Growth responses of paddy rice to an ammonia-excreting mutant cyanobacterium at elevated CO2 concentration

Applied Soil E c o l o g y ELSEVIER

Applied Soil Ecology 1 (1994) 199-206

Growth responses of paddy rice to an ammonia-excreting mutant cyanobacterium at elevated CO2 concentration F. Kamuru", S.L. Albrecht", J.T. Baker", L.H. Allen, Jrb'*, K.T. Shanmugamc "Department of Agronomy, USDA,Agronomic Physiology Laboratory, Building 164, Box 110840, Universityof Florida, Gainesville, FL 32611- 0840, USA bAgriculturalResearch Service, USDA,Agronomic Physiology Laboratory, Building 164, Box 110840, University of Florida, Gainesville, FL 32611-0840, USA CDepartment of Microbiology and Cell Science, Universityof Florida, Gainesville, FL 32611, USA

Accepted 9 May 1994

Abstract

An ammonia-excreting mutant strain ofAnabaena variabilis (strain SA-1 ) was studied as a N supplier for the growth of rice (Oryza sativa L., cultivar 'IR-30') plants at near ambient (330/zmol mol -~ ) and elevated (660 /tmol mol- l ) carbon dioxide (CO2) concentrations. Rice performance with N supplied by the mutant cyanobacterium was compared with that of three other N treatments: rice plants inoculated with the parent strain (A. variabilis, strain SA-0), plants fertilized with 75 kg N ha -z as urea, or control plants (uninoculated, with no nitrogen fertilization). Plants at elevated CO2 concentration had a higher dry matter content and accumulated more nitrogen than at near ambient CO2. At either CO2 concentration, the mutant strain enhanced the shoot and grain dry matter accumulation more than did the parent strain. Total N concentration of the shoot and grain fractions was significantly greater (P< 0.05 ) in plants inoculated with the mutant than with the parent strain of the cyanobacterium. The benefit of inoculation with the ammonia-excreting mutant strain was equivalent to or greater than the response observed in plants receiving 75 kg N ha-~. A combined response of both the rice plants and the cyanobacterium to high CO2 levels probably accounted for the higher dry matter and N at the elevated than at the ambient CO2 concentration. The potential of the ammonia-excreting mutant cyanobacterium as a N source for sustainable, low-input rice production in the predicted high CO2 atmosphere of the next century is promising.

1. Introduction Carbon dioxide is a substrate for photosynthesis in both green plants and cyanobacteria, Stoichiometrically, the concentrations of carbon dioxide and water (reactants) determine the amount of carbohydrates (product) formed at *Corresponding author. Tel: (904)392-6180, Fax: (904) 374 5852.

the end of the photosynthetic reaction. The photosynthetic reduction of carbon dioxide in plants is influenced by the concentration of CO2 available at the site of carboxylation. Carbon dioxide concentration (CO2) in the atmosphere has been increasing steadily since the industrial revolution from about 265-285 #mol tool -1 (Gammon and Fraser, 1985) to the present level of more than 355 /~mol tool -1 (Keeling et al., 1989). As global population increases and in-

0929-1393/94/$07.00 © 1994 ElsevierScienceB.V. All rights reserved SSD10929-1393 (94) 00025-5

200

F. Kamuru et aL / Applied Soil Ecology I (1994) 199-206

dustrialization expands, the CO2 of the atmosphere is expected to double in the next century (Trabalka et al., 1986 ). High CO2, together with other climatic factors, will affect plant growth and development and are the focus of substantial research worldwide. The effects of elevated COz on plant growth and productivity have been extensively reviewed by Acock and Allen (1985), Allen (1990), and Allen and Boote ( 1992 ). Increasing CO2 generally causes an increase in photosynthetic CO2 fixation rates of leaves and plant canopies, especially in plants with the C3 carbon assimilationpathway (Kimball, 1983;Carter and Peterson, 1983). The present atmospheric CO2 is limiting to the photosynthesis of C3 plants (Pearcy and Bjorkman, 1983) which explains the greater photosynthetic response from this group of plants at elevated CO2. The cyanobacteria-rice interaction commonly occurs in paddy rice production systems (Roger and Watanabe, 1982). This interaction has some agronomic benefits that derive from the cyanobacteria contributing to the N economy of this agroecosystem (Watanabe et al., 1987). Estimates of the amount of N contributed by cyanobacteria in paddy fields vary from 20 to 70 kg N h a - ~ year- 1 (Sankaram, 1967). The cyanobacteria have been credited with maintaining roodcrate rice yields in paddy fields for long periods of time. (Roger and Watanabe, 1982; Sankaram, 1967; Venkataraman, 1972, 1979 ). The growth response of rice plants and cyanobacteria at elevated CO2 has been separately investigated. However, there is little information on the response of both the cyanobacteria and rice plants grown together at elevated CO2. Studying the response of the cyanobacteria-rice interaction to elevated CO2 may help us predict how paddy rice production in low input systems may be affected by the projected high CO2 in the next century, The objectives of this experiment were to evaluate the effects of four different N treatments on dry matter and total N accumulation in rice plants grown under two levels of CO2. More specifically, the objectives of the N treatments were to compare the effects of a paddy culture inocu-

lated with either the cyanobacterium Anabaena variabilis(strain SA-0) or an ammonia-excreting mutant strain of the organism (strain SA-1 ).

2. Materials and methods

2.1. Rice plants andgrowth chamber conditions Rice (Oryza sativa L., cultivar 'IR 30') seeds were pre-germinated at 27°C and grown for l0 days in a growth chamber with a 12/12 hour light/dark cycle. The seedlings were transplanted on 8 June 1990, grown to maturity and harvested on 18 September 1990 (109 days after planting). The seedlings were planted in large plexiglass tubes (four seedlings per tube) measuring 60 cm in diameter and 24 cm deep. Eight tubes were located in the rooting zone of each of six outdoor, naturally-sunlit, computer-controlled plant growth chamber at the Irrigation Research and Education Park at the University of Florida, Gainesville, FL. The lower compartment was an aluminum vat, measuring 1.9 m × 0 . 8 m in cross section and 0.5 m deep, filled with Monteocha loamy sand (sandy, siliceous, hyperthermic ultic haplaquods soil). Water was added to provide a flooded root environment. The upper chamber was made with a clear cellulose acetate roof with mylar film walls, measuring 2 m X 1 m in cross section and 1.5 m in height. A detailed description of this controlled environment facility was presented by Jones et al. (1984). The main rice crop in each chamber was planted in 11 rows 18 cm apart. The plant population was thinned on 17 June (at the second leaf stage) to 235 plants m -2. The CO2 concentration in three of the chainbers was elevated and maintained at 660/tmol tool- 1, while the remaining three chambers were maintained near ambient at 330 amol tool- 1. Day and night air temperatures in all the chainbers were 29 °C and 21 ° C, respectively, while the paddy water temperature was maintained at 25 ° C. Dewpoint temperature was maintained at 17 ° C throughout the study.

F. Kamuru et al. / Applied Soil Ecology I (1994) 199-206

2.2. Cyanobacteria Two strains of the cyanobacterium A. variabilis were used in this study. The parent strain (SA0) is the wild-type A. variabilis Kutz (ATCC 29413 ), while the mutant strain (SA- 1 ) is a derivative of the parent strain produced by ethyl methyl sulphonate (EMS) mutagenesis (Spiller et at., 1986 ). Cyanobacterial cultures were grown at 27 °C in 3.0 1 flasks using 1.5 1 half-strength nutrient medium of Allen and Arnon ( 1955 ). The cultures were grown at low light intensity (about 120/tmol photon m-2 S - 1 ) to prevent photo-oxidation of the photosynthetic pigments. The growth medium was supplemented with 6 ml of 1.67 M fructose and 3 ml of 1.51 M ammonium sulphate 1- 1 to promote faster growth of the cultures and a higher cell yield. The cultures were gently agitated using magnetic stirrers to prevent sedimentation of the filaments, The cyanobacteria were harvested after 5 days of growth by allowing the filaments to settle down from the growth medium. The upper portion of the medium, which was free of filaments, was decanted and the cells were collected by centrifugation at 5000 x g for 5 min at 25 ° C in a Sorvall RC-SB centrifuge. The cells were washed three times in sterile deionized water before inoculating the floodwater in the pots. The tubes were inoculated twice at 19 and 68 days after transplanting. A total of 600/tg chlorophyll a per tube of cyanobacterial biomass was applied in the two inoculations. Equal amounts of cells of either the parent or mutant cyanobacterium were added to the tubes based on their chlorophyll a content. The concentration of chlorophyll a in the cyanobacterial cultures was determined from the harvested cells which had been washed and suspended in water following the method of Talling and Driver ( 1963 ).

201

tant strain of the cyanobacterium, plants fertilized with 75 kg N ha -1 applied as urea (1.52 g urea per tube), and uninoculated, unfertilized control plants. The CO2 treatments (330 and 660 #mol mo1-1 ) were replicated three times. A completely randomized design was used and the two COe were randomly assigned to six growth chambers (three growth chambers at 330/~mol mol- 1, and the other three chambers at 660/tmol mol-l C O 2 ). Nitrogen treatments were also randomly assigned in each growth chamber, without blocking.

2.4. Analyses of samples Rice plants were grown to maturity (109 days after planting), harvested, and the root, shoot and grain components separated. The panicles containing ripe grains were carefully removed with a knife, while the shoot was harvested by cutting the stems at the soil level. The roots were removed from the pots, spread over a metal gauze and the soil was carefully washed off with water. The plant materials were dried at 60°C for 72 h, weighed and separately ground using a Tecator cyclotec sample mill (Hendon, VA) with a screen mesh size of 1.0 mm. Subsamples of the ground material (0.5 g) were further dried for 15 h at 105 ° C and weighed for computation of plant dry matter. The result was expressed as percent dry matter. The total dry matter of each plant fraction was calculated from the percent dry matter of the subsamples. Total nitrogen was estimated from sub-samples used for dry matter determination. The 0.5 g sub-sample dried at 105°C for 15 h was digested using the aluminum block digestion procedure of Gallaher et al. (1975). Ammonia in the digestate was determined by semi-automated calorimetry (Hambleton, 1977).

2.5. Statistics 2.3. Experimental design Four sources of N and two CO2 levels were investigated in this study. The nitrogen treatments were replicated six times and consisted of rice plants inoculated with either the parent or mu-

The data collected were analyzed as a two-factor experiment (N and CO2 treatments) by analysis of variance using the general linear model of the statistical analysis system (Statistical Analysis Systems Institute, 1988 ). Mean separation of

F. Kamuru et al. / Applied Soil Ecology 1 (1994) 199-206

202

the nitrogen treatments were conducted within each COz using Duncan's multiple range test at the 5% level of significance,

3. Results Root dry matter (aggregated over N treatments) at the elevated CO2 was not significantly different from that obtained at the near ambient CO2. Within either CO2, root dry matter of all plants, except the controls, was not different regardless of N treatment. However, shoot and grain dry matter of all plants grown at the elevated CO2 was significantly greater ( P < 0.05 ) than those of plants grown at near ambient COz. As shoot and grain dry matter were significantly different at the two CO2 levels, analyses of the effects of N treatments were separately conducted for each CO2 level. The root dry matter of plants inoculated with either strain of the cyanobacterium was not significantly different at 330 and 660/tmol m o l - ~ CO2 (Fig. 1 ). There 26 [ ] SASA-10 '©

20

.o

rILlr

15

<

>.

75 kg N ha4

a

a

a -~

~-.!

i

or"

a

4O

L%J SA-0

,,'~=o

5

30

tr

~ b

~

< ~

K~ .,,

a

75 kg N ha"

,'-x

a

"'

~¢

330

OONTROL

[~-] SA-1

~

10

rr O O

a

[ ~ CONTROL []

~=

was also no significant difference in the root dry matter of the inoculated and N fertilized plants at either COz, whereas the control plants had a lower root dry matter than the plants that received a nitrogen treatment or cyanobacterium. Shoot dry matter was significantly higher in the rice plants inoculated with the mutant strain than in those that received the parent strain (Fig. 2). Plants inoculated with the mutant produced about 35% more shoot biomass at 330 /tmol mol- ~ CO2 than those inoculated with the parent strain. At the elevated CO2, the shoot biomass of rice plants inoculated with the ammonia-excreting mutant strain was 37% greater than plants inoculated with the parent strain. The amount of shoot dry matter produced by the fertilized plants was not significantly different ( P > 0.05 ) from that produced by the plants inoculated with the mutant strain. The grain dry matter accumulated in the rice plants inoculated with the mutant cyanobacterium was significantly greater ( P < 0.05 ) at either CO2 than the grain dry matter produced by the plants inoculated with the parent strain (Fig. 3 ). Inoculation with the mutant strain resulted in 41% more grain dry matter at near ambient CO2 than plants inoculated with the parent strain. In-

b

~'~

20

~

>-

660

CO 2 C O N C E N T R A T I O N (~tmol mol 4)

O

o

10

~

"r

co ~ 0

Fig. 1. Average root dry matter (grams per tube) office plants grown to maturity with different sources of nitrogen at 330 and 660/tmol m o l - t CO2. Bars with the same letter within each CO2 are not significantly different at the 5% level based on Duncan's multiple range test. SA-0 and SA-I indicate inoculation with the parent and mutant strains ofA. variabilis, respectively. The control plants were neither inoculated nor fertilized.

.

.

.

.

330

.

660

CO 2 C O N C E N T R A T I O N

(}xrnol rnol~) Fig. 2. Average shoot dry matter (grams per tube) of rice plants grown to maturity with different sources of nitrogen at 330 and 660 #mol m o l - ~ CO2. Legends are the same as in Fig. i.

F. Kamuru et al. / Applied Soil Ecology I (1994) 199-206 40

1

[ ] sA.0 .O~

¢~ tr I-'" ~<

30

SA.1

[] []

CONTROL 75 kg N ha1

20

a

330

0.8 O

b ~

b ~×~ ~~

~

~ ~.i

~

.... ~:~

~

10

0

[ ] sA-0

[]

><--z

203

KXJ

I.tr i

c

a

-1~

SA-1 CONTROL 75 kg N ha"1

0.6

5O

~

[] [] []

ab

a

a

0.4

0.2

0

660

330

CO2 CONCENTRATION (~mol tool 1)

Fig. 3. Average grain dry matter (grams per tube) of rice plants grown to maturity with different sources of nitrogen at 330 and 660/zmol mol-1 CO2, Legends are the same as in Fig. 1.

660

CO~ CONCENTRATION (p,rnol mol 1)

Fig. 4. Root:shoot ratio of rice plants grown with different sources of nitrogen at 330 and 660 pmol mol-~ CO2. Legends are the same as in Fig. 1. 250

oculation of rice plants with the mutant strain produced more grain dry matter than plants that received 75 kg N h a - ~ at near ambient CO2. The harvest index (ratio of grain to total above

groundbiomass) of all plantsthat receiveda hitrogen treatmentwas not significantlydifferent at either CO2. The inoculated and fertilized plants had a harvest index that was greater than 0.5 at the elevated CO2, but less than 0.5 at the near ambient C O 2. The control plants at both CO2 had a harvest index of less than 0.5. The root:shoot (above ground biomass) ratio of the control plants and plant inoculated with the parent strain was greater that the same ratio in the N fertilized plants and those inoculated with the mutant strain (Fig. 4). Roots of plants grown at 660 #mol m o l - 1 C O 2 had 28% more nitrogen than those of plants grown at the ambient CO2. The total nitrogen accumulated in the roots of the inoculated and fertilized rice plants was not significantly different at near ambient CO2 (Fig. 5 ). At the elevated CO2, plants inoculated with the mutant strain had 19% more nitrogen in the roots than plants inoculated with the parent strain, The N concentration of the shoot of all rice plants combined was 38% greater at the elevated

[ ] sA.0 [ ] $A-1 [ ] CONTROL zLM

200

o ___x~ 15o z~ .
5o 0

[]

a

75kg N ha"1

b

aa ~ l i ta ~ K

×,

:~: ~b ~ ~A ~', ~ ::: y/~ 330 660 c02 CONCENTRATION (ktmol rnol1)

Fig. 5. Average root total nitrogen (milligrams N per tube) of rice plants grown to maturity with different sources of nitrogen at 330 and 660 #mol mol-l CO2. Legends are the same as in Fig. 1.

CO2 than at the near ambient CO2 level (Fig. 6 ). The shoot N concentration of plants inoculated with the mutant strain was 68% greater than that of plants inoculated with the parent strain at the near ambient CO2. At the elevated CO2, shoot N concentration of plants inoculated with the mutant strain was 74% greater than that of plants

204

F. Kamuru et al. / Applied Soil Ecology 1 (1994) 199-206

sA-o

400

Z

tu otr

~ Z'~ I'-"

~ sA.~ ~ ] CONTROL ~ 7skgNh~"

300 200

I

E

o o

7" r.r)

a

I,t

"7.

-J-=z

a f:/i ~

that accumulated in plants grown at the near ambient C O 2 (Fig. 7 ). At near ambient COz, plants inoculated with the mutant strain had 35% more N in the grain than plants that were inoculated with the parent strain. At the elevated CO2, plants

',,

Ilia

f ~ i !i!i[~ ~b ~ ....

. li!l l ii!i !~~

c~ lOO

]

rl

330

660

inoculated with the mutant strain had 360/0 more N in the grain than those inoculated with the parent strain. At both CO2, the grain N concentration of the fertilized plants and plants that were inoculated with the mutant strain were not significantly different ( P > 0.05 ). 4. Discussion

CO2 CONCENTRATION

(~tmolmol1)

Fig. 6. Averageshoot total nitrogen (milligramsN per tube) of rice plants grownto maturity with differentsourcesof nitrogenat 330 and 660/tmol tool- 1CO2.Legendsare the same

asinFig.I.

;:5:J sA-0

500 z ILl ¢,.9 400 ot V A --- % z ~= 300 7,

O~

200

~ sA.1 ~~ 75kg CONTROL N hat a a ~ --i

a ' ' b

b

c

I~!

~

ic

~

100 o --

, __ 330

660 CO2 CONCENTRATION

(~tmol mo1-1)

Fig. 7. Averagegrain total nitrogen (milligramsN per tube) of rice plants grownto maturity with different sourcesof nitrogenat 330 and 660/zmoltool- 1CO2.Legendsare the same as in Fig. 1. inoculated with the parent strain. Total shoot N of the fertilized plants and plants inoculated with the mutant strain were not significantly different at the high CO2. The amount of N in the grain of rice plants grown at the elevated CO2 was 19% greater than

Inoculating rice plants with the mutant cyanobacterium resulted in increases in both shoot and grain dry matter, and these increases were equivalent to or greater than the response obtained by application of 75 kg N h a - 1. The enhancement in dry matter of rice plants resulting from inoculation can be attributed to the supply of nitrogen by the cyanobacteria. Unpublished data (Shanmugam, personal communication, 1990) have shown that both the parent and mutant strain populations survive well but may decrease by about 50% at 35 days after inoculation. The greater increase in dry matter in plants inoculuted with the mutant strain compared with plants that were inoculated with the parent strain reflects the amount of nitrogen that was made available to the rice plants by the ammonia-excreting mutant cyanobacterium. Spiller et al. (1986) reported on the ability of the mutant cyanobacterium (A. variabilis, SA-I ) to excrete newly fixed nitrogen to its growth environment. Plants grown with the mutant strain accumuluted more nitrogen in both shoot and grain biomass than plants grown with the parent strain. The increase in total N of plants inoculated with the mutant as compared with the parent strain is 232 mg and 308 mg at 330/~mol mol- 1 CO2 and 660/zmol mol- 1 CO2, respectively. In both cases, the amount ofcyanobacteria added was about 35 mg dry wt., which translates into about 1.5 mg N per tube. If the mutant strain merely decomposed completely and released N to the plants, this cannot account for the over 100 fold in-

F. Kamuru et al. /AppliedSoilEcology I (1994) 199-206

crease in plant N. These result show that the NH~ produced by the mutant strain is utilized by the rice plants. Furthermore, the mutant strain is incapable of assimilating NH + produced by nitrogenase (Spiller et al., 1986), any substantial growth to increase its biomass can be discounted. Growth enhancement of rice plants by the mutant strain of A. variabilis (SA- 1 ) under gnotobiotic conditions has been reported (Latorte et al., 1986) to be greater than the growth response obtained by inoculation with the parent strain. Spiller and Gunasekaran (1990) observed that the same mutant cyanobacterium enhanced growth of wheat plants that were grown hydroponically, The hypothesis implicating plant growth hormones as the major mechanism involved in inoculation with some free-living N2-fixing bacteria, as in Azotobacter sp. in association with tropical grasses (Dobereiner et al., 1972 ) and in some other species of cyanobacteria (Goyal and Venkataraman, 197 l; Subrahmanyan, 1972; Venkataraman, 1979 ), may not be valid for the cyanobacteria used in this study. There was no significant gain in the root biomass office plants inoculated with the mutant cyanobacterium when compared with root biomass of the plants inoculated with the parent strain or of those to which 75 kg N h a - ~ was applied. However, the root: shoot ratio, which is a good index for determining the effect of phytohormones on plant growth (Tien et al., 1979; Kapulnik et al., 1981 ), was significantly greater ( P < 0.05 ) in plants inoculated with the parent strain than plants that were inoculated with the mutant strain at both CO2. This difference in the root:shoot ratio was due to the higher shoot biomass accumulated in plants inoculated with the mutant cyanobacterium compared with the shoot biomass obtained in the plants that received the parent strain, Exposure of rice plants to an elevated CO 2 favoted dry matter production and the incorporation of fixed nitrogen into the plant biomass as compared with the near ambient CO2. Dry matter of the root, shoot and grain and the total nitrogen content were higher at 660/lmol molCO2 than at the near ambient level. The harvest index of all rice plants, except the controls, was

205

greater than 0.5 at the elevated CO2, but less than 0.5 at the near ambient level. This observation suggests that the high CO2 concentration favored grain over straw production probably by increasing the partitioning of assimilates to the reproductive parts of the rice plants. Rice plants have a 'C-3 pathway' for the assimilation of carbon and their rate of carbon reduction has been reported to increase with CO2 enrichment (Baker et al., 1990). Increasing levels of atmospheric CO2 had a positive effect on the cyanobacteria-rice interaction, resulting in a higher growth rate and yield when compared with plants grown at near ambient CO2. In addition, the contribution of nitrogen from cyanobactefia for growth of the rice plants was favored by high CO2. The observed enhancement in the growth of inoculated rice plants at the elevated CO2 may be due to a combined response of both the cyanobactefia and the rice plants. The benefit of inoculating rice plants with the mutant strain in an atmosphere of high CO2 is clearly greater than inoculating with the parent strain, or even the application of 75 kg N ha- 1. The potential of the mutant strain as a nitrogen source for sustainable, low-input rice production in the next century, when the CO2 of the atmosphere is predicted to double, is promising. Improving the survival of the inoculated cyanobacteria deserves further investigation in order to maximize the benefit of the cyanobacteria-rice interaction. Acknowledgement This study was supported in part by the U.S. Agency for International Development through a PSTC grant (No. 7.065, DPE-5542-G-SS-801900), the U.S. Department of Agriculture, Agricultural Research Service, and by the U.S. Department of Energy's Interagency Agreement No. DE-AI05-88ER69014 with the USDA-ARS. Florida Agricultural Experiment Station Journal Series No. R-03366. References Acock, B. and Allen, Jr., L.H., 1985. Crop responses to ele-

vated carbon dioxideconcentration.In: B.R. Strain and

206

F. Kamuru et al. / Applied Soil Ecology 1 (1994) 199-206

J.D. Cure (Editors), Direct Effects of Increasing Carbon Dioxide on Vegetation. Report DOE/ER-0238, U.S. Depannment of Energy, Carbon Dioxide Research Division, Washington D.C., pp. 53-97. Allen, Jr., L.H., 1990. Plant responses to rising carbon dioxide and potential interaction with air pollutants. J. Environ. Qual., 19:15-34. Allen, Jr., L.H. and Boote, K.J., 1992. Vegetation, effect of rising CO2. In: W.A. Nierenberg (Editor), Encyclopedia of Earth System Science, Vol. 4. Academic Press, New York, pp. 409-416. Allen, M.B. and Arnon, D.I., 1955. Studies on nitrogen-fixing blue-green algae I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol., 30: 366-372. Baker, J.T., Allen, Jr., L.H., Boote, K.J., Jones, P. and Jones, J.W., 1990. Rice photosynthesis and evapotranspiration in subambient, ambient and superambient carbon dioxide concentrations. Agron. J., 82: 834-840. Carter, D.R. and Peterson, K.M., 1983. Effects of a CO2 enrichment atmosphere on the growth and competitive interaction of C3 and C4 grasses. Oecologia, 53: 188-193. Dobereiner, J., Day, J.M. and Dartn, P.J., 1972. Nitrogenase activity and oxygen sensitivity to Paspalum notaturnAzotobacter paspali association. J. Gen. Microbiol., 71: 103-116. Gallaher, R.N., Weldon, C.O. and Futral, J.G., 1975. An aluminum block digester for plant and soil analysis. Soil Sci. Soc. Am. Proc., 39: 803-806. Gammon, R.H. and Fraser, P.J., 1985. History of carbon dioxide in the atmosphere. In: J.R. Trabalka (Editor), Atmospheric Carbon Dioxide and the Global Carbon Dioxide Cycle. Report DOE/ER-0239, U.S. Department of Energy, Carbon Dioxide Research Division, Washington, D.C., pp. 25-62. Goyal, S.K. and Venkataraman, G.S., 1971. Response of high yielding rice varieties to algalization. Phykos., 10:32-33. Hambleton, L.G., 1977. Semiautomated method for simultaneous determination of phosphorus, calcium and crude protein in animal feeds. J. Assoc. Off. Anal. Chem., 60: 845-852. Jones, P., Jones, J.W., Allen, Jr., L.H. and Mishoe, J.W., 1984. Dynamic computer control of closed environment plant growth chambers. Design and verification. Trans. Am. Soc. Agric. Eng., 27: 879-888. Kapulnik, Y., Kigel, J., Okon, Y., Nur, I. and Henis, Y., 1981. Effects of Azospirillum inoculation on some growth parameters and nitrogen inlet of wheat, sorghum and panicum. Plant Soil, 61: 65-71. Keeling, C.D., Bacastow, R.D., Canner, A.F., Piper, S.C., Whorl, T.P., Heimann, M., Mook, W.G. and Roeloffzen, H., 1989. A three dimensional model of atmospheric CO2 transport based on observed winds: Analysis of data. In: D.H. Peterson (Editor), Aspects of Climate Variability in the Pacific and the Western Americas. Geophys. Monog. 55, Am. Geophys. Union, Washington, D.C., pp. 165-232. Kimball, B.A., 1983. Carbon dioxide and agricultural yield: An assemblage of 430 prior observations. Agron. J., 75: 779-788.

Latorre, C., Lee, J.H., Spiller, H. and Shanmugam, K.T., 1986. Ammonium ion-excreting cyanobacterial mutant as a source of nitrogen for growth of rice. A feasibility study. Biotech. Lett., 8:507-512. Pearcy, R.W. and Bjrrkman, O., 1983. Physiological effect. In: E.R. Lemon (Editor), Carbon Dioxide and Plants. The Response of Plants to Rising Levels of Atmospheric Carbon Dioxide. A.A.A.S. Selected Syrup. 84, Westview Press, Boulder, CO. Roger, P.A. and Watanabe, I. 1982. Research on algae, bluegreen algae, and phototrophic nitrogen fixation at the International Rice Research Institute (1963-1981 ), summarization, problems and prospects. IRRI Research Paper Series, 78:1-21. Sankaram, A., 1967. Blue-green algae: Role in rice culture. Farmer and Parliament, 11: 9-10. Spiller, H. and Gunasekaran, M., 1990. Ammonia excreting mutant strain of the cyanobacterium Anabaena variabilis supports growth of wheat. Appl. Microbiol. Biotechnol., 33: 477-480. Spiller, H., LaTorre, C., Hassan, M.E. Hassan and Shanmugam, K.T., 1986. Isolation and characterization of nitrogenase derepressed mutant strains ofcyanobacterium Anabaena variabilis. J. Bacteriol., 165:412-419. Statistical Analysis Systems Institute Inc., 1988. Introductory Guide to Personal Computers, Release 6.03, SAS Institute, Cary, NC. Subramanyan, R., 1972. Some observations on the utilization of blue-green algal mixtures in rice cultivation in India. In: T.V. Desikachary (Editor), Taxonomy and Biology of Blue-Green Algae. Madras University Press, Madras, India, pp. 281-293. Tailing, T.E. and Driver, D., 1963. Some problems in the estimation of chlorophyll a in phytoplankton. In: Proc. Conf. Primary Productivity Measurements in Marine and Freshwater Habitats. University of Hawaii, pp. 142-146. Tien, T.M., Gaskins, M.H. and Hubbell, D.H., 1979. Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetum americanurn L. ). Appl. Environ. Microbiol., 37:10161024. Trabalka, J.R., Edmonds, J.A., Reilly, J.M., Gardner, R.H. and Reichle, D.E., 1986. Atmospheric COz projections with globally averaged carbon cycle models. In: J.R. Trabalka and D.E. Reichle (Editors), The Changing Carbon Cycle, a Global Analysis. Springer, New York, pp. 534560. Venkataraman, G.S., 1972. Algal Biofertilizersand Rice Cultivation. Today and Tomorrow's Printers and Publishers, New Delhi, India. Venkataraman, G.S., 1979. Algal inoculation in rice fields. In: IRRI, Nitrogen and Rice. International Rice Research Institute, Los Banos, The Philippines, pp. 311-324. Watanabe, I., DeDatta, S.K. and Roger, P.A., 1987. Nitrogen cycling in wetland rice soils. In: J.R. Wilson (Editor), Advances in Nitrogen Cycling in Agricultural Ecosystems. CAB International, UK, pp. 239-256.