In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections

Abstract

A histochemical procedure was developed to visualize and estimate the proportion of arbuscular mycorrhizal infections showing fungal alkaline phosphatase activity, as compared to the total amount of fungal tissue (trypan blue staining) and of living mycelium, indicated by succinate dehydrogenase activity. In roots of Allium porrum and Platanus acerifolia, only a small proportion of living intraradical mycelium showed alkaline phosphatase activity during early infection but this increased greatly just before the mycorrhizal growth response of the host plant. Infection revealed by all three stains reached a maximum at 6 weeks after inoculation, after which the level of trypan blue stained infection remained constant but the proportion showing succinate dehydrogenase and alkaline phosphatase activity declined as the infection aged. Alkaline phosphatase activity was absent from virtually all abortive entry point hyphae formed on roots of a resistant myc−, nod− mutant of Pisum sativum, although succinate dehydrogenase activity was detected. Observations suggest that the alkaline phosphatase activity is induced by colonization of host roots and that this fungal enzyme could provide a useful marker for analyzing the symbiotic efficiency of arbuscular mycorrhizal infections.