Myrica gale L. populations growing in central Massachusetts were observed throughout the ice-free season. Nitrogenase activity appeared in mid-May shortly after budbreak, was at its maximum between late June and mid-August, and disappeared in late October after all leaves had fallen. Growth of overwintering nodules began in early May and was largely complete by mid-July. Most nodules (88%) lived for 3 years or less and 61% of the nodule biomass present in autumn was produced during the current season.Colonizing hyphae of the Frankia sp. endophyte were seen throughout the year in partially expanded cortical cells near the nodule lobe apex. Vesicles first appeared in mature cortical cells coincident with the onset of nitrogenase activity in mid-May, occupied the bulk of the infected tissue during the summer, and disappeared as nitrogenase activity ceased in late October. Evidence is presented that the vesicles are the site of nitrogenase activity and are newly produced each season in freshly formed nodule lobe tissue. Sporangia frequently formed in mature infected cells in nodules at one site but were rare at another. The processes described here in M. gale are probably typical of winter-deciduous actinorhizal plants.

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The rate of acetylene reduction was measured as a function of time after addition of 10% acetylene in Alnus, Casuarina, Ceanothus, Datisca, and Myrica. The maximum rate occurred after 45 to 60 seconds and was maintained for an additional 0.5 to 4 minutes before a decline in rate to 30 to 90% of the maximum. The rate then recovered to a value of 63 to 98% of the maximum. Removal of the shoot and lower roots did not affect nodule activity.The reduction of acetylene to ethylene has been widely used as an assay for nitrogenase activity in root nodules. However, in many legume nodules, there is a decline in the rate of ethylene formation after only a few minutes of exposure to acetylene. It appears that the predecline rate of ethylene formation gives the most accurate measurement of nitrogenase activity (3). Detopping of plants and removal of the rooting medium by shaking can also reduce activity (2).We undertook the present studies to see if similar phenomena occur in actinorhizal nodules, which are structurally different from legume nodules and contain a different endophyte (4). We found that actinorhizal nodules differ greatly from legume nodules in their response to both disturbance and exposure to acetylene. MATERIALS AND METHODSPlants were grown either in water culture (250 ml wide-mouth plastic bottles) or in tubes filled with vermiculite (30-ml plastic syringe barrels), using one-fourth strength, nitrogen-free Hoagland's solution. The water level in the water cultures was kept below the region of nodules, with a gas space of about 125 ml and liquid volume of 150 ml. Myrica gale was inoculated with Frankia strain LLR161101, Alnus rubra with strain HFPArI3, Casuarina cunninghamiana with strain HFPCcI3, while Datisca gomerata and Ceanothus americanus were inoculated with crushed nodules of Ceanothus. The plants grown on vermiculite were kept in a growth chamber at a constant temperature of 23°C with a photoperiod of 17 h and light intensity of 300 ,imol m-2 S-1 (400-700 nm). The conditions for the water cultures were the same, except the day temperature was 22°C and the night temperature was 20°C.For the cumulative acetylene reduction assay (Fig. 1), plants were grown in small pots (6.5 cm diameter, 5 cm height of vermiculite) which were sealed in 600 ml beakers with a plastic lid ' Supported by National Science Foundation grant DMB-8315415, and by United States Department of Agriculture grant 84-CRCR-1-1433. and modeling clay. After adding 10% acetylene, gas mixing was maintained by sliding and shaking of the potted plants within the beaker before gas sampling.For flow-through measurements of acetylene reduction, gas mixtures of 10% acetylene, air, and 02 were made in Saran bags. Sufficient 02 was added to keep the 02 concentration at the atmospheric level of about 20%. A peristaltic pump (Cole-Parmer No. J-7520-25) pumped the gas mixture from the bag through a humidifier and then past the root system. A flow rate of 210 ml min-' was used unless otherwise noted. Gas exiting the root system was sampled w...

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