Effect of pretilachlor on nitrogen uptake and

Acta Physiol Plant (2015) 37:177 DOI 10.1007/s11738-015-1923-7

ORIGINAL ARTICLE

Effect of pretilachlor on nitrogen uptake and assimilation by the cyanobacterium Desmonostoc muscorum PUPCCC 405.10 D. P. Singh1 • J. I. S. Khattar1 • Gurdeep Kaur1 • Meenu Gupta1 • Yadvinder Singh1 Arvind Gulati2

•

Received: 31 December 2014 / Revised: 10 April 2015 / Accepted: 23 July 2015 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2015

Abstract Tolerance limit, kinetics of nitrogen uptake and activity of nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) enzymes of cyanobacterium Desmonostoc muscorum PUPCCC 405.10 were studied under the regime of chloroacetanilide herbicide pretilachlor. The organism was isolated from the paddy field under application of the herbicide and tolerated pretilachlor up to 10 ppm under laboratory conditions. The incubation of the cyanobacterium in 2.5 ppm pretilachlor did not cause significant effect on N uptake while supplementation of 5–10 ppm pretilachlor in medium reduced the rate of nitrate and nitrite uptake by 35–73 % with 50 % decrease in Vmax and no change in Km. These results indicated that herbicide did not affect the affinity of uptake system to nitrate and nitrite. In presence of herbicide, the activity of nitrogen Communicated by G. Klobus.

assimilation enzymes was inhibited by 16 % for NR and 18 % for NiR. Unchanged Km and decreased Vmax (50–60 %) in presence of herbicide indicated non-competitive-type inhibition of the enzymes. A 50 % decrease in Vmax and Km of ammonium uptake indicated un-competitive type inhibition of the ammonium transport system by the herbicide. GS activity also exhibited non-competitive inhibition in presence of pretilachlor with 21 % decrease in activity with unchanged Km and 40 % decrease in Vmax. Likewise, decreases in nitrogenase activity by 43 % and heterocyst formation by 40 % were recorded in presence of herbicide. Results indicated that pretilachlor affected nitrogen assimilation at uptake level and interacted with nitrogen-assimilating enzymes in a non-competitive manner. Keywords Cyanobacteria Desmonostoc muscorum Enzyme kinetics Herbicide Nitrogen assimilation Nitrogen uptake Pretilachlor

& D. P. Singh [email protected] J. I. S. Khattar [email protected]

Introduction

Gurdeep Kaur [email protected]

Nitrogen, the fourth most abundant nutrient element in plants, is an essential component of proteins, nucleic acids, hormones, chlorophyll and several other important primary and secondary plant constituents. Most plants obtain the bulk of their nitrogen in the form of nitrate, nitrite or ammonium from the soil pool. Plants compete with soil microorganisms for available nitrogen in natural and agricultural ecosystems. More than half of nitrogen consumed in agri-ecosystems is derived from the native soil nitrogen pool maintained through biological nitrogen fixation by both soil heterotrophs and autotrophs (Roger and Ladha 1992). The Gram-negative cyanoprokaryotes occurring across the world have the ability to fix atmospheric nitrogen in association with oxygenic

Meenu Gupta [email protected] Yadvinder Singh [email protected] Arvind Gulati [email protected] 1

Department of Botany, Punjabi University, Patiala, Punjab 147002, India

2

Plant Pathology and Microbiology Laboratory, CSIRInstitute of Himalayan Bioresource Technology, Post Box No. 6, Palampur, Himachal Pradesh 176 061, India

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photosynthesis. These are also an important component of paddy field ecosystems and contribute to soil fertility as natural biofertilizers (Fernańdez-Valiente et al. 2000; Mishra and Pabbi 2004; Singh and Datta 2006, 2007). Annual contribution of nitrogen to soil ecosystems by cyanobacteria is estimated to be between 25 and 30 kg N per hectare (Kannaiyan 1990). These microbes also improve organic content, water holding capacity, release vitamins, plant stimulating hormones, extracellular polysaccharides, and solubilize phosphates in the soil (Tiwari et al. 1991; Whitton and Potts 2000). Paddy is an important staple food crop with more than half of the world population, including about 80 % population of Asian countries, depending upon rice for the dietary needs (Way 1976). Paddy crop is attacked by more than 70 species of insects, pests and fungal diseases leading to decrease in crop yield. In India, yield losses due to weeds alone account for 45 % of the total loss of the crop (Yaduraju 2003). In Punjab, the granary state of India, paddy is cultivated in about 2.8 million ha, with the total production of about 10.8 million tons during 2010–2011 and average yield of 5787 kg per ha (Gill and Bajwa 2012). A large number of herbicides such as anilofos, butachlor, pendimethalin, pyrazolsulfuron ethyl, oxadiargyl and pretilachlor are recommended for weed control in the rice fields (Gill and Bajwa 2012). Indiscriminate use of herbicides is a great threat to environment and beneficial soil microflora (Adhya et al. 2000; Irisarri et al. 2001; Kaur et al. 2002). Successful utilization of cyanobacterial strains in rice fields as biofertilizer requires their tolerance to various agrochemicals including herbicides. Therefore, screening and selection of naturally occurring populations of herbicide-resistant cyanobacteria from paddy field ecosystems, and their use as biofertilizers may play an important role in the establishment of soil fertility. The effects of pretilachlor, propanil and glyphosate on Anabaena fertilissima (Inderjit and Kaushik 2010), bispyribac sodium, 2,4 dichlorophenoxy acetic acid and methyl chlorophenoxy acetic acid on Anabaena sp. (Legane´s and Fernańdez-Valiente 1992; Okmen and Ugur 2011; Okmen et al. 2013), bentazon and molinate on Nostoc muscorum and Anabaena cylindrica (Galhano et al. 2010a, b), paraquat on Anabaena oryzae and Nostoc ellipsosporum (Pandey et al. 2011) and anilofos on Anabaena torulosa, Synechocystis sp. PUPCCC 64 and Oscillatoria simplicissima (Singh and Sandhu 2010; Singh et al. 2012, 2013) have been studied. Many herbicides whose effects on cyanobacteria have been studied earlier are either banned or their use has been discontinued. Carbon and nitrogen metabolism are important metabolic pathways that regulate growth and biomass yield of cyanobacteria. Thus, the present investigation was undertaken to understand the effect of chloroacetanilide herbicide pretilachlor, a newly launched and extensively used agrochemical in Punjab state of India, on nitrogen uptake and assimilation by

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Acta Physiol Plant (2015) 37:177

Desmonostoc muscorum PUPCCC 405.10. The present study is important since the microorganism can be used for biofertilizer technology in India.

Materials and methods Isolation, purification, culture conditions and maintenance of the microorganism Desmonostoc was isolated from rice fields of village Koom Kalan (30°540 4800 N; 76°30 5600 E), Ludhiana district of Punjab state, India. Isolation and purification of the microorganism were performed by serial dilution and plating method (Stanier et al. 1971) on modified Chu-10 medium (Safferman and Morris 1964), where calcium nitrate was replaced with an equimolar amount of calcium chloride. The composition of Chu-10 medium was (g L-1): CaCl2.2H2O, 0.232; K2HPO4, 0.01; MgSO47H2O, 0.025; Na2CO3, 0.020; Na2SiO35H2O, 0.44; citric acid, 0.0035; ferric citrate, 0.0035; micronutrients (mg L-1): B (H3BO3), 0.5; Zn (ZnSO47H2O), 0.05; Mn (MnCl24H2O), 0.5; Cu (CuSO45H2O), 0.02; Mo (MoO3), 0.01; Co (CoCl2), 0.04. The pH of the medium was adjusted to 7.5. Stock and experimental cultures were propagated and maintained in this medium in a culture room at 28 ± 2 °C. The cultures were illuminated for 14 h a day with daylight fluorescent tubes providing a light intensity of 44.5 lmol photons m-2 s-1 (lE) on the surface of culture vessels unless otherwise stated. The cultures were agitated regularly by manual shaking 2–3 times daily to keep them in a homogenous state. Exponentially growing cultures (8 days old) were used in all the experiments. Identification of cyanobacterium isolate The cyanobacterium was identified on the basis of phenotypic characters (Desikachary 1959; Koma´rek and Anagnostidis 1989; Koma´rek and Hauer 2013) combined with partial 16S rRNA gene and rbcL gene sequencing. Genomic DNA extraction was done by HiPurATM plant genomic DNA Miniprep Purification Spin Kit (HIMEDIAÒ, Mumbai, India). Cyanobacterial 16S rDNA was amplified using 5 pmol of the cyanobacteria-specific primers (Nu¨bel et al. 1997).The total PCR mixture was 50.0 lL, comprising 200 lmol L-1 dNTPs, 50 lmol L-1 each primer, 19 PCR buffer, 3 U Taq polymerase, and 100 ng genomic DNA. The thermo-cycling conditions involved an initial denaturation at 94 °C for 4 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 52 °C, 2 min at 72 °C and final extension at 72 °C for 8 min. The rbcL gene was amplified using the primers GF-AB (50 -GARTCTTCIACYGGTACYTGGAC-30 ) and GR-E (50 -


AACTCRAACTTGATTTCYTTCC-30 ) (Tomitani et al. 2006). PCR mixture of 25 lL comprised 50 ng DNA template, 19 Go TaqÒ Green Master Mix (Promega Corporation, Madison, USA) and 25 lmol L-1 each primer. Amplification was done by initial denaturation at 94 °C for 5 min, followed by first 10 cycles of 10 s at 94 °C, 20 s at 50–60 °C and 40 s at 72 °C, and next 25 cycles of 30 s at 94 °C, 1 min at 52 °C and 1 min at 72 °C and final extension at 72 °C for 7 min. The gel-purified product was obtained using GeneJETTM Gel Extraction Kit (Fermentas, Vilnius, Lithuania). The sequencing was done using BigDyeÒ Terminator v3.1 cycle sequencing kit and an ABI Prism 310 Genetic Analyzer (Applied Biosystems, CA, USA). The sequences were analyzed using BLASTn for 16S rRNA gene and BLASTx for rbcL gene (http://www.ncbi.nlm.nih.gov/Blast) search algorithm and aligned to near neighbors. The phylogenetic trees were constructed from the multiple-aligned data using the neighbor-joining (NJ) algorithmic Kimura’s two-parameter as implemented within the MEGA version 6.0 (Tamura et al. 2013). Nucleotide sequences obtained during present study have been deposited in the NCBI GenBank database with accession number KM 225636 for 16S rRNA and KM 225637 for rbcL gene sequences. The cyanobacterial species is being maintained in our culture collection under code PUPCCC 405.10. Tolerance limit of the microorganism to pretilachlor Tolerance limit of the test strain against pretilachlor was determined by studying its growth in graded concentrations of pretilachlor (2.5–15 ppm). These concentrations were prepared in 250-mL Erlenmeyer flasks containing 100 mL modified Chu-10 medium from the stock solution of commercial grade herbicide. Exponentially growing stock cultures, after two washings with sterilized double distilled water, were inoculated in flasks containing pretilachlorsupplemented media to get 0.1 initial absorbance of cultures at 720 nm. At regular intervals of 2 days, extending up to 12 days, 10 mL cultures were withdrawn and growth was measured as increase in absorbance of the cultures with spectrophotometer (Spectronic 20 D?, USA). Data of 6 d were taken to calculate percent growth inhibition. Generation time of the test organism in graded concentration of pretilachlor was determined from the linear portion of growth curve following Forlani et al. (2008). Nitrogen uptake Nitrogen (nitrate, nitrite and ammonium) uptake by the test organism was studied by measuring its depletion from the medium with time. Cyanobacterial suspension, after two washings with double distilled water, was added separately

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in media containing either nitrate as potassium nitrate, nitrite as sodium nitrite or ammonium as ammonium chloride and incubated under light. At regular intervals of time, cells were separated by centrifugation at 5000g and residual amounts of nitrate, nitrite and ammonium were determined according to Robinson et al. (1959), Nicholas and Nason (1957) and Solarzano (1969), respectively. N uptake kinetics was studied by determining N uptake at varied concentrations of each nitrogen source and by plotting Lineweaver–Burk double reciprocal plots. Enzyme assay Nitrate and nitrite reductase activity The whole cell nitrate reductase (NR) activity was assayed as nitrate reduction with sodium dithionite and reduced methyl viologen as the electron donor (Herrero et al. 1981). Ten ml cell suspensions in nitrate containing basal medium supplemented with 10 ppm pretilachlor were incubated at 28 ± 2 °C for 24 h under fluorescent tube light giving a photon flux of 44.5 lE. At regular intervals, one mL cell suspension in triplicate after repeated washings with double distilled water was suspended in NaHCO3– Na2CO3 buffer and agitated with toluene (20 lL) for 3 min. Toluene-treated permeabilized cells were used as enzyme extract. The reaction mixture contained in a final volume of 1 mL, 0.2 mL enzymes extract; NaHCO3–Na2CO3 buffer (pH 10.5, 100 lmol); KNO3, 20 lmol; methyl viologen, 4 lmol; and 10 lmol of sodium dithionite (freshly prepared in 0.3 mol L-1 NaHCO3). The reaction mixture was incubated for 10 min at 30 °C and nitrite formed was estimated. One unit (U) of enzyme is defined as lmol nitrite formed mg-1 protein min-1. The same procedure was followed for measuring nitrite reductase (NiR) activity except that KNO3 was replaced with NaNO2 and decrease in the amount of nitrite was estimated. One unit of enzyme is expressed as lmol nitrite decreased mg-1 protein min-1. The kinetics of these enzymes was studied at different concentrations of substrate, nitrate and nitrite, and by plotting Lineweaver–Burk double reciprocal plots. Glutamine synthetase activity Whole cell glutamine synthetase (GS) activity was assayed following Shapiro and Stadtman (1970). A known volume (10 ml) of cell suspensions in nitrate containing basal medium supplemented with 10 ppm pretilachlor was incubated at 28 ± 2 °C for 24 h under fluorescent tube light giving a photon flux of 44.5 lE. At regular intervals (6, 12 and 24 h), one ml cell suspension in triplicate washed 2–3 times with double distilled water was suspended in same volume of imidazole–HCl buffer

123


Vapor pressure at 25 °C

Herbicide

Activity

Solubility at 25 °C

(50 mmol L-1, pH 7.0) and agitated with toluene (20 lL) for 3 min which served as enzyme extract. The reaction mixture in a total volume of 2 mL consisted of 0.8 mL 50 mmol L-1 imidazole–HCl buffer (pH 7.0), 1 mL assay mixture (50 mmol L-1 Imidazole buffer, 0.1 mol L-1 glutamine, 0.1 mol L-1 manganese chloride, 0.01 mol L-1 ADP, 1 mol L-1 sodium arsenate and 2.0 mol L-1 hydroxylamine hydrochloride neutralized with 2 mol L-1 sodium hydroxide), and 0.2 mL enzyme extract. This solution was incubated at 37 °C for 30 min, and 4 mL stop mixture (prepared by adding 4 mL ferric chloride (10 %), 1 mL of trichloro-acetic acid (24 %), 0.5 mL of 6 mol L-1 HCl and 6.5 mL of double distilled water) was added to it. The absorbance of brown color was measured at 540 nm. L-glutamic acid c-monohydroxamate was used to construct a standard curve. One unit of enzyme is defined as lmol of c-glutamyl hydroxamate produced mg-1 protein min-1.

50 mg L-1

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0.133 MPa

177

123

C17H26ClNO2 51218-49-6

Formula CAS registry number

311.85

All chemicals used in media preparation and enzyme assays were obtained from Merck, India. Commercial grade pretilachlor (Rifit 50 %) manufactured by Syngenta Chemicals Limited, Maharashtra, India was used in the present study. The physicochemical properties of pretilachlor are given in Table 1 (Roberts 1998).

2-chloro-20 ,60 -diethyl-N-(2propoxyethyl) acetanilide

Chemicals

Molecular weight

Heterocyst frequency was calculated by counting the number of heterocysts present in trichomes per hundred vegetative cells under light microscope. Protein content was determined following Lowry et al. (1951).

Chemical name

Heterocyst frequency

Table 1 Physico-chemical characteristics of pretilachlor (Roberts 1998)

The nitrogenase activity was estimated by acetylene reduction assay following Stewart et al. (1968). The assay was carried out in 15-mL glass tubes sealed with rubber plugs containing 5 mL cyanobacterial suspension in presence of 10 ppm pretilachlor. The cultures tubes were incubated at 28 ± 2 °C under fluorescent tube light giving a photon flux of 44.5 lE. The acetylene 10 % (v/v) was injected in cultures tubes 30 min before measuring the nitrogenase activity. A portion of gas phase was withdrawn at desired time and the amount of ethylene produced was determined using a Gas Chromatograph (Shimadzu Scientific Instruments, USA) fitted with Porapak Q column. The amount of ethylene produced was calculated by integration of the peak and by comparing with a standard curve developed from injected amount of standard ethylene. One unit of enzyme is defined as nmol ethylene produced mg-1 protein h-1.

Structure

Nitrogenase activity


Page 5 of 14

Statistical analysis All the data are the average of three independent experiments ± standard deviation (SD). Data were statistically analyzed by applying one-way analysis of variance and Tukey’s honest significance difference test. All statistical analyses were tested against probability value at 95 % confidence level (p \ 0.05) using GraphPad Prism 6.0 version 6.0 (http://www.graphpad.com).

Results Selection and identification of test organism In the preliminary experiments, 45 cyanobacterial isolates were grown in presence of 2.5 ppm pretilachlor which was 3.3 times the field application dose. Of these, 25 isolates grew in this concentration of pretilachlor and were further Table 2 Growth of selected cyanobacterial strains in presence of pretilachlor on day 4 S. no.

Organism

177

grown in 5.0, 7.5 and 10 ppm of the herbicide (Table 2). Only one isolate (Nostoc sp. KK26) showed positive growth in 10 ppm of pretilachlor and was selected for the present study. This cyanobacterium is filamentous with thin sheath, heterocystous; trichome 3–4-lm broad; cells short barrel shaped to cylindrical, up to twice as long as broad; heterocysts spherical, pale yellowish, 5–7 lm in diameter; akinetes subspherical to oval, 6.4–9.6-lm long, 4.8–8.0-lm wide. On the basis of its morphological features isolate Nostoc sp. KK26 was identified as Nostoc muscorum. For molecular characterization, partial 16S rRNA gene sequence (638 bp) and 1038 bp nucleotide sequence of rubisco large subunit (rbcL) gene were obtained. BLASTn result of 16S rRNA gene sequence revealed that Nostoc sp. KK26 showed 100 % similarity with Nostoc sp. PCC 7120 (HM573458) and 98 % similarity with Desmonostoc muscorum (AJ630452), where as BLASTx result of rbcL gene sequence revealed that Nostoc sp. KK26 had 100 % similarity with Nostoc sp. PCC 7120 (NP485564). The phylogenetic tree of 16S rRNA gene and rbcL gene sequences aligned with sequences of reference strains obtained from NCBI GenBank grouped into a supported sequence cluster (Figs. 1, 2).

Pretilachlor (ppm) 2.5

5

7.5

10

1

Anabaena sp. JL7

?

?

-

-

2 3

Anabaena sp. JL14 Anabaena sp. KK12

? ?

? ?

? -

-

4

Anabaena sp. KK15

?

-

-

-

5

Anabaena sp. TM7

?

?

?

-

6

Anabaena sp. TM6

?

?

-

-

7

Anabaena sp. TM9

?

-

-

-

8

Calothrix sp. JL21

?

-

-

-

9

Calothrix sp. KK20

?

?

-

-

10

Calothrix sp. TM22

?

-

-

-

11

Calothrix sp. TM23

?

?

-

12

Gloeotrichia sp. JL32

?

-

-

-

13

Gloeotrichia sp. KK30

?

-

-

-

14

Gloeotrichia sp. TM34

?

-

-

-

15

Nostoc sp. JL2

?

?

?

-

16

Nostoc sp. JL4

?

?

-

-

17 18

Nostoc sp. JL5 Nostoc sp. TM24

? ?

? -

-

-

19

Nostoc sp. TM28

?

?

?

-

20

Nostoc sp. KK16

?

?

-

-

21

Nostoc sp. KK25

?

-

-

-

22

Nostoc sp. KK26

?

?

?

?

23

Scytonema sp. JL44

?

-

-

-

24

Scytonema sp. KK10

?

?

-

-

25

Scytonema sp. TM40

?

-

-

-

Growth (?), no growth (-) Bold one is identified further to species level only

Tolerance level of test organism to pretilachlor Tolerance limit of Desmonostoc muscorum PUPCCC 405.10 to pretilachlor was determined by growing it in basal medium supplemented with graded concentrations (2.5–15 ppm) of the herbicide. Pretilachlor affected the growth in a dosedependent manner with 15.8, 31.09, 65.0 and 76.09 % decrease in growth in 2.5, 5.0, 7.5, and 10 ppm of the herbicide in 6 days compared to control culture (Fig. 3). The cyanobacterium did not survive in 15 ppm pretilachlor as nearly 98 % cells were lysed and became colorless/hyaline with pigments released into the medium (Figs. 3, 4). The growth data also revealed 50.9 h doubling time in culture medium without pretilachlor and concentration-dependent increase in the generation time from 57.7 h at 2.5 ppm to 144 h at 10 ppm of the herbicide (inset Fig. 3). Effect of pretilachlor on N uptake and its assimilation Short-term experiments of 6 h duration were performed to study the effect of pretilachlor on N uptake and assimilation. Linear uptake of nitrate/nitrite/ammonium was observed up to 6 h (data not shown). Thus, the data of only 6 h are given. Nitrate as N source The results revealed that 2.5 ppm herbicide did not significantly affect nitrate uptake but pretilachlor higher than this

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Fig. 1 Phylogenetic tree showing relationship of Desmonostoc muscorum PUPCCC 405.10 with closely related taxa based on partial (638 bp) 16S rRNA gene sequence. The percentage of replicate tree with the associate taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using Kimura twoparameter method and analysis conducted in MEGA 6.06 software

Fig. 2 Phylogenetic tree showing relationship of Desmonostoc muscorum PUPCCC 405.10 with closely related taxa based on partial (1038 bp) rbcL gene sequence. The percentage of replicate tree with the associate taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using Kimura twoparameter method and analysis conducted in MEGA 6.06 software

concentration inhibited nitrate uptake by the cyanobacterium in a dose-dependent manner. The control cultures were able to take up 0.86 lmol nitrate mg-1 protein in 6 h from the medium while in the presence of 5.0, 7.5 and 10 ppm of the pretilachlor, nitrate uptake was decreased by 46, 58 and 73 %, respectively (Table 3). Effect of 10 ppm pretilachlor on kinetics of nitrate uptake was studied. A Lineweaver–Burk plot revealed that Vmax value for

123

nitrate uptake decreased to 1.6 lmol nitrate taken up mg-1 protein h-1 in presence of pretilachlor compared to 3.3 lmol nitrate taken up by control culture, while Km value was 0.3 mmol nitrate L-1 for both conditions (Fig. 5). Nitrate is converted to nitrite by the enzyme NR. Thus, the effect of pretilachlor on NR activity and its kinetics was studied. A reduction of 15.9 % in NR activity was observed in the pretilachlor-supplemented cultures as compared to


Fig. 3 Growth inhibition of Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor (inset effect of pretilachlor on generation time). Generation time was calculated from the growth data between 4 and 8 days. All data in the figure are significantly different at 95 % confidence level (p \ 0.05)

control cultures after 12 h (Table 4). The kinetics of NR revealed 25 U and 16.6 U as Vmax values for control and pretilachlor-supplemented cultures, respectively, and the same Km value of 10 lmol nitrate L-1 for both control as well as pretilachlor-containing cultures (Fig. 6). Nitrite as N source Rate of nitrite uptake by the cyanobacterium significantly decreased with the increase in pretilachlor concentration above 2.5 ppm in the medium. The control cultures removed 0.99 lmol nitrite mg-1 protein from the medium in 6 h while in the presence of 5.0, 7.5 and 10 ppm herbicide, the rate of nitrite uptake decreased by 33, 62 and 68 % respectively, compared to control (Table 3). The kinetics for nitrite uptake revealed Vmax values of 0.33 lmol and 0.16 lmol nitrite mg-1 protein h-1 for control and pretilachlor-supplemented cultures, respectively. The Km value remained same (0.5 mmol nitrite L-1) for both control as well as herbicidecontaining cultures (Fig. 7). NiR activity of test organism in presence of pretilachlor decreased by 18.12 % in 12 h compared to control cultures (Table 4). The kinetics of NiR revealed Vmax value of 12.5 U for control culture and 6.25 U for pretilachlor-containing cultures. The Km value was 10 lmol nitrite L-1 for both control and pretilachlor-containing cultures (Fig. 8). Ammonium as N source Low concentration of pretilachlor (2.5 ppm) did not cause significant decrease in ammonium uptake by the organism

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but higher concentration of herbicide exhibited significant negative effect on uptake of ammonium. In 6 h, the control cultures removed 1.1 lmol ammonium mg-1 protein from the medium while in the presence of 5.0, 7.5 and 10 ppm pretilachlor, the uptake of ammonium was 0.95, 0.84 and 0.70 lmol mg-1 protein, respectively (Table 3). The kinetics of ammonium uptake by the cyanobacterium revealed 3.3 lmol ammonium uptake mg-1 protein h-1 as Vmax for control cultures and 2.0 lmol ammonium uptake mg-1 protein h-1 for the herbicide-containing cultures. Km value for control culture and herbicide-containing cultures was 1 and 0.55 mmol ammonium L-1, respectively (Fig. 9). GS activity of the test cyanobacterium grown in N2-free medium supplemented with 10 ppm herbicide showed 21 % decrease after 12 h (Table 4). The kinetic studies of GS revealed 0.5 U for control cultures and 0.2 U for pretilachlor-containing cultures. Km value for both control and pretilachlor-containing cultures was 5 mmol ammonium L-1 (Fig. 10). Nitrogen fixation Nitrogenase activity of control cultures did not change significantly with time (14 U) but in 10 ppm pretilachlor the nitrogenase activity decreased by 48 % within 6 h and afterwards there was no significant change in activity (8 U) up to 24 h (Table 4). Low doses of pretilachlor did not significantly affect the heterocyst frequency. The heterocyst frequency of 4-day-old control cultures was 6.9 % which showed 7.4, 13.0, and 40.5 % decrease in presence of 5, 7.5 and 10 ppm herbicide, respectively (Table 5).

Discussion The diazotrophic cyanobacterium Nostoc sp. KK26 was isolated from a paddy field where the pre-emergence herbicide pretilachlor is used on a large scale. On the basis of morphological features, Nostoc sp. KK26 was identified as Nostoc muscorum. BLASTn result of partial 16S rRNA gene sequence revealed 100 % similarity with Nostoc sp. PCC 7120 (HM573458) and 98 % similarity with Desmonostoc muscorum (AJ630452), where as BLASTx result of rbcL gene sequence revealed its 100 % similarity with Nostoc sp. PCC 7120 (NP485564). Recently, Hrouzek et al. (2013) had described Desmonostoc gen. nov. which included the common species Nostoc muscorum Agardh ex Bornet et Flahault and several other cyanobacterial strains previously assigned to the genus Nostoc. Hence, the present stain was named as Desmonostoc muscorum PUPCCC 405.10. Since the cyanobacteria, especially diazotrophic ones, are important biofertilizer microorganisms in soil

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Fig. 4 Photomicrographs of Desmonostoc muscorum PUPCCC 405.10 on day 4 in the presence of different concentrations of pretilachlor a control, b 5 ppm, c 10 ppm, d 15 ppm (scale bar 10 lm) Table 3 Nitrogen source uptake (lmol mg-1 protein) by Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor (ppm) after 6h Nitrogen source

Control

2.5

5.0

7.5

10.0

Nitrate

0.86 ± 0.15a

0.79 ± 0.06 (8.2)a

0.46 ± 0.17 (46.51)

0.36 ± 0.15 (58.13)

0.23 ± 0.14 (73.25)

Nitrite

a

0.99 ± 0.16

0.91 ± 0.08 (8.1)a

0.66 ± 0.18 (33.33)

0.38 ± 0.15 (62.0)

0.32 ± 0.17 (67.67)

Ammonium

1.10 ± 0.18a

1.0 ± 0.09 (9.1)a

0.95 ± 0.14 (13.6)

0.84 ± 0.15 (23.6)

0.71 ± 0.16 (35.72)

Percent inhibition of nitrogen uptake over control is given in parentheses All data presented are the mean values of three independent experiments ±SD Data within each row, except with same lowercase alphabet, are significantly different from each other at the 95 % confidence level (p \ 0.05)

ecosystem, it is necessary to examine the effect of agricultural pollutants such as pesticides on the growth, development and nitrogen assimilation of cyanobacteria (Singh et al. 2011). Desmonostoc muscorum PUPCCC 405.10 tolerated herbicide up to 10 ppm which is 13.3 times the recommended field application dose (Fig. 3). The tolerance level of microorganisms towards herbicides

123

depends upon the species, nature and mode of herbicide action. Growth inhibition in other cyanobacteria by herbicides has been reported such as by anilofos in Anabaena torulosa (Singh et al. 2012) and Synechocystis (Singh et al. 2013), by butachlor, bensulfuron-methyl in Nostoc sp. (Chen et al. 2007; Dowidar et al. 2010), glyphosate in Anabaena sp., Leptolyngbya boryana, Nostoc punctiforme,

8.36 ± 0.2 (43.12)

177

All data presented are the mean values of three independent experiments ±SD

Percent inhibition of enzyme activity over control is given in parentheses

54.8 ± 2.8

Data of control and 10 ppm-treated cells of each enzyme are significantly different from each other at 95 % confidence level (p \ 0.05)

14.7 ± 1.1 14.2 ± 1.2 (19.96) 17.7 ± 1.4 50.6 ± 1.1 (18.12) 61.8 ± 2.4

6.9 ± 0.3 (48.5)

24

46.8 ± 1.6 (14.59)

13.9 ± 1.0

13.6 ± 1.0 10.86 ± 1.0 (16.8)

12.2 ± 1.0 (21.28) 15.5 ± 1.0

13.06 ± 0.9 48.9 ± 1.3 (18.06)

50.6 ± 1.3 (18.12) 61.8 ± 2.0

59.68 ± 1.9 29.9 ± 1.2 (17.4) 36.2 ± 1.3

54.1 ± 2.5

6

12

45.5 ± 1.3 (15.89)

Control 10 ppm Control Control 10 ppm Control

10 ppm

Nitrogenase (U) Glutamine synthetase (U) Nitrite reductase (U) Nitrate reductase (U) Time (h)

and Microcystis aeruginosa (Lo´pez-Rodas et al. 2007; Forlani et al. 2008), and by molinate and bentazon in Anabaena cylindrica and Nostoc muscorum (Galhano et al. 2010b, 2011). The mode of action of these herbicides was on photosynthesis (bentazon), cell division and inhibition of synthesis of long-chain fatty acids (anilofos, butachlor and molinate), the shikimate pathway (glyphosate), and acetolactate synthase (bensulfuron-methyl) of the target weeds (Mallory-Smith and Retzinger 2003). Few studies related to herbicide mode of action on cyanobacteria are available in the literature. Glyphosate acts on shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase in cyanobacteria (Forlani et al. 2008). Mutagenic studies on Synechococcus sp. PCC 7942 revealed that bentazone interacted with acceptor side of PS-II system (Bagchi et al. 2007). Narusaka et al. (1998) suggested that herbicide atrazine acted on binding site of D1 protein in cyanobacterium Synechocystis sp. PCC 6803. Monosulfuron exhibited its mode of action on acetolactate synthase enzyme in cyanobacteria (Shen et al. 2009). Terbutryn was found to bind via two hydrogen bonds to QB site in the D1 subunit of PS-II in thermophilic cyanobacterium Thermosynechococcus elongatus (Broser et al. 2011). The concentration-dependent increase in generation time indicated herbicide affected cell division of Desmonostoc muscorum PUPCCC 405.10. (Inset Fig. 3). Cyanobacteria use nitrate, nitrite and/or ammonium as nitrogen source for growth and development, though many cyanobacteria, especially the heterocystous forms, are also able to fix atmospheric nitrogen (Flores and Herrero 2005). Studies on nitrogen assimilation revealed 33–73 %

Table 4 Activity of nitrogen-assimilating enzymes of Desmonostoc muscorum PUPCCC 405.10 in presence of pretilachlor

Fig. 5 Lineweaver–Burk plot for nitrate uptake by Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor. Asterisk lmol nitrate taken up mg-1 protein h-1

7.97 ± 0.3 (42.66)

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10 ppm


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Fig. 6 Lineweaver–Burk plot for NR activity of Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor. Asterisk nmol nitrite formed mg-1 protein min-1

Fig. 7 Lineweaver–Burk plot for nitrite uptake by Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor. Asterisk lmol nitrite taken up mg-1 protein h-1

decrease in the uptake rate of nitrate and nitrite by Desmonostoc muscorum in the presence of pretilachlor (Table 3). The unchanged Km value for nitrate and nitrite uptake by pretilachlor-supplemented cultures indicated that the herbicide did not affect the affinity of uptake systems of the cyanobacterium toward nitrate and nitrite. Nitrate and nitrite uptake by cyanobacteria is light dependent (Flores et al. 1983). The decreased Vmax for pretilachlor-containing cultures might be due to interference with photosynthetically generated energy. Decrease in NR and NiR activity of the test organism in pretilachlor-supplemented cultures was

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Fig. 8 Lineweaver–Burk plot for NiR activity of Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor. Asterisk nmol nitrite decreased mg-1 protein min-1

Fig. 9 Lineweaver–Burk plot for ammonium uptake by Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor. Asterisk lmol ammoniu taken up mg-1 protein h-1

only 15–18 % (Table 4). It appears that herbicide did not affect these enzymes directly but the decrease in activity may be due to less availability of substrate or may be due to inhibition in production of photosynthetically reduced ferredoxin which is electron donor for nitrate reduction in cyanobacteria (Flores et al. 2005). The progressive decrease in NR and NiR activity in N. muscorum by carbaryl (5–50 ppm), in Anabaena. fertilissima, Aulosira fertilissima and Westiellopsis prolifica by endosulfan (3–12 ppm), in Anabaena by trichlorfon and tebuconazole has been reported (Marco and Orus 1993; Bhunia et al. 1994; Kumar et al. 2012).


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Fig. 10 Lineweaver–Burk plot for GS activity of Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor. Asterisk lmol glutamate hydroxamate formed mg-1 protein min-1

Ammonium uptake by Desmonostoc muscorum was also significantly affected by pretilachlor in a dose-dependent manner (Table 3). The kinetics of ammonium uptake revealed a decrease of 66 % in both Vmax and Km in herbicide-supplemented cultures (Fig. 9). These observations indicated that the herbicide affected the affinity of ammonium uptake system. It has been demonstrated that ammonium uptake in cyanobacteria is a membrane potential-driven transport process (Montesinos et al. 1998). Ammonia produced by the action of NR and NiR or taken up directly is assimilated by the activity of glutamine synthetase (GS) through GS-GOGAT system (Flores and Herrero 2005). The GS activity of the test organism decreased by 21 % in 10 ppm pretilachlor-supplemented culture compared to control culture (Table 4). This decrease may be correlated with the less enzymatic reduction of nitrate/nitrite within the cells or slow ammonium uptake rather than the direct toxic effects of pretilachlor on the enzyme glutamine synthetase. The decrease in GS activity by other pesticides in cyanobacteria has been reported (Bhunia et al. 1994; Jha and Mishra 2005; Singh et al. 2012). The kinetic studies on GS revealed decreased Table 5 Heterocyst frequency (%) of Desmonostoc muscorum PUPCCC 405.10 in the presence of pretilachlor

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Vmax and unchanged Km in the presence of pretilachlor indicating non-competitive interaction of pretilachlor with GS in this cyanobacterium. Since Desmonostoc muscorum PUPCCC 405.10 is nitrogen fixer, the effect of pretilachlor on nitrogen fixing capacity of the test organism was also studied. Pretilachlor at 10 ppm decreased nitrogenase activity of the organism by 48 % within 6 h with no further significant decrease in activity up to 24 h (Table 4). However, within 24 h there was no significant change in heterocyst frequency in presence of pretilachlor. A decrease of 20.6 and 40.5 % in heterocyst frequency was observed in presence of the herbicide on day 2 and day 4, respectively (Table 5). It indicated that pretilachlor initially affected nitrogen fixation by the organism rather than heterocysts. The effect of herbicide on nitrogen fixation may be due to direct interaction with nitrogenase or indirectly through the effect on photosynthesis since nitrogen fixation in cyanobacteria has been shown to be dependent upon photosynthesis (Flores and Herrero 2005). This needs further investigation. Heterocyst development depends upon algal growth, thus herbicide inhibiting growth of the organism also inhibits heterocyst formation (Ahluwalia and Dahuja 1997). Pesticides such as bensulfuron-methyl, carbaryl, carbofuran, chlorpyrifos and endosulfan have been reported to affect nitrogen fixation in cyanobacteria (Bhunia et al. 1994; Jha and Mishra 2005; Kim and Lee 2006). Nitrogen-fixing cyanobacteria act as natural biofertilizer leading to improved crop production since they increase both carbon and nitrogen status of soil (Lange et al. 1994). A number of reports are available on use of Nostoc as biofertilizer for improving the yield of crop plants. Nostoc rivulare in association with wheat roots significantly improved wheat growth (El-Shahed 2005; El-Shahed and Abdel-Wahab 2006). Application of 20–40 % less fertilizer than recommended dose combined with cyanobacterial inoculum (1 kg ha-1) containing Nostoc spp. and other species of cyanobacteria significantly improved the yield of rice crop (Begam et al. 2011). In pot experiments, biofertilization by Nostoc muscorum and Nostoc rivulare

Days

Control

5 ppm

7.5 ppm

10 ppm

0

5.38 ± 0.12a

5.38 ± 0.12a

5.38 ± 0.12a

5.38 ± 0.12a

2

6.3 ± 0.10

6.08 ± 0.10 (3.49)

6.0 ± 0.13 (4.76)

5.0 ± 0.12 (20.63)

4

6.9 ± 0.14

6.4 ± 0.13 (7.4)

6.0 ± 0.15 (13.0)

4.1 ± 0.10 (40.5)

8

8.7 ± 0.15

8.11 ± 0.16 (6.78)

7.0 ± 0.19 (19.5)

5.32 ± 0.11 (38.85)

12

9.2 ± 0.15

8.4 ± 0.17 (8.7)

8.2 ± 0.17 (10.9)

6.08 ± 0.15 (33.9)

Data in parentheses indicate percent inhibition of heterocyst frequency All data presented are the mean values of three independent experiments ±SD Data in each row with same lower case letters are not significantly different from each other at the 95 % confidence level (p \ 0.05)

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significantly increased shoot length, leaf size and spike weight of maize and wheat when applied alone or in combination with N fertilizer (Fadl-allah et al. 2010; Sholkamy et al. 2012). Desmonostoc muscorum tolerated herbicide up to 10 ppm which is 13.3 times the recommended field dose, thus it can become a part of biofertilizer program in Punjab state of India.

Conclusion Higher concentrations of pretilachlor affected N uptake and assimilation by the Desmonostoc muscorum. Data on kinetics of nitrogen uptake suggested that pretilachlor did not change the affinity of nitrate and nitrite uptake systems but influx of nitrate and nitrite significantly changed. NR, NiR and GS were not affected directly but low activities of these enzymes were observed due to the reduced uptake of nitrate/nitrite and ammonia. There is a need to study the interaction of herbicide with membrane-bound nitrogen uptake system of the cyanobacterium. The herbicide also affected the development of heterocysts. Thus, pretilachlor affected N assimilation at uptake level rather than reduction level. Since pretilachlor at 2.5 ppm which is 3.3 times than recommended dose of field application did not affect significantly N assimilation by Desmonostoc muscorum, it can be a component of cyanobacterial biofertilizer in pretilachlor-applied rice crop. Author contribution statement Conceived and designed the experiments: D.P. Singh and J.I.S. Khattar. Performed the experiments: Gurdeep Kaur, Meenu Gupta, Yadvinder Singh. Analyzed the data: D.P. Singh, J.I.S. Khattar and Arvind Gulati. Contributed reagents: D.P. Singh, J.I.S. Khattar, and Arvind Gulati. Materials/analysis tools: D.P. Singh, J.I.S. Khattar and Arvind Gulati. Wrote the paper: Gurdeep Kaur, Meenu Gupta and Yadvinder Singh. Edited and corrected the ms: DP, JIS and AG. Acknowledgments The authors thank Head and Coordinator, DRS SAP-II of UGC, FIST of DST, Department of Botany, Punjabi University, Patiala, for infrastructure and laboratory facilities. Authors also acknowledge the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur, for providing laboratory facilities for molecular biology work. The authors are grateful to Prof. Radha Prasanna, Department of Microbiology, IARI, New Delhi for acetylene reduction assay.

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