Nitrogen Fixation Associated with the Rice Plant Grown in Water Culture

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1979, p. 373-378 0099-2240/79/03-0373/06$02.00/0 Vol. 37, No. 3 Nitrogen Fixation Associated with the Rice Plant Grown in Water Culture IWAO WATANABE* AND DELFIN R. CABRERA The International Rice Research Institute, Los Banos, Laguna, Philippines Received for publication 5 Jaunary 1979 Acetylene reduction activity of intact rice plants was measured in closed assay chambers with plants grown in water culture. Acetylene was added to the liquid medium, and the ethylene formed was measured from both gas and liquid phases. After cutoff of mineral nitrogen supply and inoculation of fresh soil, rice plants grown from the seedling stage in water culture exhibited acetylene reduction activity after a lag period. However, rice plants grown in a paddy field and transferred to water culture were more suitable for N2 fixation studies because of their higher, less variable acetylene reduction activity. The time course of acetylene reduction was monitored by continuous circulation of gas between the gas phase and the liquid phase, and the result showed an initial 2- or 3-h period of lower activity, followed by increased and almost constant activity up to 24 h. The effects on acetylene reduction activity of aeration, ammonium, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea addition are reported. Ammonium was inhibitive at 0.33 mm, and its depressive effect was alleviated by ammonium uptake by the plants. Dommergues and associates (5), and Yoshida and Ancajas (14) studied nitrogen fixation in the rhizosphere of wetland rice by acetylene reduction assay. To assay acetylene reduction activity (ARA) of the rhizosphere of rice and other graminaceous plants, the intact-core method (4), a small-tube method (2, 9), an in situ chamber method (1, 3, 6, 7, 12), and the excised-root method (11, 14) have been used. Each of them has advantages and disadvantages. In particular, the excised-root method with anaerobic preincubation as used by Von Bulow and Dobereiner (11) leads to an overestimate of ARA (3). It is reasonable to assume that the in situ chamber method or the intact-core assay may give a more realistic estimate of nitrogen fixation associated with graminaceous plants. In wetland rice, however, there are problems in the use of the in situ assay method because of slow gas transfer in the water-saturated soil and the low recovery of the gas solubilized in water. Lee and Watanabe (7) discussed the problems of the acetylene reduction assay in water-saturated paddy soils. To avoid an overestimate and anomalies resulting from excision of the roots and the difficulties in the transfer and recovery of gases, and to examine the effect of environments in the rooting media to N2 fixation, we tried a water culture technique. MATERIALS AND METHODS Growth of rice in water culture and its ARA rates. In a greenhouse 10 seedlings of rice variety 373 IR26 were grown to the 10-leaf stage in 15-liter pots containing rice culture solution as described by Yoshida et al. (13). Then, the plants were transferred to 3-liter pots with nitrogen-free mineral nutrient solution. At the same time, about 10 g of fresh soil taken from the rice root zone in a paddy field were inoculated into each pot. The plants (one per pot) were supported by 2-cm-thick styrofoam plates, and the lower 5 cm of the plant and the surface of the styrofoam plate were covered by aluminum foil to avoid algal activity (Fig. 1A). The plants were grown further without aerating the rooting media. The ARA rates were measured at 0, 7, 14, and 21 days after the transfer of plants to N- free media. Transfer of field-grown rice plants to water culture. Variety IR26 (unless otherwise stated) plants were grown in an experimental field at the International Rice Research Institute where no nitrogen fertilizer had been applied for 3 years. The surface soil was removed from around each hill to avoid algal contamination, and plant hills were removed together with soil above the plow pan. Each hill was gently washed in tap water with aerating N2. After removal of most of the soil, each hill was transferred to a 3- liter pot containing nitrogen-free mineral culture solution and grown in the greenhouse without aerating the rooting media. ARA rates were measured at various days after the transfer to liquid media. Measurement of acetylene reduction rates in rice grown in liquid media. The assay chamber is diagrammed in Fig. 1A. A known fraction of the liquid medium in the pot was removed, aerated with acetylene gas for 1 min to saturate it with acetylene, and returned to the pot. Then, plastic bag covers were tightly attached to the pot, and air was evacuated. Unless otherwise stated, a known volume of a mixture

374 WATANABE AND CABRERA A plastic sponge styro foom plate Wagner pot ; moagnetic stirring rod ME aos anlet and outlet rac PlastcC plant assay bag aluminun foil rubber bond -Curlte solution h_<iquod sanpinag tube FIG. 1. Diagram of acetylene reduction assay chamber used for rice grown in water culture. (A) Plastic bag chamber; (B) acryl resin chamber with gas circulating and cooling devices. of acetylene and ambient air was introduced into the assay chamber. The quality, size, and fabrication of the bags (polyethylene bags) were described previously by Lee et al. (6). Before gas and liquid samples were taken from the chamber, the liquid medium was vigorously stirred with a magnetic stirrer to make the ethylene concentration in the liquid uniform and to release a part of the solubilized ethylene to the gas phase. After 15 min of stiring, gas and liquid samples were taken for gas chromatograph analyses. Gas samples from within the plastic bags were collected in preevacuated 10-ml glass tubes, while 20 ml of liquid was put gently into a 55 ml-glass vial, which was immediately plugged with a serum stopper. The vial was vibrated vigorously for 3 min to equilibrate the ethylene into both phases. The reported value of the solubility of the ethylene in water at the known temperature (Bunsen coefficient 0.098 ml/ml of = water at 30 C) (8) was used to correct the ethylene content remaining in the liquid. From the gas phase in the vial, a known volume of APPL. ENVIRON. MICROBIOL. the gas was taken for determination of ethylene content by gas chromatography (6). To determine the effect of phases of acetylene addition on ARA, the rice plants from the field were grown in liquid medium for 1 day, and acetylene was added either in the gas phase, in the liquid phase, or in both phases. Twenty percent of the gas above the water was exchanged with acetylene. Also, 20% of the liquid was saturated with acetylene. After 5 and 24 h, the liquid and the gas samples were taken without disturbance to determine the distribution of ethylene in both phases. To determine the optimum concentration of acetylene, 2.5, 5, 10, and 20% of the air was replaced with acetylene. The same percentages of liquid were saturated with acetylene. The plants transferred from the paddy field and grown on liquid media for 1 day were used. A control without acetylene was also used. Time course of acetylene reduction in the circulated chamber. Rigid chambers (Fig. 1B) were used. Circulation was at a flow rate of 15 liters/min. The plastic tubing from the circulating pump was divided, with one tube leading to the gas phase and another leading to the culture solution. Thus, the liquid phase was continuously bubbled during the assay. Plants from the field were grown in culture medium for 1 day before assay. Three experiments were done with slight modifications. The uncirculated assays were run in parallel in experiments 1 and 2. In uncirculated assays, acetylene was added to 10% of both the liquid and the gas phases, and the ethylene content of both phases was analyzed after 5 and 24 h. 02 concentration in the liquid was determined by a Yellow Springs Instruments model 5000 dissolved oxygen meter. In experiment 3, a cooling condenser and water dripper were installed as shown in Fig. 1B. During the circulation of the gas, the gas samples for analysis were taken periodically only from the gas phase. Effect of ammonium addition. Three experiments were conducted. In experiment 1, rice plants at the heading stage were transferred from the field to water culture solutions containing 0, 50, or 10 mm NH4Cl. After growing the plants for 1 and 3 days, ARA assays were conducted over the following 24 h without changing the culture solution. In experiment 2, the rice plants at the maximum tillering stage were transferred from the field to deionized solution containing either NaCl or NH4Cl at 1.67 or 0.33 mm. After 1 and 3 days of growth, ARA rates were measured over the following day without changing the liquid medium. In experiment 3, the rice plants at the panicle initiation stage were transferred from the field to deionized water containing either 0.33 mm NH4Cl or 0.33 mm CaSO4 and grown for 24 h. Then the culture solution of each treatment was changed to either 0.33 mm NH4Cl or 0.33 mm CaSO4, and ARA rates were assayed for the following 24 h. In this experiment, ammonium nitrogen content was determined before and after ARA assay. Contribution of roots. Before ARA assay started after 1 day of growth on liquid medium, the roots of some plants were cut either at the base of the root or 5 cm below the base of the root. When the roots were

VOL. 37, 1979 cut at the base, the surfaces of the stems were scraped to remove rootlets. Effect of chloramphenicol and 3-(3,4-dichlorophenyl)-,1-dimethylurea (DCMU). After the growth of the rice plant at the heading stage in liquid media for 1 day, 50 jg of chloramphenicol per ml was added to the liquid phase, and ARA was assayed for 5, 10, and 24 h. The liquid phase was stirred before the gas and liquid were sampled. Samples of roots were also taken from field-grown plants for laboratory ARA assay. The roots were cut into 1-cm segments, and 1 ml of a solution of 50 jlg of chloramphenicol per ml was added per g of fresh tissue; 3 g of tissue was put into a 50-ml plastic syringe, and the gas phase was exchanged to 10% C2H2-90% Ar. The tissues were incubated at 30 C. In another trial, the plants at heading stage were grown in the liquid medium with ltm 10 DCMU for 1 day, after which ARA was assayed over the following day without changing the liquid medium. RESULTS AND DISCUSSION Changes of ARA rates after transfer to water culture. Changes in 24-h ARA rates of rice plants after transfer to nitrogen-free mineral culture solution are shown in Table 1. After the rice plants grown in a nitrogen-containing water culture were transferred to a nitrogen-free culture solution with soil inoculum, ARA rates increased, indicating the establishment of N2-fixing microflora by soil inoculum, by natural contaminants, or by both. Field-grown rice plants showed ARA just after the transfer to the water culture, and ARA rates did not change significantly up to 20 days of growth. Although the water culture-grown rice showed N2-fixing activities for 2 weeks after the transfer to N-free medium, the activities fluctuated greatly among pots, and their activities were much less than those of field-grown rice. In terms of higher activity and less fluctuation, the field-grown rice plants were more suitable for the studies using water culture. Consequently, we discuss below only the results of experiments with field-grown rice plants. Phases of acetylene addition and the effect of acetylene concentration. When acetylene (20%, vol/vol) was added either in the gas phase, in the liquid phase, or in both phases, the total amount of ethylene formed during 5 and 24 h did not differ significantly (Fig. 2). When acetylene was added only in the liquid phase, the product of acetylene reduction was found in the gas phase after a 5-h incubation. When the acetylene was added only in the gas phase, ethylene was found in the liquid phase after a 5-h incubation. This indicated a rapid exchange of acetylene and ethylene between root and top of the rice plant, as reported previously (7). Despite NITROGEN FIXATION IN WATER CULTURE RICE 375 TABLE 1. Changes ofara rates after transfer to N-free media Days after transfer to 24-h ARA rate N-free me- (panol of C2H4/hill)a dia Water culture 0 0 (4) grown 7 1.1 ± 0.27 (4) 14 14.7 ± 4.7 (4) 21 10.6 ± 5.3 (4) Field grownb 0 27.0 ± 7.5 (4) 1 39.7 ± 3.8 (4) 3 50.0 ± 2.4 (4) 6 34.1 ± 3.8 (4) 12 65.1 ± 8.8 (4) 20 38.9 ± 2.3 (4) a Mean ± standard deviation of the mean; numbers in parentheses are numbers of replicates. b Differences in ARA were not statistically significant. a IN E 0 4 a C-) 3 g - i 2 gas phase Liquid gos + phase liquid phase FIG. 2. Effect of phases where acetylene is introduced. Vertical bars indicate standard deviation of mean of total ethylene. little difference, acetylene was added to both phases as a standard procedure. In a trial to determine the effect of acetylene concentration, 24-h ARA rates were 11.3 ± 1.2 (mean ± standard deviation of the mean of four pots), 13.3 ± 4.3, 23.6 ± 5.9, and 33.0 ± 5.2,umol of C2H2 per pot at 2.5, 5, 10, and 20% acetylene concentrations, respectively. The endogenous ethylene evolution was almost negligible-less than 0.5 Amol/pot per day. Therefore, 20% acetylene concentration was used as a standard procedure, unless otherwise stated. Time course of ethylene formation during circulated gas assays. When an equilibrium is established between gas and liquid

376 WATANABE AND CABRERA phases by circulation, only 3% of the ethylene is calculated to remain in the liquid phase. The ethylene formnation was therefore monitored only from the gas phase. The results of three experiments are shown in Fig. 3. The formation of ethylene showed a slight lag for 2 or 3 h. After that, an almost linear increase of ethylene accumulation was observed except for replicate 2 in experiment 2. The reason why the curve showed nonlinearity only in this replicate is not known. In experiment 1, unaerated assays gave 10.3 2.0 (mean ± standard deviation of the mean of four pots) and 46 ± 6.7,Lmol of C2H4 at 5- and 24-h determinations. The values were almost equal to those of circulated assays. In experiment 2, unaerated assays gave 5.5 ± 1.3 (three means) and 40 ± 7.4 pumol at 5- and 24-h determinations. The values were lower than the circulated assay values. In uncirculated assays, 02 concentration in the liquid medium was already about 1 to 0.5 pl/liter at the start of the ARA assay, and similar concentrations were maintained at the end. Low oxygen concentration in the uncirculated assays might be explained by the presence of the styrofoam plate on the surface of the pot, which blocked the gas transfer from the water surface. This fact may suggest that the nitrogen-fixing system associated with rice is stable to aeration. The suggestion, however, must be confirmed in experiments with varying oxygen concentrations in the circulated air apparatus. Effect of ammonium. The presence of ammonium depressed the acetylene reduction associated with the rice plants (Table 2). In both experiments shown in Table 2, the liquid media were not changed before the 24-h assay. The less depressing effect after 3 days of growth on liquid media with an initial 0.33 mm concentration of NH4Cl was probably due to the depletion of ammonium by plant uptake. In experiment 3 (Table 3), nitrogen concentration before and after assays was determined. NH4 content after the assay decreased to a concentration below 0.07 mm. The presence of ammonium during the assay depressed ARA more strongly than pretreatment with ammonium followed by absence during the assay. This means that the depressing effect of ammonium in the rooting environment to N2 fixation is alleviated after NH4+ removal by the plant. Contribution of roots. The elimination of the roots during the assay did not completely eliminate the ARA associated with rice plants (Table 4). The contribution of top parts was found also by field assays (12). The activity ascribed to the roots was calculated after sub- APPL. ENVIRON. MICROBIOL. FIG. 3. Time course ofethylene accumulation during the circulated air assay. (A) Experiment 1, with 10% C2H2 and 90% ambient air in the gas phase. The plants were at maximum tillering stage. (B) Experiment 2, with 10% C2H2 and 90% ambient air in the gasphase. Theplants were atpanicle initiation stage. (C) Experiment 3, with a gas mixture of 10% C2H2 83% He, and 7% 02 introduced in the gas phase. The plants were at the panicle initiation stage. The cooling condenser and water dripper were set. TABLE 2. Effect of 10 and 50 mm NH4CI on 24-h ARA rates associated with lowland rice grown in water culture ARA rate (jmol of C2H4/hill) at Treatment before as- the following time before assay: say 1 Day 3 Days Expt 1 None 43.1 ± 10.9a 38.1 ± 9.1 10 mm NH4Cl 8.7 ± 2.4 6.4 ± 2.7 50 mm NH4C1 2.1 ± 1.0 1.3 ± 0.53 Expt 2 1.6 mm NaCl 26.1 ± 5.2 31.7 ± 2.2 1.6 mm NH4C1 6.0 ± 0.2 8.9 ± 0.7 0.33 mm NaCl 21.0 ± 6.3 32.2 ± 6.1 0.33 mm NH4Cl 11.2 ± 0.8 22.5 ± 2.8 a Mean of five replicates ± standard deviation of the mean. tracting the value associated with rootless plants. There was no difference in ARA between the basal and tip portion of roots. Effect of chloramphenicol and DCMU. The protein synthesis inhibitor chloramphenicol progressively retarded acetylene reduction dur-

VOL. 37, 1979 ing assay in water culture and in excised tissues (Table 5). Chloramphenicol was most inhibitory to the acetylene reduction of the excised tissues incubated anaerobically. In the excised roots, ethylene formation was barely detectable, and no increase of ethylene was found after 5 h of incubation. Barber et al. (3) reported an overestimate of ARA of the excised roots due to the multiplication of nitrogen-fixing bacteria during the assays. The strong inhibition of chloramphenicol on ARA rates of the excised root is compatible with the multiplication of nitrogenfixing bacteria during assay. The mechanism of chloramphenicol inhibition of N2 fixation needs further elucidation. The pretreatment of plants with DCMU was not inhibitory to ARA in water culture, indicating no involvement of algal N2 fixation. At the concentration tested, the rice plant did not show TABLE 3. Effect of NH4Cl pretreatment for 1 day and treatment during assay on 24-h ARA rates associated with lowland rice grown in water culture Pretreatment Treatment during ARA (umol of 0.33 mm CaSO4 0.33 mm CaSO4 39.1 ± 3.3 (3)a 0.33 mm CaSO4 0.33 mm NH4C1 10.9 ± 2.0 (4) 0.33 mm NH4C1 0.33 mm CaSO4 27.2 ± 5.0 (4) 0.33 mm NH4CI 0.33 mm NH4C1 13.6 ± 0.2 (3) Deionized water Deionized water 38.2 ± 3.0 (4) a Mean + the standard deviation of the mean; number of replicates is given in parentheses. TABLE 4. Effect of root removal on 24-h ARA rates ARA ARA (lanol Treatment (jmol of Dry wt of of C2H4 per C2H4/hil) C2H4/hi11) roots (g) g of dry NITROGEN FIXATION IN WATER CULTURE RICE 377 ~~roots)a Intact 22 ± 0.8b 3.2 ± 0.6 5.4 Roots removed 4.8 ± 0.5 0 0 Roots 5 cm long 12.3 ± 1.9 1.4 ± 0.2 5.3 a The ARA associated with root-free plants was subtracted from ARA of intact plants and divided by the root dry weight. b Mean of four replicates ± standard deviation of the mean. TABLE 5. any sign of adverse effect of DCMU during 2 days. Trolldenier used a water culture technique to study nitrogen fixation of rice (10), but he used excised root for nitrogenase assays and did not use field-grown plants. The water culture technique that we propose here may have the following advantages: (i) it eliminates the restriction of slow transfer of acetylene gas and low recovery of ethylene in a water-saturated system; (ii) it excludes nitrogen fixation in the rhizosphere soil; and (iii) it enables manipulation of nutritional conditions, gas composition in the rooting media, inoculum, and inhibitor effects. The results of these kinds of manipulations were shown in this paper. Water culture technique provides a useful tool to study the nitrogen fixation associated with rice and other graminaceous plants. ACKNOWLEDGMENT This research was supported by the United Nations Development Programme. LITERATURE CITED 1. Balandreau, J., and Y. R. Dommergues. 1973. Assaying nitrogenase (C2H2) activity in the field. Bull. Ecol. Res. Commun. Stockholm 17:247-254. 2. Balandreau, J. P., G. Rinaudo, M. M. Oumarov, and Y. R. Dommergues. 1975. Asymbiotic N2 fixation in paddy soils, p. 611-628. In W. E. Newton and C. J. Nyman (ed.), 1st International Symposium on Nitrogen Fixation. Washington State University Press, Pullman, Washington. 3. Barber, L E., J. D. Tjepkema, S. A. Russel, and H. J. Evans. 1976. Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum. Appl. Environ. Microbiol. 32:108-113. 4. Dobereiner, J., J. Day, and P. J. Dart. 1971. Nitrogenase activity of the Paspalum notatum-azotobacter paspali association and oxygen sensitivity. J. Gen. Microbiol. 71:103-116. 5. Dommergues, Y., J. Balandreau, G. Rinaudo, and P. Weinhard. 1973. Non-symbiotic nitrogen fixation in the rhizosphere of rice, maize, and different tropical grasses. Soil Biol. Biochem. 5:83-89. 6. Lee, K. K., B. V. Alimagno, and T. Yoshida. 1977. Field technique using acetylene reduction method to assay nitrogenase activity and its association with the rice rhizosphere. Plant Soil 46:127-134. Effect of chloramphenicol on intact plants and isolated roots ARA of intact plants ARA of excised roots Time of as- (pnol of C2H4/hill) (,umol of C2H4/3 g [fresh wt] of roots) say (h) Control Treated Control Treated 5 3.4 + 0.2a 2.3 + 0.2 0.2 ± 0.07 0.15 ± 0.01 (67) (75) 10 7.0 ± 0.6 3.4 ± 0.4 1.3 ± 0.10 0.16 ± 0.02 (49) (12) 24 19.2 ± 2.8 6.3 ± 0.6 2.0 ± 0.23 0.16 ± 0.02 (33) (8) a Mean ± standard deviation of the mean of four replicates. The figures in parentheses are percentages of the control.

378 WATANABE AND CABRERA 7. Lee, K. K., and I. Watanabe. 1977. Problems of the acetylene reduction technique applied to water-saturated paddy soils. Appl. Environ. Microbiol. 34:654-660. 8. Perry, J. H. (ed.). 1950. Chemical engineers handbook, p. 2001. McGraw-Hill Book Co., New York. 9. Raimbault, M., G. Rinaudo, J. L. Garcia, and M. Boureau. 1977. A device to study metabolic gases in the rice rhizosphere. Soil Biol. Biochem. 9:193-196. 10. Trolldenier, G. 1977. Influence of some environmental factors on nitrogen fixation in the rhizosphere of rice. Plant Soil 47:203-217. 11. Von Billow, J. F. W., and J. Dobereiner. 1975. Potential for nitrogen fixation in maize genotypes in Brazil. APPL. ENVIRON. MICROBIOL. Proc. Natl. Acad. Sci. U.S.A. 72:2389-2393. 12. Watanabe, L, K. K. Lee, and M. de Guzman. 1978. Seasonal change of N2 fixing rate in lowland rice field by in. situ acetylene reduction technique. H. Estimate of nitrogen fixation associated with rice plants. Soil Sci. Plant Nutr. 24:465-471. 13. Yoshida, S., D. A. Forno, and J. H. Cock. 1971. Laboratory manual for physiological studies of rice, p. 61. International Rice Research Institute, Los Bafios, Philippines. 14. Yoshida, T., and R. R. Ancajas. 1971. Nitrogen fixation by bacteria in the root zone of rice. Soil Sci. Soc. Am. Proc. 35:156-157. Downloaded from http://aem.asm.org/ on September 18, 2018 by guest