Effects of selected rice-field herbicides on photosynthesis, respiration, and nitrogen assimilating enzyme systems of paddy soil diazotrophic cyanobacteria

PESTICIDE

BIOCHEMISTRY

AND

31, 120-128 (1988)

PHYSIOLOGY

Effects of Selected Rice-Field Herbicides on Photosynthesis, Respiration, and Nitrogen Assimilating Enzyme Systems of Paddy Soil Diazotrophic Cyanobacteria J.

LAISRAM Algal

Research

Laboratory,

Centre

SINGH’

AND

D. N.

TIWAR?

of Advanced Stady in Botany, Varanasi 221 005. India

Banaras

Hindu

University,

Received August 5. 1987; accepted March 24. 1988 Two preemergence herbicides, butachlor and fluchloralin, showed little effect on the photosynthetic oxygen evolution of Nostoc rnu.scorum and Gloeocapsa sp. Propanil, a postemergence herbicide, inhibited photosynthetic 0, evolution in both organisms. These herbicides, on the other hand, showed a stimulatory effect on the respiratory oxygen uptake. All three herbicides did affect nitrogenase, nitrate reductase, and glutamine synthetase activities in N. mascorwn but in Gloeocapsa sp. butachlor and fluchloralin caused a stimulatory effect on nitrogenase and glutamine synthetase activity. 0 1988 Academic PESS. 1~. INTRODUCTION

Herbicides have been reported to inhibit photosynthesis in various ways (1, 2) but the available information is mostly based on higher plant systems. The effects of herbicides on cyanobacterial photosynthesis, which closely resembles that of chloroplast photosynthesis of eukaryotic cells (3, 4) is not well documented. Although the herbicides butachlor (a-chloroacetamide) and fluchloralin (a dinitroaniline compound) are not inhibitors of higher plant chloroplast photosynthesis as has been reported by many investigators, propanil is known to inhibit the Hill reaction (5, 6) and its site of action is believed to be cytochrome 553 (7). It is also reported to inhibit photosynthesis in cyanobacteria like Anabaena cylindrica, Tolypothrix ten&, and Nosk entophylum (8). Most of the information available on the effect of herbicides on plant respiration is based on studies carried out with excised plant tissues and isolated plant mitochoni Present address: Department of Botany, Manipur College, Imphal 7951 001, India. ’ To whom correspondence should be addressed. ’ Abbreviations used: a.i.. active ingredient; TCA, trichloroacetic acid.

dria. Propanil is reported to inhibit oxidative phosphorylation in soybean mitochondria (9) and in excised soybean hypocotyls (IO). Various authors have expressed different views on the importance of respiratory mechanism as a site of action of dinitroaniline compounds. Negi et al. (11) reported that trifluralin, which is a chemical analog of fluchloralin, inhibited O2 uptake and Pi esterification by mitochondria isolated from several species. Gruenhagen and Moreland (IO), however, failed to observe the inhibitory effect of trifluralin (0.2 mM> on ATP level of soybean hopocotyls (in vitro). Until now, very little work has been done on the effects of rice-field herbicides on nitrogen fixation and the studies carried out, so far, provide a preliminary idea about the inhibitory or stimulatory effect of the herbicides on diazotrophic growth in cyanobacteria. Many workers have reported the inhibition of cyanobacterial N, growth by various herbicides (12-16). The effect of herbicides on cyanobacterial nitrogen assimilating enzyme systems like nitrate reductase and glutamine synthetase are not clearly known. In this paper, we report the experimental findings obtained on the effect of three rice 120

0048-3575/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

HERBICIDES

AFFECTING

PHOTOSYNTHETIC

herbicides, butachlor , fluchloraline, and propanil, on the photosynthesis, respiration, and nitrogen metabolism of two diazatrophic cyanobacterial strains, namely Gloeocapsa sp. and Nostoc muscorum. MATERIALS

AND

METHODS

Organisms and culture conditions. Gloeocapsa sp., an isolate of the local rice fields of Varanasi (15) was raised to axenic clonal culture in combined nitrogen-free (conveniently called N2 medium) Hughes’ medium (17). When tested for aerobic photoautotrophic growth, in herbicidesupplemented growth medium, it showed several-fold tolerance to both butachlor and fluchloralin herbicides (15). Another herbicide sensitive strain, namely N. muscorum ISU (formerly known as Anabaena ATCC 27893), grown in combined nitrogen-free Chu-10 medium (18), was also used in the present study. For the nitrate grown culture, the growth medium used was supplemented with 1 mM calcium nitrate or 1 mM sodium nitrate. Cultures were grown in an air-conditioned growth chamber maintained at 26 -+ 2°C and under continuous illumination with fluorescent light intensity of nearly 46 PE m-* set-‘. Herbicides. Two preemergence herbicides, butachlor (2-chloro-2’,6’-diethylN-(butoxymethyl)acetanilide: a.i.3 0.5 g/ml) and fluchloralin (N-propyl-N(2’-chloroethyl)-2.6-dinitro-N-trifluoromethyl aniline; a.i. 0.48 g/ml) and one postemergence herbicide, propanil (3’,4dichloropropionanilide; a.i. 0.36 g/ml) were used during the present investigation. The three formulated herbicides were obtained from Monsanto Chemicals Pvt. Ltd. (Missouri), BASF Aktiengesellschaft (West Germany), and Indofil Chemicals Ltd. (India), respectively. Different concentrations of the respective herbicides were prepared by appropriate dilution in precooled double-distilled water and were filter-sterilized through millipore membrane filter (size 0.22 PM; Millipore Filter Corp., Bedford, MA)

N2

FIXATION

121

and finally added to the precooled sterilized growth medium at desired concentrations. Herbicide treatment. The organisms were grown in their respective N, medium in several parallel replicates. On the sixth day of growth (exponential phase), herbicides were added at desired concentrations and their effects on photosynthesis, respiration, nitrogenase activity, nitrate reductase activity (in N2 and NO, grown cultures), and GS activity were measured at specific time intervals (hours). Parallel sets of cultures grown in combined N, or NO, medium without herbicide supplementation served as controls. Measurement of photosynthetic oxygen evolution and respiratory oxygen uptake. Photosynthetic and respiratory activities of intact culture suspension were measured by a polarographic Clark electrode. A 3-ml cell suspension containing l-2 mg protein ml- ’ was placed in the reaction chamber and maintained at 28°C. Photosynthesis was measured as 0, evolution under saturating white light (light source: cool fluorescent light giving 55 PE m-’ set- ‘) and respiration as O2 consumption in the dark. Nitrogenase activity. Nitrogenase activity was determined by estimating the acetylene reducing activity of experimental cultures according to the method of Singh et al. (19). Measurement of nitrate reductase activity. For the estimation of cellular nitrate reductase activity an in situ assay procedure of Herrero et al. (20) was used. Cyanobacterial samples were centrifuged and washed with Tris-HCl buffer (50 mM, pH 7.5) for two to three times followed by the addition of 10% toluene and shaken vigorously for 3 min. A known volume of the preparation was immediately added to a reaction mixture for nitrate reductase activity determination. The reaction mixture contained in a final volume of 1 ml: NaHCO,-Na,CO, buffer, pH 10.5, 100 pmol; KNO,, 20 p,mol; methyl viologen, 4 or 10 kmol of Na,S,O, in 0.1 ml of 0.3 M NaHCO,. The reaction mixture

122

SlNGH

AND

including the cyanobacterial sample was incubated for 5 min at 30°C. The reaction was stopped by the addition of 10 ml of 1 M zinc acetate and nitrite was determined by following the azo-coupling method of Snell and Snell (21). Measurement of glutamine synthetase activity (in vivo) (Mn’+ dependent y-glutamyl transferase). Glutamine synthe-

tase activity was estimated in terms of y-glutamyl hydroxamate produced during the reaction following the method of Sampaio et al. (22). The cyanobacterial sample was centrifuged and washed successively with buffers A (50 mM Tris-HCl, pH 7.5) and B (Buffer A supplemented with 5 mM HgCl,, 10 mM Na glutamate, 5 mM mercaptoethanol, and 1 mM EDTA). The pellet was then treated with 0.01% C-Tab (hexadecyl trimethyl ammonium bromide, Sigma, St. Louis, MO) for 10 min at 30°C. Treated cultures were then mixed with 0.5 ml of reaction mixture which contained 40 mM Tris-HCl, pH 7.0; 3 mM MnCl,; 20 mM potassium arsenate; 0.4 mM ADP (NaS alt); 60 mM hydroxylamine, and 30 mM glutamine. The reaction was allowed to proceed for 15 min at 30°C in the dark. Finally, 2 ml of stop mixture (4.0 ml of 10% FeCl, + 1.0 ml of 24% TCA + 0.5 ml of 6 N HCl + 6.5 ml of distilled water) was added to the reaction mixture. The readings were taken at 540 nm against a blank which contained all the components except ADP and hydroxylamine-HCl with the help of a Model 250 Gilford spectrophotometer. Chlorophyll estimation. The chlorophyll content of various cultures were estimated by the method of Mackinney (23). Protein estimation. The Lowry method (24) was used with bovine serum albumin as standard. RESULTS

Oxygen evolution and O2 uptake. The short-term effects (cultures treated with herbicides for 24 hr) of butachlor, fluchloralin, and propalin on photosynthetic oxygen evolution and respiratory oxygen up-

TIWARI

take in N. muscorum and Gloeocapsa sp. were studied. In the case of N. muscorum, semilethal doses of herbicides were used as their minimum lethal doses were found causing lysis of the cyanobacterial cell walls. In the case of Gloeocapsa sp., which is comparatively an herbicide-resistant strain, a high concentration of herbicide, i.e., 100 pg ml-‘, which is nearly 5-10 times that of field applied doses, was used in the experiments. The extent of sensitivity of both organisms toward butachlor differed considerably, i.e., 6 Kg ml- ’ concentration of butachlor was lethal for a particular inoculum size (optical density 0.03 of homogeneous cultures) of N. muscorum while a comparative inoculum size of Gloeocapsa sp. could grow in butachlor concentrations as high as 2 mg ml-‘. Similarly, 25 pg ml- ’ fluchloralin was lethal concentration for N. muscorum while Gloeocapsa sp. could grow in fluchloralin as high as 300 pg ml - ’ . It is noteworthy that the response of both organisms toward propanil did not show any marked difference as its 5 pg ml-’ was found lethal for N. muscorum and the same concentration induced a prolonged lag in the growth of Gloeocapsa sp. It is of interest that Gloeocapsa incubated for as long as 45 days in N2 medium supplemented with 100 pg ml- ’ propanil still grew when transferred to fresh NZ medium free of herbicides. The above-mentioned lethal concentrations of butachlor and fluchloralin are taken here as minimum lethal doses and concentrations lower than these are considered as semilethal doses. Figure 1A shows that in N. muscorum, a slight depression of oxygen evolving activity was observed with 1 pg ml-’ and subsequent activity remained almost constant, i.e., about 80% of the control. Similarly, an almost constant level of oxygen evolving activity (i.e., about 70% of the control) was also maintained in cultures treated with concentration of fluchloralin ranging from 8 to 20 kg ml-’ (Fig. 1B). On the other hand, propanil significantly suppressed the activ-

HERBICIDES

AFFECTING

PHOTOSYNTHETIC

N2

123

FIXATION

700

700

0” S-‘r 600 -7 ; y500

600 500

s 0

i0

400

400

4

;Ln 300

300

o?

0”

.-E 3 ‘02 0

c*

z

200

200

$

100

1 100

0

1

2

3

Bu tachlor

4

50

1

2 Fluchloraiin

3

4

50

1

2

3

4

.! f-i g

.--? ‘y 7’ 7’ 5 F. i

; tL

5O

Propanil

(fw.ml-‘) FIG.

propanil. (reudings

1. Effect

of different concentrations of herbicides, (A) butachlor, (B) jluchloralin, on the photoevolution (0) and respiratory uptake (a) of oxygen in Nostoc recorded at intervals of 24 hr). Each point is the mean of tetraplicates.

ity in a dose-dependent manner, and, at a dose of 4 pg ml-‘, the activity was only about 38% of the control (Fig. 1C). The suppression of photosynthetic O2 evolution by butachlor and fluchloralin was insignificant in the case of Gloeocapsa sp. (Fig. 2). However, propanil (100 p,g ml-‘) significantly suppressed the oxygen evolving rate to about 34% of the control. The herbicides caused stimulatory effect on respiratory oxygen uptake rate in both N.

Herbicides

(iJg.ml-‘)

FIG. 2. Effect of different concentrations of butachlor (O), fluchloralin (O), and prop&in (A) on the photoevolution (---) and respriatory uptake (-) of oxygen in Gloeocapsa sp. (readings recorded at intervals of 24 hr). Each point is the mean of tetraplicates.

and (c’) muscorum

muscorum and Gloeocapsa sp. (Figs. 1, 2) in a dose-dependent manner. Nitrogenase activity. In N. muscorum, the sublethal doses of butachlor and fluchloralin slightly suppressed the nitrogenase activity in a dose-dependent manner and, interestingly, nitrogenase activity was recorded in cultures treated with the lethal concentrations of both herbicides as long as the vegetative cells remained intact (up to 12 hr), i.e., all the vegetative cells disintegrated within 24 hr of treatment with the lethal herbicide doses (Figs. 3A and 3B). In contrast, propanil concentrations as low as 4 pg ml-’ were sufficient to inhibit the nitrogenase activity in N. muscorum completely (Fig. 3C). The aerobic nitrogen fixing ability of the unicellular cyanobacterium Gloeocapsa sp. was studied (Table 1). When grown under continuous illumination of 46 PE me2 set-‘, an average acetylene reducing activity of 18.01 pmol pg chl a-l min-’ was maintained. In contrast, the acetylene reducing activity was completely lost in dark incubated parallel cultures within IO-12 hr of incubation. In addition, while butachlor (100 pg ml-‘) did not seem to influence the nitrogenase activity, fluchloralin (100 kg ml-‘), on the other hand, inhibited the activity within 6 hr of incubation period.

124

SINGH AND TIWARI

Time

(h)

FIG. 3. Effect of different concentrations of herbicides (kg ml- ‘) on the aerobic acetylene reducing activity of &day-old exponentially growing cultures of Nostoc muscorum. (A) Butachlor, control (0). 0.5 (III), 2 (0) 4 (a), 8 (A); (B)f7uchloralin, control (0) 0.5 (O), 6 (O), 12 (a), 24 (A): (C) propalin, control (0), 2 (O), 4 (A). Each point is the mean of tetraplicates.

However, the almost normal rate of nitrogenase activity of butachlor-treated cultures and the enhanced nitrogenase activity in fluchloralin-treated cultures did not correspond to their growth rate which remained sightly lower than the control cultures (results not shown). Nitrate-reductase activity. Propanil caused a progressive decrease in in vivo nitrate reductase activity with its sublethal doses as a propanil (4 Fg ml-‘) treatment for 36 hr resulted in complete loss of nitrate reductase activity in N. muscorum (Fig. 4). Parallel experiments with fluchloralin and butachlor on N. muscorum showed the suppressive action of these herbicides on the in vivo nitrate reductase activity but their suppressive effect was never 100%. Similar studies were carried out with

Gloeocapsa sp. and the results are shown in Table 2. Propanil, at 100 pg ml - ’ concentration, individually or in combination with butachlor (100 p,g ml ~ ‘) or fluchloralin (100 pg ml-‘), inhibited the in vivo nitrate reductase activity in both N2 and NO, grown cultures. However, butachlor and fluchloralin, individually or in combination, did not significantly change the nitrate reductase activity in Gloeocapsa sp. GS activity. Butachlor, fluchloralin, and propanil suppressed the activity of the in vivo enzyme in N. muscorum in a dosedependent manner and, as such, the combined effect of these herbicides on the activity was not tested (Table 3). In the case of Gloeocapsa sp., both butachlor and fluchloralin, individually or in combination, enhanced the in vivo glu-

TABLE 1 Effect of Light (46 FE m ~’ set ‘) vs Dark and Various Herbicides on Aerobic Acetylene Reducing Activity of Exponentially Growing Nz Cultures (6 Days old) of Gloeocapsa sp. (Acetylene Reduced pmol +g chl aa’min -1 ) Time W

Light

Dark

Light + butachlor

0 6 12 18 24

12.60 18.90 18.58 20.63 19.36

11.58 4.02 0.00 0.00 0.00

12.30 16.01 22.45 22.50 23.02

Light + fluchloralin 12.20 18.56 28.60 27.79 27.58

Light + propanil 11.82 0.00 0.00 0.00 0.00

Note. Each reading is the average of four independent readings. Herbicide used at 100 kg ml- ’ concentration each.

HERBICIDES

0

AFFECTING

12

24

36

PHOTOSYNTHETIC

48

12

24

Time

36

46

125

N1 FIXATION

12

24

36

68

(h)

FIG. 4. Effect of different concentrations of herbicides (kg ml-‘) on in vivo nitrate reductase activity of &day-old exponentially growing cultures of Nostoc muscorum. (A) Butachlor, control (0). 2.5 (O), 5 (a): (B)f[uchlora/in. control (0), 10 (0). 20 (~3): (C) propalin, control (0). 2 (0). 4 (A). Each point is the mean of tetraplicates.

tamine synthetase activity (Table 3). However, propanil (100 p,g ml-‘) individually reduced the in vivo glutamine synthetase activity to about 70% of the control and, in combination, blocks the stimulatory effect of butachlor and fluchloralin on this activity (Table 3). DISCUSSION

The negligible effect of butachlor and fluchloralin on the photosynthetic oxygen evolution processes is consistent with the earlier findings that photosynthesis is not the primary site of action of these herbicides. Alachlor, a chemical analog of butachlor, has been shown not to inhibit Hill reaction catalyzed by the wheat chloroplasts even at concentrations as high as 0.5 n-&I (25). Fletcher and Kirkwood (26) have also concluded that photosynthesis may not be the site of action of dinitroaniline com-

pounds (fluchloraline). The significant inhibitory effect of sublethal concentrations of propanil on the photosynthetic oxygen evolving activity in both organisms support the earlier indications of propanil being an inhibitor of photosynthesis. The present observations which show stimulation of respiratory oxygen uptake activity in the presence of the three herbicides suggest that the respiratory electron transport chain is not inhibited by these herbicides. The overall increase in respiration rate may be due to triggering of a series of mechanisms which the microorganisms are believed to possess to counter the effect of the toxic chemicals. These mechanisms may include uptake, accumulation, biodegradation, and transport of chemicals out of the cell (27-31). Moreover, any possible suppressive activity on the photosynthetic ATP generation might compel the organ-

TABLE 2 Effect of Herbicides (100 kg ml-’ each) on in Vivo Nitrate Reductuse Activity of Gloeocapsa sp. Grown for 6 Days Medium _~ Nz medium” NO, mediumb

Control

Butachlor

4.72 12.75

4.97 12.46

Fluchloralin 4.15 13.30

Propanil 0.00 0.00

a Cultures grown in N, medium. ’ Cultures grown in NO, medium. Note. Values expressed as nmol NO, produced mg chl a- ’ min- ‘. Readings are the average of four independent experiments.

SINGH

126

AND

isms to rely more on endogenous carbon reserves (polyglucose and poly-c+hydroxybutyrate) and oxidative phosphorylation to meet the extra energy demands under the stress conditions. The nitrogen fixing ability of Gloeocapsa sp. 1430/3 varied under the light and dark conditions (32). On the other hand, culture of Gloeocapsa sp. 143013 grown under constant illumination evolved oxygen and fixed nitrogen simultaneously (33). The present Gloeocapsa strain was grown under constant illumination so as to avoid the effect of dark-light shifts and to correctly assess the effect of herbicides on nitrogenase activity. The range of aerobic nitrogenase activity shown by the present strain under photoautotrophic condition is almost similar to that reported in Gloeocapsa (Gloeothece) sp. CCAP 1430/3 (35). In the dark (i.e., under the nonphotosynthesizing conditions) the reducing power and ATP needed to sustain nitrogen fixation must presumably arise from the oxidation of endogenous substrates or at the expense of accumulated glycogen reserves (34,36, 37). Thus, the inhibition of nitrogenase activity after 10 hr of incubation in the dark, in the present Gloeocapsa strain, may be due to the gradual depletion of endogenous carbon reserve which occurs as a glycogen-type storage polysaccharide in Gloeocapsa (37). In bracken cell preparations of Gloeocapsa sp. LB 795, the presence of oxygen tension (0.005 atm) is known to completely

TIWARI

inhibit

nitrogenase activity suggesting that nitrogenase is extremely sensitive to oxygen and the inhibition by oxygen is an irreversible process (38-40). Gallon et al. (39) have shown that nitrogenase activity in whole cells of Gloeocapsa is maximum when the oxygen evolution is depressed and also suggested respiratory protection of nitrogenase and the possession of an “uptake hydrogenase” which will consume oxygen and contribute to the protection of nitrogenase from damage by oxygen particularly under conditions of carbon deficiency in Gloeothece sp. ATCC 27152. Thus, the high respiratory rate under the influence of butachlor and fluchloralin might have also eased the oxygen tension on nitrogenase, thereby stimulating its activity in Gloeocapsa sp. Moreover, the normal and enhanced nitrogenase activity suggests that nitrogen fixation is not the limiting factor for the suppressed growth of Gloeocapsa sp. under butachlor and fluchloralin stress. The inhibition of nitrogenase activity by propanil(lO0 p,g ml ~ ‘) in Gloeocapsa sp. might have resulted from inhibition of photosystem II electron transport system. In heterocystous cyanobacteria, the photosynthetically fixed carbon which is essential for nitrogen fixation is supplied to the heterocysts by the vegetative cells (40, 41). The reduced nitrogenase activity under the increasing concentration of butachlor and fluchloralin in N. muscorum may possibly Gloeocapsa

TABLE

Effect

of Herbicides

on itI Vi,,0 Glutumine Syrlthetase for 6 Days in N2 Medium

--

3

Activit.v of N. muscorum With or Without Herbicides

and

Gloeocapsu

sp. Grown

Propanil

Organism

Control (without herbicide)

N. Muscorum

1451.47

GiOl?OCllpSL2 SP.6

808.70

Butachlor 974.46 (2)”

Fluchloralin 941.16 (4)

1136.15

1151.80 (IO) 1149.13

Propanil 908.75 (20)

656.01 (2)

Butachlor + propanil

fl”CfhlO-

808.37

898.58

ralin

Butachlor + fluchloralin

601.50 (4) 570.30

y Figures in parentheses indicate herbicide concentrations in kg ml ’ each. ’ In the case of Gloeocupsa sp., 100 pg ml-’ of each herbicide is used either individually Note. Values expressed in u-transferase activity, nmol product mg protein-’ min- ‘. Each

or in combination. reading is the mean

1332.34

of tetraplicates.

HERBICIDES

AFFECTING

PHOTOSYNTHETIC

be related to the marginal reduction in photosynthetic activity under the same herbicide concentrations. Similarly, the total inhibition of nitrogenase activity in N. muscot-urn by the sublethal concentrations of propanil may be an indirect effect arising from the inhibition of photosynthetic process by the chemical. The enzyme nitrate reductase is membrane-bound and its activity depends on the reduced ferredoxin produced during oxygenic photosynthesis (42, 43). Thus nitrate reductase activities in the presence of butachlor, fluchloralin, and propanil in N. muscorum and Gloeocapsa sp. might have corresponded to the different levels of interference of these herbicides with their photosynthetic mechanisms. The marginal suppression of glutamine synthetase activity in herbicide-treated culture of N. muscorum may be related to the suppressed nitrogenase and nitrate reductase activities under similar conditions. Glutamine synthetase is reported to be the most active under the nitrogen-fixing conditions in Gloeocaspa sp. (35) and the activation of GS gene (gln A) requires the nitrogen fixing conditions (44). Thus, the enhanced nitrogenase activity of Gloeocapsa sp. in the presence of butachlor and fluchloralin may be responsible for the enhancement of glutamine synthetase activities under the same conditions. Likewise, the suppression of nitrogenase activity in Gloeocapsa sp. by propanil may also cause indirect suppression of glutamine synthetase activity. ACKNOWLEDGMENTS

We thank the Head, Centre of Advanced Study in Botany, Banaras Hindu University, Professor H. N. Singh, School of Life Sciences, and University of Hyderabad for providing laboratory facilities. One of us (L.J.S.) is grateful to the Government of Manipur for granting study leave. REFERENCES

I. D. E. Moreland and J. E. Hilton, Actions in photosynthetic systems, in “Herbicides: Physiology, Biochemistry, Ecology” (L. J. Audus,

N1

FIXATION

127

Ed.), Vol. 1, p. 493, Academic Press, New York/London, 1976. 2. D. E. Moreland, Mechanisms of action of herbicides Annu. Rev. Plant Physiol. 31, 597 (1980). 3. A. Trebst and M. Avron, “Encyclopedia of Plant Physiology,” Vol. 5, “Photosynthesis 1.” Springer, Berlin, 1977. 4. A. San Pietro, Ed., “Methods in Enzymology,” Vol. 69, Academic Press, New York, 1980. 5. N. E. Good, Inhibitors of Hill reaction, Plant Physiol. 36, 788 (1961). 6. D. E. Moreland and K. L. Hill, Inhibition of photochemical activity of isolated chloroplasts by acylanilides. Weeds 11, 55 (1963). 7. M. Nishimura and A. Takamiya, Energy and electron transfer system in algae photosynthesis. 1. Action of two photochemical systems in oxidation-reduction reaction of cytochrome in Porphyra. Biochem. Biophys. Acta 120, 45 (1966). 8. S. J. L. Wright, A. F. Stainthorpe, and J. D. Downs, Interactions by the herbicide propanil and a metabolite. 3.4-dichloroaniline, with bluegreen algae. Actu Phytopathol. Hung. 12, 51 (1977). 9. G. Hofstra and C. M. Switzer. The Phytotoxicity of propanil, Weed Sci. 16, 23 (1968). 10. R. D. Gruenhagen and D. E. Moreland, Effects of herbicides on ATP levels in excised soybean hypocotyls, Weed Sci. 19, 319 (1971). Il. N. S. Negi. H. H. Funderbunk, D. P. Schultz. and D. E. Davis, Effect of trifluralin and nitralin on mitochondrial activities, Weed Sci. 16, 83 (1968). 12. A. N. Ibrahim, Effect of certain herbicides on growth of nitrogen-fixing algae and rice fields. Symp. Biol. Hung. 11, 445, (1972). 13. E. J. Dasilva, L. E. Henriksson, and E. Henriksson, Effects of pesticides on blue-green algae and nitrogen fixation, Arch. Environ. Contam. Toxicol. 3, 193 (1975). 14. D. N. Tiwari, A. K. Pandey, and A. K. Mishra, Action of 2,4-dichlorophenoxy acetic acid on growth and heterocyst formation in the bluegreen alga Nostoc linckia. J. Biosci. 3, 33-39 (1981). 15. L. J. Singh, D. N. Tiwari, and H. N. Singh. Evidence for genetic control of herbicide resistance in a rice-field isolate of Gloeocapsa sp. capable of aerobic diazotrophy under photoautotrophic conditions. J. Gen. Appl. Microbial. 32, 81 (1986). 16. A. Maule and S. J. L. Wright, Physiological effects of chloropropham and 3-chloroaniline on some cyanobacteria and a green alga, Pestic. Biochem. Physiol. 19, 196 (1983). 17. E. D. Hughes, P. R. Gorham, and A. Zehnder, Toxicity of a unialgal culture of Microcystis aeruginosa. Canad. J. Microbial. 4,255 (1958).

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18. R. S. Safferman and M. E. Morris, Growth characteristics of the blue-green algal virus LPP-1, J. Bacreriol. 88, 771 (1964). 19. H. N. Singh, U. N. Rai, V. V. Rao, and S. N. Bagchi, Evidence for ammonia as an inhibitor of heterocyst and nitrogenase formation in the cyanobacterum Anabaena cycadeae. Biochem. Biophys.

Res.

Commun.

111, 180 (1983).

20. A. Herrero. E. Flores, and M. G. Guerrero, Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. Strain 7119, and Nostoc. sp. Strain 6719. /. Bacferiol.

145, 175 (1981).

21. F. D. Snell and C. T. Snell. Nitrites by sulfanilamide and N-(1-naphthyl) ethylene diamine hydrochloride, in “Calorimetric Methods of Analysis (Ill/d),” Vol. 2, Part D. p. 804, Van Nostrand Co., Princeton, NJ, 1949. 22. M. J. A. M. Sampaio, P. Rowell, and W. D. P. Stewart, Purification and some properties of glutamine synthetase from the nitrogen-fixing cyanobacteria Anabaena cylindrica and Nostoc sp. J. Gen. Microbial. 111, 181 (1979). 23. G. Mackinney. Absorption of light by chlorophyllsolutions. .I. Biol. Chem. 140, 315 (1941). 24. 0. H. Lowry, N. J. Rosebrough. A. L. Farr, and R. J. Randall, Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265 (1951). 25. J. M. Chandler, L. I. Croy. and P. W. Santelman, Alachlor effects on plant nitrogen metabolism and Hill reaction. J. Agric. Food Chem. 20, 661 (1972). 26. W. W. Fletcher and R. C. Kirkwood, in “Herbicides and Plant Growth Regulators” (W. W. Fletcher and R. C. Kirkwood, Eds.). p. 2. Granada Publishing, London, 1982. 27. B. D. Vance and W. Drummond. Biological concentration of pesticides by algae. J. Amer. Water Works

Assoc.

61, 360 (1969).

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