ORIGINAL PAPER E. Ferna ´ndez Valiente · A. Quesada · C. Prosperi M. Nieva · F. Legane ´s · A. Ucha Short- and long-term effects of ammonium on photodependent nitrogen fixation in wetland rice fields of Spain Received: 28 January 1996 E. Ferna ´ndez Valiente ( ✉ ) · A. Quesada · C. Prosperi 1 M. Nieva · F. Legane ´s · A. Ucha Department of Biology, Faculty of Science, Universidad Auto ´noma de Madrid, Cantoblanco, E-28049-Madrid, Spain Current address: 1 Ca ´tedra de Diversidad Vegetal I, Instituto Multi- disciplinario de Biologı ´a Vegetal, Universidad Nacional de Co ´rdoba, Casilla de correos 495, 5000-Co ´rdoba, Argentina Biol Fertil Soils (1997) 24:353–357 © Springer-Verlag 1997 Abstract Short- and long-term experiments were con- ducted in the rice fields of Valencia, Spain, to determine the ecological significance of ammonium on nitrogen fixa- tion. A significant inhibition of nitrogenase activity by am- monium, at concentrations higher than 0.5 mM, was ob- served after 8 h of incubation in short-term experiments done with a bloom of the N 2 -fixing cyanobacterium Ana- baena sp. In a second set of short-term experiments for in situ assays of nitrogenase activity in the field, a significant correlation between nitrogenase activity and the number of N 2 -fixing cyanobacteria in soil was found. No significant inhibition of nitrogenase activity by ammonium at concen- trations up to 2 mM was observed in these assays after 24 h of incubation. This lack of inhibition was probably due to the rapid decrease in ammonium content in the flood water. Only 5% of the ammonium initially added re- mained in the water 24 h later. In the long-term experi- ments, nitrogenase activity was assayed in plots fertilized with 0, 70 and 140 kg N ha –1 , over the cultivation cycle, for 5 years. A partial inhibition of nitrogenase activity by deep-placed N fertilizers was observed. Differences were only significant in 2 years. Mean results from 5 years only showed significant differences between plots fertilized with 0 and 140 kg N ha –1 . The partial inhibition of nitro- genase activity by ammonium increased over the cultiva- tion cycle. Inhibition was only significant in September, at the end of the cultivation cycle. Key words Acetylene reduction assay · Anabaena sp. · Ammonium · Cyanobacteria · Nitrogen fixation · Wetland rice fields · Nitrogenase activity Introduction N 2 -fixing cyanobacteria are the main contributors to photodependent N 2 fixation in rice fields (Roger and Kula- sooriya 1980). It is well known, from laboratory experi- ments, that ammonium represses nitrogenase activity and heterocyst differentiation in cyanobacteria (Guerrero and Lara 1987). However, the amount of ammonium used in laboratory experiments is usually higher than that observed in most rice fields (Quesada et al. 1989). At ammonium concentrations in the range found in rice fields, a low inhi- bition of nitrogenase activity was observed in laboratory experiments with cyanobacteria isolated from rice fields (Prosperi et al. 1992). The application of N fertilizers in the field reduces cya- nobacterial nitrogen fixation (Watanabe et al. 1978). How- ever, inhibition can be largely overcome by deep place- ment of the N fertilizer (Roger et al. 1980). In an ecologi- cal study of the rice fields of Valencia, Spain, no signifi- cant correlation was found between soil N content and ni- trogenase activity. A weak negative correlation was, how- ever, observed between the N content in water and nitro- gen fixation (Quesada et al., in press). From these reports we may conclude that, although the inhibitory effect of ammonium on nitrogenase activity is indisputable, its ecological significance in rice fields as a major factor regulating nitrogen fixation is questionable. In this report we present data from field experiments con- ducted to determine the ecological significance of ammo- nium in nitrogen fixation in rice fields. Materials and methods Field experiments were done in the experimental fields of the Rice Department (Instituto Valenciano de Investigaciones Agrarias at Sueca, Valencia, Spain). Short-term experiments with blooms The first set of short-term experiments was done with a bloom of the N 2 -fixing cyanobacterium Anabaena sp., developed in the experi-