Blitox-resistant mutants of the N2-fixing blue-green algae Nostoc linckia and Nostoc muscorum

Environmentaland ExperimentalBotany, Vol. 22, No. 4, pp. 427 to 435, 1982.

0098~472/82/040427~9 $03.00/0 ~ 1982. Pergamon Press Ltd.

Printed in Great Britain.

BLITOX-RESISTANT MUTANTS OF THE Nz-FIXING BLUE-GREEN ALGAE NOSTOC LINCKIA AND NOSTOC MUSCORUM A. VAISHAMI~AYAN and A. B. PRASAD*

Mutagenesis and Cytogenetics Laboratory, University Department of Botany, University of Bihar, Muzaffarpur-842001, India ( Received 15 June 1981; revision received6 October 1981 ; acceptedin revisedform 3 April 1982)

VAISHAMPAYANA. and PRASADA. B. BuTox-resistant mutants o.fthe N2-fixing blue-green algae Nostoc linckia and N ostoc muscorum. ENVlRON~tENTALANn EXPERIMENTALBOTANY22, 427--435, 1982.-BLxTox-resistant mutants of N. linckia and N. muscorum, defective in N 2- and NO~-metabolism were produced by growth in media containing 45.5 ppm BLXTOXfungicide (copper oxychloride). The mutant resistant to 100 ppm BuTox occurred under conditions of growth inhibition in N 2 or NO~ medium. This mutant showed the characteristics of defective nitrogenase and nitrate reductase activity in N 2 and NO 3 media, respectively, coupled with an induction of heterocyst formation in NO~ medium. Such characteristics indicate defects in nitrogenase (or heterocyst) and nitrate reductase enzyme systems.

INTRODUCTION

PADDY microflora include a majority ofheterocystous and filamentous blue-green algae that contribute significantly to soil fertility ~6'24"3z'33) through fixing nitrogen both photosynthetically and photoheterotrophically. ~3°) A large n u m b e r of pesticides which eradicate pests and weeds from the crop-fields has been found toxic to these N zfixing blue-green algae. (9'19'22'23'31) A paradoxical situation arises when the beneficial effects of the pesticides are c o m p a r e d with the depletion of natural biological nitrogen resources. O n e possible solution for making these N2-fixators of paddy-fields a viable biofertilizer is to raise pesticide-resistant mutants of blue-green algae. Copper and copper compounds are widely used as fungicidal and biocidal agents, ~14~out of which BLITOX (copper oxychloride), a Cu-fungicide, is commonly applied to the rice-fields of North India. Studies on fungal strains resistant to m a n y heavy metals t1'13'~5~ and saturated solution of

copper sulfate, ~3'7'26'27) tempted us to raise algal populations resistant to this Cu-fungicide at a dosage level normally applied to the fields. Nostoc linckia and N. muscorum have been used as test materials for such studies as they can grow and fix nitrogen in a simple medium consisting of inorganic ions. U n d e r these conditions the interactions that are known to occur between Cu ions and constituents of m a n y microbiological media are minimized. ~z'l 5) MATERIALS A N D M E T H O D S

Organisms T h e wild-type parents Nostoc muscorum G and Nostoc linckia were obtained from the Botany Department of Banaras H i n d u University, Varanasi, India. Both algae are filamentous and neither form heterocysts nor fix nitrogen in inorganic nitrogen media (NO 3 or N O ~ ) . But when transferred from combined nitrogen media to a fresh nitrogen-free (Nz) medium they form

* Department of Botany, L.N.M. University, Darbhanga-846004, India. 427

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A. VAISHAMPAYAN and A. B. PRASAD

heterocysts within 20-24 hr with a frequency of 56%, and fix nitrogen. The identical concentration of NO~- or NO~- (required for complete suppression of heterocysts) consistently used during the present series of experiments is 5 and 20 m M for N. muscorum and N. linckia, respectively. These two Nostoc species are non-mucilage forming and are easily fragmented into unicellular bits. This property of these two algae makes them eminently suited for genetic and mutational work. These algae were grown routinely in Chu 10 medium as modified by Gerloff et al. (s) under CO2 enriched conditions (v) at a temperature of 28 + 20C and a continuous light intensity of 2800 lux in an airconditioned culture chamber (which are optimal growth conditions for the two algae). All experiments in the liquid medium were done under these culture conditions. Inhibitory effects of thefungicide BLITOX is a powdery green chemical with 50% active ingredient. The other 50°/0 consists of the silicates and carbonates of sodium and calcium, that are also constituents of the Chu 10 medium used here. Thus, the selection of BiIvox as a Cufungicide for the present study minimized the action of inert ingredients. Different concentrations of the fungicide were prepared by appropriate dilutions with sterile glass distilled water. NO3--grown log-phase non-heterocystous cultures of the two algae were harvested and washed with sterile distilled water (by repeated centrifugation and decantation) for removing the traces of NO~-. Washed cultures of both Nostoc species were divided into three equal parts. One was left as such and the remaining two were treated for 1 hr with a filter sterilized solution of 10 or 50 p p m BLITOX (BLITOXsolution was not autoclaved). After the BLiTOX-treatment, the treated samples were harvested and washed with sterile distilled water. Each of the treated sets of the two algae (untreated, l0 and 50 p p m treated) was then inoculated separately into fresh N2, N 2 + 50 ppm FeSOa, N O ~ , N O ~ + 50 p p m FeSO4, N O 2 , or N O 2 + 5 0 p p m FeSO 4 medium (each with five replicates in corning 50 ml conical flasks) and incubated in the culture chamber under previously mentioned sterile conditions. FeSO 4 was used because of the fact that Cu had been found earlier to cause Fe-deficiency in higher

plants. (4'5'1°'s4) On the ninth day of the inoculation of the entire set, the maximum growth, heterocyst frequency and nitrogenase activity of all untreated and treated samples _+FeSO4 of the two species were recorded (it having been earlier found that N. muscorum and N. linckia show their maximum growth, heterocyst frequency and nitrogenase activity on the ninth day of inoculation).(ls) Growth was measured by optical density determinations at 663 nm in a 'Systronic' spectrocolorimeter (increase in O.D. on the ninth day of inoculation); heterocyst frequency was measured by microscopic observations in terms of the number ofheterocysts per hundred vegetative cells (each individual reading was the mean of 12 randomly assorted algal filaments); nitrogenase activity was measured in terms ofnmoles ethylene #g chlorophyll-a-1 hr-1 by the gas chromatograph method of Stewart et al. t29~ Nitrate reductase activity of all samples was determined on the second day of inoculation (in earlier trials it was found by the authors that within 24-48 hr ./V. linckia and N. muscorum exhibited their maximum nitrate reductase activity) in terms of#g nitrite #g protein-1 by the biochemical method of nitrite estimation. (25) It was found that the fungicide exhibited its inhibitory effect(s) only on the N 2- and N O 3mediated cultures. Thereafter, the whole set of experiments was repeated by using similar concentrations of CuSO4 instead of BLITOX for assessing whether Cu was responsible for causing the observed anomalies in the two Nostoc species. Isolation and characterization of BLtTox-resistant mutants The concentration of BLITOX in the upper ½ inch of the rice-field soil is 15.5 to 45.5 ppm (report from Plant Protection Department, Govt. of India, Muzaffarpur). The intention was, therefore, to isolate mutant strains ofN. linckia and N. muscorum resistant to 45.5 p p m BLITOX. The general procedure of mutagenesis in N. muscorum and N. linckia earlier used by Singh and his group tas'19,22) has been followed here for the isolation of Birrox-resistant mutants of these two algae. For this, NO~--grown (non-heterocystous) log-phase cultures of both Nostoc species were harvested and washed with sterile N 2 medium.

BLITOX-RESISTANT MUTANTS The washed cultures were gently shaken in glassdistilled water with sterilized glass beads on an electrically operated shaker until the filaments were uniformly fragmented into unicellular bits (on the basis of periodical microscopic examination). The cultures were again harvested and spread on separate sets of solid (1.2% agar) N 2 or NO~ medium supplemented with 45.5 ppm BLITOX (100 Petri dishes were used for each set). Suspensions of the same populations of the two algae, diluted 1 : 1000, were spread on BLITOXfree N 2 medium (50 Petri dishes) for scoring the total number of colony-forming units. All sets were incubated in the culture chamber for colony growth. After one week the populations of two algae inoculated on BLiTOX-supplemented N2 Petri dishes started dying out and exhibited a loss of their typical blue-green color. At this stage half of this set (50 Petri dishes) were overlayed with NO~ agar medium. A significant colony growth of the two algae on all four sets occurred after 2 weeks (control as well as BLiTOX-treated N2, NOg and NO~- Petri dishes). Frequencies of formation of BHTox-resistant colonies were determined by counting the total number of putative mutant colonies on each set (N2, NO~ or NO~-) in proportion to the total number of colony-forming units multiplied by 1000. Similar experiments were performed five times for facilitating statistical analysis. Isolates were characterized for growth, nitrogenase/nitrate reductase activity and maximum heterocyst frequency in N 2 or NO~ medium by the methods earlier discussed. BLtTOx-resistant mutants were found to have defects in their nitrogenase and nitrate reductase enzyme systems. Thus, a prototrophic reversion analysis of these mutants was made: a thick suspension of each of the BLiTOx-resistant mutants was harvested, washed with sterile N 2 medium and spread on fresh solid minimal (N2) medium gelled with 1.2% agar (100 Petri dishes were used for preparing this set in each case). A 1000 times diluted suspension of the same population was spread on solid NO~ medium (because the mutants grew well on NO~) for scoring the total number of colony-forming units. After two weeks of incubation colony growth was obtained. Prototrophic reversion frequency was determined by counting the number of revertant colonies in

429

proportion to the total number of colony-forming units multiplied by 1000. Similar experiments were performed five times for facilitating statistical analysis. The pesticide BLiwox was obtained from the Plant Protection Department, Govt. of India, Muzaffarpur. All other chemicals used were of BDH grade and agar was obtained from Centron Laboratories, Bombay. The experiments were done in five replicates and results were analyzed statistically, tz2~ RESULTS

BLITOX or CuSO# treatment could inhibit N 2or NO~-mediated growth ofN. muscorum and N. linckia (Table 1) leading to massive fragmentation

and chlorosis of the filaments. However, the treated cultures grew at a good rate in NO~medium, showing that the organisms were metabolically protected in NO2 medium (Table 1). BLITOX or CuSO4, in contrast to the control cultures, allowed the formation of heterocysts in NO~- medium in both Nostoc species with a significant frequency (Table 1). A similar effect, i.e. induction of heterocyst formation, was not found in NO~- medium. Nevertheless, these treatments resulted in complete inhibition of nitrogenase and nitrate reductase activities of the two algae (Table 2). Remarkably, when FeSO 4 was applied to the medium, the treated cultures of both Nostoc species showed significant nitrogenase activity (Table 2) and consequently grew well in N2 medium (Table 1). Heterocyst frequency (Table 1) and nitrate reductase activity (Table 2) of the untreated and treated cultures remained unchanged with the addition of FeSO 4. Mutant colonies resistant to 100 ppm BLITOX were isolated from a population inoculated on 45.5 ppm BLITOX-supplemented mineral medium. These mutant colonies appeared with significant frequencies on N 2 and NO~- media in .N. muscorum, and only on NO~- medium in N. linckia (Table 3). However, such mutant colonies did not appear in NO~- medium in either of the two Nostoc species. The mutant of aV. muscorum isolated from N z medium had het + n i f + n i t phenotypic expression, i.e. heterocystous, nitrogen-fixing and non-nitrate metabolizing. Since this mutant had been produced as a result of

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Table 1. Data* on growth'~ and heterocystfrequency~ of parent Nostoe muscorum and Nostoc linckia in N2, NO~- or N O r medium -+ FeSO4 ( 50 ppm ) , untreated or treated with different concentrations 0fBtaTOX or CuSO,

Growth

fifostoc

species 333FeS04

Treated with

N. muscorum Nil

-FeSO 4

10 ppm 50 ppm 10 ppm 50 ppm

N2

NO~-

BLITOX BLITOX CuSO4 CuSO4

0.640___0.015 0.7153-0.012 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

W. muscorum Nil

Heterocyst frequency NO~-

0.755-t-0.025 0.745+0.015 0.750-1-0.020 0.760___0.010 0.755+0.013

N2

NO~-

5.6+0.8 5.6+0.3 5.5-t-0.4 5.5-t-0.2 5.5-t-0.5

0.0 4.9+0.5 4.4-1-0.6 4.6-t-0.8 4.5-t-0.4

+FeSO,

I0 ppm 50 ppm 10 ppm 50 ppm

BLITOX BLITOX CuSO4 CuSO4

0.655-t-0.017 0.715+__0.014 0.765+_0.010 0.435+0.034 0.0 0.760-t-0.025 0.4253330.012 0.0 0.7603330.014 0.4203330.014 0.0 0.7653330.015 0.405_+0.016 0.0 0.770___0.012

5.6-t-0.4 5.5___+0.2 5.6-t-0.3 5.4___0.6 5.4+0.5

0.0 4.33330.8 4.8-t-0.5 4.8-t-0.3 4.5-t-0.6

N. linckia

Nil 10 ppm 50 ppm 10 ppm 50 ppm

BLITOX BLITOX CuSO, CuSO4

0.660-t-0.014 0.0 0.0 0.0 0.0

0.775_+0.015 0.7603330.012 0.7553330.018 0.755--+0.017 0.765+0.013

5.6_+0.8 5.3 +0.5 5.63330.4 5.83330.5 5.7___0.3

0.0 3.83330.3 4.0-t-0.6 4.1 -+0.4 4.2-+0.7

Nil I0 ppm 50 ppm 10 ppm 50 ppm

BLITOX BHTOX CuSO4 CuSO4

0.720+0.018 0.765___0.016 0.785___0.013 0.4253330.014 0.0 0.7703330.016 0.4303330.013 0.0 0.7753330.015 0.4353330.017 0.0 0.7353330.018 0.4103330.015 0.0 0.775+0.017

5.6___0.4 5.6-+0.2 5.9+0.3 5.8-+0.6 5.6___0.8

0.0 3.83330.3 4.2-+0.8 4.03334.5 3.93330.4

-FeSO4

,N. linckia

+ FeSO4

0.705_0.014 0.0 0.0 0.0 0.0

* The values are means of five independent readings with their respective standard errors. ~"Increase in optical density at 663 nm on ninth day after inoculation (initial optical density was uniformly 0.005 for all the samples). Number of heterocysts per hundred vegetative cells. Heterocysts were not found in N O r medium. BLITOx-treatment of 2¢. muscorum it was designated as a M her + n i l + nit m u t a n t strain. The mutants of both N . muscorum and N . linckia isolated from N O ] medium, had a het + n i f - nit- phenotypic expression, and hence they were designated as M het+ n i f - n i t - and L het + n i f - nit- m u t a n t strains, respectively (Table 3). The M het + n i l + nit- m u t a n t strain in the presence or absence of 100 p p m BLITOX, grew well in N O ~ medium, failed to grow in NO3- medium and showed weak growth in N 2 medium (Table 4). It exhibited the characteristics of heterocyst formation in N O 3 medium similar to that found in N 2 medium (Table 4). This m u t a n t had shown a reduced nitrogenase activity (almost half that of the parental strain) and no nitrate reductase activity (Table 4). The M her + n i l - nit- and L her + n i f - nit-

m u t a n t strains grew neither in the presence o f N 2 nor N O 3 , but showed normal growth with NO~(Table 4). Both mutants had heterocyst frequencies approaching their respective parental strains in N 2 as well as NO~- media, but appeared to exhibit a highly reduced and insignificant nitrogenase activity and no nitrate reductase activity (Table 4). This table further shows that all the three mutants were spontaneously revertible to their prototrophic characters (BLITOXsensitivity n i f + nit +) with frequencies of 1 × 10 - 5 or 10- 6 (Table 4). DISCUSSION

The inhibitory effects of the fungicide BLITOX on nitrogenase activity and consequently on N 2mediated growth of N. muscorum and 2¢'. linckia

B L I T O X - R E S I S T A N T MUTANTS

431

Table 2. Data* on nitrogenaset and nitrate reductase~ activities of parent Nostoc muscorum and Nostoc linckia, untreated and treated with different concentrations of BLXTOXor CuSO4 ( the activities were detected in medium containing or lacking 50 ppm FeSO4) Wostoc species 4- FeSO,

Treated with

Nitrogenase activity (N 2 medium)

Nitrate reductase activity (NO~ medium)

N. muscorum -FeSO 4

Nil 10 ppm 50 ppm 10 ppm 50 ppm

BLITOX BLITOX CuSO 4 CuSO4

6.25+0.18 0.0 0.0 0.0 0.0

0.151 +0.022 0.0 0.0 0.0 0.0

dV. muscorum + FeSO,

Nil 10 ppm 50 ppm 10 ppm 50 ppm

BLITOX BLITOX CuSO, CuSO 4

6.68_ 0.14 4.65 4-0.26 4.58 4- 0.14 4.36 4- 0.17 3.89 4- 0.13

0.149___0.034 0.0 0.0 0.0 0.0

N. linckia --FeSO 4

Nil 10 ppm 50 ppm 10 ppm 50 ppm

BLITOX BLXTOX CuSO, CuSO 4

6.574-0.16 0.0 0.0 0.0 0.0

0.1164-0.018 0.0 0.0 0.0 0.0

W. linckia + FeSO 4

Nil 10 ppm 50 ppm 10 ppm 50 ppm

BLITOX BLITOX CuSO, CuSO 4

6.82+0.15 4.89 4- 0.32 4.394-0.88 4.62 4- 0.34 4.364-0.18

0.122+_0.025 0.0 0.0 0.0 0.0

* The values are means of five independent readings with their respective standard errors. 1"n moles ethylene/#g chlorophyll-a/hr (nitrogenase activity was found only in N 2 medium and not in NO~ or NO2 medium). /~g nitrite//~g protein (specifically detectable during NO~ metabolism). suggest t h a t it is an i n h i b i t o r of N 2 fixation processes. T h e r e is only one r e p o r t on the direct inhibition of N2 fixation processes by the pesticides 2 - m e t h y l - 4 - c h l o r o p h e n o x y a c e t i c acid a n d 2,4-dichlorophenoxyacetic acid in Nostoc muscorum, dV. punctiforme a n d Cylindrospermum sp. ~ta) I n h i b i t i o n of N O r - m e d i a t e d g r o w t h together with the f o r m a t i o n of heterocysts in N O ~ m e d i u m , clearly d e m o n s t r a t e s that the N O ~ m e t a b o l i z i n g factor, i.e., n i t r a t e reductase, has been i n a c t i v a t e d by the fungicide t r e a t m e n t , a n d since active n i t r a t e reductase is the enzyme which represses heterocyst f o r m a t i o n d u r i n g N O 3 metabolization, ~ls'2t~ its i n a c t i v a t i o n n a t u r a l l y relieves the repressed heterocysts in N O 3 m e d i u m ,

as previously shown by SINGH et al.~2t) Consequently in the present case, despite the inability of the BLITOx-treated organisms to use an exogenous nitrogen source (NO~-), heterocysts differentiated on the filaments to the same extent as in controls even though the filaments b e c a m e chlorotic a n d exhibited massive f r a g m e n t a t i o n due to nitrogen starvation b r o u g h t a b o u t by inhibition of b o t h nitrogenase a n d nitrate reductase enzyme systems. H o w e v e r , BLITOXtreated m a t e r i a l was still alive a n d results of an assay of the two e n z y m e systems i n d i c a t e d that the fungicide caused a simple inhibition of their activities r a t h e r t h a n a general loss of their function. BLITOX, therefore, a p p e a r e d to be oper-

A. V A I S H A M P A Y A N and A. B. P R A S A D

432

Table 3. Data* on frequencies of mutation for BLITOx-resistance in Nostoc muscorum and Nostoc linckia, scored on 45.5 ppm BLITOX-supplemented mineral medium

Strain

Nitrogen source in the mutant selection medium

Phenotypic expression of the resulting mutants and their designations

Mutation frequenciest

N. mascorum N. muscorum N. muscorum

N2 NO~ N2, overlayered with N O 2

M het + nif + nitx

2.8-t- 1.2 x 10 . 6 x

M h e t + nef- nit-

3.74-0.9 x 10 - s

N. linckia N. linckia N. linckia

N2 NO 3 N2, overlayered with NO~-

X x

X x

L het + n i f - nit-

2.74-0.7 x 10 -7

* The values are means of five independent readings with their respective standard errors. "~Total number of colony-forming units were counted and multiplied by 1000 to give the real population size (as it was a 1000 times diluted suspension of the thick culture spread on 45.5 ppm BLiTOX-supplemented solid mineral medium for scoring BLxTox-resistant mutants). The number of mutant colonies which appeared in proportion to this real population size was considered to be the mutation frequency in the particular nitrogen source. These mutants were found stable through their repeated transfer to fresh media. Their reversion analysis was, however, made and recorded in Table 4. Table 4. Data* on growth,t heterocystfrequency,~ nitrogenase activity,§ nitrate reductase activityll and prototrophic reversion frequency¶ of different classes of BLiTOx-resistant mutants ofN. linckia and N. muscorum BLiTOx-resistant mutants Characteristics Growth in N2 Growth in NO~Growth in NO~Het. freq. in N2 Het. freq. in N O ~ Nitrogenase activity Nitrate reductase activity Reversion frequency to het + nif + nit + prototrophy

M het + nif + nit-

M her + n i f - nit-

L het + n i f - nit-

0.4354- 0.025 0.0 0.725 4-0.010 4.94-0.7 4.54-0.3 3.6 4- 0.8 0.0

0.0 0.0 0.735 4-0.025 4.94-0.3 4.54-0.8 1.2 4- 1.1 0.0

0.0 0.0 0.720_+0.015 5.74-0.4 4.6-t-0.5 0.7 4- 0.9 0.0

2.5+_ 1.3 x 10 -5

2.1 4- 1.6 x 10 -6

2.84- 1.4 x 10 -6

* The values are means of five independent readings with their respective standard errors. ~"Increase in optical density at 663 nm on ninth day of inoculation (initial optical density was uniformly 0.005 for all the three strains). Number of heterocysts per hundred vegetative cells. Heterocysts were not found in N O ~ medium. § (n moles ethylene//tg chlorophyll-a)/hr. Nitrogenase activity was found only in N 2 medium and not in N O ~ or N O ~ medium. IIug nitrite#tg protein, ¶ Number of revertant colonies in proportion to the total population size. These mutants no longer had any resistance for BLITOX and behaved just like the parental strains.

BLITOX-RESISTANT MUTANTS ative at the level of the two enzyme systems, nitrogenase and nitrate reductase. Since identical inhibitory effects were exerted by copper sulfate, it was strongly believed that the observed inhibitory effect(s) of BLIa'ox were the result of Cu-toxicity in N . muscorum and N . linckia. The observation that FeSO 4 could reverse the inhibitory action of BLn'ox and CuSO4 on nitrogenase activity and consequently N2-mediated growth, clearly suggested that these Cu salts caused a Fe-deficiency in the two Nostoc species and as a result inactivated the F e - M o (ironmolybdenum) co-factor that is essentially responsible for N 2 fixation, t12'xT) Such effects of Cu on Fe-deficiency in higher plants have been established, t4'5'x°'34) but is first reported here for the species used in the present research. The M het + n i l + nit- mutant strain indicated a reduced nitrogenase and no nitrate reductase activity with heterocysts in N O ~ medium. The reduced nitrogenase activity may not be due to a defect in the nitrogenase enzyme complex, because this strain had a very high reversion frequency to prototrophy. Apparently there has been some defect in the heterocysts as a result of a mutational event which is responsible for such a reduced nitrogenase activity in this mutant. Further biochemical studies are in progress to locate the heterocyst disorders in the M het + n i f + nit- mutant strain. The loss of nitrate reductase activity coupled with the formation ofheterocysts in NO~ medium suggest that the mutant lacks active nitrate reductase (the enzyme responsible for the repression of heterocysts in N O ~ medium), tts'2x) Although showing a lower magnitude of N2-mediated and no NO~-mediated growth, this strain can be considered of some applied value as it shows a slower but significant nitrogenase activity in the presence or absence of BLIa'OX fungicide and can be inoculated into BLITOX-treated paddy-fields. It is puzzling and difficult to explain that the frequency of double ( n i l - nit- ) mutant formation by N. muscorum ( M het + n i l - nit- mutant) is almost ten times the frequency of single (nit-) mutant formation ( M het + n i l + nit- mutant). However, studies on the genetics of N . muscorum have shown for the last 7-8 years that this organism has a high degree of genetic elasticity in comparison to all other filamentous cyano-

433

bacteria, and this may be one of the reasons why a double mutation rather than a single one is prevalent in this alga. SINGH and Some ~2°~ while isolating a nitrate reductase-less mutant of N . muscorum, found n i l - nit- colonies with greater frequencies than n / f - or nit- alone. The M het + n i f - nit- and L het + n i f - nitmutants have shown insignificant nitrogenase activity. The marked abnormal reduction in nitrogenase activity of these two mutants may not be correlated with a defect in heterocysts, as interpreted for the M het ÷ n i f + nit- mutant strain, as their reversion frequency to prototrophy is almost ten times lower than that of the M het + n i l + nit- mutant. Additionally, nitrogenase activity of these two mutants is highly reduced and insignificant, and reflects a general loss of the function of the enzyme in question. Similarly, an absence of nitrate reductase activity, as shown by these two mutants, suggests that a mutation for BLia'ox-resistance is accompanied with a complete failure of nitrate reductase in addition to the suppression of nitrogenase activity; thus, all B~.xa-ox-resistant colonies could not be derived from the N O ~ medium. Brill and his co-workers have recently demonstrated that Mo co-factor is responsible for nitrate reductase activity, and an F e - M o co-factor is responsible for the activation of nitrogenase in Azotobacter vinelandii. ~x2"16"xT) It can be implied, therefore, that Cu has acted not only at the level of Fe-uptake (causing inactivation of Fe--Mo co-factor, i.e. nitrogenase) but also probably at the level of Mo co-factor, rendering the nitrate reductase enzyme inactive. These findings strongly suggest that the observed inhibition of nitrogenase and nitrate reductase activities is due t~ Cu-toxicity and that there is a relationship between Cu-resistance and defects in nitrogenase and/or nitrate reductase. This is further confirmed by the finding that the prototrophic ( het + n i f + nit +) revertants o f M het + n i f + n i t - , M het + n i l - nit- or L het + n i f - nitmutants could no longer resist BLn'ox and acted like the Cu-sensitive het + n i f + nit + parental strains of N . linckia and N. muscorum. Further detailed studies on Cu-interactions (in vitro) with nitrogenase/nitrate reductase or F e - M o / M o cofactor(s) are in progress to confirm these postulates. These studies suggest the necessity of screening

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A. VAISHAMPAYAN and A. B. PRASAD

other metallic pesticides for their effects on various metal ion-dependent life processes. Such studies will bridge the existing gap in our knowledge regarding the relationship between life sciences and inorganic chemistry. T h e possibility of the isolation of pesticide-resistant m u t a n t strains of N2-fixing blue-green algae in the present study, raises a hope for an isolation of m u t a n t strains resistant to those pesticides that might not injure the nitrogenase system. Such strains will be able to survive and continue fixing nitrogen at a useful rate in pesticide-treated fields. Therefore, it seems probable that isolation of such resistant

m u t a n t strains m a y help in maintaining a balance between the use of pesticides in eradicating pests and weeds and maintaining N 2 fixation capability of p a d d y microflora. Experiments in this direction are in progress. Acknowledgements--The authors feel thankful to Mr. T. BAOeHI, ICRISAT, Hyderabad, for carefully running the samples on gas chromatograph and assessing their nitrogenase activity. The grant of financial assistance for this work to one of them (A.V.) in the form of a PostDoctoral Research Fellowship by C.S.I.R., Govt. of India, New Delhi- 110001, is gratefully acknowledged.

REFERENCES 1. ASHIDAJ. (1965) Adaptation of fungi to metal toxicants. Annu. Rev. Phytopathol. 3, 153-174. 2. BALDRYM. G. D., HOOARTHD. S. and DEANA. C. R. (1977) Chromium and copper sensitivity and tolerance in Klebsiella ( Aerobacter) aerogenes. Microbios Lett. 4, 7-16. 3. BASUS. N., Bose R. G. and BHATTACHARYYAJ.P. (1955) Some physiological studies on a copper tolerant PeniciUium species, a7. Sci. Ind. Res. 14, 4653. 4. BROWNJ.C. and AMBLERJ.C. (1973) 'Reductants' released by roots of Fe-deficient soybeans. Agron. ft. 65, 311-314. 5. BROWNJ.C. and JONESW. E. (1977) Fitting plants nutritionally to soils. I. Soybeans. Agron. aT. 69, 399-404. 6. FOGGG. E. (1974) Nitrogen fixation. Pages 560582 in W. D. P. STEWART,ed. Algal physiology and biochemistry, Blackwell Scientific Publications, Oxford. 7. GADD G. M. and GRIFFITHSA. J. (1978) Copper tolerance of Aureobasidium puUulans. Microbios Lett. 6, 117 124. 8. GERLOFFG. C., FITZGERALDG. P. and SKOOGF. (1950) The isolation, purification and culture of blue-green algae. Am. aT. Bot. 37, 216-218. 9. HAWXBVK., TUBEAB., OWNBVJ. and BASLERE. (1977) Effects of various classes of herbicides on four species of algae. Pestic. Physiol. Biochem. 7, 203209. I0. LINGLEJ. C., TIFFINL. O. and BROWNJ. C. (1963) Iron uptake-translocation of soybeans as influenced by other cations. Plant Physiol. 38, 71-76. 11. LUNDQVXSTI. (1970) Effects of two herbicides on nitrogen fixation by the blue-green algae. Sven. Bot. Tidskr. 64, 460-461.

12. PIENKOSP. T., SHAHV. K. and BRILLW.J. (1977) Molybdenum co-factor from molybdoenzymes and in vitro reconstitution ofnitrogenase and nitrate reductase. Proc. Natl Acad. Sci. USA 74, 5468-5471. 13. Ross I.S. (1975) Some effects of heavy metals on fungal cells. Trans. Br. Mycol. Soc. 64, 175-193. 14. ROTHJ. A., WALLIHANE. F. and SHARPLESSR. G. ( 1971 ) Uptake by oats and soybeans of copper and nickel added to a peat soil. Soil Sci. 112, 338-342. 15. SADLERW. R. and TRUDINGERP. A. (1967) The inhibition of micro-organisms by heavy metals, Miner. Deposita 2, 158-168. 16. SHAHV. K. and B~LL W.J. (1977) Isolation of an iron-molybdenum co-factor from nitrogenase (nitrogen fixation). Proc. Natl Acad. Sci. USA 74, 3249-3253. 17. SHAHV. K., CHISNELLJ.R. and BRILLW.J. (1978) Acetylene reduction by the iron-molybdenum cofactor from nitrogenase. Biochem. Biophys. Res. Commun. 81, 232-236. 18. SINGn H, N., LADHAJ. K. and KUMAR H. D. (1977) Genetic control of heterocyst formation in the blue-green algae Nostoc muscorum and Nostoc linckia. Arch. Mikrobiol. 114, 155-159. 19. SINGHH. N., SINGHH. R. and VAISHAMPAYANA. (1979) Toxic and mutagenic action of the herbicide alachlor (lasso) on various strains of the nitrogen-fixing blue-green alga Nostoc muscorum and characterization of herbicide-induced mutants resistant to methylamine and L-methionine-DLsulfoximine. Envir. Exp. Bot. 19, 5-12. 20. SINGHH. N. and SONIEK. C. (1977) Isolation and characterization of chlorate-resistant mutants of the blue-green alga Xostoc muscorum. Mutat. Res. 43, 205-212. 21. SINGHH. N., SONIEK. C. and SINGHH. R. (1977)

BLITOX-RESISTANT MUTANTS

22.

23.

24.

25.

26.

27.

28.

Nitrate regulation of heterocyst formation and nitrogen fixation in the blue-green alga Nostoc muscorum. Murat. Res. 42, 447-452. SINOH H. N. and VAISHAraPAYAN A. (1978) Biological effects of rice-field herbicide machete on various strains of the nitrogen-fixing blue-green alga Nostoc muscorum. Envir. Exp. Bot. 18, 87-94. Slsori P. K. (1974) Algicidal effect of 2,4dichlorophenoxyacetic acid on the blue-green alga Cylindrospermum sp. Arch. Mikrobiol. 97, 69-72. SmQH R. N. (1961) The role of blue-green algae in nitrogen economy of lndian agriculture, p. 175, Indian Council of Agricultural Research Publications, New Delhi, India. SNELL F. D. and SNELL C. D. (1949) Nitrites by sulphanilamides and N- (1-naphthyl) ethylene diamide hydrochloride. Pages 804--805 in D. VAN ROSTRAND, ed. Colorimetric methods of analysis, I I I edn, Vol. 2, John Wiley, New York. STARKEr R. L. (1973) Effects o f p H on toxicity of copper to Scytalidium sp., a copper tolerant fungus, and some other fungi. 07. Gen. Microbiol. 78, 217225. STARKEYR. L. and WAKSMANS. A. (1943) Fungi tolerant to extreme acidity and high concentration of copper sulphate. 07. Bacteriol. 45, 509-519. STEWART W. D. P. (1975) Biological cycling of nitrogen in intertidal and supralittoral marine

29.

30.

31.

32.

33.

34.

435

environments. Pages 637-660 in H. BARNES, ed. Proc. 9th Europ. Mar. Biol. Syrup., Aberdeen University Press. STEWARTW. D. P., FITZOE~LD G. P. and BURRIS R. H. (1967) In situ studies on N 2 fixation using the acetylene reduction technique. Proc. aVatl Acad. Sci. USA 58, 2071-2078. STEWART W. D. P., HAYSTEAD A. and DHARMAWARDENE M. W. N. (1975) Nitrogen assimilation and metabolism in blue-green algae. Pages 129-159 in IBP Nitrogen fixation by free-living micro-organisms, Vol 6, Cambridge University Press. VAISHAMPAYANA., SINGH H, R. and SINGH H. N. (1978) Biological effects of rice-field herbicide stare f-34 on various strains of the nitrogen-fixing bluegreen alga Nostoc muscorum. Biochem. Physiol. Pflanz. 173, 410-419. VENKATARAMANG. S. (1972) Algal biofertilizers and rice cultivation. Today and Tomorrow's Publications, New Delhi, India. V~NKATARAMANG. S. (1981) Blue-green algae: a possible remedy to nitrogen scarcity. Curt. Sci. 50, 253-256. WALt~eE A. and DE KOeK P. C. (1966) Translocation of iron in tobacco, sunflower and bush bean plants. Pages 3-9 in A. WALLACE, ed. Current topics in plant nutrition. J. W. Edwards, Ann Arbor.