Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18.Protease activity increased in nodules from harvested plants, reached a maximum at day 7 after harvest, and then declined to a level almost equal to the control by day 22 after harvest. Soluble protein and leghemoglobin decreased in nodules from harvested plants in an inverse relationship to protease activity.Nitrate reductase activity of nodules from harvested plants increased significantly within 24 hours and was inversely associated with acetylene reduction. The difference in nitrate reductase between nodules from harvested plants and control plants became less evident as shoot regrowth occurred and as acetylene reduction increased in the harvested plants.No massive loss of nodules occurred after harvest as evidenced by little net change in nodule fresh weight. There was, however, a rapid localized senescence which occurred in nodules of harvested plants. Histology of nodules from harvested plants showed that they degenerated at the proximal end after harvest. Starch in the nodule was depleted by 10 days after harvest. The meristem and vascular bundles of nodules from harvested plants remained intact. The senescent nodules began to regrow and fix nitrogen after shoot growth resumed.Harvesting the shoots of legumes, such as is periodically done in alfalfa and other forages, removes a primary source of energy for maintaining N2 fixation (15), nodule structure and function (3,25), and for the initiation of new nodules. The ability of nodules to remain functional after harvesting of the shoot may depend upon plant species, microsymbiont, nodule morphology, rate of shoot regrowth, nutrient availability, and competition between plant parts for energy. The interrelationships among some of these factors have been reported for several annual grain legumes (7,8 Roots from a portion of each replicate were shaken gently to remove sand and then washed in buckets containing cold water.Nodules were removed and placed in 20-ml beakers on ice. On each sampling day there were three replicates. At least two subsamples from each replicate were assayed for N2(C2H2) reduction3 2 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by either the U.S. Department of Agriculture or University of Minnesota and does not imply its approval to the exclusion of other products that may also be suitable.3Abbreviations: N2(C2H2) reduction; nitrogenase-dependent acetylene reduction.

Nodule structure, nodule enzymes of ammonia assimilation, nodule phosphoenolpyruvate carboxylase, nitrogenase-dependent acetylene reduction, and soluble protein were studied in four plant-determined ineffective genotypes and in one effective genotype of alfalfa (Medicago sativa L.) grown in a glasshouse. Ineffective nodules that formed on genotypes MnAg(In) and MnSa(In) were similar to effective nodules in early development. However, these nodules had smaller bacteroids and senesced much more rapidly than effective nodules. In contrast, nodules that formed on ineffective genotypes MnNC-3226(In) and MnNC-3811(In) were tumorlike and had few infection threads and bacteroids, and host cells were filled with starch. Nodules from plant-determined ineffective genotypes had reduced glutamine synthetase, glutamate synthase, phosphoenolpyruvate carboxylase, and nodule soluble protein when compared with effective nodules. There were differences between genotypes for all biochemical parameters assayed.

Root nodules from control and defoliated alfalfa (Medicago sativa L.) were compared for a period of 18 days following defoliation. Bacteroid containing cells of the nodules decreased markedly for the first 10 days following defoliation, but then increased during the next 8 days. Nodule length increased continuously during the 18 days in both control and defoliated plants. Cellular integrity of vascular bundles, meristems, and infection threads was retained in nodules from treated and control plants throughout the experiment. The sequence of senescence in bacteroid containing cells was similar in nodules from both treatments. Senescence was initially observed in the cytoplasm of bacteroid containing cells. Membranes surrounding bacteroids showed degenerative changes as bacteroids senesced. Bacteroids aggregated within nodule cells. Infection threads and bacteria inside infection threads did not disintegrate. Bacteroids ultimately disappeared in senescent cells.Nodules of alfalfa appear to respond to foliar harvesting by a partial but temporary senescence from which they recover by regeneration of bacteroid containing cells.

Events in development of powdery mildew fungi from spore germination to formation of the first haustorium and hypha, including host cell responses, were cataloged using a binary pathway system. This system branches at "event-points" which represent sites of "on–off" alternatives for the occurrence of an event or sites where one of two alternative events occur. The system permits statistical comparisons of probabilities for different events within a given host–parasite combination and for specific events in different host–parasite combinations. Data from the binary pathway system can be converted to percentage of total spores to show the size of the fungal population which passes each event-point, or the data can be resorted to determine how many germlings are associated with a given combination of events.The binary system was used with data for powdery mildew of barley obtained from whole leaves harvested 1 and 2 days after inoculation, which were fixed and then stained with acid fuchsin and aniline blue. Approximately 5000 germinated spores were observed for each host–parasite combination. In compatible combinations, 6–7% of spores failed to produce a normal germ tube and (or) appressorium after germination, and 9–11% of spores failed to produce a penetration peg at the first appressorial lobe. In epidermal cells distant from stomates, only 2–3% of the fungal population produced haustoria and hyphae. The fungus had a high probability of inducing papillae in such cells and a low probability of passing through papillae. In cells near stomates, papillae were less frequent and 46–52% of the population produced haustoria and hyphae.In an incompatible host–parasite combination (with the M1a gene), most penetration pegs terminated in papillae regardless of host cell location. In cells distant from stomates, virtually no germlings grew beyond papillae. In cells near stomates, 11% of the fungal population either penetrated a papilla or entered a host cell without inducing papillae. Of these, about half induced host cell collapse either before or after haustorium formation and half stopped growing after haustorium formation without inducing cell collapse. Thus, incompatability was expressed at several stages in fungal development.

Nodule structure, nodule enzymes of ammonia assimilation, nitrogenase-dependent acetylene reduction, and nodule soluble protein were studied during vegetative regrowth of detopped birdsfoot trefoil (Lotus corniculatus L.) seedlings grown in the glasshouse. Nodules senesced rapidly for a period of 14 days following shoot removal, but then pink nodule populations increased as shoot regrowth occurred. The structural sequence of senescence was similar in nodules whether the result of either aging or shoot removal. Membranes surrounding bacteroids showed degenerative changes as bacteroids senesced. Bacteroids aggregated within the nodule cells and ultimately disappeared in senescent cells. Infection threads and bacteria inside infection threads did not disintegrate.Nodule senescence as a result of shoot removal was accompanied by a marked decline in acetylene reduction, nodule soluble protein, nodule host plant glutamine synthetase (GS), and glutamate synthase (GOGAT). Nodule enzyme activity, soluble protein, and acetylene reduction activity recovered to initial values as shoot regrowth occurred and pink nodule populations increased. Nodule host plant glutamate dehydrogenase (GDH) did not change after shoot removal.This study shows that birdsfoot trefoil nodules respond to shoot removal by an increased senescence. Recovery of nodule function is associated with the formation of a new nodule population. The data also indicate that host plant GS and GOGAT function to assimilate fixed N. Spherical nodules with determinant growth may be less efficient than elongate nodules with indeterminant growth in nodule maintenance and function.