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Soil Science and Plant Nutrition

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Nitrogen fixation in Azolla-Anabaena symbiosis as
affected by mineral nutrient status

Michihiko Yatazawa , Naoki Tomomatsu , Noriyo Hosoda & Katsunori

To cite this article: Michihiko Yatazawa , Naoki Tomomatsu , Noriyo Hosoda & Katsunori Nunome
(1980) Nitrogen fixation in Azolla-Anabaena symbiosis as affected by mineral nutrient status, Soil
Science and Plant Nutrition, 26:3, 415-426, DOI: 10.1080/00380768.1980.10431227

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Published online: 29 Mar 2012.

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5 mmol·liter-t. 12. On the other·liter.10. day-I.. 415 .)·hr-l. Cu.5. Ltd. ~nd 0. quantitative determinations of the nitrogen- • Present address: Yakult Honsha Co. and 30 . and B. The threshold levels of Fe. This effect is discussed in relation to the saturation point in photosynthesis. Deficiency of anyone of the micronutrient elements.). and Katsunori NUNOME** Department of Agricultural Chemistry. The nitrogen-fixing capacity measured from the increase in total nitrogen in Azalia plants cultured with complete nutrient solution lacking only combined nitrogen. Full development of nitrogenase activity was not realized at concentrations below 0. The threshold levels of P..4.W. Mn.l. and 0. Soil Sci. had an unfavorable effect on growth and developing nitrogenase activity. Tokyo Metropolitan Government. 17). Fe and Mo were very sensitive for developing nitrogenase activity.50 mg N . However. Azalia. 16. 26 (3). greatly enhanced nitrogen fixation. Additional Index Words: nitrogen fixation. B was shown to affect growth specifically. 5. Mn.l (D. About six extant species are known globally. 464 Japan Received December 20. Fe. Considerable numbers of practical experiments have been reported on this symbiosis based on its potential importance in agriculture (4.03. K. 11.umole ~H.g-l (D. 0. Mo. On the other hand.. Illumination (up to 6. Fujisawa Factory.3.6. and 20 . produced·g. Mo. Co. 0. •• Present address: Bureau of Public Health. Zn.* Noriyo HosoDA. Mg. 1979 The growth and nitrogen-fixing capacity of Azalia imbricata were studied with special reference to the effects of the mineral nutrient status in the medium. respectively. 15). 415-426. Combined nitrogen was unfavorable for growth and nitrogen fixation.W. and Ca in the medium for Azalia growth were ca. Plant Nutr. Naoki TOMOMATSU. 0. Nitrogen fixation in Azalia-Anabaena symbiosis has long been known from the work of several pioneer investigators (2. 0.03. Nagoya University. the acetylene-reducing capacity of the plants was·liter-" and those for nitrogenase activity were 20. respectively.000 lux) of the plants during the period of acetylene reduction. The host plant Azalia occurs widely on the surface of bodies of fresh water in the tropical and temperate regions. 0.5 mmol'liter-t. 1980 NITROGEN FIXATION IN AZOLLA-ANABAENA SYMBIOSIS AS AFFECTED BY MINERAL NUTRIENT STATUS Michihiko YATAZAWA. respectively. Nagoya. was 6. 0. 20. The very low nitrogen-fixing capacities reported by pioneer workers were found to be raised considerably when the data were recalculated on the basis of the daily increase ratio in growth.6 . These two values are not so different from one another. I. and B for growth were 50.

The pH of each solution was adjusted to 6. The Azolla plants employed were originally collected from a pond in Chita. The water for making the culture solutions was prepared by passing glass distilled water through ion- exchange resins. = fresh weight of harvested plants/fresh weight of initial explants) was calculated. where t is the period (day) of the culture. K 2SO. 15-17).'5H 20 0.. Japanese taxonomists always classify it as a different species from A. and the growth value (G.V. CaCI 2 3.00. 3.o4H 20 1.1): NaH 2 PO. pinnata (13).o7H 20 0. Measurement of growth.N) solution contained the fol- lowing macro nutrients (in mmoHiter. Aichi Pref.N) or its modi- fied nutrient-deficient culture solutions. YATAZAWA. Nutrient-deficient solutions were prepared by reducing one component to various levels from that in the AII( .000 lux at the surface of the desk unless otherwise indicated. The daily increase ratio (R) was also calculated from the equation. A. However. Precultured ex plants of about 50 mg fresh weight were incubated with 200 ml of nitrogen-free nutrient solution (AII( . CuSO. was measured with a gas-chromatograph (Shimadzu GC-4CPF) under the . where polyethylene beakers were used. plus the following micronutrients (in pmoHiter. and HaBOa 5. ZnSO. Japan. The light intensity was 4.) were placed in a 50-ml conical flask under light of 8.o7H 20 4.3 g (F. and K. TOMOMATSU. 2.05.W.13. imbricata is sometimes used as a synonym of A. Harvested plants were placed on a dry filter paper to remove adhering culture solution. pinnata. N.08.V. Culture methods. The amount of C 2H. (I + R)t=G. The culture period was 3 weeks unless otherwise indicated. MnSO. They were placed on a desk in a phytotron with 12 hr light at 30°C and 12 hr darkness at 25°C. and MgSO.77.19. The culture vessels were 500 ml Tystone glass beakers except in the case of B-deficient culture experiments. N. and information concerning the levels of mineral nutrients required for sufficient display of nitrogen fixation in the symbiosis is almost lacking. This paper reports quantitative relations between mineral nutrient status and nitrogen-fixing capacity in Azolla imbricata-Anabaena symbiosis measured by acetylene reduction assay and the Kjeldahl analytical method. and no special purification of them was performed. Several plant colonies were taken from a stock culture in a small pond and precultured with a dilute complete nutrient solution lacking only combined nitrogen for a month.00. The AII( . The chemicals used were of analytical grade. produced in an atmosphere containing 10% C2 H 2 during an incubation period of 150 min. NUNOME fixing activity in the symbiosis are rather limited (1. The Azolla was identified as Azolla imbricata (13) associated with Anabaena azollae. Harvested plants weighing about 0.00.416 M. 1. MATERIALS AND METHODS Azolla.00.33. 2.000 lux.. The fresh weight was measured. 12. NaMo0 4 H 20 0. HOSODA.N) solution. Measurement for nitrogenase activity.5.1): FeSO. They were covered with glass or plastic trans- parent dishes to avoid mineral contamination from dust materials.

Effects of macronutrient elements The growth and nitrogenase activity of Azolla plants cultured with macronutrient- deficient solutions for 21 days at the first transference are shown in Table 1.g-1(D.J <. nitrate- N.or K-deficient plants obtained at harvest of the first transference culture were reinoculated into modified AII( .J c( 90 120 150 Period of inCUbation (min) Fig.)·hr. Inocu- lated explants on Ca. After chromatographic measurement. Analysis of combined nitrogen. To obtain more accurate data for the effects of macronutrient elements in the medium on growth and nitrogenase activity in Azolla. Ammonium-N. .. This was thought to be due to carry-over of P and K with the inoculated explants. 1.bO ~cU ~o 40 -oE CI) ::t. considerable growth was obtained with P.N) solution 100 o .W... the plant samples were dried and weighed.1.o -c. RESULTS Nitrogenase activity under different light intensities Since light affords energy for nitrogen fixation. illumination of the plants during the period of acetylene reduction greatly enhanced the nitrogenase activity in the plants. The total nitrogen of the dried plant materials and of the culture solution was measured by the Kjeldahl method. Eo ::::. Nitrogenase activity was expressed in most cases in pmole C2H4 produced. 1. As shown in Fig. On the other hand..::: 80 '"03:. although acetylene reduction was still recognized even in the dark. Nitrogenase activity in Azolla-Anabaena symbiosis as affected by light intensity. P. Nitrogen Fixation in Azolla-Anabaena Symbiosis 417 same conditions as described previously (7).60 >1 .or K-deficient plants. ~~ E N 20 "'U <.. presumably due to the utilization of reserved carbohydrates which had been synthesized in daytime. the effect of light intensity on the acetylene-reducing activity of Azolla was examined by exposing the cultures to different light intensities during the period of incubation with acetylene.or Mg-deficient solution grew only to a slight extent. and nitrite-N were analyzed by standard methods (6).

418 M. Mg.5 mmol· liter-I.0 0 All (-N)-Mg 4.0 -x.4 ~ . Plant colonies deficeint in Mg were liable to dis- integrate into small colonies. 2. N.0 69 13.s= 0.5 mmol·liter.. As regards deficiency symptoms.8 'B. o 0. K. On the other hand.5 100 All (-N)-P 13.. plants deficient in P became slightly pale.6 Mean of 4 replicates.6 ~ ~ gj.5 22 0.)·hr-l) control All ( .Mg ..4 !: 'c 0:: . 3 34 All (-N)-K 14... and 0. The nitrogenase activity of Azolla decreased sharply through the reduction of macronutrient elements. K 1.5.2 0.03.W. Growth and nitrogenase activity in Azolla-Anabaena associates as affected by the level of macronutrients in the culture solution.. TOMOMATSU. 2). N.8 4 0. Full development of nitrogenase activity was not realized at concentrations below 0. NUNOME Table 1. the decrease of growth was very sharp with Ca-deficient plants. The effects of combined nitrogen were examined by supplementing ammonium . Culture solution Growth % of Nitrogenase activity % of value control (pmole C1H. and K. 0. and 0. K. 0. Nitrogenase activity -O-P ..6.4. 0.. 0. Growth and nitrogenase activity in Azolla-Anabaena associates cultured with macronutrient-deficient solution. respectively.::-:.4 100 33. fresh explants were cultured with other modified All( .8 . and the roots elongated extraordinarily.03.N): control 20. or Mg. 0. containing various levels of P or K.4...----'-----:----'=""'-:---'-----'0. 0. Necrosis on the fronds was also recognized in plants deficient in K.6 . The threshold levels of P.0 -A-Ca 1... The results showed that elimination of carry-over of P or K resulted in sufficient reduction of growth and nitrogenase activity in the plants (Fig. HOSODA.3 65 11.0 10 Concentration of macronutrients (mmole) Concentration of macronutrients (mmole) Fig.2 0. 0.N) solutions containing various levels of Ca or Mg. 0. Below the threshold value.·g-1 (D.2 ~ Qj 0:: '::----. 0.1 for P.7 41 All (-N)-Ca 0. Mg. and Ca. respectively. Ca. and Ca for Azolla growth were found to be about 0. YATAZAWA. i .

Other plants grew similarly to the first transference plants.8 43 22. he ' ) control All (-N): control 15.7 68 24.W. produced· g-l (D. green algae grew intensely on the surface of the culture solutions.8 67 All (-N)+N 20 ppm 9. the growth and nitrogenase activity were measured (Table 3B). Mn. B-. The growth and nitrogenase activity of the harvested plants were measured (Table 3A). and Mo in the medium for the growth and nitrogenase activity of Azolla. . On the other hand. Effects of micronutrient elements Azolla plants were cultured with micronutrient-deficient solutions for 21 days. while B may affect growth rather specifi- cally. The data suggest that Fe and Mo are characteristically sensitive for developing nitrogenase activity. A considerable decrease in growth value was observed at higher concentrations of combined nitrogen (Table 2).9 100 All (-NHN 2 ppm 11.0 57 24. the nitrogenase activity decreased considerably in plants deficient in Fe.6 67 30. Culture solution Growth %of Nitrogenase activity % of value control (pmole C 2 H.2 60 Mean of duplicate. At the first transference. and B in that order. Mo. To examine the comparative im- portance of each micronutrient element in the development of nitrogenase activity and the amount of growth.N) solution at various levels of nitrogen. part of the harvested plants of the first transference (Table 3A) was reinoculated into each micronutrient-deficient culture solution. In an attempt to reduce the carry-over of micronutrient elements from the inocu- lating explants. Nitrogen Fixation in AzalIa-Anabaena Symbiosis 419 sulfate to AU( .).-N) on the growth and nitrogenase activity in Azalia-Anabaena associates. Effect of combined nitrogen (NH. To obtain information on the threshold levels of Fe. the Azolla plants grew reasonably well except for B- and Fe-deficient plants. and Mn-deficient solutions. This may have been due to inevitable contamination of these micronutrient elements from the culture medium and explants. This was thought to be due to carry-over of micronutrient elements from the inoculated explants and by contaminants from the reagents and water employed. ratios for relative nitrogenase activity/relative growth value were calculated (Table 3). explants which had been slightly deficient in each of the micronutrient elements were reinoculated into culture solutions Table 2. Mo-. Further decreases in growth value and specific nitrogenase activity were obtained for plants cultured with Fe-.8 81 All (-NHN 5 ppm 10. After 21 days incuba- tion.6 67 All (-NHN 40 ppm 6. B. 1 70 29.5 83 All (-N)+N 10 ppm 10.8 100 36. Under these conditions. The nitrogenase activity was also decreased to some extent.

H. Newly developed fronds became strongly discolored. The results indicated that.9 26 2.)· hr. respectively.8 67 10.52 All (-N)-B 6.0 78 1.4 100 33. respectively.3 79 1.84 All (-N)-Mo 15.7 33 0. A. YATAZAWA.8 25 10.)·hr-l) (B) . First transference culture Growth % of Nitrogenase activity % of Culture solution value control (pmole C.0 or 7.3 60 O.2 100 32.22 30 13.4 80 27. 73 All (-N)-Mo 12.8 77 25. 00 All (-N)-Fe 12.0 and incubated for 17 days.00 All (-N)-Fe 4.3 75 13. NUNOME Table 3.4 80 26.2 78 0.W. Mo.N) culture solution. under the experimental conditions employed.98 All (-N)-Cu 16.8 77 19. which was prepared by replacing Fe2+ with Fe3 + in All( . 01 All (-N)-Zn 16. N. During the course of the culture. at pH 4. and 20 pg·liter. N. 9 37 0.7 83 1. chlorotic symptoms appeared in Fe-deficient plants. --~---~~-~ All ( . Fe-deficient plants obtained after 19 days culture were transferred to Fe3 + solution. 1. and K. the growth and nitrogenase activity were measured. for nitro- gen-fixing activity.23 All (-N)-Mn 11.0 78 26. Plants grown at pH 4. 1 58 11. After 21 days incubation. 0. TOMOMATSU. containing various levels of micronutrient elements. 01 All (-N)-Zn 14.N): control 19. produced. 1 59 9. Growth and nitrogenase activity in Azolla-Anabaena associates cultured with micronutrient-deficient solution. the threshold levels of Fe.47 All (-N)-Mn 16.04 All (-N)-Co 16. 20. Second transference culture Growth % of Nitrogenase activity %of Culture solution control (pmo)e C 2H.49 All (-N)-B 4.3 32 1.23 28 0. 33 B.2 100 1.2 60 O.0 became dark green and the growth value was restored . 10. and B in the culture solution were roughly 20.3 80 22. g-1 control (B)/(A) (A) (D.5 100 1. HOSODA.0 6 0. Iron also showed another characteristic response in Azolla nutrition.W.N): control 20. 28 Mean of 4 replicates.1.8 82 19.64 All (-N)-Co 14. g-1 control (B)/(A) value (A) (D.3. Mn.5 40 1.4 67 0. produced.1 39 0. and the levels for growth were about 50. 78 All (-N)-Cu 15. and 30 pg·liter-t.420 M.' ) (B) All ( .

.50 )0S:(ifowth value) R: (I +R)t = growth value.8 7.6 6.6 4 20 40 499 28.187 1.W.56 O. with a light intensity of 8.6 13.umole C2H 4 produced" g-I(D. 61 42. No. value (1 ) (mg) F. Nitrogen Fixation in Azolla-Anabaena Symbiosis 421 to 10. 39 42. The values obtained ranged from 25 to 45 .000 lux. 53 41.5 6 22 32 582 32. On the other hand.2 .umole C 2H 4 produced" g-I(D.).)" hr.W.82 o. 154 42. and the nitrogen-fixing activity in the dark is 10% of that in daytime.5 5 21 31 512 28.1 5.) " hr. 23 43. 20 41.6 and the acetylene-reducing capacity to 36. then the nitrogen-fixing capacity in Azolla imbricata calculates at 4. per culture Culture period in Explants Harvested Growth Exp.6 18.2 Total N in N concentration Daily harvested in harvested N-fixing capacity increase crops crops (D) (RxD) ratio (R) (mg) (mg N·g-l (D.0.l (average 34.)" hr-l. Nitrogen-fixing capacity of Azolla-Anabaena associates as determined from the increase in total nitrogen in the culture.)" 0.88 0.23 0. Plant wt.N) culture solution was measured with 31 cultures.135 1.000 lux during the period of acetylene reduction" Assuming that the conversion ratio for C2H 4 produced: N2 reduced is 3.5 3 17 43 671 37.01 Aver. the average light intensity in daytime is 8.1 2 21 31 512 29.W.6).» (mg N·g-l (D.2 16.175 1. 143 1. Nitrogen-fixing capacity as estimated from the nitrogenase activity The nitrogenase activity in plants grown on AII( .2 6. R = 10 t -1.6 15.W.W. (mg) (mg) 15 50 653 36.26 mg N"g-I(D. D. Nitrogen-fixing capacity estimated from the increase in total nitrogen Confirmatory evidence of nitrogen fixation can be obtained from the increase in Table 4. days F.l .0 showed intensified deficiency symp- toms and gave a smaller growth value of 4.umole C 2H 4 produced "g-I(D.49 0.2 16. 8 7.W.w. 143 1.19 41. the day length is 12 hr.6 6. plants grown at pH 7.9 12.141 1. 2 5.2 and a lower nitrogenase activity of 3.W. O.

The magni- tude of the value was about the same as those obtained by other workers. A few previous papers (5.56-7.422 M. the amount of Kjeldahl nitrogen in the cultures was measured.177. N. The nitrogen-fixing capacity indicated by the increase in mg N· g-l(D.50) under the experimental conditions employed. The composition of the culture solution in the present study was basically the same as that of the medium used by JOHNSON et al.) on average. DISCUSSION Culture solution. The daily increase ratio of 0. Table 4 gives some examples of the nitrogen-fixing capacity as measured by the Kjeldahl method. The dou- bling time estimated by BROTONEGORO and ABDULKADIR (3) was 63-105 hr. HOSODA.2 mg N·g-l(D. The growth of Azolla with AII( . especially the light intensity employed.)·day-l was calculated by mUltiplying the nitrogen content of the harvested plant~ in mg N·g-l(D. Various kinds of culture solutions have been used by previous authors.(J6) and JOHNSON et al.N) solution were therefore analyzed for ammonia. and that of WATANABE et al.N) culture solution was reasonable (Table 4). 15) have reported that considerable amounts of combined nitrogen were found in culture solutions of Azo/la pinnata.W. respectively.82 mg N . but a somewhat lower concentration of macronutrient elements would be preferable under the present experimental conditions. YATAZAWA. N. nitrate. since values for dry matter of the explants were not available.154 corresponds roughly to 116 hr as doubling time. The daily increase ratios calculated from the data of TUZIMURA et al. therefore. Several residual culture solutions after the harvest of crops grown with All( . and K. (9) were 0.141 to 0. in which nitrogenase activity was not reduced under a 65% reduction of full sunlight for 5 hr.187 (average 0.W.179 and 0.) by the daily increase ratio in growth on a dry matter basis. TOMOMATSU. nitrate.W. (9) with only small amendments.N) solution for various periods of incubation therefore lay within the range of 5.)·day-l (aver- age 6. and nitrite. NUNOME total nitrogen of the culture. the R value was found to be within the range of 0. The present results clearly indicated that there is a positive effect of light intensity in enhancing the nitrogenase activity in Azolla plants (Fig. The nitrogen content in the harvested crops was 42. Light intensity as affecting the nitrogenase activity. In the present study. No indication of the presence of ammonia.W. This finding was seemingly different from Broto- negoro's results. To evaluate the results obtained by the acetylene reduc- tion method. The nitrogen-fixing capacity in Azolla imbricata-Anabaena associations cultured with AII( . or nitrite in the culture solutions was found under the experimental conditions employed. (17) was 3-5 days. This daily increase ratio on a dry matter basis was assumed to be the same as that of the daily increase ratio on a fresh weight basis (R). 1). The slightly lower value in the present experiment may be attributable to the lower light intensity during the incubation period.154). even though further reduction of the light intensity led to a .g-l(D. Kjeldahl analysis of these culture solutions also gave only negligible amounts of combined nitrogen.

000 lux would thus have been supplied to the plants under 65% reduction of full sunlight. The importance of macronutrient elements including P.000 lux. as has been postulated by PETERS (14). This. Ca under certain natural freshwater environmental conditions. The value. 16).17). Avail- able information is confined to the requirements for the following elements: Fe (10. Effects of micronl!trient elements. Ca. and consequently Anabaena azo/lae can be expected to have a similar value of light saturation point in photosynthesis as that observed in these shade plants. This was clearly shown in the present experiment in which Azolla was cultured repeatedly with a macronutrient- deficient solution (Fig. but significantly positive capacity for nitrogen fixation observed in Azolla in the dark is presumably supported by photosynthates supplied from the algae themselves or their host plant Azolla. the light intensity of full sun- light was about (2-6) x 104 lux. The situa- tion of Anabaena azollae in cavities of the Azolla leaf appears to be similar for getting light as that of such common shade plants. The saturation intensity would lie at about 1 x 104 lux. since further reduction of carry-over nutrients from the explant should surely result in a smaller yield of harvested crops. suggests that nitrogen fixation in Azolla- Anabaena symbiosis operates principally by being supplied with reducing energy di- rectly from the algae. The degree of reduction in growth together with that in nitrogenase activity of plants deficient in a particular nutrient element thus tells only the liability of establishing deficient conditions with the particular element. at least under light conditions. in turn. The unfavorable effect appears to be due mainly to the vigorous growth of green algae under nitrogen-rich conditions. K. Combined nitrogen was unfavorable for growth and nitrogen fixation in Azo/la (Table 2). Effects of macronutrient elements. The combined experimental data suggest therefore that there may be two phases of nitrogen fixation in response to light intensity: one would proceed under a higher light intensity than the saturation intensity above which no increase in nitrogen fixation would be expected. the degree of reduction in growth of plants cultured with macronutrient-deficient solutions varies with different authors (16. The reasons for this variation may derive mostly from differences in pre- culture conditions. However. More than 7. The smaller. although somewhat contradictory favorable effects have been described by a few (2. coincides well with the light saturation point in photosynthesis usually observed in shade plants. . 17). in fact. and the other would operate at a lower light intensity under which a proportional increase in nitrogen fixation would occur with increasing light intensity. In their experiment. Inside the Azolla plant. would represent the first among the four macronutrients to become short for Azolla. Nitrogen Fixation in Azolla-Anabaena Symbiosis 423 decrease in nitrogenase activity. 2). and Mg in supporting the growth of Azolla has been universally recognized by previous authors (16. In this sense. Very few papers have dealt with the effects of micronutrient elements on the growth and nitrogen-fixing capacity of Azolla. the saturation intensity would take a somewhat smaller value than that. Similar effects have been reported by other authors (3.12. 17). The results of the present study were obtained under light intensities below 6.10.

)·day-l in Azolla pinnata when he applied 15N excess dinitrogen to Azolla-Anabaena symbiosis for 19 days. produced·g-l(D. the nitrogen-fixing capacity of Azolla increased to 3. HOSODA. These values also differ greatly from that referred to by MOORE (11) for Saubert's results.W. . this value would corre- spond to 5. Moore himself obtained direct evidence to suggest a nitrogen-fixing capacity of 222 pg N·g-l(D.W. produced unfavorable effects on growth and nitrogen fixation in Azolla.N) solution was 34.)· hr. N. Co (9). In the present experiment. The first determination was made by OES (12) using the Kjeldahl method. SAUBERT'S results (15) indicated a nitrogen-fixing capacity in Azolla pinnata of about 2. Several papers have dealt quantitatively with the nitrogen-fixing capacity in Azolla-Anabaena symbiosis. N. Quantitative value of nitrogen-fixing capacity in the symbiosis. the acetylene-reducing capacity of Azolla imbricata cultured with All (. in which the daily increase ratio of Azolla in his 11 experiments was 0.)·day-l on averge. who may have made calcu- lations using a simple mean of the increased nitrogen. Cu.W. amounting to as much as 7. and Mn were more liable to become deficient. His value would be increased by a recalculation made on the basis of the daily increase ratio in growth.082 and a mean N content of 3.).72 mg N 'g-l(D.W.)·hr.7 pm ole CaH4 produced·g-l(D. If the moisture content of Azolla was 94%.424 M. WATANABE et al.)·hr-l in daytime. In the present experiment. B.l . 16). When a recalculation was made to include the released nitrogen. Mo. In his experiment.7 mg N 'g-l(D.W.59 on average and the N content was 2.2 mg N·g-l(D. the nitrogen-fixing capacity of Azolla imbricata was estimated to be 6. (17) reported that Azolla fixed 340± 102 nmole C 2H.hrl. The capacity was so estimated at about 1. deficiency of the micro- nutrient elements. Co. Mn. In our case. Azolla pinnata reduced acetylene at a rate of 3-6 nmole'mg. the depression in some micronutrient-deficient plants was sustained at a definite level.I as an average of 31 determinations. produced· g-l(D.)·day-l. basis.W.1 corre- sponding to 36-72 pmole C 2H.W. and B. The nitro- gen-fixing capacity was recalculated by us from his data.84% on a D. This value is more than 4 times that given by MooRE (11). On the other hand. and K. NUNOME 16. basis.1 protein'min. This value is consider- ably larger than those obtained by the above pioneer workers. TOMOMATSU.W. and Mo (2. Mo. The threshold levels of these elements for the growth of Azolla were almost the same as those found for common angiosperm crop plants (8).50 mg N·g-l(D. the nitrogen-fixing capacity determined by the acetylene reduc- tion method has been reported to take somewhat larger values.W.)· day-I.W.W. when recalculated on the basis of a mean daily increase ratio of 0.6 pmole' g-l(D. presumably due to the unavoidable contamination of micronutrient elements from reagents and water under the present experimental conditions. Fe. produced·g-1(D. Fe. How- ever.W.67 mg N·g-l(D. a certain amount of combined nitrogen was released into the culture solution. According to BECKING (1).)·hr. YATAZAWA. Repeated transfer of the culture to micro- nutrient-deficient solution increased the degree of depression of both activities.1.)·day-l.W.24% on a D.)·day-l. 10. Zn. BROTONEGORO and ABDUL- KADIR (3) reported that Azolla pinnata fixed dinitrogen at a rate of 60-90 pmole C 2 H. 17).

The problem of the use of Azolla as fertilizer in the Democratic Republic of Vietnam..26 mg N2 fixed·g-1(D.. I. Philippines. K. Nogaku. The value of 34. Bureaux.g-l(D. Science and Culture of Japan. 41. on Nitrogen Fixation. Nutman. 155-186 (1940) 3) BROTONEGORO. 107-108 14) PETERS.6.C. pp.. 2. MAYEUX.umole C2H.. and SOBACHKIN. Plant Nutr. 94. Soil Sci. 74-85. Ann.8). Ober die Bedeutung des Molybdiins fUr die stickstoffbindende Nostocaceen.S. C. Bot.. 35. 852-855 (1966) 10) LE VAN. N. Takakura High School. Newton and C. Ober die Assimilation des freien Stickstoff durch Azolla. 7). by P. 113-119 (1978) 8) HEWITT.J. Akad. J. 539-550 2) BORTEl. G. S. 6(2). 592-610 . Azolla pinnata. I... Rev. Cambridge Univ. 24. and ABDULKADIR. 56-66 7) HosoDA.V. ed. Studies on the Azolla-Anabaena symbiosis. 1966. Mikrobiol.. Washington State Univ. Hamashima. 1965. E. pp.. and YOSHIDA.W. In Symbiotic Nitrogen Fixation in Plants (Int. A cobalt requirement for symbiotic growth of Azolla filiculoides in the absence of combined nitrogen.. Bot.. Nagoya. Very large variations in nitrogen-fixing capacity have sometimes been noted be- tween the results of the pioneer workers as referred to by MOORE (11) and results which have been published recently. H. for his kind provision of information on recent studies. Pullman. and is not so differ- ent from the value of 6. Z. Vol.50 mg N .. Press. I. Arch. im.s. produced. G.). Growth and nitrogen-fixing activity of Azolla pinnata. A.H. and YATAZAWA. H. Smithsonian Inst..)·day-l determined by the Kjeldahl method in the present study. Thanks are due to Mr.g-1(D.. for his gift of Azolla imbricata plants.W. Oxford. Bogorienses. P. Effect of carbon dioxide.. 238-242 9) JOHNSON. The decomposition of Azolla pinnata in moist and flooded soil. J.17-34 (1969) 12) OES. Black- well Sci. Cambridge.1 would corre- spond to 4. and to Dr.)·day-l as mentioned above. ed. H. 11. 93-97 (1963) (in Russian) 11) A. 361-363 (1947) (in Japanese) 6) GOLTERMAN. BioI. M. pp.. K. 1976. I.. F. A. Methods for Chemical Analysis of Fresh Waters (IBP Handbook No.). Prog.E. A. 1976.W. Washington D. Flora of Japan. Publ. by W. 169-175 (1978) 5) FUJIWARA. 69-77 (1976) 4) BROTONEGORO. especially the use of a much lower light intensity during incubation.. S. and the difference is presumably attributable to the different experimental conditions. TSUBOI. and ABDULKADlR.A. IRRI.. Nitrogen fixation in some natural ecosystems in Indonesia. Watanabe.W. In Proc. and EVANS.. Ann. Common- wealth Agr.A. Press. S. oxygen and light on nitrogen- fixing activities in Japanese clover (Kummerowia striata S. Sand and Water Culture Method Used in the Study of Plant Nutrition. Plant Physiol.. Timiryazeva Dakl. S. This research was supported in part by a grant from the Ministry of Education.. S.. Such variations may well be cancelled when the data of the pioneer workers are recalculated on the basis of the daily increase ratio as men- tioned above. 1971. England... Fixation of free nitrogen in non-leguminous plants. 6(4). Acknowledgements.. pp.J.J. 5.A. Symp.K. REFERENCES 1) BECKING.. Bucks. Nitrogen Fixation in Azolla-Anabaena Symbiosis 425 This value is somewhat smaller than those obtained by BECKING (1) and BROTONEGORO and ABDULKADIR (3). Azolla: Biology and agronomic significance. 1st Intern.L. Bogorienses. 145-163 (1913) 13) OHWI.. A. Newman. pp. LEE.

HOSODA. Bot.. F. K. G... B. 51.P.R.. and K. Utilization of the Azalia- Anabaena complex as a nitrogen fertilizer for rice. ESPlNAS. Soil Manure.. J.. 275-278 (1957) (in Japanese) 17) WATANABE. C. IKEDA.G. No. YATAZAWA. I.S. Gardens Buitenzorg. N. and TUKAMOTO. Azalia as a green manure in paddy field. Paper Ser. K. Jpn. BERJA.. N. Provisional communication on the fixation of elementary nitrogen by a floating fern. N. IRRI Res. Ann. Roy. NUNOME 15) SAUBERT.. 177-197 (1949) 16) TUZIMURA.426 M. Sci.. 28. 11. 1-15 (1977) . TOMOMATSU.Y. and ALIMAGNO.