Plant and Soil 48, 613-619 (1977) Ms. 3246/58

N I T R O G E N F I X A T I O N T H R O U G H O U T G R O W T H ,

A N D V A R I E T A L D I F F E R E N C E S IN N I T R O G E N

F I X A T I O N BY T H E R H I Z O S P H E R E OF R I C E

P L A N T E D IN POTS

by KUK-KI LEE, TERESITA CASTRO and TOMIO YOSHIDA*

The International Rice Research Institute, Los Bafios, Laguna, Philippines

SUMMARY

The nitrogen-fixing activity in the rhizospheres of various rices was mea- sured by the acetylene-reduction method throughout plant growth in green- house pots. The activity began to increase 4 weeks after transplanting, in- creased unti l heading stage, then decreased. The concentrations of exchange- able ammonium and sugars in the soil were not related to the variation of nitrogen-fixing activity during rice growth.

The nitrogen-fixing activities in the rhizospheres of 41 rice varieties in pots were measured to discover varietal differences. Levels of nitrogen fixation were highly correlated with the rices' dry root weight at heading stage.

INTRODUCTION

The maintenance of nitrogen fertility in tropical paddy fields has been thought to be due mainly to autotrophic nitrogen fixers, particularly to the well-known blue-green algae. However, Y o s hi d a and Ancaj as 6 recently found that the rice rhizosphere could also fix a considerable amount of atmospheric nitrogen under flooding, and attributed the nitrogen-fixing activity to heterotrophic bac- teria around the roots.

It is important to investigate the variation in nitrogen-fixing activity throughout growth in order to clarify the relationship be- tween rice and bacteria. Although reports 8 6 on nitrogen fixation at different growth stages are available, no attempt has been made to

* Present address: National Institute for Environmental Studies, P.O Yatabe, Ibaraki 300-21, Japan

614 KUK-KI LEE, TERESITA CASTRO AND TOMIO YOSHIDA

measure nitrogen-fixing activity using the entire plant and soil system. In this study, nitrogenase activity in the rhizosphere of one rice variety (IR26) was measured in the plant-soil system at dif- ferent growth stages. The relations between nitrogenase activity and exchangeable ammonium in the soil, and between nitrogenase acti- vi ty and sugars in the soil were also examined.

Y o s h i d a and A n c a j a s 5 showed that nitrogen-fixing activity in the root zone of one variety, IRS, differed from that in another variety, Peta. The finding led to an extensive investigation of dif- ferences in nitrogen fixation in the rhizospheres of various rice va- rieties. The present paper deals with an experiment in which the nitrogen-fixing activities in the rhizospheres of 41 rice varieties planted in pots were measured by the acetylene reduction method.

MATERIALS AND METHODS

Nitrogen-fixing activity in the rhizosphere of the rice variety IR26 was measured at different growth stages. The seed was germinated on net floating on tap water. One week after germination, three seedlings were transplanted into a pot containing 500 g of Maahas soil. The pot surface was covered with black cloth to prevent algal growth, and the pot was watered two or three times each day to maintain flooding until harvest.

To examine varietal differences, 4 ! rice varieties were given the t reatment described above. Each variety occupied 10 pots in the greenhouse. Five pots were collected from each variety 4 weeks after transplanting, the other five at heading stage.

Since the detachment of the aerial part immediately before acetylene re- duction assay did not affect acetylene-reducing activity 2, the top of the plant was detached. The pot containing the root and soil was placed in a plastic bag, and 3 liters of a gas mixture (20% acetylene, 80% air) was introduced into the bag 2. After 24 hours of incubation, 1 ml of the gas was analyzed for ethylene by gas chromatography (Varian Aerograph Model 2700; column, aluminum 1.85 m long and 0.32 cm ill diameter packed with Porapak 1R; column temperature, 50°C; detector temperature, 70°C; injector tempera- ture, 35°C).

Soil cores were taken from each pot and extracted with 2N KCL solution. The extrac~ was analyzed for ammonium and for anthrone sugars 4.

RESULTS

Nitrogen-fixing activity and concentrations of exchangeable ammonium and of anthrone-reactive sugars at different stages of growth are shown in Fig. 1.

N 2 FIXATION BY RICE AND VARIETAL DIFFERENCES

C2H4 formed (z/moll24 hr )

615

1.5 Heading

Panicle initiation / [ ~

1.0

0.5

/Unplanted 0 $ ~ I ~ I I I I G ~ i i b i ~ ,

Exchangeable NH~, {ppm) 5O

pal

I0

0 I I I T ~ ~_ ' t ' '

Anthrone reactive sugars(p pm, equiv, glucose )

jUnplanted _ E,._J,-"

- I I I I - I I . I ~t

20 i ~j~l~ ~'- ,~ ~ ~ n t e d E ~ I~

O I I I I I I J L I t 1 I I I I I I I I

0 5 I0 15 20

Weeks after transplanting

Fig. l. Acetylene-reducing activity in the IR26 rice rhizosphere, and ex- changeable NI-I4 + and anthrone-reactive sugars in the soil. Vertical lines show

standard error of means.

Ni t rogen f ixa t ion in the rhizosphere began to increase 4 weeks

af ter t r ansp lan t ing and a t t a ined m a x i m u m at heading stage, then decreased rapidly . A pot of u n p l a n t e d soil showed no act iv i ty . Y o s h i d a and A n c a j as 6 earlier r epor ted t h a t f rom abou t 5 to 7 weeks af ter t r ansp lan t ing , the ni t rogen-f ixing ac t iv i ty of the rhizo-

6 1 6 K U K - K I L E E , T E R E S I T A CASTRO AND TOMIO Y O S H I D A

TABLE 1

Nitrogen-fixing ac t iv i ty in the rhizospheres of 41 rice varieties

Variety Days to heading (no.)

C2H4 formed***(~mol.pot-1.24 h - i t )

4 wk after Heading t ransplant ing stage

CTG 1612 42 0.00 0.89 i 0.40

DV 20 49 0.00 0.47 4- 0.04 HBJ Aman 11 54 0.33 4- 0.07 0.70 :~ 0.05 DZ 60 54 0.00 0.79 :t: 0.07 CO 30 57 0.18 :t: 0.18 0.72 4- 0.06 Magsanaya 59 0.76 4- 0.14 0.69 4- 0.08 Kaluheenat i 60 0.I3 4- 0.06 0.73 ± 0.12

H B J Aman V 61 0.44 ± 0.14 0.71 ± 0.11 Latisol (Dacca 17) 64 0.18 ~ 0.18 0.43 4- 0.05 Ifugao Rice* 64 0.06 4- 0.05 0.20 ± 0.08 Imminon 64 0.02 -4- 0.02 1.76 -4- 0.17 Pankhar i 64 0.00 0.84 ± 0.09 Pokkal i 66 0.80 4- 0.10 1.94 4- 0.23 MLYCXVI 1 283 67 0.36 ± 0.18 1.56 4- 0.22 T-141 67 0.02 4- 0.02 0.72 4- 0.10 Shinriki 68 0.00 0.83 4- 0.05

Basmat i 370 68 0.01 4- 0.01 0.70 4- 0.04 JBS 236 70 0.19 4- 0.16 2.63 ± 0.40 Minagara 1 74 0.63 4- 0.20 1.36 -4- 0.10 GEB 24 74 0.00 1.70 4- 0.10

Pe Bi Hun 74 0.00 0.57 4- 0.10 Ifugao Rice* 75 0.44 4- 0.25 1.17 4- 0.13 BPI-76 77 0.01 4- 0.01 1.26 4- 0.07 t t 105 78 0.00 0.92 4- 0.07 Puan Nahk 16 79 0.40 4- 0.16 0.75 4- 0.10 Nang Tay C 79 0.00 1.10 4- 0.12 Honduras 81 1.01 4- 0.21 2.07 4- 0.28 Tjere Mas 83 0.00 1.68 4- 0.23 Perurutong NB-B 89 0.00 2.11 4- 0.19 IR8 90 0.09 4- 0.03 1.87 4- 0.09 IR26 90 0.00 1.60 q- 0.11 Peta 91 0.52 4- 0.05 0.82 4- 0.07 Bengawan 92 0.09 4- 0.07 1.02 ± 0.16 IR5 92 0.17 4- 0.08 2.08 -b 0.39 Baok 3 94 0.22 4- 0.18 1.04 4- 0.15 Paray tarabid 95 0.05 4- 0.03 1.93 4- 0.40 Radin Ebos 33 99 0.00 1.42 ± 0.14 DIMA 99 0.00 1.10 4- 0.08 Gendjah Beton 1101 102 0.15 4- 0.07 0.87 ± 0.02 81B-25 102 0.04 4- 0.04 1.78 -t- 0.14 Bali Kambang 37 127 0.26 4- 0.25 2.36 4- 0.36 Unplanted soil 127"* 0.00 0.00

* These are different varieties ** No. of days to assay *** 4- Standard error of mean

N2 FIXATION BY RICE AND VARIETAL DIFFERENCES

C 2 H 4 formed (z~ mol * pot-I * 24 hr-J )

3.0

617

2.5

2.0

1.5

1.0

0 .5

0 0

Fig. 2.

r = 0 . 6 0 5 ~

0 0

I

0

I I I I

0.5 1.0 1.5 2 .0 2,5

Root wt. ( gr. dry wt. )

Acetylene-reducing ac t i v i t y and root weight of 41 rice varieties. Each va lue is t he mean of 5 replications.

sphere increased. Similarly, the present tests showed higher activity at later stages until heading.

The concentration of exchangeable ammonium in planted soil decreased remarkably with the start of nitrogen fixation, and was negligible (or very small) 6 weeks after transplanting.

The concentration of anthrone-reactive sugars showed no sharp

6 1 8 KUK-KI LEE, TERESITA CASTRO AND TOMIO YOSHIDA

fluctuation in either planted or unplanted soils. After panicle initi- ation, the concentration of sugars in planted soil was higher than in unplanted soil, but the difference was slight.

Nitrogen-fixing activity in the root-soil systems of 41 varieties is shown in Table 1. Unplanted soil showed no activity, as mentioned previously. The maximum activity was obtained at 2.63 ~mol/pot in 24 hours in variety JBS 236. That value is very low compared with values (2,000 or 6,000 ~mol/g/h) obtained by D o m m e r g u e s et al. 1 with IR8, a rice variety that we also used.

There was a high correlation (r =- 0.605***) between root weight and nitrogen-fixing activity in the various varieties at heading stage (rig. 2).

DISCUSSION

Y o s h i d a and B r o a d b e n t 7 reported that the rice plant can transport molecular nitrogen from its aerial parts to the roots, and that the movement of molecular nitrogen at flowering stage is great- er than at tillering stage. Y o s h i d a et al. s also showed that the quanti ty of nitrogen gas in the paddy soil increases as the rice plant ages. These findings seem to imply that the increase of nitrogen- fixing activity in the rice rhizosphere is associated with the supply of substrate for nitrogenase in the rhizosphere.

It is a well-known fact that sugar favors, and ammonium in- hibits asymbiotic nitrogen fixation. In the present study, however, the concentrations of ammonium and sugars were not related to the variation in levels of nitrogen fixation during growth. It is thought that endogenous ammonium in the Maahas soil was taken up by the rice plant before the ammonium affected the activity of bacteria inhabiting the rhizosphere, and that endogenous anthrone-reactive sugars were not consumed by bacteria in the rhizosphere.

As mentioned in the section on Results, the nitrogen-fixing activi- ty we observed was not so high as the activity reported by Dom- m e r g u e s et al. 1. However, in our experiment each pot was covered with black cloth to prevent algal growth. That may have inhibited proliferation of the photosynthetic bacteria which are responsible for the accumulation of organic compounds and for the nitrogen fixation at the soil surface. Since R i n a u d o et al. 3 reported that nitrogen fixation in the rice rhizosphere differed in different soils,

Ne F IXAT ION BY RICE AND V A R I E T A L D I F F E R E N C E S 6 19

it should be considered that the Maahas soil in our experiment might have contained microflora different from those in the soil which D o m m e r g u e s et al. 1 used.

The high correlation between the root weights of the various rice varieties and the nitrogen-fixing activity in their rhizospheres can- not be fully explained. But the amount of organic material supplied by roots to the rhizosphere seems to be one of the factors responsible. Received 10 August 1976

R E F E R E N C E S

1 D o m m e r g u e s , Y., B a l a n d r e a u , J., R i n a u d o , G. and W e i n h a r d , P., Non-sym- biotic ni trogen fixation in the rhizosphere of rice, maize and different tropical grasses. Soil Biol. Bioehem. 5, 83-89 (1973).

2 Lee, K. K. and Y o s h i d a , T., An assay technique of measurement of ni trogenase ac- t iv i ty in root zone of rice for varietal screening by the acetylene reduction method. Plant and Soil 46, 127-134 (1977).

3 R i i i a u d o , G., B a l a n d r e a u , J. and D o m m e r g u e s , Y., Algal and bacterial non- symbiotic nitrogen fixation in paddy soils. P lant and Soil (Spec. Vol.), 471-479 (1971).

4 ¥ o s h i d a , S., F o r n o , D. A., Cock, J. H. and G o m e z , K. A., Laboratory Manual for Physiological Studies of Rice. 2rid ed. The Internat ional Rice Research Inst i tute , Los Bafios, Philippines, 70 pp. (1972).

5 Y o s h i d a , T. and A n e a j as, R. R., Nitrogen fixation by bacteria in the root zone of rice. Soil Sci. Soc. Am. Proc. 36, 156-157 (1971).

6 Y o s h i d a, T. and A n t aj as, R. R., Nitrogen-fixing activi ty in upland and flooded rice fields. Soil Sei. Soc. Am. Proc. 37, 42-46 (1973).

7 ¥ o s h i d a , T. and B r o a d b e n t , F. E., Movement of atmospheric ni trogen in rice plants. Soil Sci. 120, 288-291 (197S).

8 Y o s h i d a , T., T a k a i , Y. and De l R o s a r i o , D. C., i~{olecular ni trogen content in a submerged rice field. Plant and Soil 42, 653-660 (1975).