IMPROVED METHOD FOR OBTAINING VEGETATIVE GROWTH OF MYCORRHIZAL AND OTHER SLOW GROWING FUNGI

With fungi which do not sporulate when grown in vitro, the usual method of propagation is by the transfer of a few mycelial fragments to a fresh medium. Fungi which grow slowly and do not fragment during growth thus are provided with relatively few growing points for the development of new hyphae. In order to increase the growth rate and to attain a greater mycelial mass of slow growing fungi, the following method was developed. The mycorrhizal fungi (Boletus luteus, B. bicolor, Amanita rubescens, A. caesaria, A. muscaria, and Cenococcum graniforme) were used as examples, since their rate of vegetative growth is very slow, they do not sporulate when grown on laboratory media, and their hyphae do not normally fragment during growth. To 6-ounce prescription bottles provided with screw caps were added enough glass beads (Kimble 6 mm solid glass) to cover the wall of the bottle when laid on its side. These bottles also contained 15 ml of a medium (Fries, Arch. Mikrobiol., 12, 266, 1941) composed of, per L: malt extract, 5 g; glucose, 5 g; K2HP04, 0.5 g; MgSO4 7H20, 0.5 g; NH4C1, 0.5 g; 1 per cent ferric citrate chelate, 0.5 ml. The bottles were inoculated with the total growth from 7-day-old slants of mycorrhizal fungi growing on the same medium and the bottles were shaken vigorously to fragment the mycelium. The bottles were incubated on their sides for 7 days at 21 C, then again shaken vigorously to fragment mycelial growth. Fragmented mycelium plus medium from these bottles were transferred aseptically by pipette to 32-ounce prescription bottles, screw cap, prepared in a manner similar to that for the 6-ounce bottles. Each bottle contained 45 ml of a medium (Melin and Rama Das, Physiologia Plantarum, 7, 851, 1954) composed of, per L: glucose, 20 g; KH2PO4, 1 g; MgSO4-7H20, 0.5 g; ammoniuin tartrate, 0.5 g; ZnSO4, 2.5 mg; 1 per cent ferric citrate ebelate, 0.5 ml; thiamine HCl, 50,jAg. These bottles were incubated on their sides at 21 C for a period of 7 to 12 days, at which time a considerable amount of aerial and submerged growth was evident (figure 1). In most instances