Measurement of development of endomycorrhizal mycelium using three different vital stains

The extraradical hyphae of Glomus intraradices Schenck & Smith associated with a mixture of alfalfa (Medicago sativa L. cv. Saranac) and bromegrass (Bromus inermis Leyss. cv. Tempo) were tested for metabolic activity by three enzyme staining procedures. Mycorrhizal plants were grown for 6, 9 or 12 weeks and the vital staining methods were evaluated and compared at each date. Succinate dehydrogenase activity was assessed by the reduction of nitro blue tetrazolium (nitro-BT), and NADH diaphorase activity by the reduction of indonitrotetrazolium (INT). Fluorescein diacetate (FDA) hydrolysis was used to assess the activity of esterases in the extraradical hyphae. Percentages of intraradical hyphae that were active in alfalfa and bromegrass were also determined after staining root samples with nitro-BT and chlorazol black E. The percentage of live intraradical infection declined with the maturing symbiosis. In contrast, no reduction with time in the percentage of extraradical hyphae that were metabolically active was shown by nitro-BT, suggesting a different turnover for extra- and intraradical hyphae. The FDA method gave the most precise estimates of enzyme activity (low SE) followed by the INT method, while the nitro-BT method was the least precise. Different percentages of extraradical hyphae that were metabolically active were obtained with the different methods. The nitro-BT assay deviated from the others as it did not show declining percentage of active extraradical hyphae as the symbiosis aged.