Growth of Epichloe/Neotyphodium and p-endophytes in leaves of Lolium and Festuca grasses

Epichloe spp. (Clavicipitaceae) and their close asexual relatives, Neotyphodium spp., form systemic endophytic associations with Pooideae grasses. Interactions between Lolium and Festuca host grasses and fungal endophytes were examined in studies focusing on leaves of natural associations and also of plants into which the endophyte was introduced by seedling inoculation. Light microscopy as well as fungal isolation was used to locate the position of hyphae, while transmission electron microscopy was used to examine host/fungus interactions at the cellular level. These studies provide support for synchronised plant and endophyte growth. This characteristic pattern of growth was maintained when both Epichloe and Neotyphodium spp. were introduced by seedling inoculation into new host species. Hyphae, with few exceptions, grew rapidly as leaves grew and ceased when leaf growth ceased, This pattern of growth offers an explanation for the characteristic appearance of hyphae in leaf sheaths of host grasses; seldom branched and for all species other than N. occultans, aligned parallel to the leaf axis. Hyphal growth of a second group of endophytic fungi, referred to as p-endophytes, was not regulated in the same way, with growth continuing as leaves aged. This pattern of growth gives rise to high concentrations of branched ramifying hyphae in old leaf sheaths. Although host genotype did not effect the basic pattern of hyphal growth of Epichloe/Neotyphodium endophytes, it strongly influenced the concentration and distribution of hyphae throughout leaves. Examination by TEM revealed no evidence that penetration of hyphae into dense tissue was aided by the secretion of pectic enzymes that loosen the middle lamella connecting host cells. Instead it appeared that penetration occurred by hyphae physically pushing between cells. The absence of enzymatic loosening of the middle lamella during penetration could explain why hyphae of these endophytes apparently do not elicit host defence reactions in natural associations. However, subtle interactions between the hyphae of Epichloe /Neotyphodium and host cells in natural associations were observed.