Epiphytic cyanobacteria on Chara vulgaris are the main contributors to N(2) fixation in rice fields

Abstract

The distribution of nitrogenase activity in the rice-soil system and the possible contribution of epiphytic cyanobacteria on rice plants and other macrophytes to this activity were studied in two locations in the rice fields of Valencia, Spain, in two consecutive crop seasons. The largest proportion of photodependent N(2) fixation was associated with the macrophyte Chara vulgaris in both years and at both locations. The nitrogen fixation rate associated with Chara always represented more than 45% of the global nitrogenase activity measured in the rice field. The estimated average N(2) fixation rate associated with Chara was 27.53 kg of N ha(-1) crop(-1). The mean estimated N(2) fixation rates for the other parts of the system for all sampling periods were as follows: soil, 4.07 kg of N ha(-1) crop(-1); submerged parts of rice plants, 3.93 kg of N ha(-1) crop(-1); and roots, 0.28 kg of N ha(-1) crop(-1). Micrographic studies revealed the presence of epiphytic cyanobacteria on the surface of Chara. Three-dimensional reconstructions by confocal scanning laser microscopy revealed no cyanobacterial cells inside the Chara structures. Quantification of epiphytic cyanobacteria by image analysis revealed that cyanobacteria were more abundant in nodes than in internodes (on average, cyanobacteria covered 8.4% +/- 4.4% and 6.2% +/- 5.0% of the surface area in the nodes and internodes, respectively). Epiphytic cyanobacteria were also quantified by using a fluorometer. This made it possible to discriminate which algal groups were the source of chlorophyll a. Chlorophyll a measurements confirmed that cyanobacteria were more abundant in nodes than in internodes (on average, the chlorophyll a concentrations were 17.2 +/- 28.0 and 4.0 +/- 3.8 microg mg [dry weight] of Chara(-1) in the nodes and internodes, respectively). These results indicate that this macrophyte, which is usually considered a weed in the context of rice cultivation, may help maintain soil N fertility in the rice field ecosystem.

FIG. 1.

Acetylene reduction rates (means ± standard deviations; n = 11) associated with the different parts of the rice soil system at two sites in a Valencian rice field during the 1999 and 2000 crop seasons.

FIG. 2.

Optical micrographs of the main genera of N₂-fixing cyanobacteria that grew in petri dishes after field samples of Chara were grown in N-free BG11 medium. (A) Anabaena sp. (B) Nostoc sp. (C) Calothrix sp. Scale bars = 10 μm.

FIG. 3.

Scanning electron micrographs of Chara. (A) Carbonate aggregates were abundant in Chara from field samples. Scale bar = 50 μm. (B) Filamentous epiphytic cyanobacteria attached to the Chara surface. Scale bar = 5 μm.

FIG. 4.

Micrographs of Chara viewed under the fluorescence microscope with a violet (A and C) or green (B, D, E, and F) filter. The violet excitation distinguished carbonate particles (green fluorescence) from Chara chloroplasts (red), while the green excitation distinguished cyanobacterial phycobiliproteins (bright red). Carbonate aggregates were abundant in Chara from field samples (A) but not when the organism was maintained in the laboratory (C). Epiphytic cyanobacteria were abundant on the Chara surface with carbonate particles (B) but were scarce in sections without carbonate (D). Heterocystous cyanobacteria were observed on Chara from field samples (E and F). The arrows indicate heterocysts. (A, B, E, and F) Scale bar = 40 μm. (C and D) Scale bar = 150 μm.

FIG. 5.

Micrographs of field samples of Chara viewed under the confocal scanning laser microscope. Microcolonies of unicellular and filamentous cyanobacteria were present on the surfaces of internodes (A) and verticiles (B) of Chara. The arrows indicate heterocysts. (A) Scale bar = 100 μm. (B) Scale bar = 70 μm.

FIG. 6.

Differential colonization of Chara by epiphytic cyanobacteria in field samples collected during the 2000 crop season in Valencian rice fields. The values are percentages of the surface area (means ± standard deviations; n = 54).