Shunji Kitoh

Effect of Mineral Nutrients and Combined Nitrogen Sources in the Medium on Growth and Nitrogen Fixation of the Azolla-Anabaena Association

Abstract The effect of the mineral nutrient status and combined nitrogen sources in the medium on the growth and nitrogen fixation of Azolla japonica was investigated. The threshold concentrations of K, Mg, Ca, P, and S in the medium for growth were about 0.3, 0.3, 0.4, 0.08, and 0.08 ml, and those for the nitrogen-fixing capacity were about 0.07, 0.06, 1.0, 0.4, and 0.05 mM, respectively. Ammonium in the medium inhibited strongly both growth and nitrogen fixation. The total nitrogen budget, as a consequence of the balance between nitrogen inputs from the atmosphere and medium, was positive at concentrations of ammonium below 10 mM, whereas above this concentration, it was negative. Nitrate in the medium was less effective than ammonium. The addition of urea to the medium resulted in a substantial increase in the growth rate and nitrogen content although nitrogen fixation was appreciably reduced.

The growth and nitrogen fixation of Azolla filiculoides Lam. in polluted water

Abstract The growth and nitrogen fixation of Azolla filiculoides Lam. cultured in water of a polluted pond and two types of secondary effluent were investigated. The gradual decreases in the growth and acetylene reduction activity (ARA) of the plants in pond water were due mainly to a phosphorus deficiency. In secondary effluent, ammonium was an important factor limiting growth and ARA. The ARA was inhibited by ammonium in the medium even at 0.1 mM, whereas the growth rate in up to 1 mM ammonium was comparable with that of plants grown without ammonium. Nitrite and volatile organic acids in culture media were very toxic to Azolla plants. In secondary effluent containing nitrite, the plants shed their roots, followed by the fragmentation of fronds, and eventually died.

Response of six Azolla species to transient high-temperature stress

Abstract Response of eight Azolla strains from six species to transient exposure to high-temperature stress (above 40°C) was examined. Each Azolla strain showed differential tolerances to the stress. Based on growth and survival after the treatment, we concluded that the order of tolerance of six species to transient high-temperature stress was as follows: A. pinnata>A. microphylla, A. mexicana>A. caroliniana, A. filiculoides>A. rubra. Treatment with high-temperature stress also caused the rapid abscission of root and branches of Azolla. The temperature and time that caused rapid abscission depended on the species. In A. filiculoides and A. microphylla, the abscised branches were alive and proliferative when the temperature stress was short-term.

Response of 19 Azolla strains to a high concentration of ammonium ions

The response of 19 Azolla strains from five species to 20 mM ammonium ions was examined. The response varied even among strains of the same species. The tolerance indexes of both relative growth rate and nitrogen fixation [(values for plants grown in the presence of ammonium ions)/(values for plants grown in the absence of ammonium ions)] showed that ammonium ions did not always simultaneously inhibit growth and nitrogen fixation of individual strains. The tolerance of each Azolla–Anabaena association to ammonium ions is assumed to be determined separately by both the host fern and the symbiotic Anabaena. The inhibitory effects of ammonium appeared predominantly in the mature region of Azolla–Anabaena associations. In the sensitive strains, both chlorophyll content and the number of cyanobionts were reduced only in the mature region when ammonium ions were present. Possible strains for treatment of wastewater, which contains a high concentration of ammonium ions, are discussed.

Culture of Azolla in a pond, nutrient composition, and use as fish feed

Abstract The aquatic fern Azolla can grow without nitrogenous nutrients because of the presence of the N2-fixing ability of symbiotic Anabaena. Azolla filiculoides Lam. was cultured in a pond and its potential as a fish feed was examined. The extent of growth in the pond (Komoike pond, Sakai) of 1,200 kg/l00 m2 was less than that in a synthetic nutrient solution and in a diluted secondary treated effluent. The gradual decrease of the growth rate in the pond water was mainly due to phosphorus deficiency. The high level of crude protein of Azolla and higher lysine concentration indicated that Azolla plants may be a good source of fish feed. A significantly higher content of dehydro-ascorbic acid (1,909 mg kg-1 dry weight) than that of L-ascorbic acid (10 mg kg-1 dry weight) in Azolla grown for 3 d was observed. There was a gradual increase in the ratio of linolenic acid with aging in the composition of fatty acids in Azolla. In a feeding experiment with Tilapia nilotica, a diet containing 20.7, 34.4, and 48.2% of the total weight of dried Azolla, given for 3 weeks, led to a decrease of the fish weight gain of 5.2, 16.8, 17.1% compared with the control, respectively.

Differences in growth rate, nitrogen fixation and numbers of cyanobionts and heterocysts among three Azolla pinnata var. pinnata strains

The differences in nitrogen fixation, growth rate and numbers of cyanobionts and heterocysts among three Azolla pinnata var. pinnata strains were examined. The relative growth rate (RGR) and nitrogen fixation of PP7002 and PP7003 were significantly low compared with those of PP7005. The application of ammonium ions at 0.2 mM or more increased the growth rate of PP7002 and PP7003, but not PP7005. The numbers of cyanobionts and heterocysts in the mature region of PP7002 and PP7003 were statistically lower than those of PP7005. The low nitrogen-fixing activity of PP7002 and PP7003 as compared with PP7005 might be related to the restricted number of heterocysts. In PP7002 and PP7003, nitrogen fixation might be insufficient for full growth.

Aspects of the Very Rapid Abscission of Azolla Branches: Anatomy and Possible Mechanism

Sodium azide caused the rapid abscission of branches only in the mature regions of Azolla filiculoides. The abscission started at or after 5 min of exposure to 0.5 mM sodium azide. The presence of ethylene at 70 ppm neither induced rapid abscission within 3 h nor influenced the rapid abscission of Azolla branches caused by sodium azide. Cycloheximide or actinomycin D had no inhibitory effect on the abscission that was caused by sodium azide. Light and electron microscopy revealed that large, flattened cells, present at the base of each branch, formed an abscission layer. Sequential observations of large flattened cells from the apical to the mature region of Azolla indicated that the middle lamella between the cells in the mature region had already been partially degraded, even when a branch was still attached to a plant. Sodium azide induced the rapid dissolution of the small amount of middle lamella that remained between the large, flattened cells in the mature region, as well as modification of the primary wall of these cells. These events appeared to weaken both the cell walls and the adhesion between the cells, causing cell expansion and rounding. The expansion and rounding of the large, flattened cells facilitated the complete separation of these cells and the subsequent separation of the branch. Possible mechanisms for the rapid abscission of Azolla branches upon treatment with sodium azide are discussed.

Disappearance of Symbiotic Algae in the Azolla-Anabaena Association Subjected to Transitory Exposure to Ammonium

Abstract When Azolla japonica was grown on media containing ammonium at high concentrations, the ammonium levels within the Azolla tissue increased rapidly and reached the same levels as those in the media. When these Azolla fronds were transferred to a N-free medium, yellow spots were observed. In the crushed yellow spots, hardly any filaments of symbiotic algae could be detected under a microscope. Furthermore, the yellow spots cut from whole plants were able to regenerate to normal fronds only on a medium containing combined nitrogen at low concentrations. These regenerated fronds were free from Anabaena, did not exhibit acetylene reduction activity and failed to survive on a N-free medium.

Preparation of cyanobionts from the apical and mature regions of Azolla

Abstract A procedure has been developed to isolate apical segments and their endophytic cyanobionts from Azolla filiculoides var. japonica . Apical segments were isolated by enzyme treatment and flotation in 15% (v/v) Percoll solution. Anabaena filaments from the tips were purified by passing them through nylon meshes and then subjecting them to Percoll density gradient centrifugation. The cyanobionts obtained from the apical segments were morphologically distinct from those in the mature region. Heterocyst frequency of cyanobionts from the apical segments (2–3% of the total cells) suggested that those cells were from leaves corresponding to the apical 2–3 leaves in the main stem axis. This procedure yielded sufficient material for enzyme assay. By applying this method, the activity of glutamine synthetase in cyanobionts from the apical region was determined.

Decrease in the numbers of cyanobionts and heterocysts and nitrogen-fixing activity in Azolla leaves upon transfer to low light intensity

Abstract The numbers of cyanobionts and heterocysts in successive leaves from the apex along the axis were larger in the Azolla plants grown at a high light intensity than in those grown at a low light intensity. When the plants grown at a high light intensity were transferred to a low light intensity, the numbers of cyanobionts and heterocysts in the mature region gradually decreased. On the other hand, the nitrogenase activity per leaf sharply decreased immediately after the transfer of the plants to a low light intensity and then remained at a low level during the examination. Nitrogenase activity per heterocyst also decreased immediately after the transfer of the plants to a low light intensity. However, the activity gradually increased to the level of the plants grown at a high light intensity. These results suggest that the nitrogenase activity of Azolla changes in two ways. The low nitrogen-fixing activity in each leaf immediately after the transfer and after acclimatization was due to a low nitrogenase activity in each heterocyst and to the small number of heterocysts, respectively.