at the same speed as the host (i.e., through their attachment to elongating plant cells). Hyphal branching only occurs in tissues where cells are dividing, and this explains why the number of hyphae present does not increase as leaves age. The characteristic of pronounced hyphal convolutions observed in N. coenophialum (Fig. 14-1) in plants can be explained by continued intercalary extension in hyphae beyond the expansion zone of tall fescue leaves.

The cessation of hyphal growth when the surrounding plant tissue ceases dividing and enlarging also provides an explanation for the presence of endophyte free tillers that can be found in some plants. Initially axillary buds that give rise to tillers are part of the dividing mass of cells at the shoot apex and so can be colonized by hyphae. With time, axillary buds are displaced away from the SAM and so become separated from this zone by nondividing cells. If they are not colonized when associated with SAM, these developing tillers will forever remain endophyte free. A further consequence of hyphae being unable to penetrate through nondividing tissue is that individual tillers of plants infected with two Epichloë or Neotyphodium endophytes usually are colonized by just one of the two strains. This has been shown in plants naturally infected with two Epichloë strains (Meijer and Leuchtmann, 1999) and in dually infected associations made either through callus inoculation using mixed inoculum (Wille et al., 1999) or by the inoculation of infected seedlings (Christensen et al., 2000). Each axillary bud is infected by a small number of hyphae, and the chance of these hyphae being from both strains is low. Eventually a single strain predominates. Thus, although an endophyte in a tiller can infect each leaf, axillary bud, and inflorescence being formed from the SAM, it is confined within the tiller by the inability of hyphae to grow through tissue composed of nondividing cells.


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